O. J. EiGST!
Pierre DUSTIN,
By :-;-i^Ari;.'.^i-ii:;;-
In Agriculture, Medicine, Biology, and Chemisty
{:i;i:;;.;!'::!r<;';ti':i:-ffl''H'-wif'i!!H:f;'g::iK:i;i-f;iiiiot
:-:'.:-b[^:t':^-: x-%---<:^i-\;\'
Colchicine —
in Agriculture, Medicine,
Biology, and Chemistry
Colchicine
in Agriculture
Medicine
Biology
and Chemistry
O. J. EIGSTI, PhD.
Colchicine Research Foundation, Inc.
Normal, Illinois, U. S. A.
Pierre DUSTIN, Jr., md.
Department of Pathology
University of Brussels, Belgium
I > C l» The Iowa State College Press, Ames, lowa^, [T. S.A..
~~\
All rights resemed. Composed and printed by
The loica State College Press, Ames, loica. U.S.A.
Copyright. /9ii, by The loica State College Press.
Library of Congress Catalog Card Xuniber: 54-7657
To tlie mefnory of Albert Pierre Dust in, i88^-ip^2,
whose concepts concerning the regulation of mitotic
activity prepared a foundation for the broad scope of
biological research t/iat folloiued the rediscovery in
^934 "^ t^>(^ effects of colchicine upon mitosis.
■ v
Preface
\\aien an American botanist and a Belgian pathologist collaborate
in writing a book, the obstacles to be encountered are necessarily
numerous, and this is true of the present work even though the subject
is limited to the single substance, colchicine. Our collaboration has
required intercontinental travel, hours spent together in discussing
factual materials from plant and animal sciences, countless days
assembling a vast bibliography.
Finally, our cooperative project made it necessary to overcome
barriers inherent in our widely different research fields, to resolve
problems arising from the use of different languages, and to recognize
the dissimilar perspectives of the American and European educational
systems. But a common ground of interest was maintained, irrespec-
tive of personal interests, through a constant realization of the re-
markable and singular properties of colchicine as a mitotic poison
and as a tool for experimental work. Moreover, research programs in
mitotic problems which each of us had developed prior to the work
with colchicine provided a basis of mutual interest.
This work actually had two beginnings when in 1912, almost
simultaneously, two scientists commenced manuscripts, each without
knowledge of the other. One of them was A. P. Dustin, Sr., of Brussels,
whose untimely death occurred in the \ear his review was started.
The task of completing this study fortunately passed to Dr. Dustin's
son, and in 1917 the botanical writing done in America by the senior
author and the medical studies under way in Europe were brought
together into one joint project. It was decided to integrate the many
lines of research with colchicine into one study. Ihis book is the
result of that cooperative effort.
A survey of the chapters comprising this study will indicate the
many lines of research that have been included. The modern litera-
tme on colchicine is vast. The references to gout alone would require
LviiJ
viii Preface
pages. Rather than catalog titles, we have brought together significant
contributions and have attempted to correlate the various lines of
research. Whenever possible, we summarize the basic contribution,
point out differences of opinion, and, most important, call attention
to work that needs to be accomplished. Finally, in retrospect over the
modern period of studies of colchicine, one of our purposes has been
to point out the progress made, rather than to predict what is to come.
For the shortcomings, the errors of interpretation, statements of
viewpoints not pleasing to all specialists, which ma) be found in any
portion of this book, the authors assume full responsibility. We who
have assembled as many as possible of the important facts about col-
chicine welcome corrections and comments concerning the conclusions
which we have reached.
The modern period of research with colchicine began in 1889,
when Pernice described metaphasic arrest produced by this drug.
Until Pernice's report was rediscovered, Dixon and Maiden were cited
as the pioneers. Thus, our search for all references to colchicine was
rewarded. Special recognition is due to Nancy Gay-Winn, whose
diligent cjuest led to this classic work by Pernice.
Colchicine in its present role as a mitotic poison and as a tool
for biological research was discovered in 1931 at Brussels, Belgium,
in the laboratory of Professor A. P. Dustin, Sr.. who for a long time
had been investigating means of altering mitosis. WHien colchicine
was suggested by a Brussels medical student, F. Fits, the characteristics
of colchicine were quickly measured. Our review covers the period
from 1934 to the middle 1950's.
In 1937 botanical research began in several countries, generally
following descriptions or reports of unusual observations from animal
cells. In this same year, the scientists at Brussels included Alliu?n root
tips for their tests. Other botanists chose Alliirni root tips or plant
materials to illustrate the action of colchicine. In this year the role of
colchicine as an agent for the induction of |:)ohploid\ was conclu-
sively demonstrated.
The horizons of colchicine research widened quickly when bota-
nists learned how effectively the drug could be used in their work.
Laymen became interested in the drug as references to cancer entered
the discussions and as the creation of new varieties of plants stimulated
new programs in agriculture. A broad scope of research was opened
up by this single substance.
Organic chemists realized that Windaus' concept of the structural
formula for colchicine needed revision. In 1940 definite evidence was
at hand. 1 here followed an unusually large \olume of research on
Preface ix
the chemistry of colchicine. In 1947 we realized the need for specialized
help. Fortunately, Dr. James D. Loudon of Glasgow University,
Scotland, who worked with the group that began the revision of col-
chicine structure, generously contributed to this aspect of the study.
We express our gratitude to him for the writing of Chapter 6.
Colchicum, which is a drug plant of antiquity, has a long history
in the annals of pharmacy. Professor F. Santavy of the Medical Insti-
tute of Olomouc, Czechoslovakia, pro\ided special materials for
Chapter 5. Many facts about the pharmacognosy of Colchicum were
compiled by Mr. Ikram Hassan of the University of Panjab, Lahore,
Pakistan. We appreciate their special aid in the preparation of
Chapter 5.
However, the authors, and not the contributors mentioned, assume
full responsibility for the material published. We are gi^ateful for
help from our jniblishers, the Iowa State College Press, and particu-
larly its Chief Editor. Mr. AVilliam H. Van Horn.
Financial aid is necessary for a project of this proportion not
designed specifically for return of investment. We have received
support from organizations whose contributions were made without
consideration of a future financial return.
Some grants-in-aid were made to each author and some jointly
to this project. Without citing specific contributions it is our pleasure
to acknowledge with thanks the following organizations, foundations,
and agencies providing funds. But quite as important as the financial
aid. ha\e been the approval and encouragement given to us in our
efforts.
These contributors are listed herewith: Carnegie Corporation of
New York, Century Fund, Northwestern University, Colchicine Re-
search Foundation, Fonds National de la Recherche Scientifique (Brus-
sels) , Funk Brothers Seed Company, Genetics Society of America,
General Biological Suj^ply House, Graduate Committees on Research
of the University of Oklahoma and Northwestern University, John
Crerar Library, Lady Tata Memorial Fund, National Cancer Institute
of the National Institute of Health, U. S. A., Rosenheim Foundation,
Pakistan, United States Educational Foundation, Pakistan, United
States Educational Foundation, India, United Nations Educational
and Scientific Organization, University of Oklahoma Research Insti-
tute, University of Oklahoma, Department of Plant Sciences, Univer-
site libre de Bruxelles, Faculte de Medecine, Belgium.
Contributions in preparing the manuscript were made during the
course of our work. For illustrations, photographs, typing, photo-
micrography, bibliography, and reference work we express our thanks.
X Preface
C. A. Berger, A. M. Brues, Joseph Carlson, George L. Cross, Agnes
W. Eigsti, M. Fauconnier, M. E. Gaulden, Tilman Johnson, H. Kihara,
Carol S. Lems, A. Lonert, E. Lotens, Marjorie Lindholm, Elizabeth
McKee, Portia M. Mercier, Leona Schnell, Barbara Tenney Sherman,
Marselda Scarff, Harvey Smith, Herbert Taylor, Atlee S. Tracy, Ruth
VV^itkus, Vera Williamson, Nancy Gay-Winn.
Scientists around the world gave us unpublished materials, refer-
ences, and specific aid toward the manuscript. We acknowledge the
help of the following: John Beal, C. A. Berger, P. Bhaduri, Muriel
Bradley, James Brewbaker, Max E. Britton, Meta S. Brown, A. M.
Brues, Otto Bucher, Joseph Carlson, Belayet H. Choudhury, Jens
Clausen, J. W. Cook, Geo. H. Conant, Alan Conger, Geo. L. Cross,
George Darrow, Haig Dermen, Sam Emsweller, Rob't. K. Enders, K.
Frandsen, D. U. Gardner, Mary E. Gaulden, Pierre Gavaudan, C. J.
Gorter, Ake Gustafson, A. Hecht, E. K. Ammal Janaki, Tilman John-
son, A. Josefson, Theo Just, H. Kihara, Peo Koller, Ernest Lahr, Hans
Lettre, Albert Levan, S. Lodhi, James Loudon, P. Maheswari, G. P.
Majumdar, Ralph G. Meader, Arne Muntzing, A. Mohajir, B. R.
Nebel, Fredrich Nilsson, I. Nishiyama, Gosta Olsson, Joseph O'Mara,
Gunar Ostergren, B. Pal, Barbara Palser, Joseph Peters, S. Ramanu-
jam, F. Ramirez, M. L. Ruttle, Leona Schnell, E. R. Sears, Paul
Sentein, Barbara Tenney Sherman, H. Shimamura, H. Slizynska, B.
Slizynski, Harold H. Smith, Paul F. Smith, Leon Snyder, Leon Steele,
G. Ledyard Stebbins, Jr., S. G. Stephens, Robert N. Stewart, R. R.
Stewart, Betty Thomson, Geo. Tischler, Paul Voth, B. Wada, Hanford
Tiffany, L E. Jeffs, S. J. Wellensiek, M. Westergaard.
O. J. Eigsti
Pierre Dustin, Jr.
October, 1954
Table of Contents
1 . The Parent Plant 1
1.1: The Knowledge of Colchicum in Ancient Civilizations 1
1.2: Botanical Studies of Colchicum From Dioscorides to
rwentieth-Century Investigators 4
1.3: Medical Applications of Colchicine 11
1.4: Chemical Studies of the Pure Substance Colchicine 14
1.5: New Biological Uses for Colchicine 16
2. Nucleus and Chromosomes 24
2.1 : Original Concepts 24
2.2: The Original Statements 26
2.3: Prophase 31
2.4: Colchicine Melaphase 35
2.5: Processes Leading to Interphase 50
2.6: Alterations of Chromosome Structure 52
2.7: Reiteration of the C-mitosis 55
3. Spindle and Cytoplasm 65
3.1 : Colchicine and Spindle Fibers 65
3.2: Spindle Inhibition 68
3.3: Destruction of the Spindle Fibers 69
3.1: Changes in Spindle Form 78
3.5: The Arrested Metaphase and Spindle Mechanisms 81
3.6: Spindle Disturbance and Cytological Standards 86
3.7: Cytoplasmic Division 86
3.8: Reversible Characteristics of the Spindle 91
3.9: Summary 98
4. Cellular Growth 102
4.1 : Colchicine Tumors in Roots, Hypocotyl, and Stems 103
4.2: Effects of Colchicine on Pollen Tubes, Hair Cells,
and Other Parts of Plants 107
4.3: Colchicine-Meiosis and Gametophytic Development 110
4.4: Microbiological Data '-0
4.5: Differentiation Processes 125j-v
4.6: Metabolism and Colchicine 131
[xi]
71036
xii Table of Contents
5. Sources of the Drug 140
5.1 : Scope of Study 140
5.2: Problems in Pharmacognosy 141
5.3: Plants Containing Ciolchicine 141
5.4: Cultivation, Collection, and Preparation 150
5.5: The Crude Drug 151
5.6: Compounds Isolated From Coldiicuin 153
6. Chemistry 159
6.1: Extraction and Ccneral Properties 159
6.2: The Functional Groups 160
6.3: The Structural Problem 161
6.4: Comparison AV'ith Tropolones 168
6.5: Structure of Colchicine 169
6.6: Miscellany 169
7. Pharmacology 175
7.1: Colchicine in Medical Therapeutics and Forensic Practice .175
7.2: Colchicine Poisoning in Man 176
7.3: Disturbances Unrelated to Mitotic Poisoning 178
7.4: Disturbances Possibly Related to Mitotic Poisoning 183
7.5: Nonspecific Toxic Changes 190
7.6: Metabolism of Colchicine 194
7.7: The Treatment of Gout 196
8. Embryonic Growth in Animals 202
8.1: Action on Gonads and Early Development 202
8.2: Colchicine-induced Malfoniiations 206
8.3: A Tool for the Study of Embryonic Growth 209
9. Experimental Growth in Animals 214
9.1: Endocrinological Research 214
9.2: Theoretical Considerations 216
9.3: Cellular Multiplication in Normal Growth 219
9.4: Hormone-stimulated Growth 224
9.5: Regeneration and Hypertrophy 236
9.6: Wound Healing 246
9.7: The Action of Chemicals on Mitotic Growth 247
10. Neoplastic Growths — in Animals and Plants 255
10.1: Colchicine in Cancer Research 255
10.2: Experimental Study of Neoplastic Cells 258
10.3: Cancer Chemotherapy 260
10.4: Chemotherapy of Human Neoplasms 263
10.5: A Tool for the Study of Cancer Chemotherapy 265
10.6: Plant Tumors ' 265
10.7: Colchicine and X-rays Associated 266
10.8: The Study of Carcinogenesis 269
1 1. The Experimental Polyploids 274
11.1: 1937 — Beginning of a New Era in Polyploidv 274
1 1 .2: Terminology [ 276
11.3: Catachsmic Origin of Species 277
Table of Contents xiii
11.4: Classification of Polyploids 280
1 1.5: Principles of Polyploid Breeding 282
1 1 .6: The Scope of Research 286
12. The Amphiploids 292
12.1: Amphiploidy and Im|)licaiions 292
12.2: Amphiploidy in the Graiiiincae 294
12.3: Gossypium ' 302
1 2.4 : Nicotiaua 307
12.5: Dysploidv Combined With .Amphiploidy 309
12.fi: Other Interspecific Hybrids and Amphiploids 310
13. The Autoploids 318
13.1: Autotetraploids 318
13.2: Triploidy 326
13.3: Monoploids and Autodiploids 333
13.4: Conclusion 334
14. The Aneuploids 345
14.1: Aneuploids Among the Treated Generation 345
14.2: Mixoploidy From Colchicine 347
14.3: Chimeras Induced by Colchicine 348
14.4: Sex Determination and Polyploidy 351
14.5: Aneuploids and Colchicine 354
15. Criteria for Judging Polyploidy 362
15.1: Sterile Hylirids Made Fertile 362
15.2: Appearance of Polyploids 363
15.3: Fruit and Seed . . ! 363
15.4: Physiological Differences 367
15.5: Microscopic Characteristics 368
15.6: Ecological Considerations 370
15.7: Fertility 371
16. Techniques of Colchicine Treatment 373
A. In Animals 373
16A.1: Solutions 373
16A.2: Temperature 374
16A.3: The Study of Mitosis 374
16A.4: Polyploidy 380
B. In Plants 383
16B.1 : Solutions Used 383
16B.2: Seed and Seedlings 384
16B.3: Root Systems and Special Structures 384
16B.4: Special Techniques for Studying the .\ction of Colchicine .385
16B.5: Chromosome Studies 386
17. Mechanism of Colchicine-Mitosis 391
17.1: Introduction 391
17.2: Metabolic .\ctions of Colchicine 396
17.3: Physical Action 399
17.4: Chemical Action ^03
17.5: Synergists and .Antagonists 116
17.6: Conclusion: The Singularity of Colchicine 420
Author Index 429
Subject Index 441
CHAPTER 1
The Parent Plant
1.1: The Knowledge of Colchicum in Ancient Civilizations
The history of Cvlcliiciim, the drug of ancient and modern materia
medica, is rooted in the myths and the written records of ancient
Egypt, India, and Greece, and runs its course through the ages into
the world of today. Not only do modern formularies admit Colchi-
cum, the producer of the pure substance colchicine, but this plant is
probably one of those mentioned in the Ebers Papyrus. This Egyp-
tian document was prepared al:)out 1550 b.c.^ and is our oldest medical
text. Colchician could be one of the saffron plants of the Papyrus.
From this early age through thirty-five centuries of medical history to
the compilation of the modern pharmacopeias, very few drug plants
have survived. In fact, only eighteen, among seven hundred plants^^
originally listed as material for ancient Egyptian practitioners,
achieved such historical fame.
The Egyptian civilization developed a code for practicing medi-
cine in which plant products played an important role, and the Ebers
Papyrus summarized this accinnulation of knowledge. Egyptian doc-
tors were advised in the Papyrus to give various seeds to their patients
for relief from aches and pains. The seeds were administered on
bread. ^ While pure colchicine was not given in these doses, we can
assume that the drug was used in treating rheumatism and gout, ail-
ments which then and even yet afflict the human race. It is probable
also that, if seeds were used, a large quantity would have been ad-
ministered to the patient.
A danger associated with using colchicine in the crude form is
the poisonous projicrty of the drug. Enough active substance can be
given to cause death in warm-blooded animals. Dry seeds may have
as much as four parts of the drug j^er thousand of dry raw material.
Perhaps some patients died from the colchicine prescription, for
severe piuiishments were said to be meted out to ancient doctors when
a patient succumbed. In some instances the jjhysician even paid with
[1]
2 Colchicine
his life.-^ Since gout and rheumatism were common aihiients among
the noble and the wealthy, the attending physicians, who were often
servants of the court, must have held a rather precarious position.
Yet, in spite of its poisonous natvne, ColcJiicum in correct dosage was
capable of relieving pain if administered as seed, powdered corm,- or
even dried flowers. It is probable that substitutes for Colcliicmn, as
well as similar plants containing very small amounts of colchicine,
were employed.
Plants were frequently used in ancient days without sound basis,
and more magic than medicine was practiced; in fact, magic and the
medicine man have been associated through the ages. Our modern
word pharynacy originates-^ from an Egyptian term pharmaki and the
Greek pharmakon. These terms are in turn related to another Egyp-
tian word pharmagia, which means the art of making magic.
Another civilization, the Hindu, developed a medical system inde-
pendent of the Egyptian and the Babvlonian. This period is known
as the Vedic,-'"' and extends from 2U()U b.c. to 800 b.c. Much informa-
tion about treating diseases with plants is transmitted in the Vedic
text.--' Although in this book specific plants are mentioned and cer-
tain diseases noted, and while Colchicum luteum, a producer of pure
colchicine, is common in the Indus River area of the Himalayas, the
present Indian ColcJiicum cannot be deciphered from this book.
At some time during the Vedic period a traffic in drugs was estab-
lished between the Orient and Arabia. Good evidence is at hand to
show that Hindu medicine had an influence upon Arabian medical
knowledge. There was a serious decline in Hindu medicine, but the
traffic in drugs continued. This exchange reached such proportions
that Pliny the Elder complained about his money being drained to
the Orient for drugs. Two species, known as the Kashmir hermodac-
tyls,''' could have been among these drugs. They are identified as
ColcJiicum Juteum and Merendera persica. Although both contain
colchicine, the respective quantities diff^er markedly, as will be de-
scribed later.
Botanical historians-^ tell of an ancient class in Greece known as
the Rhi/oiomi, or root gatherers. They were pharmacobotanists prac-
ticing their art in the pre-Hippocratic era; their powers resembled
those of inagicians, associating all manner of ritual with the collec-
tion, preparation, and dispensing of roots. Such details as the wind
direction, time, season, as well as astronomical signs were observed.
Since foods were primarily grain and leaves, the roots must have
served other purposes such as medicine. Driving away evil spirits
that caused disease may have been helped by using underground plant
parts, and the trade in roots by the Rhizotomi flourished.-^
More than fifty species containing colchicine are native to the
region where the Rhizotomi practiced. ^^ The most notable species is
The Parent Plant 3
Col( hinini aiit iniiiKile.^'^ tlial )jroduces flowers in autmuii followed by
leaves, triiits, and seeds the next spring. Siuli an unusual habit must
have attracted these pharniacobotanists.-^
Perhaps the best link between ancient and modern medicine is
seen in the two drugs tound in Oriental ba/aars: the Surinjan-i-talkh
and the Sininjan-i-chirrin." These corms are distinguished as bitter
and sweet surinjan and are obtained from the Kashmir hermodactyls
growing in the northwest Himalayan foothills.' Botanically the drugs
are identified as (1) Colchicum luteum. the bitter, and (2) Meren-
dera persica, the sweet; both contain colchicine, 0.2 per cent and 0.02
per cent, respectively.-^" Pharmacists advise their use for rheumatism
as well as for aching joints.
If these same hermodactyls entered the drug trade from the Orient
to Arabia, then early Arabian physicians may have borrowed their
ideas for treating gout from this source. It is difficult to determine
how many centuries have passed since the Hindu specialists began
collecting the hermodactyls and other plants useful in medical prac-
tice. But their knowledge of herbs has been handed down for count-
less generations to their successors of the jjresent day.
The ancient usage of ColcJiicum. along with an antiqiuty in medi-
cine, can be established through several somces: the Ebers Papyrus,
a drug traffic from the Orient, and the evidence about a pharmaco-
botanical trade practiced by the Rhizotomi. Present-day surinjan
may link the past to modern medicine.
Our discussion of the knowledge of Colchicum in the ancient
world turns for a moment to Greek history and mythology, and it is
in Greece that the jjeriod we are examining will close with the or-
ganization of medical knowledge aroiuid the system of Hippocrates.
Colchicinii is named for the land of Golchis at the eastern tip of
the Black Sea.^'- -- In this area the plants are most abundant. When
Colchis was mentioned to the Greek, visions of sorcery immediately
arose. This was the land where Jason secured the Golden Fleece.
Here he met the sorceress Medea, famous for her powerfid life-giving
brews. She was said to have rejuvenated Jason's aging father by sub-
stituting a special potent mixtine for his blood. Many of her direc-
tions for poisonous mixtines recjuired iniderground roots. Magic
powers were associated with these ingredients that figured in Medea's
sorcery.^'
Among the instruc lions for making a certain mixture were specilic
details for collecting the poisonous plants.'' In one instance, only
during a hoarfrost could roots be dug. While boiling the juices in
a pot, it was said olive branches touching the brew woidd immediately
bring forth flowers and fruits.
The ancient Colchian kings had gardens containing ])()is()nous
species. Undoubtedly the knowledge of the toxic projjerties of jilants
4
Colchicine
was at their disposal. Such phints might have served their intrigues
and provided means lor the elimination ot competitors or persons
convicted of crime.
1.2: Botanical Studies of Colchicum From Dioscorides to Twentieth-
Century Investigators
In the land of Colchis, along the Black Sea, an autumn-flowering
crocus-like plant occurs in abundance (Fig. 1.1). Dioscorides, first
century botanist-physician, knew about this particular species from
either personal observations in the area or through reports by travel-
ers to this region. This fall-blooming meadow saffron was named the
Fig. 1.1 — Flowers of Colchicum autumnale showing only the floral parts above ground.
(Photograph, courtesy of General Biological Supply House, Chicago, III.)
The Parent Plant 5
Colchiconr- a name which has been continued in its Latinized form
to the present time.
Dioscorides made very carefid descriptions dealing with such
phases as growth, development, and morphology of the plant. His
drawings involving two plants (Fig. 1.2), one with fruits, seeds, and
leaves, the other with flowers only, clearly show that he associated
2p2 PcdaciiDiofcoridij'5ttrt<fi?95U(^/
Fig. 1.2 — Diagrams showing the seed-producing portion of Colchicum autumnale, and the
flower stalk appearing in autumn. A, fruiting; B, flowering. (After drawings by Dioscorides)
autunnial flowering with sjjring fruiting, both having the same under-
ground portion. This was a careful scientific observation for his day.
Such great detail was gi\'en to the corm, bud, leaf, flower, and seed
that writers copied his observations and drawings for the next fifteen
centuries.
Since the botanical and medical professions were closely allied in
the times of Dioscorides, it was natural that the ()l)jccti\e of his study
6 Colchicine
should extend beyond strictly botanical descriptions and that his
primary interest should be in the medical ajjplication of plants. He
warned that Colchicon was a dangerous poison and compared it with
the mushroom that causes death (Fig. 1.3) . He was concerned that
this plant might be used by practitioners unaware of its poisonous
nature, and the effect of his careful descriptions and stern warnings
^vas so profound that many followers avoided the use of Colchicon.
5pcrbftb(umcn/ ^pinubdimcil/ Colchicon, Buibus
Agrcftis. <Sa\>. (vrjf.
Cv> "p niiMujticn/t^.KhfHumcn/ /:»cib(IMumm / &\wfnf(h Colchicon, ^u^.Ufiii^^J^'^"''
^Bulbus AgreftiSjfiiiPttvctiikcbtc ^SMiimai/ Dni tovJ)fi\?n^2»lattfrnc^n(:fl)/viiiiD
^■'latfcrDfr^^urpaii/ Dteman@riccl)ircNnD;uiinc:n cngcntlicbBuIbosnatnt/ ciufiina
nommcnKif; ficfcn|1crfinDt : toKh.»bcn kotcn<^d ana (^r^p.inncn f\oci^imittohtm^M
mfn / robtlccfjfc iBumlnJ DjcbcflciDct finDe mu braumobt i atvas fchwarttfdrbi^cn Dvin?
ten I rrcnn miin t>ie Ovtnbc abtf^ut I fo fmDt Die ^urijdn tvaf; I ^art / fiifj / t>oUcr to jfff / jbre
5C!tri?c(f<iif m Dcr miftcanrincr (Socmen PonwnDcn auff cin.^frffoDcrDvii? / DiirPurcbbiC
«2«{umctvacI>(1onbauf;bnrf)t. ^cv S^xrbilblumm wc\ci^Unvi(l m ^(^cma vnb ^olib\i\
S)tcij3uri;clngc|Tcn/ tdDtmwicDiegifTttgctodjmamm/miftrur^npnDcrflccFcn.^Kfa^KM bre
Swut Kibftt trir aucf? aUctn Durumb bcfchncbcn / Pamit njcmjiiDt DJlTclbu^c / ohy |'(iiic'^"'"'^'i'''
^urpcltit?inri|Tcn(Ucb'jn f?vUt Dcr '^»ll(t>cnt^nll■(?dnc)Tc / Dcnnctlicf>c turch )hrc fuff^lfnt
ti^ir^u »rcrDcn ^rrcidt. ^tDcrbic(c6(iSi)ft bnnicbf ttiiin bcqucmiicb Die ,?(r«ncn/Du DicKn
UMDcr Pic gtfftige ©chrdmm bcfcf>ricbcti troitcn finPt /.^iibmilcb i(ltiucb jiut PiinriCici;
Qctruncfcn/ alfoDa^ mv<nfcmfrvnit»frn^(rpnci) bcDvirrf/u'i? ilnhmilcbvcrbviti5cni(^.
^:^b i; ^^?nv
Fig 13 — Dioscorides' description of Cokhicum taken from the Kraoterbuch of Pedanius
Dioscorides, printed by J. Bringern, Frankfurt, 1610. Reproductions obtained tnrough court-
esy of John Crerar tibrary, Chicago, III.
In spite of such warnings, Dioscorides believed plants were very
useful in the medical practice. Accordingly, other less poisonous
species were recommended. In one case he suggested the EpJiemeron
instead of the ColcJiicoii, particularly for those tumors that had not
yet spread into the body. The EplicDirron is now identified as Colchi-
cum linnulatutn. •*! which contains less colchicine than C. autum)wle,
the autvmui-flowering plant, his Colchicou."^' There can be no doubt
that his careful attention to species ditference distinguished him as a
great botanist.
The Greek physicians at the beginning of the Clhristian era de-
veloped a distrust for Oriental medicine, notably the plants that were
used in drug traffic.-- This suspicion had been aroused as early as
the time of Hipjioci ates. Perhaps diere was some basis for their
doubt. If our assumption was correct that Kashmir hermodactyls
were introduced into this drug traffic from the Orient to the West,
The Parent Plant 7
tlicn two \er\ similar thugs Avould have appeared. These arc C.olclii-
( uiii hiteuin and Merendera persica, which were described in the last
section. AVhile the alkaloid contents of these two plants differ con-
siderably, it is jMobable that then as now they were sold under the
name surinjau. A carclul worker like Diostorides would not have
been misled by these substitutions, but not all Cireek physicians were
skilled in distinguishing botanical specnnens. and they undoubtedly
appreciated the excellent services rendered by Dioscorides through
his botanical investigations.
In the tollowing fiiteen centuries, down through the period ol the
Herbalists, nothing dillerent was added to the description of Colchi-
coii. In fact, the Herbalists merely copied and repeated what Dios-
corides and several other botanists of his period had written.*" The
great contributions matle during the fifteenth to seventeenth centuries,
of coinse, were the translation, copying, and j^rinting which made
book production easier than at any previous period in history.
The Herbalists-- collected interesting names that became associ-
ated \vith dolclnc <))iJ' These ustially refer to the poisonous features
or to some unusual habit such as fall flowering and spring fruiting.
The plants were called "mort an chien," or "death to dogs.^' The
name "hit I bus arrest is." or "wild bidb," was commonlv used.^' Since
the flowers appeared in clusters out of the ground without leaves
associated, a descriptive name "naked ladies" was given. Probably
the most involved name was the Latin "Filiiis ante patre/n," trans-
lated "son before the father," meaning a deviation from established
biological laws.^' Ihis is imderstandable, for ^vhen they associated the
spring seeds and fruiting with the Hoovers that came up the same
year in autumn, several months later, it was an instance of the off-
spring preceding the parents. However, Dioscorides had made the
correct interpretation because his diagrams (Fig. 1.2) clearly associ-
ated buds, flowers, leaves, and fruits at the correct season and he
realized that the flowering plants of autumn put forth fruits the
next spring. Some Herbalists devoted much chscussion to the growth
habits involving flowering and fruiting. Finally, the common name
Hermodactyl caused confusion for a long time initil it was clearly
shown that the CoJchicoJi and Hcrtnoddciyl were the same plant. •^•*
Linnaeus kept the original name given by Dioscorides, changing
it from the Greek ColcJiicoji to Latin Colchiciim . when he devised his
extensive system of nomenclature. .\ binomial ailixed to the autunni
crocus was published in Species Pltintaruin. 1753: Colchicum aiiiimi-
nale L. The species describes the fall-flowering character, and the
genus retains the original reference to the land of Colchis. Very few
changes were made in descrijjtions as originally given by the Greek
botanist. Linnaeus m;ule an important contiibution in showing re-
8 Colchicine
lationships between the Colchicuni group and other iauiihes of
plants.*!
The genus Colchicum L. belongs to the tribe Colchiceae, which
also includes Merendera Ram., Bulbocodium L., and Synsiphoyi Regel.
This tribe is a part of the subfamily Melanthoideae. The family
Liliaceae shows many relationships to the species Colchictim; hence
their correct position is within the lily family. At one time the family
Colchicaceae was on the same level of importance that was given the
Liliaceae, but this became changed to the system listed above.
An excellent monograph*^ dealing with Colchicum was published
by Stefanoff in 1926. Considerable revision has been made and ten
new species have been added. The text is in Bidgarian, but the de-
scriptions and keys are printed in Latin, thus making this information
available to specialists of any nationality. Useful distribution maps
are attached to the monograph. ^^
The genus is divided into two subgenera:*^ (1) Archicolchicum
including seven sections, and (2) Eucolchicum with a single section.
An Indian species, C. Jtiteum Baker, official in the Indian Pharmaco-
poeia belongs to the first subgenus, whereas the most notable drug
species, C. autumnale L. is placed in the subgenus Eucolchicum. All
species belonging to the latter subgenus flower in the autumn, while
the members of the first subgenus have many members that bloom in
the spring.
A total of 64 species are described and extensively reviewed for
their geographical distribution. All belong to the Northern Hemis-
phere and are primarily indigenous to the Mediterranean region,
although many species range over Europe and North Africa and ex-
tend eastward into India along the northwestern Himalayan ranges.
Thirty-six species flower in the months of September to November.
Except for several unknown, the remaining twenty-five species bloom
during the spring, early in January, or late in June. These character-
istics are noted in the list of species given in Table 1.1.
Cytological investigations include eleven species for which exact
chromosomal determinations have been made.-"- ^'^ There is no evi-
dence that speciation has proceeded along a polyploidy series with
or without hybridization. In fact, the number for these at hand is
entirely heteroploid. No correlation exists between taxonomic posi-
tion and chromosome number. Certainly the diploid numbers rang-
ing from 36 to 54 are not exceptionally high. In light of the poly-
ploidizing effect of colchicine on many plant cells, the suggestion has
been made that perhaps within tliis group high numbers may be
found. Chapters 4 and 17 deal with this problem and show by re-
sistance to the drug how polyploidy could not be developed. Further-
more, there is no indication that other species of plants found in the
TABLE 1.1
The Genus Colchicum Linnaeus
(After Stefanoff)
Family: Liliaceae
Subfamily: Melanthoideae
Tribe: Colchiceae
!
Species Name
Authority
Flowering Date
Chromosome
Number
Subgenus 1. Archicolchicum:
In = 38
Section 1 . Luteae
C. luteum Baker Feb. -May
C. regelii Stef. Feb. -March
C. hissariciim Stef. .July
C. robustum Stef. Feb. -May
Section 2. Bulbocodiae
C. szovilsii F. M. Jan. -April
C. crocifolhim Boiss. Feb. -March
C. Jascicidare Boiss. Jan.— Feb.
C. Ubanoticiim Ehrenb. June
C. rtlchii R. Br. Nov.-Jan.
C. schimperi Janka Dec.
C. tauri Siehe Feb
C. serpentinum Woronow ap. not given
Mischenko
C. hydrophiliim Siehe May-June
C hirsutum Stef. April-May
C. nivale Boiss. et Huet April June
C. biebersteinii Rouy Feb. -March
C. davidovi Stef. Feb. -April
C. catacuzenium Heldr March-May
C. hungaricum Janka Dec. -April
C. doerjleri Hal Feb. -April
C. macedonicum Kosanin .June
C. triphvllum Kze March
C. kurdicum Stef. .June
C. caucasicum Spreng, March-May
C. sobolijirum Stef. Feb. -April
C. atticiim Sprun. Nov. -March
C. jordanknhim Stef. not given
C. sieheanum Hausskn. Sept.
C. procurrinx . Baker Oct.
Section 3. \'ernae
C. vernum Ker-Gawl. March-May
Section 4. Montanae
C. monlarium L. Sept. -Oct.
Section 5. Cupaniae
C. cupani Guss. Sept.— Dec.
C. psaridis Heldr. Sept. -Dec.
C. boissieri Orph. Sept.— Dec.
In = 54
(fotiliinicd on next jxii^t')
10 Colchicine
Tabk" 1 . 1 [continued)
Chromosome
Species Name Authority Flowering Date Number
Section 5. Cupaniae {continued)
C. pusillum Sieb. Oct.-Nov.
C. hiemale Freyn Dec. -Jan.
C. troodt Kotschy Oct.
C. steveni Kunth. Sept.-Jan.
C. parlatoris Orph. Aug.-Nov.
Section 6. Filifoliae
C. fili folium Stef. Oct.-Nov.
Section 7. Arenariae
C. arenarnim W. K. Sept. -Oct.
C. alpinum Lam. et DC. Aug.-Sept.
Subgenus 2. Eucoichicum:
Section 8. Aiitumnales
C. cursicum Baker Sept.
C. micranlhum Boiss. Sept.
C. borisii Stef. Aug.
C. umhrosum Stev. Aug.-Sept.
C. laetum Stev. Sept.
C. kotschyi Boiss. Aug.-Nov.
C. decaisnei Boiss. Oct.
C. neapoUtanum Ten. Aug.-Sept. 2« = 38
C. longifolium Cast. Aug.-Oct.
C. kochii Pari. Aug.-Sept.
C. lingidatum Boiss. et Sprun Sept. -Oct.
C. haynaldii Heuff. Sept. -Oct.
C. autumnale L. Aug.-Oct. 2n = 38
C. lusitanum Brot. Sept. -Nov.
C. tenorii Pari. Sept. 2n = 40
(C. byzanlium Ten.)
C. levied Janka Sept.
C. visianii Pari. Sept.
C. turicum Jka Aug.-Oct.
C vnriegatum L. Sept.-Oct. 2«=44
C. latifoUum S. S. Aug.-Oct. 2« = 54
C. speciosum Stev. Aug.-Oct. 2v=38
C. bivonae Guss. Sept.-Oct. 2/? = 36
regions where Colchic inn is abundant are unusually high in chromo-
.sonie numbers. This question was raised alter the cytological work re-
vealed an action on mitotic processes in plants.
Additional lelerences and details concerning the botanical fea-
tures ol the official di iig-producing species are given in Chapter 5.
The Parent Plant 1 1
1.3: Medical Applications of Colchicine
Hippocrates louiulccl modern medicine; lie swept away many
mystical concepts, introduced new explanations tor disease, and lelt
a profound inlluence upon the medical profession. About three oi"
four hundred drugs were kept in his materia medica, some of them
introduced from the East where he was a visitor. The ritual of magic
and charm was eliminated as much as possible, but his direct con-
tacts with Hindu medicine did leave impressions. He made no refer-
ence to a specific treatment for gout, although he was familiar with
the ailment called podagra'^'^ in various aspects. It is possible that the
bitter hermodactyls were a part of his materia medica.
A History of Plan is j)rej)ared by Theophrastus (.872?-285 r..c.) de-
scribed five hundred plants'" for medicinal use. This study marks a
new age. \\hich continued the advancement of medicine started by
Hippocrates. Gout was a familiar disease in Theophrastus' day, but
he does not record specifically the form of drug for treating the dif-
ficidty. However, Theophrastus gave stern warning that the bitter
hermodactyls were jjowerful poisons. There can he no doubt that
the practice of medicine was enlarged by the work of Theophrastus.
I he first materia medica with accurate descriptions was firmly
established by Dioscorides in the first century a.d. He showed an ac-
quaintance with the studies of Theophrastus and gave many new
details from his private observations that became useful to j>rac-
ticing doctors. Colchicon was very poisonous and in its place the
Ephemeyo)! was recommended for those "tumors" that had not yet
"spread into the body." This same plant, the Ephemeron, was advo-
cated by Galen in the second century a.d. The Colcliiciim treatment
for gout may have been advocated by Galen because the bitter hermo-
dactyls were listed in his materia medica and he was well acquainted
with gout. The heiinodactyls and Ephemeron are both members of
the Colchiciitn genus.
Aretaeus, the Cappadocian, contemporary with Galen, clearly
recognized podagra and ncjticed that many remedies were advocated.
He obser\ed innumerable remedies were suggested for gout; in fact,
this calamity usually made the jiatient "an expert druggist." ^•*
Many j^lants were dispensed from the pharmacist. In light of the
widespread distribution of colchicine-j)roduc ing sjiecies, a large selec-
tion might have been in the hands of the druggists.
About this same time, the "Doctrine of Signatures" was j>romoted
by Pliny, ^'■' who also made his mark upon medical thought. Plants
were chosen for a specific disease by means of suggestive associations.
For instance, saxifrages grew among rocks; iheielore kidney stones
12 Colchicine
could be dissolved by juices from this plant. Solomon's seal in cross
section ot the root looked like the King's seal; hence the plant
should be used to seal wounds. Perhaps gout, frequently attacking
the fingers, was treated by the hermodactyls since these flowers came
up like the fingers of a hand. Recalling that a translation of hermodac-
tyl means "fingers of Hermes," the doctrine woidd have provided
good basis for treating these ills and aches.
Emperors, rulers, and the wealthy were most frequently afflicted
with gout and arthritic rheumatism. One medical councilor, J.
Psychriste, who was attached to the court of the Byzantine rider Leon
the Great (457-475 a.d.) , used one single dose of bitter hermodactyl
to cure gout.i^ Doctors attached to riding classes found gout a preva-
lent disease among these personages, though specific directions for
curing gout have not been recognized in most historical records.
Colchicuni, or the bitter hermodactyls are usually mentioned as first
used in the sixth century.
Alexander of Trallcs (ca. 560 a.d.) has been credited as the first
to advocate fritter hermodactyP'* to alleviate the pains from gout. He
used a drastic purgative combining scammony, colcynth, aloes, hermo-
dactyls with anise, myrrh, peppers, cinnamon, and ginger. His twelve
books on medicine include many references to drug plants.
The seventh century physician, •'^'^ Paul of Aeginata, recommended
the hermodactyls when treating gout or other arthritic complaints.
His record is likewise well established by the medical historians.
Following him. two Arabian doctors, Rhazes and Avicenna, specifi-
cally proposed hermodactyls in cases of gout. The latter wrote from
traditional belief and personal experience about the "Souradjan"
from Arabia. Undoubtedly this is the same as the surinjan, or bitter
hermodactyl, Colchicum liiteum of the Indus River area. The com-
bined periods of Paul of Aeginata, Rhazes, and Avicenna extend from
the seventh century to 1037 a.d. The translations made by these
physicians included many documents dealing with science and medi-
cine,^'' and they exerted a profound influence upon medicine generally
as well as upon the specific knowledge passed on about gout.
An extensive treatise on gout dedicated to the Emperor Michael
Paleologus was prepared by a famous thirteenth century Greek physi-
cian, Demetrius Pepagomeus.'^^ In this account, specific directions
were stated for making a pill of hermodactyl, aloes, and cinnamon,
to be used in treating podagra.
From the thirteenth to the sixteenth century, records about gout
and drugs are scarce. Confusion embroiled the Greek doctors be-
cause of the widespread distrust for Arabian medicine and advice
from the East. Others suggest that the stern warnings noted about
the toxic property of Cohhicoii . beginning with Theophrastus and
The Parent Plant 73
Dioscorides, discouraged its uses. While reliel was obtained quickly,
the dangers associated with treatment were always present. As some
writers believe, the chance ol death was so great the gamble wasn't
"worth the candle."
A German writer, Wirtzimg (1500-1571) , revived interest in l)itter
hermodactyl by his discussions <jn treating gout, and about this time
joined in the call lor retinn to ColcJiicum as a treatment tor gout.-^'-'
Later John Quincy pid^lished a Complete EngJisJt Dispensatory and
called attention to hermodactyls, identifying these drugs with ColcJii-
cunt. Accordingly, the British iormularies carried both Hermodactyl
and Colchiciim in the 1618 edition. •*'• This practice was continued
in subsequent editions of the London Pharniacopoeia: 1627, 1632,
1639; but both j>lants were dropped in 1650. The omissions con-
tinued for 149 years— until 1788, when Colchicinn was admitted as
official. Hermodactyl was droj>j:)ed, never to be heard from again in
materia medica."''' This revival, after such a long period without
recognition, requires some explanation.
Without doid^t the renewal in the eighteenth century was largely
due to the thorough studies by Baron Anton von Storck^'^ (1731-
1803) . who experimented with Colchiciim in a Vienna hos]:)ital. His
own body was used for testing sensations as well as bodily changes
intluced by Colcliicinn. Students joined him in experiments that in-
volved rubbing the tongue with some of the drug to experience the
numbness, then recording the time necessary to render the tongue
"void of sensation."
Dr. \on Storck determined lethal doses for dogs, observing that
"two chams killed the animal in 13 hotirs." Post-mortem studies es-
tablished the changes induced t)y the drug, particularly among the
internal organs. These tests aided in formidating correct dosages such
as the oxymel colchici, used by many practitioners throughout Britain,
France, and Germany. Undoubtedly the place gained for Colchiciim
in materia medica by the middle eighteenth ccntiny ^vas a direct re-
sult of \on Storck's eifort.
While debates were going on as to the elficacy of Colchiciim,
Husson,-'-' a military officer in the pay of the French king, gave out
a vinous prej^aration called "Eau Medicinale," especially useful for
gout. The identity of the effective ingredient was kept secret, known
only to Husson. There arose quack preparations, i.e., Wilsons Tinc-
ture, Reynolds Specific, and others. Their true nature \vas always
kept secret, but an English pharmacist discovered in 181 1 that the
active ingredient in Husson's preparation was Colchiciim.
The combined research by I^r. von Storck and the popular suc-
cess achieved by the "Eau Medicinale" preparations established
Colchiciim in modern materia medica as a spetidc for gout.
14 Colchicine
During the latter eighteenth and beginning nineteenth centmies,
many English and French physicians wrote extensively about gout,
recommending Cohliic iini lor reliel. The great nineteenth century
doctor, Thomas Sydenham, who styled himself as the English Hippoc-
rates,^-' was a martyr to gout. He offered theories tor its natine and
cause, and advocated treatment with Colcliiciu)}. Another successful
student and physician was Alfred Baring Garrod, whose books^'-^* and
papers contained \aluable data about the changes indticed by gout.
In the nineteenth centiuy almost every prominent doctor with a
knowledge of gotit had a j^artictdar theory as to its origin and natme.
The forty-seven cases studied by Garrod are classic examples of soiuid
scientific investigation. Like others, he stood behind the Colchicum
treatment even though the poisonous nattue of this crtide drug was
well known.
An application of (olchicine reported in modern medical prac-
tice is the treatment of Hodgkin's disease in which instance remis-
sions were obtained.-'
1.4: Chemical Studies of the Pure Substance Colchicins
Accuracy in treating gout and in j^erforming critical experiments
demanded j)ure substances. Until the chemists' analysis and ex-
traction of crystalline compounds from corm and seed, only the crude
material was axailable to provide the active )jrincij)les in the drug.
A toxic principle invoh ing ptue colchicine was detected in substance
from Colchicum seed in 1(S2(),-^- but the compoiuid was confused with
veratrine. Later the name colchlciuc'^^' was jjroposed for a crystalline
material extracted by chemical procedures developed for this pin jiose.
Thus, the first steps were taken toward solving the problems in the
chemistry of colchicine. C^hapter 6, devoted to the chemistry of this
substance, illustrates the exceedingly complicated analytical work
necessary to tmderstand colchicine chemistry, much less to contribute
to its development, liut the rewards in a broad field of biology appear
promising for experimenters who can obtain derivatives of known
chemical organi/atif)n and apjjly the same to critical biological test
cjrganisms.
Thorcjugh descriptions characleii/ing crystalline colchicine were
prepared by Zeisel in 1883, and by Houdc- in 1884.^ The formula
G22H2,;0,;N was proposed. •^■'^ These analytical developments formed
the groundwork for later work. Pharmacological studies using colchi-
cine and its derivatives coidd then jjroceed on a sounder basis, as
shown by the work done dining the next several decades from the
laboratories of Jacobj and Fuhner.^
One of the first derivatives studied was colchiceine, obviously
demonstrating different biologicaH- activity from that of colchicine.
The Parent Plant 15
This intorniation lias been linked with nuxlei n concepts ol specific
biological activity associated with certain chemical structures.^ The
.Svnii)osiuni on the Chemistry ot Colchicine at the 1951-52 meeting
ol the American Association for the Advancement of Science at Phila-
delphia, Pennsylvania, dealt with this problem.
Advancement was made in colchicine chemistry when Adolph W^in-
ilaus. alter a long series of investigations, set forth the concept of a
three-ring structure.-^^ l^pon analysis of oxidation products, his case
was developed for three rings, A. B, and C:, each constructed of 6
carbons, respectively. The first ring A is aromatic, 6 carbon with
three associated methoxyl groups. This much of the Windaus formula
has l)een confirmed and remains as earlier constructed. •• Other parts
required modification as will be shown below and in more detail
in Chapter 6.
l^nusually high water solubility characterizes colchicine in spite
of a deficiency of the groups generally associated with this capacity.'^
To account for this feature and others, Dewar speculated that the
structural concept should include a "tropolone" system and proposed
that ring C was a 7-membered structure.'^
Earlier than this projjosal, doubts were raised by Cohen, Cook,
and Roe in 1940^ that led to changes in the central part of the struc-
ture, ring B. Changing ring B, as well as C, from a 6- to 7-membered
ring appeared necessary. This first evidence for the need to modify
Windaus" structure, which came from the Clasgow Laboratories,^ has
since led to extensive studies dealing with the structure of colchicine.
Dr. James Loudon, a member of this team, has generously contributed
the chapter on chemistry. Degradative work provided thorough evi-
dence that ring B is 7-membered instead of 6 as originally proj^osed.
Further confirmation came through synthesis -work-^^ upon dl colchinol
methyl ether, also establishing the position of the amino group on
ring B.
A compound described as octahydrodemethoxydesoxydesacetamido-
colchicine,-'" has been obtained by degradation. Such a product de-
rived from colchicine that is more or less a carbon skeleton for rings
B and C presents opportunities for making some definitive proof of
the structure of colchicine through synthesis.
Tropolone, as originally suggested by Dewar has been synthesized;!^
therefore, ring C of colchicine is essentially as jiredicted in earlier
sjK'c ulations. Much might be expected here for biological experi-
mental procedures. Interesting tests with trojjolonoid compounds
have been tried. 1 he "radiomimetic" action of a tropolonoid com-
pound is of considerable interest.^"'
Polarographic evidence supjjorts the work with colchicine and
deri\ati\es in several aspects.-'" Santavy and associates beginning in
16 Colchicine
1942 have been con iribu tors. -^"^ Other simihir resuUs comparing in
particular the infrared spectra of colchicine and its derivatives with
the tropolone structme, also offer supporting evidence for the cor-
rectness of the structure of colchicine.-'*'^
Tools for deeper insight to biological problems arise from the
many derivatives obtained with chemical studies.-"' There are also
natural compoiuids accompanying the crude product from Colchi-
ciim which can be of value for experimental work. Numerous areas
Avhere such may be introduced shall be considered in chapters through-
out this work.
When /^ocolchicine was prepared, additional c-mitotic* analysis
could be made. Significant changes in the biological activity ac-
companied changes in chemical structure. The new compoimd has
a c-mitotic activity 100 times lower than colchicine.^- In this instance,
ring C appears to be decisive through the interchanges of keto and
methoxyl groups. Another well-known derivative, colchiceine, demon-
strates little or no c-mitotic action in any concentrations tcsted.-*-
Thesc and other cases call for cooperative work between two highly
complex laboratory ojjcrations, chemistry on one hand and experi-
mental biology on the other. These areas are exceedingly difficult;
the lack of control in biology often becomes frustrating to the physical
scientist. Control or direction over life processes such as mitosis by
designing chemical striutines are intriguing fields for investigation.
1.5: New Biological Uses for Colchicine
Colchicine causes a "veritable explosion"-' of mitoses ^\•hen in con-
tact with mitotically active tissues. The sudden increase in published
reports dealing with colchicine was also described as a "veritable ex-
plosion" of publications,^*' particularly from 19.^8 to 1942. For this
reason, Wellcnsiek proclaimed a new "fad" in biological research,'*''
the "colchicine fad." An immense bibliography'*' has accunudated,
chiefly since 1934.
Accurate historical records have established the way in which
colchicine research began in new fields^"^ and chronologies--* have been
written; no attempt shall be made to review this aspect. i*^' Such sud-
den increase in research with a drug known to man for thirty-five
centuries does arouse interesting specidations as to the causes for an
immediate switch to this particular line of work. After research in
several fields had shown unusual residts, much work was soon under
way. Here we touch upon the initiation of research with colchicine;
extensive details are foLuid in subsequent chapters.
* The adjective c-mitotir is derived from r-iiiitnsis. which designates a mitosis
occurring inider the infiuence of colchicine.
The Parent Plant 17
An early experimenter with [jlants and colchicine was Sir Charles
Darwin \\ ho appHed the drug to "insectivorous" and "sensitive"
plants. 1 he reactions in leal movements were tested, but no con-
clusive results were obtained lor colchicine, nicotine, or morphine.
This work was done about 1875 and is of historical interest only. No
motlern colchicine papers cite Darwin's study.
Another report, tui touched lor sixty years, was obviously closer
to the central theme: Pernice in 1889 clearly described the action of
colchicine on mitosis. i" His figures (Fig. 1.4) showing arrested meta-
phase are remarkable even though their significance was not entirely
realized. Pernice conducted research far ahead of the knowledge at
hand in his day.
Many references credit Maiden with the first observation on mitotic
effects of colchicine because he said the drug appeared to "excite
karyokinesis" '' in white blood cells. The fidl significance was not
realized at this date, but Dixon and Afalden-^ prepared an excellent
report on the eliects of colchicine on the blood picture.
This relationship between colchicine and leukocytosis was re-
examined b) Lits,-" a student in the Pathology Laboratory, Uni-
versity of Brussels, Belgium, luider the direction of the late Pro-
fessor A. P. Dustin, Sr., in 1934. Since the mitotic effects induced by
colchicine were so similar to those previously reported by Dustin and
Gregoire^-' ■with sodium cacodylate, more than passing attention was
paid to the restdts by Lits. The situation was ideal for striking at
the basic biological issues since Professor Dustin had already devoted
much time to the study of the action of chemicals upon mitosis. i-
Colchicine was effective in much less concentration and the volimie of
arrested metaphases in a given treated tissue was an impressive sight.
The Dustin school immediately established that colchicine acts
upon mitosis whether using animal or plant tissues. ^^ Their contribu-
tion was important and significant. With regard to polyploidy in
Allium root tips they did not grasp its significance even though the
preser\ed slides today show restitution nuclei that have multiples of
chromosome sets.^^
Independently, a penetrating analysis of colchicine acting upon
mitosis was made l^y Ludford-"^' -'*'' with tissue c ulture methods using
normal ami malignant cells in xnx'o and in xnlro. His restdts showed
that metaphases were arrested. Amoroso tnged tising colchicine.
Attention was called tc^ the possibilities of colchicine as a tool for
cancer chemotherapy.'- Two c:)ther projects specifically mention the
use of colchicine as a means of attacking problems of cancer. One
was done by Amoroso in 1935 when colchicine was given to mice
bearing specific timiors." The other reported regression of a spindle-
BJERNIfE
Sulla Mriocinesi nella gas'tro-enlerite acuta
Fig A'.
i-M^'i
■'\T(fl ^
^<iSinUaAfniin, A I Fas r
liiJFSanyo.C
Fig. 1.4 — Pernice's first description of colchicine-mitoses (in dog). 1. Gastric gland.
2. Arrested metaphases at the tip of a villosity of gastric mucosa. 3. Endothelial mi-
toses in the vessels of the mucosa. 4. Lieberkuhn s gland crowded with abnormal mi-
toses. Note absence of anaphases and telophases. (After O. Eigsti, P. Dustin, et al.)
The Parent Plant 19
celled sarcoma oi a mare ihat received colchicine by intramuscular
injections.^
Reference to Dominici,-' a jMonecring investigator with irradia-
tions and treatment of cancer, is frequently made, but his original
studies have not been found except for a sentence carried in a text-
book. Dominici died in 1919, so the relation between his work and
modern studies is not as direct as many have been led to believe.
While the late Professor G. M. Smith of Yale attended the Second
International Cancer Congress in Brussels in September, 19.H6, the
work by the Dustin school came to his attention. Here an elaborate
demonstration of research with colchicine was made. Before leaving
Europe, Professor Smith purchased colchicine with the hope that
specific research could be done in his laboratory in the United
States.!*^ Along with Professor D. U. Gardner and the late Professor E.
Allen, he developed assay methods to test estrogenic hormones. Their
preliminary paper was published in 1936.
In another laboratory Dr. A. M. Brues^ and associates reported
important observations on the effect of colchicine upon mitosis in re-
generating liver. These studies struck at the basic mitotic problem.
At Cold Spring Harbor, Long Island, New York, Mr. E. L. Lahr
initiated research similar to that reported by the Yale group. An
Atlantic City A.A.A.S. sectional meeting, 1936-37, presented the work
by Allen, Gardner, and Smith, which paper was heard by Carnegie
staff scientists. Mr. Lahr performed two valuable services: first, he
informed the geneticists at the Carnegie Institution abotit research
with colchicine at the regular seminar attended by all the Datura
workers: and secondly, his excellent slides showed metaphasic stages
in tremendous numbers when colchicine was present. These results
were freely demonstrated and thoroughly discussed with all who
visited Mr. Lahr's laboratory. ^-^
One day in February, 1937, the slides were shown to the senior
author. The demonstration was so impressive that he obtained colchi-
cine for Allium root tip tests before leaving the laboratory. Appropri-
ate concentrations were determined for the experiment with plant
materials. \\'iihin 72 hours, large bulbous tips appeared cm onion
roots immersed in colchicine; the cells showed polyploid restitution
nuclei by acetocarmine methods. Since the senior author had been
privileged to attend seminars in cvtophysiology by Professor C. F.
Hottes, University of Illinois, the i)olyploid cells found in treated
root tips at the Carnegie Laboratories received more than average
passing attention.-''
The Allium root tip tests at the Carnegie Institution Laboratories
were follo^ved l)y seedling ticalments. Eadi test ])oint('d to:\;ird a
20 Colchicine
potential use for inducing polyploidy. These preliminary results
aroused discussion at Cold Spring Harbor which continued up to
April 30, 1937.15
On this date, the senior author severed connections with the
Carnegie Laboratories. Working conditions for continuing colchi-
cine research with plant materials were obtained for him May 1, 1937,
through the generosity of Dr. Geo. H. Conant in his Triarch Labora-
tories, Ripon, Wisconsin. Here the All I inn test was repeated. Datura
stram())iitnii seedlings were treated with colchicine, and the drug was
applied to the generative cell in pollen tube cultures. Remarkable
results at Wisconsin confirmed the previous oj^inion that colchicine
was an unusually etfective substance. From these experiments the
senior author developed a deep interest in colchicine research, and
he has maintained a continued contact with various phases of it
through the years.
Following the departure of the senior author from the Carnegie
Laboratories, research workers investigating cytogenetic problems of
Datura began treatments of seeds of this species with recommended
dosages of colchicine.^" Announcement of these results was made in a
publication- by the French Academy of Science in September, 1937.
By December, 1937,-' the evidence from Datura and other species
clearly established the fact that colchicine was a new and effective
tool for making polyploids experimentally. Since there are sufficient
historical notes^'^ and colchicine chronologies, -•^' ^o an elal)orate dis-
cussion does not seem needed here, except to recommend an article
from the Botanical Review,^" published in 1940, for important details
of historical significance concerning the pioneering work with col-
chicine pmsued at Cold Spring Harbor from januarv to December,
1937.
Independently. Doctors B. R. Nebel and M. L. Ruttle began re-
search in April, 1937, and concluded important experiments that year,
clearly demonstrating that colchicine acted upon mitosis.-^- Further-
more, this drug was an important tool for inducing polyploidy in
plants. •■■- Dr. D. F. Jones of Connecticut is credited with calling their
attention to colchicine; however, they also acknowledged a biljliog-
rajihy in their early publications, mentioning the work by Dustin,^-
Ludford,-"* and Brues.^
In France, Dr. P. Gavaudan and associates published the first
account-" that called attention to polyploidy induced by colchicine.
This paper was presented in June, 1937, but little notice was given
to the contribution. The text clearly described doubling of the
chromosomes along with specific figures. While Havas claims an
earlier date in publication,--^' his paper completely disregarded poly-
ploidy as a consequence of the colchicine treatment. In this regard
The Parent Plant 2 J
Gavauclan Avas more closely associated with cytogenetic asjjects than
Havas.
During the sunniier of 1937, a Swedish geneticist, Dr. A. Levan,
visited genetics lalioratories in eastern United States and was shown
by Dr. Nebel data obtained from his colchicine studies. When Dr.
Levan returned to Sweden, he began experiments with colchicine and
made basic contributions to the concepts ol jjolyploidy and colchi-
cine mitosis.-''
The Cold Sjiring Harbor studies exerted an influence that spread
around the world. These activities plus the other biological work
created an intense and wide interest that led to the "colchicine fad."^^
Many scientists went to work establishing lacts about colchicine.^*'
Generally, the cooperation was genuine, ideas were exchanged freely,
mutual problems were discussed, and knowledge advanced rapidly.
Significant contributions were made within a short time.
By 1938 colchicine was applied to man) kinds of living cells, plant
and animal, with outstanding specific reactions obtained by the treat-
ment. Cancer control continued to be injected into the discussions.
Geneticists discovered a very useful tool at their disposal for theoreti-
cal and practical work. These data were linked to ])ubli(itv that
developed a common language for layman and scientist.
In spite of volumes published, there remain imexplored problems
which appear to have promise for more discoveries. Excellent research
has been accomplished; future progress in agriculture, medicine,
l^harmacy, biology, and chemistry will be facilitated fjy the possession
of such a tool as (Dkhicinc.^i
REFERENCES
1. BiRc.NER, A. Studies on colchicine deri\atives. Cancer. 3:134—41. lO'ifl.
2. Blakeslee, a. Deciouljlement dii nombie de chromosomes chez les planies j)ar-
traitement chimi(iiie. C. R. Acad. Sci. Paris. 205:476-79. 1937.
2a.— . AM) AvERV, A. Methods of indiuins^ doubling of chromosomes in
plants, jour. Hercd. 28:393-411. 1937.
3. Broun, G., Hager, V., Goehacisen, M., Grebel, C.. Sweeney, W., and Hellman.
R. Remission in Hodgkin"s disease followina; colchicine, desoxycorticosterone
and ascorbic acid. jour. Lab. and Clin. Med. 3(i:S()3-4. 1950.
4. Bri ES. .\. 7 he ellect of colchicine on regenerating li\er. Jour. Phvsiol. 8():63-6l.
l9,S(i.
5. Br^ AN, C. The Papyrus Ebers. Appleton & Co., New York. 1931.
(i. BiLEiNCH, T. The age of fables. Thomas Crowell, New York. 1905.
7. Chopra, R. Indigenous drugs of India. .Arts Press, Calcutta, India. 1933.
K. Cohen. A.. Cook. (., and Roe, E. Colchicine and lelated coinpoiuuls. Cliem.
.Soc. London Jour.' 1910:194-97. 1940.
- 9. C;ooK. J., AND Loudon, J. .\lkaloids: colchicine. Kd. Holmes jL- Mankse. .Aca-
demic Press, .New York. 2:261-325. 1951.
10. Dermen, H. Colchicine, polyploidy and technique. Bot. Re\ . 6:599-635. 1910.
11. DoERiNc;. W., \M> K\()\, L. Svntiiesis of tropolone. Joui. Anu-r. Chcni. Soc.
72:205. 1950.
22 Colchicine
12. DusTiN, A. Conti ihulioii a Ictiule des jjoisons car\()clasi(|ues 'ui les tuineuis
animales. Bull. Atad. Rov. Med. Bcl^. 1 l:4S7-.")()L'. I9;M.
KS. , AM) Grec.orii , C;. Contiibiuioii a rctude de ratlion dcs poisons
caryoclasiques sui les luineuis animales. Bull. Acad. Roy. Med. Belg. 13:585-
92.' 1933.
1 I. , Havas, L., AM) LiTS, F. Action de la colchicine sur les divisions cellu-
laires chez les vegetaux, C. R. Assoc, des Anat. 32:170-76. 1937.
15. EiGsrr, O. A cytological study of colchicine effects in the induction of poly-
ploidy in plants. Pr'oc. Nat. Acad. Sci. 24:56-63. 193S.
16. . AND DusTiN, P. Colchicine bibliography. Llovdia. 10:65-111. 1947.
Colchicine I)il)liography III. Lloydia. 12:lS5-207. 1949.
17. , , AM) Gav-Winn, N. On the disco\eiy of the action of colchi-
cine on mitosis in 1889. Science. 110:692. 1949.
18. Gardner, D. V. Personal communication. Vale Uni\ersitv Medical School, New
Haven. Conn. 1949.
19. Garrod, a. Ciout and rheumatic gout. Longmans, Loiulon. 1876.
20. Gavaudan, p., and Po^rRIASKINSKV-KOliOZIEFF, N. Sur rinfluence de la colchicine
sur la caryocinese dans les meristemes radiculares de VAllium cepa. C. R.
Soc. Biol. Paris. 125:70,5-7. 1937.
21. Greene, E. Landmarks of botanical histor\. Sniillisonian Institution. AVash-
ington, D. C. No. 1870. 1909.
22. Gunther, R. Greek herbal Dioscorides. Oxford LTniv. Press, London. 1934.
23. Havas, L. Colchicine chronology. Jour. Hered. 31:115-17. 1940.
24. Kremers, E., and Urdang, G. History of pharmacy. J. B. Lippincott Co., Phila
delphia. 1940.
25. Letire. H. Zur koustituiion des Colchicins. Angew . Chem. A/59:218-24. 1947.
Zur Chemie und Biologic der Mitosegifte. Angew. Chem. 63:421-30. I95I.
26. Levan, a. Effect of colchicine on root mitosis in AUiuin. Hereditas. 24:471-86.
1938. Note on the somatic chromosomes of some Colrliiciint species. Hereditas.
26:317-20. 1940.
27. LiTS, F. Contribution a I'd'tude des reactions cellulaires pro\ocjuees par la colchi-
cine. C. R. Soc. Biol. Paris. 115:1421-23. 1933.
28. Li'DFORD, R. f. The action of toxic substances upon the di\ision of normal and
malignant cells /// x'ityo and in I'h'o. Arch. Exp. Zellforsch. und Mikr. Anat.
I8:4il-}1. 1936.
28a. . Chemically induced derangements of cell di\ision. )oui. Royal
Microscopical Soc. 73:1-23. 1953.
29. Majumdar, G. The history of botan\ and allied sciences in ancient India. Arch.
Internat. Hist. Sci. 14:100-133. 1951.
3(!. Mehra, p., and Khoshoo, \ . Chromosome number and effect of colchicine on
chromosomes oi Colchicinn litteuDi Baker. Curr. Sci. Bangalore. 17:242-43. 1948.
01)ser\ations on some colchicine-containing plants. )our. Pharm. and Pharma-
col. 3:486-96. 1951.
31. Moreau, F. Akaloidcs el plautes alcaloifc res. Presses l'ni\., Paris. 191;).
32. Nebel, B., and Ruttle, M. The cytological and genetical signihcance of colchi-
cine. Jour. Hered. 29:3-9. 1938. '
33. Rai'OI'ort, H., and Wu.liams, A. The degradation of colchicine to octah\-
drodcmethoxyclesox\clesacetamido-colchicine. Jour. .\mer. Chem. Soc. 73:1896.
1951.
34. . , and C;isnev, M. Fhe synthesis cll-cokiiinol methyl ether.
Jour. Amer. Chem. Soc. 72:3324. 1950.
35. Santavv, F. Polarograpln and spectrography of colchicine and its deri\ati\es.
Pidjl. Fac. Med. Brno, Republ. Tchecosl. 19:1-24. 1946.
36. Sgott, G., and Tarbell, D. Studies in the structure of colchicine. |our. .\mer.
Chem. Soc. 72:240-43. 1950.
37. Sfntein, p. Peisonal connnunication. Mc)nt|)elier, France. 1952.
38. Seris, L. a pro^Jos dc la fornude de la cokiiicine. La Rev. Sci. Fas. 88:489-93.
1947.
The Parent Plant 23
■][). Smari', G. Cokhiciim studied histoi ic;ilh. ]'li;mii. Jom. and I'hannacist,
London. 83:5-<S. 1909.
Id. Sk()(>(.. F. Plant growth sidistaiucs. I'liiv. Wisconsin I'lC's, Madison. 19.")1.
11. SiiFANoFF, B. NJonot^iaphie del (.attun,<; Culdiiiuiii I.. I'loc. Bul<^aiian Acad.
Sci. 22:1-99. 192(i.
42. Steinfoger, E., and Levan, A. Constitution and c-mitotic activity of iso-colchi-
cine. Hereditas. 83:385-96. 1947. The c-mitotic qualities of colchiceine, tri-
metlnl colchicinic acid and two phenanthrene derivatives. Hereditas. 34:193-
203. 194S.
43. Waiia. B. The effect of chemicals on mitosis studied in Tradescantia cells
in I'ivo 1. p-acetvlaminotropolone. Cytologia. 17:14-34. 1952.
44. Warrfn. L. Pharmacv and medicine in ancient Egvpt. Jour. .\mer. I'iiarm.
Assoc. 20:1065-7(1. 1931.
45. Wellensifk, .S. Ihc newc-t fad, ((ikhicinc, and its origin. C.liron. R;)i. 5:1.5-17.
1939.
Hi. Williams. T. Drills from plants. Sigma Books Ltd., London. 1947.
(7. Woodward, M. Cicrard's herball, Houghton Mifflin Co.. Boston. 1931.
CHAPTER 2
Nucleus and Chromosoines
2.1: Original Concepts
A basic and far-reaching discovery in biology emerged from the
activities--'- •^'^ of the Laboratories of Pathological Anatomy, Faculty
of Medicine. University of Brussels, under the direction of Professor
Albert-Pierre Dustin: Colchicine induced metaphasic arrest
(stathmokinesis) . Nuclear mitoses were studied experimentally at
Brussels for more than a decade, 1924-1934. chemicals being applied
by several methods. After colchicine was suggested, '^^ evaluation of
its mitotic activity came quickly, and showed that a powerful agent
had been discovered. ComjKuative tests for mitotic poisons proved
that colchicine was one thousand times more potent than sodium
cacodylate, which they had studied previously. •'^'^ Pure substance, in
minute quantity, caused metaphasic stages to accumulate in a treated
tissue far beyond the percentages found in untreated sarcomas. These
original tests with colchicine, coujiled with previous experience
with other mitotic poisons, helped to frame the idea of. metaphasic
arrest by colchicine.--'
7he original slides preserving the tissues treated wiih colchicine
were re-examined by the authors when they worked together in
1949.-'''^ From these impressive sections, new photomicrograj)hs were
made for this book (animal cells, cf. Chapter 10, Fig. 10.1; plant
tissues. Fig. 2.1C'). Ihe total effectiveness displayed by the drug act-
ing upon mitosis is re-emphasized by these pictures. Microscopic in-
spection reveals an luiusual sight. Similar impressions of this totally
different mitotic picture had been formed earlier when the senior
author, -^^ in 19.S7, saw animal cells treated with colchicine and placed
beneath the microscope (cf. Chaj)ter 1) . The jjower to sto)) mitosis
in metaphase was clear to us, and this property has been confirmed by
many experimenters. •^•'' Everyone agrees that the reaction upon nuclear
mitosis is specific, selective, and total, inider prescribed conditions. ^•'- ^^
A large bibliography^-^ has accumulated since 1934, but one of the
original conclusions, metaphasic arrest, conceived by Professor A. P.
[24]
Fig. 2.1 — Allium roots. A, untreated; B, treated; and C, photomicrograph of section from
treated root. A. Roots grown in tap water do not show enlargement. B. Colchicine solu-
tion of 0.01 per cent causes spears, or coichicine-tumors. This group was one of the orig-
inal tests run in 1937 at Co.d Spring Harbor, Long Island, N. Y., by Eigsti. C. A photo-
micrograph prepared specifically for this monograph, from a slide of sectioned root tip
made in the Brussels laboratory, 1934 to 1937, and presently with the A. P. Dustin
Collection, University of Brussels. The polyploid numbers can be seen, as well as large
multinucleate cells, amoeboid nucleate cells, and pseudospindle. Similar views were
illustrated by Havas, Dustin, and Lits in 1937.
26 Colchicine
Dustin, Sr., stands correct.-"' Almost universally, living cells respond
to colchicine after one basic pattern, and new tests extend knowl-
edge into other areas of science. The "colchicine-niitosis"^*'* (abbrevi-
ated, c-mitosis) is built upon the principle of an arrested metaphase.
A c-mitosis was conceived from experiments with plants after the
idea had been developed from animal cells. ^-- ^•''- "'• *"'- Undoubtedly,
the interest in colchicine by the biologist has stimulated an extensive
research in the chemistry of this substance.-^
Metaphasic arrest implies control over dividing cells; seemingly
then, control over cancer might be obtained from the use of this
chemical or others. This discovery raised hopes and new questions
about the problem. However, biological problems being as complex
as they are — and cancer is a major one — the answers have not come
to us as definitely as might have been hoped or expected. Neverthe-
less, basic contributions to knowledge such as the idea of metaphasic
arrest opened new frontiers in research,''-' even though magic cures
have not been produced.
Chromosomal numbers in plant cells are frequently doubled after
treatment with colchicine; polyploidy is a consequence of contact
with the drug.-"" Since many species, including those important eco-
nomically, i.e., wheat, cotton, oats, and tobacco, are natural poly-
ploids, the suggestion was frequently made that this tool would help
create new "synthetic" plants according to man's desires.-^- A revolu-
tion in agriculture was predicted when colchicine became known
for its capacity to induce polyploidy. But many were disappointed
as the heralded magic did not apj^ear with each newly created tetra-
]jloid plant. •■' Informed geneticists, acquainted with polyploidy as a
l^hint-breeding method,''- did not underestimate the difficulties, nor
did they fail to appreciate the opportunities provided by this new
tool. Unfortunately, some practical agronomists"^ have condemned
the use of colchicine for its failure to produce practical residts within
a short time; therefore, such research using induced polyploidy has
been discouraged. Nevertheless, the technique is valuable for those
able to direct such plant breeding, harmonizing theoretical and practi-
cal knowledge. For by these methods, mankintl's food and fiber supply
can be increased (cf. (;ha|jters 12 and 13).
2.2: The Original Statements
When nuclear mitoses in the grafted sarcoma of the mouse were
treated with colchicine,-"' deviations from normal division gave the
observer a j^icture of an arrested mitosis. In 1934, Professor A. P.
Dustin made the following description:
. . . after a very short prophase, the niulear membrane disappears, the cyto-
plasm swells, and the chromosomes chunp together in a strongly bas()|)hilic
mass. The mitoses remain arrested in tliat state for al)out twentv-foiu' hours.
Nucleus and Chromosomes 27
During that period, a certain ninnber of nuclei undergo degeneration. . . .
Alter tliat period . . . cells . . . (oniplete their di\ision. . . . The achromatic
figure becomes visible. . . . Chromosomes move toward the poles. . . . Cyto-
plasmic division is completed. . . . Some mitotic figures of too great size . . .
and some pluricentric divisions remain as a testimony of the nucleotoxic
eliect. . . .*
These basic statements require no change today even though knowl-
edge lias expanded in many cHrections. Admittedly, as the basic idea
becomes extended and broadened, additional points are added. For
example, the c-mitosis illustrates enlargement of the original ex-
planation, but no radical changes in concept arc necessary."'''
The Dustin school did not limit their work to animal cells. A
Himgarian scientist, the late Dr. L. Havas, treated Alliuiii root tips
with colchicine.-" His slides were a part of the Dustin collection
available to the authors in 1949. Since the arrested metaphase or
c-mitosis was so clearly preserved, new photomicrographs were made
(Fig. 2.1C), showing the increase in numbers ol chiomosomes, large
restitution nuclei, and "achromatic spheres." ^" ' Btit the original
text by the Brussels investigators did not mention the polyploid con-
ditions ol these cells. ••^
Independently, iri 1937, the senior author tested cells from treated
root tips (Fig. 2.\A and B) with acetocarmine methods; the tests
showed that polyploidy was created in many different areas of the
A Hi II III root. I he Brussels material and that used at Cold Spring
Harbor (cf. ChajJter 1) were, in every respect, similar. -^^
A third and independently conducted test with Alliiini roots and
colchicine was reported by Dr. Pierre Gavaudan and associates. They
published the first account of polyploidy induced by colchicine in
ftme, 19.87. Their rei)ort stated:^^
It is evident that in these cases there is a separation of pairs of chromosomes,
the lumiber of chromosomes of a restitution nucleus is double the normal
nimil)er. The chromosome list of Gaiser indicates that 2n-16 occin-s in Allium
crfxi. Our residts show "pseudomitoses" with more than thirty pairs. f
This original report and its significance were not mentioned in
reviews-*^' ^'^ or papers-""'^ in the period immediately following its publi-
cation. The more dramatic demonstrations that dealt with induction
of j)Cjlyj)loidy in plants overshadowed the original and what is now
realized as a classic ptdilication by the Gavatidan schocjl.
As soon as Dr. .Albert Levan returned to Sweden from America
in the autumn of 19-i7,''" experiments with Alii inn roots and colchi-
cine were started. This material formed the basis for his concept of
an arrested metaphase, as a cole hie ine-mitosis.''" Remarkable simi-
* A iranslalion of pertinent coiiimeiUs tioni tlic aiii(le cited in Reference No.
12, Chap. I.
t Iranslatecl from paper written in French 1)\ authors tiled in Reference No.
20. C'.liap, I. and Rcfeieiuc No. 11 of tliis chaptei.
28 Colchicine
larity exists between the separate desciiptions with animal cells-^ by
Professor Dustin and the plant work by Professor Levan. A colchicine-
mitosis was described by him as follows:^*'
The effect of colchicine on the course of mitosis is entirely specific. . . .
Modification in mitotic behavior . . . will be abbreviated "c-mitosis." . . .
Prophase stages take place normally: the chromosomes divide, condense, and
assume metaphase appearance. . . . They are scattered over the cell. . . . This
condition (c-metaphase) lasts . . . long . . . after the disappearance of the
nuclear membrane. . . . Formation of "c-pairs" is peculiar to material treated
with colchicine. . . . Their origin is evidently due to a delay of the division
of the centromere \fter a few hours . . . the two daughter chromosomes
are straightened out . . . like "pairs of skis." . . . Centromeres are placed
opposite one another in each pair. . . . During the c-anaphase . . . division
of the centromeres does not take place quite simultaneously within <me cell.
. . . Inactivation of the spindle ... is reversible \fter a period of 12-24
hours in pure water the spindle begins to regenerate. ... In the course of
the transition to normal spindle all kinds of aljnormalities are seen \fter
36 hours the mitoses run their normal course. At a certain moment after
transfer from colchicine . . . frequent diploid mitoses are seen. . . . Highly
polyploid giant nuclei still linger in the prophase stages. . . . Numbers as
high as five hundred were not rare.*
Simimarily. these are the interesting points covered thus far. An
untisual sight appears in a microscojiic field focused upon tissues
treated with colchicine; the nuclear mitoses are halted at metaphase,
and converted into c-mitoses.^"^- '^^' - This power to induce c-mitosis
belongs to select chemical and physical agents,'''^- ^^ of which the most
potent, in this respect, is colchicine. It acts upon mitosis with great
efficiency,'^" high specificity, and total selectivity. The obvious dif-
ference between normal nuclear mitosis and c-mitosis is the tremen-
dous accumulation of chromosomes within a given area (Fig. 2.2)
where ntmierous cells adjacent to each other are arrested in meta-
phase, a primary feature of c-mitosis activity.
Now the total or partial reaction from this drug depends upon
the interaction of (1) a specific concentration, (2) given exposure
period, (3) particular mitotic stage when chemical contacts nucleus,
(4) cellular type, and (5) environment favorable to mitosis. Under
these conditions metaphases are arrested. Consequently metaphasic
* A condensation of the concept of a cniitosis taken fioni I.cxaii. I'.):5S, Refer-
ence No. 26, Chap. 1.
fig. 2.2 — Accumulation of arrested mitoses in animals injected with colchicine and sodium
cacodylate, both spindle poisons. A. Spleen of Siredon five days after a single injection
of colchicine. The organ has increased in size, and many arrested prophase-metaphases
can be observed. These belong mainly to young red blood cells. The longitudinal split-
ting of chromosomes can be noticed at some places. (From an unpublished photomi-
crograph by Delcourt) B. Accumulation of arrested metaphases of the "ball" type in
the intestinal crypts of the small intestine of a mouse. This condition follows injec-
tion of sodium cacodylate and is identical to that observed 6 hours after injection of
colchicine. Cf. Chapter 17. (From an unpublished photomicrograph from the work of
Piton and A. P. Dustin)
»»
r
i
W ^4
t
1. •
*^.
■w i ,
•^^381
A
%■
30
Colchicine
chromosomes acciinuilatc in pairs, "colchicine-pairs," ■''*"' in cytoplasm.
Their distribution then is not the usual equatorial plate arrange-
ment. Furthermore, an arrest at metaphase reduces the number of
anaphases or telophases (Fig. 2.3) thus adding to the apparent in-
creases in this one jjarticular stage, the c-metaphase. That is why the
observer is struck by a totally different mitotic pattern as he looks
at treated tissues throtigh the microscope. Usually tissues ha\'e a tew
metaphases, some anaphases, some telophases, but mostly non-dividing
cells. Even a meristematic tisstie in plants or a sarcoma of animals, i^
Early Equator. Ana-
Prophases metaphases platss phases Telophases Reconstruction
CONTROLS ,
COLCHICINE
1 in 500 millions
1 in lOOmillions
1 in SO millions
my^mmmi \
t
■>:<-:>:mimm4
t
\<<<y^^--<-mm^m 1
t
1
\<ymm 1
1 in 40 millions
1 in 30 millions
Fig. 2.3 — Graphic representation of the percentages of mitotic stages in fibroblast cul-
tures exposed for ten hours to solutions of colchicine. With increasing concentration, the
percentage of metaphases with unoriented chromosomes increases. The displacement
to the right of the arrow, indicating the end of anaphase, demonstrates that in the most
concentrated solutions, nearly all mitoses remain arrested and do not proceed to telo-
phase. This effect is clearly related to concentration. (After Bucher, 1947)
each noted lor cell di\isi(;n. has only a limited number oi cells show-
ing chromosomes at a particidar moment. It is not smprising that the
accumulation ot metaphases impressed one pioneering investigator
who described this reaction by colchicine as "an explosion of
mitoses. ""1
Ultimately, exclusive of recovery, the restitution nucleus is formed
when the chromosomes transform-- to interphase without forming
the daughter nuclei. This transformation may start from an arrested
metaphase, thus by-passing the c-anaphase. Or, the changes-- may
begin after the chromosomes of each c-pair have fallen apart in the
(-anaphase'''' — a transition involving separate chromosomes. Some-
times the uncoiling begins as early as prophase. ''^ These different
points of origin mark three routes taken when the chromosomes "un-
ravel" and vmdergo transformations to interphase. If the number of
centromeres has doubled, a featine clearly seen at (-anaphase, then
Nuc/eus and Chromosomes 31
the (hromosomal iuiiuIki in the restitution nucleus will be twice that
ol the nucleus betore a c-mitosis began. One important consequence
ol the c-niitosis in contrast to the normal nuclear mitosis is the in-
duction of polyploidy.^'-''*' But not all restitution nuclei become
polvj)loid. since the changes-- may start from a jiiophase or meta-
phase.^' In fact, many animal cells treated with colchicine are
arrested at metaphase. 1 he transformation from this stage docs not
lead to a restitutional polyploid nucleus, for in these instances other
changes occur. -■'• '^^
Finally, the most significant biological feature basic to all these
changes is reversibility.''^' After the colchicine in concentrations creat-
ing arrest becomes dissipated, the cell may recover; that is, a bipolar
nuclear mitosis again proceeds in the same manner as before an arrest
was induced. Such recovered cells will continue to divide thus as
long as the cell lineage retains that power. No permanent damage,
with few exceptions,'" to sjiindle mechanisms or chromosomes is ac-
quired from the arrested metaphase. Of course, the arrest may have
been so severe that changes in metabolism cause the cell to degenerate
and ultimately die, but our concepts of reversibility now refer to
those cases where there is complete recovery, a reversibility to the
bipolar mitosis. These can take place among i)lant and animal cells.
The recovery pattern like the whole c-mitotic sequence is unique and
notably imiform for many subjects.
Since there is the reversibility potential, a restitution nucleus with
twice the number of chromosomes may regenerate its new spindle
mechanism. From a genetic view this is a most significant aspect of
reversibility, since the restitution nucleus with twice the number of
chromosomes gives rise thereafter to daughter cells, each with a poly-
ploid condition.
By this jjrocedure of metaphasic arrest — c-anaphase, restitutional
polyploid nucleus, and recovery — the induced polyploidy is trans-
mitted to succeeding generations. This discovery has had inqjortant
ramifications in agricidttnal research. Whereas control over cell di-
vision woidd appear to be desirable for treating certain diseases, this
same control over cell division has entirely different, broad applica-
tions in agricidtiue. That is why a basic discovery in science can be
so widely used in other fields.
2.3: Prophase
First reports said that (olchicine had no iniluence upon pro-
phase.'''' -" Later by cinematographic record, no modification at pro-
phase was noticed.'"' A general belief developed that this jK)rtion of
niulear mitosis was not changed by the drug, for data obtained by
new methods from fixed and stained cells apj^eared the same for
treated and imtreated cases.
32 Colchicine
In animal cells the prophase stages were thought to be non-
susceptible to colchicine because the drug did not penetrate the
nuclear membrane.*'- Theretore chromosomes remained as usual until
the membrane disappeared. Then the chromosomes came in contact
with the drug present in the cytoplasm. Alter this period, contraction
might take place.'^- '• "^' ^^' ^^
From plant tissues, fixed and stained, three important changes
were compared at prophase. ^^ First, chromatin threads developed
the minor spiral in both instances. Second, the major spiralization
proceeded along usual patterns. Third, chromosomes condensed into
proportioned prophasic structures as this stage ended. The two dis-
tinct chromatids were strongly cleaved, appearing as longitudinal
pairs twisted about each other in a relational coil (Fig. 2.1{)A) . On
these three points no noticeable differences among fixed and stained
cells, treated and luitreated, were observed.''^ But such opinions
about the action of colchicine at prophase required modification as
new techniques'-^' ^^- ■^'* replaced traditional cytological methods, and
a wide range of concentrations was included.
Living cells were observed continuously from prophase through
all mitotic stages in Tradescantia staminal hair cells. '-^ By this method
colchicine could be applied at any stage chosen by the investigator,
who then followed the effects from that particular stage on through
sidDsequent ones.
Strong concentrations (2 per cent) admitted dining mid-prophase
at the stage when chromosomes were condensing, caused the process
to revert back to an interphasic dispersion of chromatin.''-^ The time
schedule tor this reversion showed that a metaphasic arrest had not
been reached, but the restitution nucleus w^as formed from a mid-
prophase stage. In some cases the rcstitiuion nucleus appeared to be
doubled for chromosomal number. Similar cases were reported for
Siredou (Fig. 2.9A-D) .-^- "^^ This is one type of transformation Irom
prophase to interphase.
Time schedules for the formation of chromosomes in projjhase
have been made with Tradescantia. This phase is called the anachro-
tnasis^'^ period of chromosomes. Untreated cells require 97 minutes
from early prophase to the polar cap stage. Longer time is taken in
the presence of 0.05 per cent (121 min.) , biu a mininunn time in 0.1
per cent (84 min.) is less than control. These concentrations permit
the chromosomes to move into the arrested metaphase, whereas a
stronger solution induces interphase. Colchicine slows down the pro-
cess of anachromasis as it occurs in prophase. To contrast these de-
velopmental processes, new methods had to be developed.
The neuroblastic cells of grasshopper are used in another tech-
nique'^" with unusual possibilities for a different inspection of c-
Nucleus and Chromosomes 33
mitosis, jxirticularly at prophase. Like the Tradescantia staminal hair
cell method, the drug can be administered when mitosis reaches a
certain stage; thus a new approach is made with animal cells. Time,
gross changes, and unusual developmental sequences can be charted.
B\ this critical method the action of colchicine tipon jjrojjhase
was manifested in three distinct ways.'^^ First, strong concentrations
(50 and 25 X lO'^ M col.), applied at late and very late prophase,
caused the chromosomes already partially formed to revert to an
earlier dispersed phase. Second, lowering the concentration (2.5 X
10 •' M) induced precocious reduction in the relational coiling and
an unusual contraction of the chromosomes before the nuclear mem-
brane disappeared. At this concentration, prophase chromosomes,
normally fixed with centromeres at the polar side of the nucleus, were
disoiiented. By microdissection methods, the polar fixation at pro-
jjhase was tested."'' Colchicine, in proper concentration, destroys some
factor associated with this fixed position. Third, additional decrease
in concentration (1.9 X 1^^'' ^i) applied at prophase disposes the
chromosomes into the "star" formation as soon as the nuclear mem-
brane disappears. These stages may develop into a multij)le-star
phase, and from this formation chromosomes settle out to the bottom
of the cell. These three conditions show that colchicine induces
changes at prophase when certain concentrations are used. These
changes are revealed when continuous records can be made.-'-'
Thus colchicine may act upon chromosomes at prophase, causing
interphase loss in relational coiling, contraction, destruction of
intranuclear orientation, and predisposing the chromosomes to a star
formation. These comparisons required a special technique able to
focus attention ujK)n specific stages, using a wide range of concentra-
tions, and then following the successive development from one stage
to the next. •'''
Pollen grains planted in colchicine sucrose-agar^^- ^" provide a
special method for observing the effects of strong concentrations (1
per cent) upon prophasic stages. Each grain at the time a cidture
starts, begins with a nucleus in prophase. Pollen tubes grow and the
cell lives for a time, but the jjrophase goes into interphase and
does not move into an arrested metaphase. These unpublished data
were collected from treated and untreated cells fixed and stained at
given intervals.
Analyzing percentages of prophases, trcatetl and untreated, there
is noted a proportional decrease in the relative percentage of pro-
phase as the experiments continue."-^ Inhibition of prophase is indi-
cated with concentrations that cause arrest at metajihase (0.01 per
cent). This decrease for AUiutii begins after twenty-four hours""
(Table 2.1). At this period the c-metaphases have reached a peak.''"
34
Colchicine
TABLE 2.1
Percentage of C-mitoses for One Hundred Figures
(After Mangenot, 1942)
Root Tips of Germinating Onion Seedlings — Colchicine 0.05%
Resting stage. .
Prophase
Meta-anaphase
Telophase ....
Control
85.0
6.6
4.2
3.4
24 hi
48 hrs.
85.0
3.2
9.6
2.2
86.2
2.8
7.2
3.8
72 hrs.
90.0
1.6
6.4
2.0
96 hrs.
96.6
0.6
2.0
0.8
Onion Bulb Root Tips— Colchicine 0.05%
Resting stage. .
Prophase
Meta-anaphase
Telophase ....
Control
18 hrs.
40 hrs.
88.42
77.22
8.21
7.18
1.57
14.30
1.78
1.30
77.30
7.53
13.84
1.30
93 hrs.
88.61
1 .84
8.46
1.07
184 hrs.
95
76
0
69
3
00
0
53
Onion Bulb Root Tips— Combined Test— Heteroauxin 0.0001 %— Colchicine 0.05%
Control 24 hrs.
40 hrs.
Resting stage
Prophase
Meta-anaphase . . .
Telophase
?8.42
8.21
1.58
1.78
80.5
4.6
13.10
1.80
84 . 50
4.50
8.00
3.00
67 hrs.
89.20
2.60
5.30
1.90
91 hrs.
90.70
1.50
4.80
3.00
139 hrs.
97.30
0.4
1 .40
0.90
A similar inhibition was seen in neuroblastic cells'^-* but expressed in
somewhat ditterent manner. Cells subjected to colchicine in late pro-
phase remained arrested in jjrophase for 150 miniues before develop-
ing a meta])hase stage. ■^" This process at late pro]:)hase, a transition
from projjhase to metaphase, requires 32 minutes.-^"'
Critical time-dose relationships nuist be observed to produce maxi-
imnn arrested metaphases in regenerating liver of rat."- ^-- ^•' This
dose is one microgram per gram of body weight. Above this concen-
tration, colchicine catises reduction in the mitotic stages in metaphase.
Even before any supralethal dose kills the animal, the inhibiting
action tipon mitosis is observed. That is, the prophases do not seem
Nucleus and Chromosomes 35
to move into the arrested metaphase. This would seem to be an
inhibition at prophase. Under optimum conditions for dose-time
relations, the maxinuun mctaphasic arrest is obtained in uianinials
at 8 to 10 hours following the injection of colchicine.''^
Amoeba sphaeronucleus may grow in colchicine without notice-
able changes. When colchicine is injected into the cytoplasm by
micropipette, action upon mitosis occurs. Amounts injected when
the nucleus is in prophase cause return to interphase. Continuous
photographic records verified this process. About l^per cent strengths
are needed to induce such chromosomal changes.-^
Different cells in Allium root tips show variation in degree of
polyploidy. Pericycle cells may contain several hundred chromosomes,
vet the cells at the tip, a meristematic area, will have the diploid num-
ber. Seventy-two hours of treatment with adequate concentrations do
not induce polyploidy among restricted groups of cells.*^'^- '-' This has
been called a prophase "resistance," characteristic of younger cells.s«
Practical significance becomes attached to this feature if polyploids
are to be induced without any diploid cells accompanying the new
tissues. Prophase stages are more involved than was formerly ac-
cepted.
Two terms might be usefid in discussing prophase influences by
colchicine and other chemicals: (1) the pre-prophase poison which
prevents resting cells from entering the prophase, and (2) the pro-
phase poison, as described above, that inhibits the normal prophase
develoi:)ment and in exceptional cases causes a change to interphase.
Plants and animals differ with respect to the relative toxic action of
colchicine and these make a great difference in the inhibitions not
only of metaphase but of prophase as well.
Prophasic arrangements that are held over from the previous telo-
phase are not disturbed in plants by concentrations that induce c-
mitosis, e.g., Dipcadifi'^ Yet this arrangement is upset in neuroblast
cells with concentrations that give typical arrested mitosis, ='" while in
mammals, prophase appears to be the most resistant period. i-^- ~^- ^i- ^^
Earlier opinion regarding prophase as always normal in the pres-
ence of colchicine must be modified. More information is needed at
this critical and difficult stage. Depending upon concentration and
the particular material treated, prophase stages are influenced by
colchicine.
2.4: Colchicine Metaphase
Again and again, after experiments w'ith animals and with plant
cells, the same conclusions were reached: colchicine changed the
nuclear processes at metaphase. With few exceptions, agreement is
unanimous, and the o])inions are usually formed around the lollow-
36 Colchicine
ing exj^hiiKilions: (1) The metaphasic arrest arises when the spindle
fiber mechanisms are partially or totally destroyed.^-- ^^- -•''• -^' ^' ^'^' ^^'
77, 75, 39 ^2) Chromosomes lose their metaphasic orientation when the
spindle fibers become disengaged from the chromosomes. •^^' ^^' ^^' ^^'
7, 2G, 22 ^3^ The spindle mechanisms are inhibited by colchicine;
therefore, nuclear mitoses are arrested at metaphase.-"'' ''• •'■^' ^-^^ ^"' !• "•^- -^^
While three similar cases are presented, each thesis leads to the same
general conclusion: the metaphasic arrest. That is why agreement in
the final analysis is so excellent considering the many different bio-
logical specimens studied. Universally every one's attention is di-
rected first to the chromosomal pattern at metaphase arrested by
colchicine (Fig. 2.1(7, 2.4/;, and 2.8/1) that is quite different from
the normal metaphasic orientation (Fig. 2AA) . Spindle mechanisms
enter the discussion only after the first impressions of chromosomal
patterns have been obtained. Accordingly, our discussion is first di-
rected to the chromosomal patterns of arrested metaphase. After
these have been compared, it would appear consistent to discuss and
analyze the spindle mechanisms that must operate in the production
of c-mitosis. The spindle mechanism will be considered in Chapter 3.
2.4-1: Types of arrested meta phases. The regular metaphasic fig-
ures and equatorial plate orientations are replaced by different
chromosomal patterns (Figs. 2.1A, 2.SA, and 2.40). Such distribu-
tions are induced by colchicine, and these arrangements are not
wholly random ones.^' ''•• Characteristic stages repeat often enough
that a classification (Fig. 2.5) is possible. ^ If we disregard spindle
action lor the moment, the arrested metaphases may be grouped into
two major categories: (1) the oriented metaphase (Fig. 2.5, above),
(2) the unoriented metai)hase (Fig. 2.5, beloiv) . There are subtypes
for each group which will be considered under the special headings
that follow.
Analysis of the pattern will be made on the basis of interacting
factors that create the special type of arrested metaphase, while direct
reference to spindle mechanisms will be deferred for the moment.
The classification shown in Figure 2.5 was made from stained cells
by cytological methods not thoroughly reliable in differentiating the
fibers.i For this reason, criticism"'' has been made regarding assump-
tions involving spindle mechanisms, specifically with reference to
the distorted star metaphase. Even though this classification was de-
veloped by a chromosomal pattern, an insight into c-mitosis and the
arrested metaphasic types can be gained by such comparisons.
Colchicine penetrates the cell very rapidly. Effects may be noticed
within seconds after the drug contacts the nucleus. C-mitosis in
AUiuiii ck\elops permanently and completely within fifteen minutes.^'^
Rate of jjenetration, as well as concentration, is very important. The
A
'%
B t^
Ci
D
Fig. 2.4 — Pollen tube cultures treated and untreated. A. A metaphase of generative cell
of Lllium michiganensis without treatment. One per cent agar and 7 per cent sucrose,
stained with iron alum haemotoxylin. B. Anaphase, Polygonatum commutatum un-
treated. Stained with acetocarmine. C. Two microgametes and tube nucleus. D. Ar-
rested metaphase, c-pairs, caused by adding 0.01 per cent colchicine to culture media.
The duplications among c-pairs indicate polyploidy. There are 20 c-pairs but only 10
types for the entire group. Centromeric locus shown by incision along chromosomes.
Stained with acetocarmine. (Eigsti, 1940)
38
Colchicine
mitotic stage on hand when colchicine reaches the nucleus may de-
termine the metaphasic type.
Since the action is reversible/^'^ cells may recover from the action
of the drug. Arrested types appearing during the recovery sequence"''
on the way to complete bipolar mitosis are as significant as those
showing up ^vhen the drug is acting upon the mitosis. ^
STAR
DISTORTED STAR
EXPLODED
BALL
Fig. 2.5 — Schematic representations of the main types of arrested metaphases. (After
Barber and Callan)
Length of exposine and concentration are directly related to the
pattern that will develop.'^ A given situation must be noted with
reference to these two factors.
Then, as was mentioned before, concentration, cxposmc, mitotic
stage, kind of cell, recovery, active treatment, and general growth
conditions become critical to the formation of an arrested metaphasic
pattern whether oriented or imoriented.^ Even though the interact-
ing factors are several, the number of metaphasic types is surprisingly
Nucleus and Chromosomes 39
limited. In light of the complex interaction, it would seem that the
kinds of metaphase that could develop would be more extensive.
2.7-2; The oriented arn'sted metaphase. In 1889, Pernice^^
sketched the first star metaphase, a distinctive oriented type induced
by colchicine.'"' Next, these were reported in 1936*'i among tissues of
mice and carcinomatous tissue cultures,*"'- and since then the oriented
star metaphase has been published many times, from a great variety
of biological specimens.
The frequency of star metaphases is far too regular to be ascribed
to a random occmrence.i- "-' The chromosomes are all drawn to one
focal point with the proximal jjortions extended outward resembling
a star, and the type was named accordingly. The centromeric por-
tions of the chromosomes are congregated at this one focal point^
(Figs. 2.5, upper left, and 2.1B-F) .
Two sets of data from similar materials, Triton vulgaris'^ and
Triturus viridescens,'^ respectively, are pertinent to the matter of
origin of the star. Larval cells of Triton were kept in solutions and
were then removed from time to time, fixed, and stained for chromo-
somal pictures. The star, or oriented, metaphases, exceeded the un-
oriented types in the first fixations, at three hours (Table 2.2) . The
Triturus corneal cells, fixed and stained at intervals during recovery
from the effects of drug, do not show the star metaphases at their
peak initil twenty-four hours have elapsed (Table 2.2) .
Two critical experiments performed with neuroblastic cells in the
grasshopper explain some of these differences.-^'* Strong concentrations
applied when the cell w^as at metaphase led to a star metaphase (cf.
Chapter 3; Fig. 3.20) . This action occurred after a particular mitotic
stage had been reached. Another route was used to produce the star
in neuroblastic cells, viz., application of lower dosage (1.9 X ^^~^' ^^^)
at late prophase. Two sets of factors were operating: the concentra-
tion and the mitotic stage. In one instance a metaphasic stage was
used, and in the other, prophase. Each required a different concen-
tration. In the Triton materials, strong concentrations acted early,
yet in Triturus, the stars accunudated later as cells were recovering
from a previous strong dose. We shall return to this problem again
inider the subject of spindle mechanisms.
Multiple stars in single cells are commonly found in AlJiu7n root
tips when cells recover.'-''- *'■' In similar instances, the "multiple" stars
(Fig. 2.6) are to be seen in the Tubifex eggs."'' Among the Triturus,
recovery stages at six days show multiple stars (Fig. 2.7) . Multiple
stars are formed in connection with transition stages from the full
c-mitosis to the complete recovery of the bipolar mitosis.^*'
Distorted star metaphases'^ are asymmetrical figures (Fig. 2.5) . The
origin of distorted star metai)hase is controversial, and although they
40
Colchicine
TABLE 2.2
Arrested Met.^phases — Treatment and Recovery ,
I. Colchicine Treatment .Study: Triton vulgaris; Epidermal Cells of L.\rv.\e
(After Barber and Callan, 1943)
Frequency of Different Types of Cell (Means of Counts From 3 Larvae)
Duration
of
Treatment
(hours)
Prophase
Bipolar
Meta-
phase
Star
Meta-
phase
Un-
oriented
Meta-
phase
Total
Meta-
phase
25.0
29.7
Anaphase
0
22.3
24.0
25.0
15.7
30.7
3
7.7
6.3
20.0
6
20.3
15.0
16.3
10.7
42.0
15.7
12
27.0
12.3
20.7
66.3
99.3
8.3
24
17.7
5.0
6.7
175.3
186.0
6.7
48
12.0
0.3
1.7
83.3
85.3
4.3
72
2.3
9.7
9.7
1 .0
Differential Count Expressing Percentage of Mitotic Types During Recovery
Recovery Time
(hours)
Meta phase.
Anaphase,
Telophase
LInoriented
Metaphases
Star Metaphases
8
24
72
2 +
8 +
79 +
92 +
69 +
5 +
5 +
20 +
16 +
were among the first cases known, -^ less exact knowledge oi their
formation is at hand than ior the star metaphase.
Outside the star or the distorted star, isolated chromosomes are
regularly observed. This iormation accounts for "lost" chromosomes
frequently described in plant and animal tissue-culture cells. !■''• '^"
2.4-y. Uiioriented metaphases. Chromosomes scattered in the
cytoplasm after a nuclear membrane disappears have been thoroughly
described in plants^-- ■^^- ^''^ "•^- 5'^' ~'^'- •'*'^' -"• '^•^- ^O' "■5- •■'• '"'■ --• ^'^ and ani-
mals.-«- •'^- "-■ ^■'- -'^- •^-- «'• "'»• ^- ""• 28, 53, 39 xhe descriptive expression ex-
ploded III rl a phase is appropriate (Figs. 2.4D, 2.1 A, and 2.8^4) . There
Nucleus and Chromosomes
41
is a complete lack oi the usual equatorial metaphase orientation,
hence the epithet uuorwntcd (Fig. 2.1C. 2AD, and 2.8^).
The exploded nietaphases were described from cells of mice
treated with strong doses of sodium cacodylatc."" Therefore, a re-
appearance with colchicine tended to call attention to similarities be-
tween the two substances.-^-^
Among regenerating liver cells follo\\ing hepatectomy, the ex-
ploded metaphase is very characteristic (Fig. 2.8^) . 1 he investi-
Fig. 2.6 — Cell of Allium root tip with an excessive number of chromosomes. FixecJ
after treatment for 208 hours, with 0.05 per cent colchicine in nutrient solution. The
cells are beginning recovery; multiple star metaphases are present. Later cell plates
form between the groups reducing one large cell to a number of smaller cells. Cf. Chap-
ter 3. (After Mangenot)
gators^-- ^^ described the unusual arrangement as though the in-
dividual chromosomes "repulsed one another." These widely scat-
tered chromosomes in a single cell were equally impressive from other
animals, the tissue cultures, and special cases, e.g., Siyedoti.-'^ Triton.'^
Tritiiyiis,''' and Orlhoptera.^' With plants. Allium root tips have
been a favorite source for these types, but pollen tubes show unusual
scattering of the c-pairs through the length of a single tube (Fig.
2AD) .
A specific concentration (2.5 X 1^^'*^ ^^^) applied at late prophase
created the exploded metaphase in grasshopper neuroblastic cells.
Similarly, critical dose-time reqtiirements were necessary to jjroduce
an arrested exploded metaphase in the regenerating cells of liver
42 Colchicine
(hepatectomized rats) .^i- ^'^ Supralethal doses did not induce maxi-
mum arrested metaphases or exploded metaphases. There is then an
optimum dose required for this type. Apparently this same rule
holds for pollen tubes, because maximum scattering throughout the
tube occurred only under given conditions of concentration and
favorable pollen tube growth. -^^ There are other cases bearing on
this point.
Prophase-metaphase arrangements of chromosomes as an un-
oriented type are frequently observed (Fig. 2.2B) . The spleen of
Siredon yielded these types among the first colchicine-arrestcd mitoses
ever studied (Fig. 2.2) .-^- ^^ Perhaps a more logical descriptive term
would be arrested prophase, since the prophase orientation is main-
tained as the nuclear membrane disappears. No sign of spindle move-
ment is detected. The chromosomes may revert to the interphase
from a prophase-metaphase. During periods as long as five days after
injection, the prophase-metaphase appears in Siredoti (Fig. 2.9) .
Representative cases in animals arc noted for this type.^'^' ^- Follow-
ing anaphasic treatment the intermingling of two sets of chromosomes
leads to a similar prophase-metaphase grouping,-^-* so that treatment
at prophase or at anaphase might give this vnioriented association. ^^^
Ball metaphases^ are distinctly clumped types (Figs. 2.2, 2.5) . In
fact, the clumped c-mitosis observed in Spinacia,' Lepidiuni, and
Petroselimtni^''' are typically ball metaphases. A toxic action is un-
doubtedly responsible for the particular apparent fusion of un-
oriented chromosomes. The next step in progressive development is
either the degeneration after pycnosis or recovery to an intcrphasic
stage. Triton material was represented with more ball metaphases
than any other imoricnted type. Even though chromosomes appear
clumped, an individuality may be maintained as was pictured for
cells of mice by the lacnioid-acetic method applied to a ball meta-
phase.^^ Many of these mitoses undergo destruction eventually in
warm-blooded animals.''^ Lysis or degeneration after a ball metaphase
may account for the destruction noticed in Tiibifex.^^' ^^' ^^
Ball metaphases are regularly produced in pollen tube cultures
when the concentrations exceed .01 per cent in culturing media. -^^
Clumping at the early stages followed by pycnosis and eventual lysis
forms the regular course taken by the ball metaphase in pollen tube
cells. Similar degeneration and settling of chromosomes in neuro-
blastic cells indicate destructive action as accompanying this particular
unoriented type.
Much discussion has been directed to the distributed c-mitosis, a
type that can be clearly demonstrated in pollen tubes when the c-
pairs group into two clumps (Fig. 2 AD) . The chromosomes are
c-pairs, and separation may or may not be equal in number. The
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Fig. 2.8 — Stages of restitution in exploded metaphases in the regenerating liver of rats
injected with colchicine. Feulgen-fast green staining. A. Eight hours after colchicine.
Typical exploded metaphase, without spindle. Scattered and shortened chromosomes. B.
Sixteen hours. Chromosome agglutination and lengthening. C. Sixteen hours. Some sug-
gestion of catachromatic changes. D. Thirty hours. Formation of large micronuclei;
these originate by the catachromatic changes of agglutinated groups of chromosomes.
(Original photomicrographs. Courtesy of A. M. Brues, Univ. of Chicago)
Nucleus and Chromosomes
45
best classification for the clistrilnitcd c-mitosis, or bi-inctaphase,"'' is
a subtvpe of the exploded metajjhase. A somatic meiosis is not con-
ceiialile for the pollen tube, yet the distributed c-mitosis is like the
cases upon which evidence for somatic meiosis has been built.
Seven years after the distributed c-mitosis was first published and
illustrated'^^ the term was coined."-^ This is preferable to somatic
meiosis.-^'^ An unfortunate confusion in terms arises because one word
has been used in two different instances to describe entirely different
processes: The word pseudoauapJuise~ is used for the distributed, so-
called bipolar arrangement of the c-pairs. In another instance, pseudo-
(1)1(1 p! I ase is synonomous with colchicine-anaphase.^'"' The word should
Fig. 2.9 — Stages of recovery of arrested prophases in epidermal cells of Siredon after
colchicine treatment. (Compare with Fig. 2.2A). Acetocarmine smear. A. Slight swell-
ing of the chromosomes which have retained their prophasic disposition. B, C. Gradual
loosening of the chromatic material of similar chromosomes: catachromasis. D. Resti-
tution nucleus, formed by the fusion of the swollen chromosomes, which is already
noticeable in C. (After Ries)
46 Colchicine
be dropped in favor of (1) distribnted c-mitosis, and (2) colchicine-
anaphase. Our preference for distributed c-mitosis instead of somatic
meiosis has already been given. Since all factors related to the dis-
tributing action cannot be logically considered here, they will be re-
viewed later.
2.4-4: Chromosomal evolution in plants. Chromosomes persist
individually ten times longer when colchicine is present than during
ordinary mitosis.^^ Their intactness as measured in Tradescantia is
maintained for 23 minutes normally, but treated cases extend this
intactness period to 249 minutes. Of course, concentration plays an
important role; however, optimum doses give this extensive period
of intactness. A comparative estimate of metaphasic delay is gathered
from inspection of records that show total time chromosomes remain
intact.^-'^
Estimated time given for neuroblastic cells also indicates a delay,
but the extent of retardation is calculated in a different manner. The
interval is seven to nine times longer with colchicine. Again the con-
centrations are all-important for any calculation.^^
Specific measurements for pollen tube cultvires, with colchicine
in sucrose-agar, are from five to seven times that of the control.
Treated and untreated populations were compared for the total
period of chromosomal intactness.^^
An analogy may be drawn with normal-speed motion pictures
that are slowed down five to ten times their regular speed. Chromo-
somes normally go through metaphase, anaphase, and telophase at
a speed of 20 minutes. With colchicine, this process is drawn out to
200 minutes. Such delay affects the sequence of chromosomal evolu-
tion. The number of chromosomal changes from prophase through
telophase is not different, but the span of time which is longer, 200
rather than 20 minutes, accentuates the changes made in the longer
period. Now one begins to realize how impressive a definite sequence
of chromosomal forms becomes; this is characteristic enough to be
outlined.
This extension in time is the reason for a comparison that is
usually made between chromosomal evolution under colchicine in
plants and the "terminalization of chiasmata" at meiosis.^**
During a regular nuclear mitosis the process of chromosomal
change is so rapid that one loses sight of the uncoiling and the
straightening or evolution of the chromosome. There is a threshold
for chromosome contraction that is independent of the c-mitosis.
The contraction is related to c-mitosis but is autonomous.'' Some
studies indicated that the longer time allowed a greater contraction
since super-contraction was caused by excessive coiling.'
Nucleus and Chromosomes 47
The first sequence in chromosomal evolution is seen at the late
prophase and early metaphase, while chromosomes are strongly cleft,
and two chromatids are coiled about each other in a relational coil
(Fig. 2.10) . The entire chromosome is straightened so that relational
coiling is easily perceived. Through the whole process of uncoiling,
the delayed metaphase permits observation at each stage. Since both
arms are held at one point, the centromere, the description of un-
coiling is made easier. Uncoiling, then, is the first step and ]:)egins
when the nuclear membrane disappears, unless action takes place
earlier in a precocious uncoiling, as was reported in the section above
under actions during prophase. The first step in the evolution toward
a c-pair is passed when the major relational coiling has been removed
(Fig. 2.10).
Next, the further reduction is similar to the terminalization of
the chiasmata. The contacts of chromatids occurring originally at
several points, finally slip off at the end (Fig. 2.\0B) . The movement
begins at the centromere and proceeds to the end of each chromosome.
The last contact is at the very end of each chromosome. If both ends
are in contact, the characteristic figuie-8 obtains (Fig. 2.105) . Should
one end lose contact, and the other remain attached, a forceps type
develops (Fig. 2. IOC) . All the while uncoiling takes place, the
chromosomes are shortening. Usually the reduction is to one and
one-half times the regular length."' In one instance, actual measure-
ments for chromosomes of Petroselinum were 4.0 microns for control
and 1.5 microns for colchicine-treated chromosomes at c-metaphase."*-^
Finally the last stage is reached, when both ends separate and
move out as if there were actual repulsion of the two arms (Fig.
2. IOC) . The cruciform type has been seen a number of times in
plant,-^*^ insect,^" and mammalian cells cultured in vitro.^'^ Manuiials
receiving colchicine via injection have not generally shown cells with
the cruciform type. A maximum contraction is attained and the c-
pair is held together only at the centromere (Figs. 2 AD and 2. IOC) .
Thus the t\vo chromatids starting from prometaphase as a cleft
structure relationally coiled, are reduced until only the ends are in
contact. After these are released, there develops the typical X-shaped
structures (Fig. 2. IOC). This sequence has taken a longer time than
the control because an intactness period is ten times longer than
untreated mitosis.
A stickiness of chromosomes prevents the X-shapes, or cruciforms.
Such physical changes are important to the falling apart of the c-
pairs."'
Straightened chromosomes that are clearly marked at the centro-
mere (Fig. 2AD) improve the cytological and morphological studies
48 Colchicine
of chromosomes. Not only the comparative sizes of chromosomes
within a set can be jtidged (Fig. 2.4D) , but the relative differences be-
tween the two arms of a chromosome can be estimated.^^ For these
reasons the pretreatment of chromosomes by colchicine was sug-
gested'o and there followed an important advancement in cytological
technique which now makes it possible to study chromosomes, par-
ticularly among root tips, with much greater accuracy. i"- ''•"■*• *'» Scat-
tered chromosomes in the pollen tube led to the discovery of the
natural polyploid Polygonatum cominiitatum.^^ If the chromosome
pairs are studied, duplication of a haploid set is obvious (Fig. 2.4D) .
Since the generative nucleus is haploid, there should theoretically be
only one of each chromosomal type. But each type was repeated, typi-
cal of tetraploids (Fig. 2.4D) . Then any related diploid should have
only one of each type. This was found by extending the study to
other representatives of the genus. The colchicine technique was use-
ful for this cyto-taxonomic study.-^^.
2../-5.- Duration of colchicine-initosis hi (niinidl cells. Degenera-
tive changes are frequent in arrested metaphases of animal cells,
especially in mammals.' Their mechanism, which may be of some im-
portance when colchicine is utilized in the treatment of abnormal
growth (cf. Chapter 10) is not clearly understood. As explained
in further chapters, colchicine has been extensively used as a tool for
the study of growth. It is impossible to reach precise conclusions if
the duration of a given c-mitosis is not known. Direct observations
can be made only in limited cases excluding all sectioning materials.
From the study of sections, it appeared from the early work that
within 24 hours or less, an arrested metaphase either recovered, or
underwent destruction.-'^- *^^
In cold-blooded animals, colchicine is probably metabolized much
more slowly (cf. Chapter 7) . In Siredon, after a single injection, a
great number of arrested mitoses could be seen in the spleen (Fig.
2.2) . This was apparent five days after the injection, and lasted for
about ten days.^-* In Triturus, seven days after colchicine had been
applied to the cornea, abnormal mitoses with scattered contracted and
unoriented chromosomes have been reported (Fig. 2.7) .'^
However, a precise study of the duration of colchicine-mitoses in
the larva of Xenojnis led to the conclusion that destruction took
place much sooner. This was calculated by an indirect method.^^
From data of short treatments with colchicine and from direct ob-
servation, it was foimd that epidermal mitoses lasted about 100
minutes. It was further assumed that the normal prophase duration
of about 25 minutes was not modified by colchicine. In colchicinized
animals the relations between the numbers of prophases and colchi-
cine-metaphases and the average duration of each should be equal.
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50 Colchicine
It was found that the arrested mitoses lasted from 5 hrs. 26 min. to
14 hrs. 20 mill., and later were destroyed.
The spleen of Siredon Avas crammed with arrested mitoses five
days after colchicine treatment. It the figures given above are ac-
cepted, the correlation of the two sets of data— (1) duration of c-
mitoses and (2) the appearance of large numbers five days after
treatment — naturally raises some questions that appear important.
In Xe}ioj)us. while cellular degeneration may be rapid, the percent-
age of metaphases remains very high as long as three days after colchi-
cine. In Siredon,, it is possible that in the spleen only the intact cells
remain visible, the others being washed away by the blood stream,
so the results are not as contradictory as they seem at a first glance.
It is thus most probable, from what is known about the pharma-
cology of colchicine (cf. Chapter 7) , that in warm-blooded animals,
and "particularly in mammals, arrested metaphases are destroyed in
less than ten hours. This is in agreement with the histological evi-
dence of nuclear degeneration,--'- "i and must be kept in mind when
colchicine is used as a tool for the study of growth.
2.5: Processes Leading to Interphase
Chromosomal formation is not stopped by colchicine. Under cer-
tain conditions the process is slowed down or the delay is so pro-
nounced that there is an appearance of its formation being stopped.
For example, many prophase-metaphase types are essentially arrested
prophases. Also we pointed out how colchicine might stop chromo-
somal formation during prophase and turn the process back to inter-
phase.93' '^^
There are three ways in which chromosomes change to interphasic
dispersal under the influence of colchicine — exclusive of recovery,
which we will discuss in a subsequent section. They are: (1) the just-
mentioned prophase reversal to interphase;39- 03 (9) the changes
from any of the arrested metaphases,^' -• 34 i g., prophase-metaphase,
ball metaphase, exploded metaphase, star and distorted star meta-
phases; and (3) a full c-rnitosis through c-anaphase and c-telophase
transformations.''''^- ^^'
Basically, the physical change that takes place in the chromosome
does not differ much in either of the three routes taken. Therefore
a general description of this process shall include the changes-
common to plants and animals. Moreover, the process is not very
different from a regular telophasic transformation found in a normal
nuclear mitosis.-'-^ In all probability the unraveling, loss of chromatic-
ity, and general physical changes are very similar." Colchicine does
not prevent the return of chromosomes to interphase and similarly
Nuc/eus and Chromosomes 51
it does not prevent chromosomal formation.' But colchicine does
one thing important at this stage; it desynchronizes the separation of
the chromosomes.^-*' ^^- ~^' ^*'- -^ Or we may say the coordinated pro-
cesses of anaphasic separation of all chromosomes at one particular
moment are very badly upset.
Colchicine does not inhibit the uncoiling or the stage of katachro-
Nuisis:-'-' the return to interphase. The drug in certain concentration
does slow down the uncoiling process in Tradescantia since it takes
60 minutes for uncoiling with 0.05 per cent colchicine and 77 minutes
in 0.1 per cent contrasted with 35 minutes among untreated cells.
There is one other relation of interest: The ratio of time for chromo-
somal formation, anachroinasis, to chromosome uncoiling, hntachro-
masis. is about 2:1 in regular mitosis. Colchicine-treated mitoses main-
tain this 2:1 ratio, i.e., 121:60 in colchicine and 97:35 for untreated
cells. The significance of these corresponding figures is not under-
stood.
The loss of chromatin, dcspiralization, and vesiculating stages^-*
in the presence of colchicine are much the same as in normal plant
cells. A solid chromosome becomes perforated, and two twisted coils
appear. The chromosome is reduced to a zigzag thread. There is a
fusion of chromosomes that lie close by and the final stages appear
as a reticulated network with nucleoli'^ and a membrane surrounding
the chromatin. Whether the change begins (1) from prophase, or (2)
from arrested metaphase, or (3) through c-anaphase, the general
dcspiralization, sometimes called unraveling, dechromatization, or
katachromasis, is similar (cf. Chapter 3) .^-i- ^c 93. m. i
A full c-mitosis implies tliat the c-pairs of chromosomes "fall
apart" like "pairs of skis"'- '- in the cytoplasm (cf. Chapter 3;
Fig. 2.10). Allitini root tips (Fig. 2.10D), particularly, demonstrate
this stage except when stickiness holds them together. Thus the c-
anaphase can be observed without question.-^^, g5, i, 79 Such separation
is evidence that the restitution nucleus shall carry the tetraploid ntim-
ber of centromeres.
Desynchronization is most easily observed if the chromosomes can
be compared at a given moment. For example. Figure 3.7 shows a c-
anaphase pair at the bottom, whereas above, c-pairs are clearly in X's
and held together." This has been shown over and over, from plants
and animal's, at arrested metaphase.-^*'- -^**' ^=5 within one set, single
chromosomes, and others in c-pairs, have been noticed to revert^^ to
interphase.
C-anaphase is more distinct in some plants, but the distinction is
by no means valid for differentiating animals from plants.^"?, s^. 3, 2. 1. 70,
5« Tetraploid restitution nuclei have been observed for many kinds of
animal cells treated with colchicine.
52 Colchicine
Tetraploid numbers would also develop in animals if colchicine
hit a cell in regular anaphase, because the two groups of chromosomes
intermingle, fuse, and form a restitution nucleus. ^^ This was demon-
strated in grasshopper neiuoblastic cells. This is basic to the develop-
ment of triploid animals by treating egg cells at second maturation
anaj^hase.*^'"*
Pycnotic changes are very common ^vhen chromosomes revert to
the interphase. This is especially so in mammals where destruction
is the fate of most arrested metaphases.-^* ^■^' ^^ Toxic or strong con-
centration induces pycnosis. What structural changes occur are dif-
ficult to determine. Such changes are discussed imder the section of
chromosomal alteration. -'•> ^-^
2.6: Alterations of Chromosome Structure
The most frequent change of the chromosomes in arrested animal
mitoses is an abnormal thickness and shortness."'' This is especially
evident in arrested and exploded metaphases of mammalian cells.
The shortening may be the consequence of an excessive coiling. Very
often these chromosomes degenerate, losing all visible structure; only
irregular clumps of basophilic material remain scattered in the cyto-
plasm, and these in turn fall to pieces.^s Agglutination and fusion
are also quite freqtient (Fig. 2.85. 2.8C) .29. ci. 12. 1.3, 24, 1.5 These have
been observed in cells where the colchicine action was incomplete and
where the spindle was apj^arent,!-^ a fact suggesting that the alkaloid
modifies the chromosomes themselves.
In manmials, the colchicine-mitoses with short and clumped
chromosomes are more frequent when the dose of alkaloid is high.^i
Animals injected with colchicine show mitotic abnormalities that
vary from cell to cell. As an example, the tubules of the kidney con-
tain cells with exploded metaphases and shortened chromosomes,
while the cells of the renal pelvis show ball metaphases.'^- Short
chromosomes are seen in cells of regenerating liveri- when treated
with colchicine according to specific schedules of time and concentra-
tion. Similar shortening also appears following bile duct ligature,-'"*
and in carbon tetrachloride jjoisoning.i"* Such changes were also ob-
served in cells of human tissues poisoned with colchicine.^^ The
junior author had the luiique experience of following the successive
changes in cells of the human body in a clinical case. This occurred
when an individual suffering from an overdose of colchicine was
brought to the hospital in which the jiuiior author was a staff mem-
ber. These effects are described in detail in ChajKer 7.
There is no clear evidence that their structure is damaeed. In
mammalian cells, pycnotic, ball, or star metaphases may often pro-
ceed to normal telophase, although many degenerate, the whole cell
being then rapidly destroyed. "i There is no clear indication that the
Nucleus and Chromosomes 53
chromosomes arc the first to be involved in the cellular death. Their
eventual disintegration is probably a consequence of cytoplasmic or
metabolic changes. A better understanding ot these ^vould be of great
physiological interest, for it appears that among the warm-blooded
species of vertebrates the chromosomes are unable to remain for more
than a few hours in a cell with arrested mitosis. Quantitative data
on this problem have been given in a preceding paragraph; it would
be necessary to know what the biochemical changes are which lead
to the destruction of the nuclear structures, and in what way this is
related to the prolongation of metaphase.
Breakages such as transverse division of chromosomes in plants
have been reported. "^i A number of other observations have been
made along this line, but no tests have been performed to demon-
strate that colchicine increases their frequency. Broken chromo-
somes and fragments are observed in untreated cells.
2.6-1: The destruction of chromosomes in Tubifex. Colchicine
is regarded as a destructive mitotic poison, leading to degenerative
changes of the nucleus in Tubifex,^'-^- 5^' -'^ as opposed to the inhibitive
mitotic poisons which prevent cell division mainly by disturbing the
spindle mechanism. Tubifex is very favorable for the study of early
development and cytoplasmic division, but the "numerous and very
small chromosomes are unfavorable for cytological analysis,'"''^ so this
mav ex])lain the great discrepancies between these findings and those
of -workers using different cells.
\Vhen the egg of Tubifex is treated by colchicine during its first
cleavage, the spindle gradually fades away as it does in other objects.
Then the chromosomes become progressively pycnotic and lose all
visible structure. In the second cleavage, or after longer colchicine
treatments, a total disaj^jiearance of the chromosomes was observed.
5.3. 54. 5.-.. 9.T -phe cells became empty; no more nuclear material could
be stained by any method. More than seventy per cent of the eggs,
twelve hours after colchicine, had such empty cells. But a few hours
later, new nuclear structure appeared. First were seen protoplasmic
condensations which did not stain with the Feulgen reaction. Then
scattered Feulgen-i^ositive masses appeared in the cytoplasm (Fig.
2.11). They seemed structureless but bore some resemblance to the
small nuclei which are foiuid in the control eggs. It is suggested that
some synthesis of thymonucleic acid takes place in the cytoplasm.
The accompanying Figure 2.11 shows pseudonuclei in Tubifex.
Among AmpJiibia after colchicine, podojjhylline, and ben/anthra-
cenequinone, evidence has been presented of a "nudtiplication of
nuclear material without mitosis."-^*
One may, nevertheless, conclude that in animal cells other than
Tubifex, chromosomes disintegrate only when extensive degenerative
changes alter the whole cell. Contrary to plant cells, which may
54
Colchicine
undergo subsequently several cokhicinc-mitoses, animal cells either
remain arrested at j^rophase-metaphase or metaphase, or recover from
the action of the drug and, exceptionally, become polyploid. This is
true whether in protozoa, invertebrates, amphibians, or mammals;
tissue cultures show that colchicine is no more a chromatin poison in
animals than in plants. Nor does it appear to affect other nuclear
mm
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Fig. 2.11 — Action of colchicine on the nuclei of developing eggs of Tubifex. A. After
44 hours, no nucleus is visible. Several cytoplasmic condensations (stippled) are notice-
able. Yolk platelets are block. B, C. Formation of "pseudonuclei" (black). These are
Feulgen-positive, apparently unstructured masses. D. Numerous pseudonuclei in an egg
treated for 70 hours with colchicine. E. Control egg at the same stage as D. Note that
colchicine has suppressed the cleavage clearly visible in E. (After Woker)
Structures; there is no mention of any nucleolar changes apart from
their possible multiplication in relation to polyploidy. Changes in
the nuclear sap will be discussed later.
2.6-2: Colchicine and X-ray combined. Neoplastic tissues have
been subjected to X-ray and colchicine, ^^ but small attention was
given to the relation between c-mitosis and the pretreatments that
influence the effect of X-ray in normal cells (cf. Chapter 10) .
Allium root tips pretreated with 0.05 per cent colchicine and then
subjected to irradiation showed one-third as many chromatid aberra-
tions among colchicinized root tip cells as the controls. ^^
Nucleus and Chromosomes 55
The mutation process-" was measured by pretreating barley seed
twenty-four hours before irradiation. A series of solutions (0.1, 0.05,
0.01, 0.005, 0.001 per cent) of colchicine were used just prior to
treatment with 5000, 10,000, 15,000 r units, respectively .-^^^ A treat-
ment with colchicine prior to irradiation causes a decrease in the
viridis mutants, but an increase in the rare and very rare mutations.
There was no significant change in the albinos.--^
It was concluded that the mutation process is considerably altered
by the application of colchicine to the seedlings previous to irradia-
tions according to the schedules given above.^^
2.7: Reiteration of the C-mitosis
Cells of Alliiiin with sixteen chromosomes as the diploid number
accumulate chromosomes in hundreds, even more than a thousand
per cell. These large numbers are striking. Obviously more than
one doubling has taken place. If we plot the progression, it becomes
clear how such high numbers accumulate. If the number of basic
sets in a somatic cell is 2, then the chromosome number is 2 X t^^e
haploid number per set, i.e., 2 X 8 = 16 for Alliian. When one c-
mitosis has been completed, the doubling produces 32, or four sets of
8 each. The second c-mitosis doubling 32, creates a cell with 64
chromosomes, or 8 sets of 8 chromosomes per set. We may let 7i
equal the number of c-mitoses completed. Then 2'"^^' represents the
number of basic sets. Multiply these factors by the number of chromo-
somes per set. If cell A has completed 6 c-mitoses, then n = 6 and
the number of sets of chromosomes becomes 2<*'^^' or 2', or 128 X
8 = 1024 chromosomes after 6 c-mitoses. Therefore, the c-mitotic
cycles occur in a definite order.^''''
The number of chromosomes that may be packed into one cell is
an interesting question. When the total exceeds 500 per cell, recovery
of the bipolar mitosis does not occur.^e Divisions of 64 may recover
regularly, but numbers over 100 often show twisted spindles among
recovering cells. The high ninnbers are found most generally in the
embryonic vascular cells, notably the area where lateral root initials
develop. ^^' *'^
Short exposures of seven minutes to one hour permit one c-mitosis
while more cycles follow in the longer exposure, i.e., 24- and 72-hour
treatments.56 A tetraploid cell begins the second c-mitosis after 30
hours and an octaploid c-mitosis at 72 hours.^''
There is a correlation between the number of c-mitoses per cell
and the region of the root.^c- «-^' ■*"• ^' If an Alliiun root is divided
into five or six regions and chromosome numbers tabulated, the
greater percentage of cells with increased numbers occurs in the older
parts of the root while cells very near the tip retain diploid numbers.
56 Colchicine
A distribution study ior seven root tips showed that the regions away
from the tip contained hirgest number of polyploid cells.
Reiteration of the c-mitosis in animals is limited by other factors,
such as toxicity to cells exposed over a long time. Also the balance
may be upset by increase in chromosomes per cell, so that only cells
with tetraploidy or octoploidy may survive. High numbers per cell
in animals have not been found as a consequence of c-mitosis.
2.7-/; Recovery in plants. One remarkable feature about colchi-
cine is the ability of cells once stepped up to higher chromosome
numbers, to recover and thereafter produce new cells with the in-
creased niunber.'^*'' '^^^ ^0 In other words, tetrajjloid cells induced by
colchicine, if removed to water, will resimic nuclear mitosis with the
new increased numbers.
A second notable point in the recovery process is the change tak-
ing place when cells with high chromosome numbers begin the re-
newal of the regular mitosis. If one hundred or more chromosomes
have aggregated in one cell and colchicine is removed, soon the
chromosomes gather into small groujjs giving the effect of many star
metaphases. Each of these groups may be the focal point around
which a new cell is formed (Fig. 2.6) . By a process of multipolar
divisions the large numbers in a cell become reduced to smaller num-
bers.'^'^
The length of treatment at a given concentration determines the
speed of recovery based upon the types of metaphase chromosome
formations observed. A one-hour treatment of Spinacia in 0.25 per
cent shows complete recovery in 48 hours. A five-hour treatment at
0.25 per cent requires 63 hours for recovery.'''
2.7-2.- Recovery in animals. Interphase from star metaphase with-
out an anaphasic movement took place in corneal epithelial cells as
these tissues recovered from a strong dosage under a short exposure
period. ^^9 Multiple stars appeared after five and six days from the
time of the last application of colchicine.
Siredon cells show another phenomenon reported many times in
other material, the swelling of chromosomes and cytoplasm. The
immobile chromosomes seem to swell while in a scattered arrange-
ment."^^ This is similar to reversal of prophase; later the chromosomes
fuse into an interphasic nucleus (Fig. 2.9) . Similar reconstructions
during recovery are to be found in regenerating liver cells of the rat
(Fig. 2.12) .'-^ A progressive fusion of micronuclei reduces the num-
ber until trinucleate and binucleate cells develop. Tissue cultures
show comparatively the same micronuclear development.^^- ^^
Partial c-mitoses and multiple stars are common during recovery
as observed in neuroblasts.'^" The multiple stars are evidence that
recovery processes are imder^vay.
Nucleus and Chromosomes
57
Consequences of c-ii}itoses: polyploidy in plants. The arti-
ficial induction ol jjolyploidy by colchicine was not a new discovery
in plant science. Doubling of chromosomes was demonstrated in
jilant cells as early as 1904.'^- Dining a long and successful teaching
career, Professor C. F. Hottes, University of Illinois, repeatedly ovit-
lined cytophysiological methods for inducing polyjjloidy in root tip
12h.
18
24
48
72
1j
1
■ ■
I ■
- ■ ■ I
Fig. 2.12 — Regenerating liver of the rat, after a single injection of colchicine. Schematic
drawings of the various types of restitution nuclei: (1) exploded metaphase with scat-
tered chromosomes, (2) fusion of some of these chromosomes, (3) micronuclei, (4) fusion
of the micronuclei (compare with Fig. 2.4), (5) three nuclei, (6) abnormal mitosis with
partially inactive spindle, (7) normal mitosis. The percentages of these types of cell-
ular changes at various intervals after colchicine are expressed by the black rectangles.
Normal mitoses are only found 72 hours after the injection, and restitution appears to
proceed by the fusion of the micronuclei. (After Brues and Jackson)
cells. Specific polyploid plants were induced by regeneration tech-
niques with mosses in 1908 by the Marchals. Later, polyploids were
created among the flowering plants by Winkler in 1916 and similar
work w^as continued by W'cttstein, Jorgcnsen, Lindstrom and Koos,
and Greenleaf from 1924 to 1934. An early suggestion for inducing
polyploidy by temperature change was made by John Belling in
1925.'' The temperature shock technique was later standardized sue-
58 Colchicine
cessfully for maize in 1932,*- after which time other laboratories fol-
lowed Randolph's general method. This is a brief history of poly-
ploidy through artificial means before the colchicine era began. That
important period made work with colchicine more fruitful than it
otherwise would have been. Sudden attention to colchicine almost
blotted oiu the facts that polyploidy induced by several techniques
had been well developed before 1937.
The vast literatme-^-^ dealing with polyploidy in plants is discussed
in subsequent chapters.
2.j-^: Polyploidy in animals. Polyploidy in animals has also re-
ceived attention for a long time but success with artificial induction
has been limited. The introduction of colchicine did not achieve the
success found among many projects with plants.
Temperature shock-cold treatments with newly fertilized eggs of
Tyitunis viridescens^^ were more successful than the application of
colchicine to these animals. The procedures with colchicine were not
efficient, at least when compared with treatment of plants; much was
to be desired for work with animals.
Newly fertilized eggs of rabbits were treated with weak solutions
of colchicine. "^^ Other animals, frogs, ^^- -^"^ Triturus/'* Triton,'^ Xeno-
pns,^'^ Artemia^ silkworm,'*'* Habrobracon.^-^ Drosophila*-- ^'^ chick-
ens,"*" Amoeba,^^ were tested with colchicine for polyploidy. Gen-
erally colchicine has failed in comparison with the induction of
polyploidy in plants. ^^
One remarkable series of experiments demonstrated in Amoeba
sphaeronucleus how polyploid imicellulars could be created by colchi-
cine.-'^ This had no effect iniless injected into the cytoplasm at meta-
phase, with a micropipette. Actual counting of chromosomes was not
possible but there resulted larger cells with a larger nucleus. These,
however, at each division built one normal and one abnormal nucleus,
a fact suggesting triploidy. Supposedly polyploid nuclei were trans-
planted into enucleated fragments of normal amoebae and vice versa.
It was observed that the size of the tniicellidar was directly related to
the size of nucleus. The opposite was also true, and a normal nucleus
grafted in a "polyploid" cytoplasm was observed to swell considerably.
Cytoplasm and nucleus luiderwent several divisions and then re-
covered their normal volume of the original species. If the normal
nucleus was grafted into a fragment of a polyploid cell, growth was
resumed normally. These experiments have been illustrated by a
remarkable series of cinemicrographic documents. They have pro-
vided new insight on nuclcar-cytoplasmic relatiouship and the
possibility of observing colchicine effects in cells, the membranes
of which are impermeable to the drtig.
Nucleus and Chromosomes 59
A diflerent attack was tried by taking advantage of the fact that
colchicine coming in contact with egg cells in the second maturation
division would arrest the anaphase stage thereby creating a diploid
egg cell. If this cell imited with a haploid sperm, it could give rise
to a triploid individual. i-' The reasoning was logical enough and
colchicine coidd be introduced at the proper moment through the
admittance of sperm and colchicine by artificial insemination
methods. Whether sufficient dosage of drug was given shrouds these
tests with doubt.
Experiments with frogs in 1947^^ encouraged the trial of introduc-
ing colchicine at the time of fertilization, since larvae from eggs
treated at fertilization seemed to be polyploid judging from the size
of cells and nucleus. The idea was extended to other animals, notably
rabbits and pigs.'*'^' ^^ Certain principles were substantiated by these
tests, viz., that the application of colchicine at the precise moment
of fertilization would bring triploidy in the zygote, because a
doubled egg cell would unite with a haploid sperm.
Techniques were developed to inseminate artificially rabbits and
pigs,^^ by adding colchicine to sperm material. Proper concentrations
were determined by laboratory tests. Suspected triploid offspring were
studied cytologically and a conclusion was reached that egg cells were
doubled by this procedure. One rabbit that deviated from diploids
showed 66 chromosomes among certain mitotic cells of testicles. ^^
There were other diploid cells in this test with 44 chromosomes. Thus
the individual may have started as a triploid zygote with reduction
as development proceeded. These results were, however, by no means
conclusive. Previous accounts as weU as these above have been criti-
cized and not without some basis.
Similar experiments were done with pigs.^^- ^■'' Among 31 offspring
from artificial inseminations, one differed from the rest as well as
from dij)loid pigs. This male animal showed consistent mitotic fig-
ures with 47 chromosomes,*^^ a good triploid, that originated when a
diploid egg of 32 chromosomes and a haploid sperm carrying 15
chromosomes united. These techniques are new and merit fiuther
attention for theoretical studies of polyploidy among animals. ^'^
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Colchicins auf Furchungsmitosen und Entwicklungsleistungen des Tubifex-Eies.
Rev. Suisse Zool. ,51:109-71. 1944.
"^^caI
CHAPTER 3
Spindle and Cytoplasm
3.1: Colchicine and Spindle Fibers
More metaphases than anaphases or telophases collect in tissues
treated ■with colchicine, creating an inij^ression that chromosomes
appear stranded between the two poles. Obxiously colchicine blocks
the mechanism that regularly mo\ es them to the respective poles (Fig.
'5.1A,B) . Interference seems to be localized at the spindle fiber; con-
sequently, arrested metaphases pile up in greater numbers per given
area than do the other mitotic stages.-^' ^^- ^
A disproportion of metaphases was pictured b) Pernice in 1889. His
illustrations-"^' ^^ show many arrested metaphases with \ery few ana-
phases; the contact between the drug and intestinal cells of the dog
blocked mitosis (Fig. 1.4).
If the spindle fiber is the substrate where colchicine acts — and
there are man\- data to support this assumption — then c\ tological and
biochemical methods shoidd show us more clearly what reactions
occur. The basic cause for a mitotic arrest undoubtedh is to be found
in the chemistry and physiology of the spindle fiber and attending
mechanisms. ^1
Provisionally, let us say that colchicine alters rather than totally
destroys the spindle substance. Such assumjjtions are consistent with
cytological tests, ft is known that arrested metaphases fail to show
the usual spindle fibers as linear structures; therefore, conversion of
a fibriform element into a corpuscular one becomes a tempting sug-
gestion, with attractive possibilities for explaining, at one le\el, how
the spindle fiber and colchicine in teract.'-^- "'"• ^"- '■'•''■'-■ •^'
Molecules of colchicine reacting with a molecular system ol sjjindle
substrate ha\e been considered as one of the basic relationships be-
tween the two substances^-^' ^^' '^^' '"'■ "" Such an explanation can be
given on a quantitative basis. The destruction or inhibition of the
fiber then appears to be a quantitative reaction, because the concen-
tration of colchicine is a critical factor.
[65]
Fig. 3.1 — Photomicrographs from embryo of grasshopper, sectioned 13 microns, stained
with iron hematoxylin. A. Untreated cell at metaphase, spindle fibers difFerentiated. B.
Cell treated, 25x10" M, 30-minute exposure; spindle fibers reduced by treatment but
chromosomes not dispersed. C. Concentration of, 2.5x10"" M, 90 minutes; star meta-
phase with some spindle activity. D. Clear spherical area, which is not stained, is the
hyaline globule, that increases when spindle substance disappears as a result of treat-
ment with colchicine. E. Chromosomes outside the star, 120 minutes, with 2.5x10' M
concentration. F. Multiple stars, three in one cell, 2.5 x 10" M, 180 minutes. G. Exploded
c-metaphase derived from prometaphase treatment, 2.5 x 10~" M, 15 minutes. H. Chromo-
somes shortened after 180 minutes, 2.5x10'' M, settle to bottom of cell. (Photographs
provided through courtesy of Drs. M. Gaulden and J. Carlson. Adapted from Experi-
mental Cell Research 2:416-33, 1951.)
Spindle and Cytoplasm 67
Wide ranges of concentration induce a wide variety ot reactions.
These ransje from extremely minute chanoes inxolviny tlie spindle
orientation, the tropokinesis,^^ to the full c-mitosis, slatlniiokine-
sis, obtained by strong doses.^^. ss, 73, 25 These two reactions repre-
sent the extremes, between Avhich there can occin- many intermediate
changes.
Before proceeding further, we should recall the old argument about
spindle liber reality as opposed to "artefact." If we are dealing with a
specific molecular problem, the possibility that spindle fibers are arte-
facts woidd seriously influence oin- proposition. Perhaps the whole
concept would be annulled. Rut excellent results, obtained from
treated and untreated cells and Irom living and fixed materials, have
opened up new approaches. Hence, the argument that spindle fibeis
are not real is almost extinct. An entirely new series of studies with
phase contrast microscopes, polarization microscopes, cinematography,
and other techniques has shown that fixed and stained fibers are
similar to the living functional linear structures. ^'' Colchicine has
been employed most eff^ectively in these studies.
A high specificity can be demonstrated between colchicine and
spindle fibers.i^- ^o. ». ss. 54 Moreover, this specificity can be cjuickly
destroyed if the chemical structure of the drug is changed only slightly.
Pharmacobiologists have known for a long time that certain deriva-
tives such as colchiccine are less active pharmacologically than colchi-
cine. Numerous chemical deri\atives of colchicine are accurately
kno^\•n by chemists and these have become available to biologists.^*'
For example, isocolchicine is a transformed molecule of colchicine,
that involves a shift in the position of keto and methoxyl groups on
ring C. By this change the specificity between spindle fiber and colchi-
cine is reduced. '^^ Isocolchicine is one hiuidred times less active in
producing a c-mitosis than colchicine.
The specificity between colchicine and spindle appears to be on the
order of the enzyme and substrate specificity.
Admittedly, the spindle fiber mechanism is complex, highly orga-
nized, and delicately coordinated. But much is understood of this
mechanism in animals and plants. Cytologists agiee that two sets of
fibers are formed at each regular mitosis: the continuous and the
chromosomal.
The reaction between colchicine and the several components of
the spindle appears, then, to have a quantitative basis. Some portions
of the sj)indlc can be inactivated leaving other jiortions activated.
Such fractionating possibilities have been demonstrated,^"' and this
fact merits attention.
68 Colchicine
3.2: Spindle Inhibition
Every mitotic cycle builds anew the spindle fibers. Cytoplasmic
separation, a function of cytokinesis, is closely coordinated with the
fiber and spindle functions.-'* Colchicine prevents the formation of a
sjjindle at jMojihase, jjrecludes a nuclear mitosis, delays chromosomal
separation, inhibits daughter nuclei, and effectively blocks cleavage
processes.
Among plants, the inhibition starts at the polar cap stage when
polarity makes an appearance.-*'' The first sign that colchicine acts
ujjon a spindle is noticed at the ])olar cap stage. ••^' Among animals,
the preliminary spindle inhibition is an interference with the de\elop-
ment of the astral rays, and functioning of the centriole outside the
nucleus.^ The initial inhibiting inHuence is seen at the time nuclear
membranes are about to disappear and the centrioles begin their
movement.
The prophase orientation of chromosomes in animal cells may
or may not be destroyed by colchicine. Likewise, i)lant cells, e.g. in
Dipcadi, have a prophase orientation that is determined from the pre-
vious telophase. These arrangements are not disturbed by colchicine.
Thus, colchicine may inhibit the spindle without changing a basic
chromosomal arrangement at prophase, •''•'' although strong solutions
may interfere with the orientation before membranes disappear.
The bipolar mitosis is effectively pre\ented by colchicine acting at
late prophase, and progressive changes from interphase into prophase
are not inhibited by colchicine.
Undoubtedly there is an action upon resting cells if strong con-
centrations are used.'-^- ''^ Nuclear poisoning,^- intranuclear precipi-
tates,*'* chromatin condensation, *•"• pycnotic destruction,-'^- -^i- -* and
nuclear degeneration'"'" before mitotic arrest, are possible actions of
colchicine. Deeply stained inclusions in cells of Amphibia were ob-
served after strong treatments.*^'' In most cases concentrations abo\e
the threshold for c-mitosis induce the changes. Neuroblastic cells of
grasshopper, usually very responsive at prophase, metajjhase, and ana-
phase, recjuire a tremendous concentration (1000 X '*^ *' ^^^) 'i^ inter-
jjhase or late telophase.-''
The mitotic stage at which colchicine is most effective in lowest
concentration, is late prophase. Ihere is no doubt that colchicine
interferes with transformations of karyolymph, because the regular
linear arrangements of fibers do not develop. These structures nor-
mally are formed 20 mimites after disapj^earance of the nuclear mem-
i)rane; but in the presence of colchicine, fibers do not form. Instead,
there is formed a hyaline globule in grasshopper neuroblastic cells,
which is nonfibrous.
Spindle and Cytoplasm 69
Similarly lor Tradcscnntia, fibers do not develop at projihasc
A\ith concentrations ol 0.05 per cent or 0.1 per cent colchicine.-''' 1 here
arc other cases, bnt these two are enough to prove that the first stage
ol sj)indle inhibition sets in at j^rophase.
Full strength solutions applied at prophase cause total inhibition:
no \estige of the mitotic spindle can be observed. Partial inactiva-
tions are only foimd at the threshold le\'els.""' The continuous fibers
and astral rays rather than chromosomal fibers are then the ones in-
hibited during a partial inactivation. That is, enough colchicine is
present to inhibit the exterior spindle, but the interior spindle devel-
ops. Such partial inactivation leads to a star metaphase.
Sj)indle material may be con\erted into such bodies as hyaline
glob ides,'-'' (Fig. oAD) , the lakelike substance in Arbacia'' (Fig. 3.5),
achromatic sphere of AJUiim-^-"' (Fig. 3.6), or the deformed atracto-
plasm among Tradesanitid.'^'' All these structures are closely associated
to karyolymph; consequently, the inhibiting process of a normal
spindle fiber is in reality transformation to another form of substrate.
Electron microscojjic anahsis of colchicinc-treated polar cap stages
in Allium indicated a "solubilization"' and "fragmentation" ol fibrous
strands. These changes are interpreted as spindle fiber transforma-
tions. Submicroscopic interpretations are difficult, l>ut the evidence
is consistent with other microscopic data."^-
A jjrimary effect of colchicine is the inhibiton of a mitotic spindle."
Secondary eftects stemming from this action are colchicine pairs,
chromosomal changes, desynchroni/ation of mitotic processes, delayed
separation of chromosomes, and restitution nuclei instead of daughter
nuclei.^
Originally the term cGlchicinc-tnitosis designated an "effect of
colchicine on the course of mitosis" that is entirely specific."'"' Addi-
tionally, in a colchicine-mitosis the spindle aj:)paratus is totally in-
activated, and this causes completion of a "chromosome mitosis with-
out nuclear or cellidar mitosis." '''''
3.3: Destruction of the Spindle Fibers
That colchicine inhibits the spindle at late prophase is well estab-
lished. Less familiar are the facts about colchicine when applied to a
mitotic spindle that has developed as far as anaphase (Fig. ?).2s-v) .
Ao establish these facts, special technicpies had to be developed.
Individual cells nuist be observed at the critical stage, anaphase, and
the chemical nuist be ajjjjlied at a precise moment when the mitosis
has reached a certain stage. Fortiniatelv, several excellent methods
for i)lants and animals^'- ^^- ^^- *'■'• •''' have been develojjcd, and we may
now learn what ha|)ijens when the drug is added to a cell after a
spindle has foriiud.
Mitotic stage
treated
Colchicine
xlO-6
molar
SUCCESSIVE CHANGES
late prophase
50-25
2.5
1.9
prometaph.
25.2.5
0.2
metaphase
25
2,5
anaphase
50.25
50.25
50.25
N»
^
w
w
Spindle and Cytoplasm 71
The spindle fibers at anaj^hasc can be destroyed l)y the proper
concentration oi colchicine. Ihiis, in addition to an inhibitive action
upon a spindle at the start of the mitotic cycle, the spindle fibers can
be reduced after they ha\e been formed (Fig. 3.Li(-G) . The destruc-
ti\e action at anaphase follows a regular order, and there is a (juan-
titati\e as well as a cjualitati\e basis for the change.
3.5—/: Neuroblast cells uf grasshopper. The technique developed
bv Professor J. Carlson, University of Tennessee, and used etfecti\ely
in cooperative research with Dr. M. E. Gaulden, Oak Ridge Labora-
tories, Tennessee, has given a new insight to the relationship between
colchicine and spindle fibers. Continuous observations upon li\ing
cells, together with the application of the chemical at a s])ecific stage
and in \ariable concentrations, ha\e been a \aluable addition. In fact,
the answer to our question about anaphase and colchicine demands
this kind of special method foi" watching an action upon the fiber
(Figs. 3.1 and 3.2).
Cells at early, middle, and late anaphase were chosen. Strong
concentrations (50 and 25 X 10 "^ M) were used, and in each instance
the spindle was "imj^aired almost innnediateh' •'' (Fig. 3.2/) . The
chromosomes stopped in ihcir mo\ement to the poles; the two groups
intermingled, fused, and formed into a single telophasic nudcus (Fig.
S.2s-zi'') . This restitution nucleus was tetraploid, since the anaphasic
separation of centromeres had taken place before the drug was ap])lied.
Fom- nucleoli appeared instead of two, and the "uncoiling" ])rocesses
were only slighth delayed by colchicine (Fig. 3.2it'') . Spindle fibers
were destroyed at anaphase.
When the concentration was reduced to 2.5 X 10 " ^^^ f^^i' the
same stage, an anajjhase. no detectable restdts were obser\ed. The
chromosomes continued to mo\e to the respective poles. Vet this
same concentration in\oked a definite reaction at an earlier mitotic
stage, i.e., late prophase or pro-metaphase (Fig. 3.2c) .^'
Fig 3.2 — Mitotic stage when treatment began, shown in right colurr.n. Concentrations
are expressed in molarity. Successive stages are lettered a to i'. a and b: prophase
reversions occurring 10 to 20 minutes after treatment with this strong concentration.
Chromatin resembles early prophase, c to e: chromosomes lie at random, no spindle
formed, exploded c-metophoses, chromosomes continue to shorten, then clump together
in groups at bottom of cell, hyaline globules formed in d rise to top of cell, f to h: .he
evolution of a star metophase. i to k: star metaphase that becomes increased to mul-
tiple star and lost chromosomes. I to m: weak solutions do not fully inhibit spindle but
reduce the size, n to q: the metophasic spindle is reduced, hyaline globules form in o,
chromosomes settle to bottom and globules rise in cell, r cell divides when concentration
is too weak to destroy spindle completely. Compare figure r and c, that received same
concentration, but applied at different stages. Anaphase spindles are reduced if con-
centration is 25 X 10 ' M or more. Chromosomes fuse and intermingle in t and v, hya-
line globule forms in stages t, v, and y. Four nucleoli in w' and i' indicate a tetraploid
restitution nucleus. These stages show the interaction of concentration, stage of mitosis,
and length of exposure. (Diagrams adapted from M. Gaulden and J. Carlson, Experi-
mental Cell Research 2:416-33, 1951)
72 Colchicine
A fully formed nietajihasic spindle was reduced by weaker concen-
trations than those necessary for anaphase. Specific concentrations
applied to the fully formed metaphasic spindle led directly to a star
metaphase (cf. Chapter 2) . These stars formed by treated metaphases
persisted for five or six hours. Dining this time the Brownian move-
ment shown by the mitochondria was actively increasing. While
the activity of the protoplasmic material was increasing, the meta-
phasic spindle fibers were being reduced.
With finther reduction of concentrations and with application to
metaphase, no obvious reduction of the spindle was obtained. This
concentration (2.5 X 1^^"" ^^) l^'^^' 'i'> effect on anaphase, but produced
a slight retardation of the spindle at metaphase. Yet this same con-
centration applied to earlier stages, the prophase, induced visible and
truly inhibiti\e effects. No visible changes were observed at full meta-
phase by the concentration 1.9 X 1^^"" ^^•
Pro-metaphase, an earlier stage than metaphase, responded (Fig.
3.2/-/>) innnediately to a strength (2.5 X 10 "^ ^^) t^^^t was without
detectaijle action at anaphase. The s])indlc formed at late proj)hase
was innnediately reduced, and the chromosomes scattered in the cyto-
plasm: a typical exploded metaphase. Doses without inliuence at
anaphase and with only slight effectiveness at metaphase were totally
effective at pro-metaphase, or late prophase (Fig. 3.2r-e) .
Reduction to a concentration of 1.9 X 10 ^' M, effective at meta-
phase and now ajDplied at prophase, created the star meta])hase.
Under these conditions, sexeral focal j^oints for the star remained after
treatment (Fig. 3.2/,g) . Hence, this concentration usually led to
the multiijle star metai)hase (Fig 3.2/) . The particular concentra-
tion inducing stars was effective only at prophase. Now, compare the
difference between an effective concentration at projihase, .2 X 10" M,
with the concentration required to reduce the anaphasic spindle,-"
25 X 10'*^ M. The difference is significant.
Since, as one approaches interphase from anaphase, corresjjond-
ingly weaker concentrations are recjuired, it becomes a point ol in-
terest to note requirements for detectable results at interphase, or
resting stage, or even late telophase. The concentration ^vas raised to
1 ()()() X 10 '■' M before any changes were noticed, and then the toxic
action as well as pycnotic changes were the only results obtained.
From all these tests there appears to l)e a critical point in ilie nntoiic
cycle when spindle fibers can be reduced with a minimum toncenira-
tion.-'" That stage is late prophase and pro-metaphase.
f hree important conclusions were reached:-" (1) Effectiveness in
destroying the spindle or interference with its further develoj^mcnt
depends upon concentration; the greater the concentration, tlu
greater the effectiveness upon the spindle, within certain limits. (2)
Spindle and Cytoplasm 73
r
A oreater concenii aiion is necessary to destroy the more adxanced
spindle, i.e., at anaphase, than a spindle at an early stage, pro-nieta-
phase. (3) The loini ol a particidar spindle is directly related to the
characteristic t\pe of nietaphasic pattern thai \vill develop alter treat-
ment such as the star, multiple star, ball, ex])lotled. or other arrested
metaphase.-''^ Configtn^ations dejjend uj^on stage at time of treatment,
concentration, and duration ot treatment or recover).
Alter sober reflection upon these conclusions no one can disregard
the importance ot a specific concentration, ihe tyj^e ot cell, and, most
interesting of all, the particular mitotic stage at the time the drug
enters the cell. Specificity between chemical and spindle fiber is sup-
ported b\ these in\estigations.
3.5— 2; 'Stamina] hair cells of Tradescantia. Techniques with the
Tradescautia material were used quite as effectively as \\'n\\ the neuro-
blastic cells just re\ iewed. The central feature and main advantage
lie in the possibilit) of applving colchicine at a particular stage and
following the progressi\e develoj^ment of mitosis thereafter. Trades-
cantia staminal hair cells ha\e been a faxorite material ior mitotic
studies in vivo for a long time. The first studies to be conducted with
colchicine and plant cells were accomplished with the stamina! hair
cells.'-
Colchicine aj^j^lied to a cell when the spindle was well de\eloped
stojiped further de\elopment and reduced the spindle within a short
time. A deformed atractoplasm ajjpeared in the cell after destruction
of fibers b\ the chemical. Stronger concentrations were necessary to
induce changes if the spindle was very far along in development.
As the drug began its actifni, Brownian mo\ement on the spindle
was increased, indicating that the colchicine was acting u})on the
fibers. This action took place suddenh, as the chemical reached
the cell.
Pliragmoplasts, which are spindle materials of cytokinesis, were
stopped in their further development and also reduced by colchicine.
A cell wall partly de\ eloped from each side of the cell can be stopjjed
by the drug.
At metaphase, aclixity ujxjn the sjjindle is immediate. Die c-pairs
are formed as the spindle fibers are destroyed. Within 1 .S minutes,
grantdar changes upon the spindle showed that action had set in.
Within I hom^ and "iWi minutes, the entire grouj) of (hromosomes
retinned bv a j^recocious re\ersion to an intei j)hase. Such quick results
required strong solutions (2 per cent) . Generally, lesser concentrations
(0.05 per cent and 0.1 per cent) were used to elfect spindle lil)ers.
Regardless of the stage from projjhase to anaj^hase, e\en as late
as the phragmoplast, an application of colchicine stopped mo\ement,
destroyed the spindle, and retinned the chiomosomes to interphase
7A Colchicine
by regular uncoiling processes, similar lo the regular tcloj:)hasic trans-
formations. During later stages a "cytoplasmatization" of spindle or
■lluidity" was created."- By this process the spindle was transformed.
Metaphasic spindles were destroyed in pollen cells of Ephedra.
The concentration was a strong one (2 per cent) . and rexersion to
interphase was rapid. The total time for a cell to proceed through
a regular mitosis was no different from the time taken for a rever-
sion. A full c-mitosis would have taken a longer time. This rapid
conversion back to interphase led to the conclusion that colchicine
did not delay the mitotic cycle. Preliminary results unptdilished by
the authors show that concentration is a most important consider-
ation for Ephedra as well as other cells. Reversions can proceed very
rapidly under the action of colchicine.^'
The data from Tradcscantia and neuroblasts confirm an opinion
stated earlier that the destructive action is cjuite as notable for col-
chicine as its inhibitive activity. The main difference lies with the
concentration. Stronger solutions arc recjuircd to destroy a fiber at
anaphase than to inhibit its formation during prophase. 1 hat is
why a broad range of concentrations is imperative to obtain a full
picture of c-mitosis.
3.3-3: Arbacia j>un( tiilata. Colchicine applied to eggs of Arbacia
at a specific time after fertilization, showed a clisintegrating action upon
the astral ray.^'^ They faded out shortly after the drug entered the cell,
and a "lakelike" body appeared at one end of the mitotic figure (Fig.
3.3) . The chromosomes were massed in the center of the cell. If the
drug entered the cell when two polar regions had already developed,
then two lakelike bodies were seen, one at each end. Finally, a still
later stage showed the chrom()sf)mes in two anaphasic chmips and a
lake area encircled the entire figure.
1 here is a critical time beyond which the colchicine does not stop
cleavage, but then a fluidity may be developed around each set of
chromosomes even though separate cells were formed.
The disintegration of amphiasters was rapid, and restitution nuclei
were formed after a scattering of chromosomal portions was obtained.
The destruction of the mitotic sjMndle at metaphase blocked cleavage
effectively. Thus, the spindle components are vitally important to
cleavage. The independence of the spindle action and a rhythm of
viscosity changes of the cortical layers, independent of mitosis, have
been demonstrated. The two processes may go on simultaneously.
These have been shown by methods for obser\ing the changes at the
outer layer of the cytoplasm.-"' "-
lliere can be no doubt that spindle fibers already formed can be
destroyed. The specificity between drug and fiber is necessary for such
action. A confirmation from materials representing diverse biological
Spindle and Cytoplasm 75
sources has been effectively concluded. Therefore, colchicine acts
either by an inhibition before mitosis or by destruction after spindles
ha\e been formed.
^.3-4: The pulayizalion micruscope. Submicroscopic structures
were followed with an improved polarization microscope adapted for
specific biological purposes. The birefringence pattern is clear because
Fig. 3.3 — Effects of colchicine upon first cleavage in Arbacia punctulato. The area where
colchicine causes spindle destruction is a "lakelike' body. Compare A, the control, with
B, a treated metaphase. A. Spindle fibers of untreated egg at metaphase. B. Colchicine
applied when egg was at metaphase, both polar areas laked and chromosomes are
clumped. 0.0002 molar concentration of colchicine in sea water applied 10 minutes
after fertilization, temperature 22 to 24.4 C. C. Prophase when treated causing lique-
faction of spindle and asters at one side. D. Spindle destroyed, chromosomes separated,
but no cleavage furrows. E. Three groups of chromosomes. F. Four groups of chromo-
somes with laked areas around each group. (Drawings adapted from photomicro-
graphs by Beams and Evans, 1940)
spindle fibers are optically anisotropic. The fibers, therefore, shine
l>rightly, as compared with a dark grey for the chromosomes.
The disappearance of the spindle was correlated with the disappear-
ance of the l)irefringcnce pattern. Therefore, as colchicine acted upon
the spindle, a reduction was noticed by a definite fading out of the
light pattern. Obviously the fibers changed their form under an attack
by the chemical. This general procedure made it possible to pci foi ni
some critical experiments.*"
Ihe first matuiation di\ision of the egg, the metaphasic sjjindle
of a marine annelid ^vorm, Chaeloplerus pergamcutnct'us. was cho.sen
76 Colchicine
for these experiments.^^ Normal metaphasic patterns are ^vell known
for this species at 25°C. Thus it was possible to judge the exact time
when a fully formed metaphasic spindle could be expected. Accord-
ingly, at this stage, the sj)indle fibers shone brighth- and chromosomes
Avere less brilliant against the light background of spindle fibers when
viewed through this polarization microscope.
An egg cell in metaphase immersed in colchicine-sea water, showed
1x10-5 3 5 IxlO-'* 3 5 1x10-3 3 5 1x10-2
MOLAR CONCENTRATION OF COLCHICINE
Fig. 3.4 — The average time for disappearance of metaphasic spindle of Chaetopterus
egg, disappearance measured by polarized light pattern. The stronger the concentration,
the shorter the time for complete disappearance of spindle. Temperature of sea water
25°C. (Adapted from Inoue, Experimental Cell Research Suppl. 2:305-18. 1952)
a Steady disappearance of the spindle. This meant that colchicine was
destroying an already formed metaphasic spindle. 7 he rate for a dis-
appearance was directly correlated with concentration. In line with
jjre\ious data, then, the greater the concentration, the more rajiid the
destruction of the spindle. Figure .S.4 shows these relationships
clearly. For example, in one test, the disappearance of spindle occur-
red in 30 minutes with the concentration 5 X 10'^ M. But an increas-
ing concentration (5 X It)-^ M) reduced the same stage of a spindle
within 3 minutes. Moreover, these observations were made by con-
tinuous records from living cells and not fixed structures.^"
By an entirely new technique the destructive action of colchicine
was traced from a fully formed metaphase spindle to the complete
disappearance. Finally, the cjuantitative relation I)et^veen concentra-
tion and disappearance supports the proposition that specificity has a
quantitative basis.
Spindle and Cytoplasm 77
Several other similar observations were made at the same time
spindle disappearance was studied. The continuous fibers are the first
to disappear along ^vith the astral rays. These observations confirmed
previous Avork. Accordingly, the last fibers to lose their birefringence
were the chromosomal fibers. 1 liese data also fit other results. The
order in which the component spindles disappear is important to an
explanation for the star metaphase. Acti\e chromosomal fibers and
supi^ressed continuous fibers create the star figure.
A\'hile stronger solutions cause the most rapid disappearance of the
spindle, the shortening of the spindle during its disapjjearance is not
the same for each strength. Rapid destruction showed very little
shortening, whereas weak solutions. Avhich rec^uire a long time, showed
much shortening during destruction. The shortening process carried
the chromosomes up to the periphery of a cell. While this reduction
in length of spindle occurred, the chromosomes were always main-
tained at a midway point between two poles. At the same time
chromosomes retained their metaphase position on the equator.
Another important detail was noticed just before the final dis-
appearance of the metaphasic spindle. The chromosomal fibers Avere
the last to disappear, and as soon as the last vestige of spindle faded
out, the chromosomes scattered. Tlieir position in the equatorial
plate exidently was maintained 1j\ chromosomal fibers. Thus chromo-
somal fibers are responsible for equatorial orientation. Chromosomal
fibers once destroyed caused a scattering of the chromosomes and a
typical exploded metaphase.
Spindle retardation, measured in millimicrons, showed that changes
in spindle measured against time, and plotted accordingly, showed a
rapid decrease at first then a sloA\ing doAvn of this process (Fig. 3.5) .
An exponential decay curve Avas obtained for this activity.
Confirmation of an action of colchicine along similar lines Avas
obtained by a phase contrast microscope in Avliich no spindle fibers
were detected 24 iiours after treating testis cells of Melanoplus difjer-
entialis Avith colchicine.'^" By other methods and Avith different chemi-
cals, the spindle fibers Avere studied as bodies that operated during
a mitosis. These could be destroyed, or transformed into other
structures. The net result was c-mitosis.^''
Fibers that appeared anisotropicallv acti\c. liiiearlv differentiated
Avith iiiicelhu- particles arranged end to end, changed in their
structural pattern. Birefringence sho\\ed that colchicine destroyed
the fil^rous arrangement progrcssivclv, step by stej). First the con-
tinuous fibers and asters disappeared, then the chromosomal fibers.
These critical tests w iih a polarization microscope deal a solid bloAV to
the argument that spindle fibers are (\ tological artefacts. Not only can
the spindle fibers be demonstrated bv a light pattern but their changes
78
Colchicine
under an influence of colchicine are traceable. Finally a quantitative
relation between concentration and rate of spindle reduction has been
established (Figs. 3.4 and 3.5) .
3.4: Changes in Spindle Form
1 he Allniin root tij> cells treated by the research group at Brussels
showed that a differentially stainable body was lornied in the col-
30
20 -
10
LENGTH
WIDTH
10
15 (min.) 20
H)
5 -
4 -
3 -
2 -
1 -
O
RETARDATION
5^,0-4M ^-
10
15 (min.) 2C
Fig. 3.5 — The shortening of spindle as it disappears differs according to the con-
centration. The strong solutions cause rapid disappearance and not much shortening
of spindle. The width does not change as much as length of spindle. Measurements
of retardation in millicrons show rapid retardation at first, then gradual slowing toward
the end. Top group shows decrease in length compared to width for two concentrations.
Bottom group indicates the sharp drop at the beginning and slower rates of retardation
until final disappearance. (After Inoue)
chicinized cells.-'' The chromosomes were clustered about this body
(Fig. 3.6) . .Such structures persist through the interphase and be-
come prominent in the large amoeboid restitution nuclei (Fig. 3.6) .
Although the relation to spindle was not suggested until later,^^' ^^
the role of the deformed spindle has been mentioned lor a number of
Spindle and Cytoplasm 79
cases. Specificall)', this was called the achromatic sphere and the
pseudospindle. Related to this same structtire from ol)ser\ations
with neuroblasts is the hyaline globule.^"
lliese bodies do not show polarity, their staining properties are
distinct from cytojjlasm. and their relationship to spindle material or
karyohniph is a good one. It was belie\ed that the c-])airs regularly
Fig. 3.6 — Cell of Allium root treated with colchicine showing the spindle substance around
which chromosomes are grouped. Another amoeboid nucleus shows the influence of this
substance. (Photomicrograph made from slide of the A. P. Dustin Collection, Univer-
sity of Brussels. An unpublished photo similar to diagrams by Havas, Dustin, and Lits,
1937)
associated around the pseudospindle, and that this structure accounted
for the cxjjloded metaphase. Indeed the chromosomes were distributed
bv this bod\, and the specific distrilnited c-mitosis was seemingly re-
lated to the pseudospindle, but no tiniher direct associations can be
made.-"' ^^' ''^ Different subjects tend to show different kinds of
material. Ihe clear area around chromosomes^'' and the lakelike
bodies of Arbada may all be related to these deformed spindle
materials.
80 Colchicine
Some materials, such as Spinacid' and Lepidiuni,''' do not show the
body. Not all cells of Allium develop the achromatic sphere. There
may be some progressi\e relational dcxelopment, or a specific con-
centration may be required for producing the achromatic sphere and
other similar bodies. That a defniitc progressive stage is followed was
carefully shown by the work with neuroblasts.
Until the final answer is obtained, our jirescnt obser\ations ha\e
led to the idea that fibriform materials, that is, substrate making the
spindle fibers, are converted into a corpuscular form instead of the
usual fibrillar arrangements. Colchicine plays a role in directing the
spindle fiber substance into these modifications noticed for many
cells. The course of development of the spindle to its disappearance
in neuroblasts and the jjrogressive enlargement of the hyaline globule
as the spmdle fibers disappear, point to the fact that a spindle
material is converted into another form and this form is shown by
the hyaline globule. Such a body has definite ojJtical characters, size
relationships, and is, in fact, a structine that must be given serious
consideration as a changed form of spindle substrate.
If the globules form at prophase, then karyolymph is suspected to
be the original material. When metajjhasic and anaphasic stages are
studied, the spindles ha\c been de\eloped and (|uite another A'iew
comes into focus. In such cases, colchicine progressively reduces or
destroys the spindle, and globules form as spindles disappear. Such
globule formation requires a longer lime at metaphase or anaphase
than at prophase. Again, both concentration and stage of spindle are
important factors in conxerting the spindle into globules-^" (cf. Sub-
section 2.4-3) .
W'hen 25 and 50 X 1^ '' ^^I colchicine solutions are ajjplied during
anaphase, the spindle disappears and a hyaline globule forms-^' (Fig.
3. ID). The globule occupies a position near one of the poles. The
formation of a globule, as the drug acts, leads to a correlation between
s|Mndle and globule. Since concentrations determine spindle de-
struction, the globular formations are likewise dependent upon con-
centration. These facts are clear.
In agreement with reports on the hyaline globule specifically noted
in treated nemoblasts, a similar structure, the achromatic sphere, has
characteristics in common with the hyaline globules. Very likely
these are similar, just as the spindle fibers of mitoses in cells of plants
and animals have certain similar projjerties. Characteristics of the
hyaline globule are: (1) it is spherical: (2) diameters vary from 3 to
15 microns; (3) rate of formation is related to speed of spindle de-
struction; (4) it is opaque, homogeneous, of high \iscosity, not sur-
rounded by membrane, and is optically indistinguishable from karyo-
lymph or spindle; (5) it tends to lodge at top of cell while chromo-
Spindle and Cytoplasm 81
somes settle to bottom.-^" Finally after all these characteristics are cited,
the fact remains that in colchicine-treated neuroblasts, the hyaline
globule increases when disorientation of chromosomes and spindle
destruction take place. Obscr\ations such as these support the idea
that, as colchicine acts, spindle structure becomes altered rather than
annihilated.
The spindle fiber analyzed by electronic microscopy can be de-
scribed as compound, measuring from 600 to 800 A at the polar cap
stage.^^- AV'hen colchicine is applied to AUium root tip cells tor 30
minutes, the fibers lose their compactness. After one-hour exposures
the fibers are disoriented and fragmented. After 2 hours the fibers
api^ear swollen as well as increasingh fragmented, fn the untreated
cell, fibers remain as such regardless of the type, whether they be
chromosomal fibers, continuous fibers, or fibers of the polar cap stage.
With long exposure to dilute solutions or short exposure to stronger
concentrations, a decided swelling and a tendency to^vard "solubili-
zation" of their substance were apparent.''-
3.5: The Arrested Metaphase and Spindle Mechanisms
Interaction between colchicine and spindle fibers ultiniatelv de-
termines the arrested metaphase. The two types, oriented and un-
oriented,- both depend upon several \ariables existing during a treat-
ment or during a reco\ery from the drug. As mentioned before, con-
centration of colchicine, mitotic stage at time of action, length of ex-
posure, recovery processes, type of cell, and conditions favorable to
mitosis, all play an important role in the production of the particular
arrested metaphase, whether oriented or unoriented."''
A pattern such as the star metaphase (Fig. 3.1C) is far too regular
to be regarded \\holly as a random occurrence. During a reco\ery,
the star is characteristic, as is also the multiple star (Fig. 3. IF) . These
types do not reach a jjeak in a reco\ery until some time has elapsed
between application and the dissipation of drug. A majority of the
bipolar mitoses follow the star metaphases, thereby indicating that
reco\erv ^\•as nearing completion. Fhe star metaphases are the last
colchicine effects to ap)X'ar during recovery. The Triton material that
was fixed- directly out of colchicine and staine'd^ at three hours and
at succeeding intervals, shows that stars appear at once and build
u\) much faster than in TritunisJ'^ When the stars reach a jK'ak in
Triton, unoricntcd tvi)es, rather tlian bipolar mitoses, become the most
j)rominent mitotic figures.
Any pattern, whether star or exploded metaphase, sliould be re-
garded as a response to colchicine, operating primarily through the
spindle fibers. Two basic comj>onents are accepted as established for
plants and animals; these are (1) continuous fibers and (2) rhromo-
82 Colchicine
somal fibers (Fig. 3.1) . Sometimes these two are called the exterior
and interior spindles, ^ or the centrosomic and centromeric spindles. '^'^
The birefringence ]jattern for a metaphasic sjiindle^' in Chae-
toptenis egg, disappearance due to the action of colchicine, registers
the fading of continuous fibers and astral rays first, while the chromo-
somal fibers are the last to disappear. Action uj^on astral ravs before
the interior jiortions has been demonstrated with other material. •**
Hence, data on the Ii\ ing cell and f)n fixed tissue are in accord as to
the action upon the several parts of the total spindle.
Acenaphthene is 1000 times slower in action upon a spindle than
colchicine. ■'"'•'' This slower activity jjermits a better analysis, because
the exterior spindle is destroyed before the interior. Colchicine acts
so totally and abruptly that this delicate difference is frequently o\er-
looked. Until the threshold concentrations are employed, a partial
action showed that colchicine in dilute solution, like acenaphthene,
destroyed the exterior spindle before the interior. That is, continuous
fibers are first to be affected. This exjicrience is like dissecting an
organism into its essential parts. •^'''
Certain concentrations of colchicine applied to the metaphasic
spindle in neuroblasts cause star formations (Fig. 3.1). The con-
tinuous fibers are inactivated, but chromosomal fibers remain intact.
The centromeric ]>ortions of chromosomes are drawn to one focal point
(Fig. 3.1). Ihere, however, is another way to j^roduce a star meta-
phase in neuroblastic cells. To obtain the correct concentration for
prophasic treatment, enough colchicine is used to inhibit the con-
tinuous fiber in its development, bin such a concentration does not
act in the same manner on the chromosomal fiber. These interactions
lead to a star metaphasc.
Now a final explanation for Triton- and Tnturus"^ appears to be
at hand. Tritoii cells removed from colchicine show star metaphases
at 3 hours, build up to a jjeak within 12 hours, and are succeeded by
unoriented metaphases. Colchicine acts progressively more strongly
as the peak is being built. During the action, continuous fibers were
destroyed before chromosomal fibers, tjivina^ cause for stars in Triton
cells. Finally, the whole spindle was inactivated when colchicine
reached full effect and unoriented types took precedence (cf. Chapter
2) . Inspection of data from Tri turns"'* leads to another observation.
The stars appear later, and after the peak is reached, the bipolar
mitoses occujjy the prominent position among dividing cells. As re-
covery was taking place, the colchicine was becoming more dilute. At
a certain point the continuous fibers were inhibited but not the
chromosomal fibers. Then at last, Ijoth continuous and chomosomal
fibers developed, and bijjolar mitosis predominated among the divid-
ing cells. Among cells of Triton the stars appear as the effect of col-
Spindle and Cytoplasm 83
chicine beains. Tlie stars ^verc the "arrivals" in this case. While
Triturus cells developed, the star showed that the effect of colchicine
Avas "departing."
\Xe may conclude that the star i'ornis when centriole, centromere,
and chromosomal fibers interact while continuous fibers are sup-
pressed. A mitotic polar metaphase appears much the same as the
star, btit the latter has very small, if any, stainable achromatic core.
The size differences have been demonstrated in several instances. '^^' ^' ^^
Chromosomes occasionally fall outside the star cluster. Lagging
chromosomes may be observed in tmtreated cells. Neuroblasts, treated
with very weak solutions of colchicine, consistently show lagging
chromosomes. The lost chromosome is confirmation that a partial
spindle inactivation takes place when these partictdar types form.''-*
Mtdtiple stars (Fig. 3.2/) are basically the same as the single star,
except for several focal centers instead of one. If two or more chromo-
somes fell outside the first star, a second could form. This type is most
common when cells are recovering in AUiuin root tips. Increasing the
ninuber of chromosomes shows a corresponding increase in the
number of multiple stars. Multiplex stars have been demonstrated in
both plants and animals, during recovery as well as during active
treatment. Triturus showed the bimetaphase and trimetaphase, c(|ui\ a-
lent to nudtipolars, five to six days after recovery. '^^
Distorted stars- are not proved as easily as the star formation. Two
explanations ha\e been given. One, the action is a response of centro-
meres and a centrosomic center, but the staining procedures did not
bear otit these assumjjtions. l\vo, the hxaline globule which forms
when sjiindle fibers disajipear. becomes ^vedged between the chromo-
somes, distorting the star.-^' Either explanation may be considered \ alid
tmtil more information is at hand.
Unoriented metaphases. such as ball, clumped, prophase-meta-
phase, or exploded types, do not show activity on the chromosomes or
any j^art thereof. The term uiioriruted is entirely appropriate- for
such figures (Fig. 3.IG, 3.2rf) .
An exjjloded or scattered arrangement has been observed in many
plants and animals (cf. Chapter 2) . It the disappearance of a meta-
phasic spindle is follow^ed by the birefringence pattern,^' one may
assume some mechanical explanation for the exploded tyj)c. for as
soon as the spindle disappears completelv, the chromosomes seem
to scatter as if they were held on the ecjuatorial jjlate to the very last
moment. Disappearance of the continuous fibers did not permit the
scattering. Not tmtil chromosomal fibers disap]:)eared did the chromo-
somes disperse. This confirms that the exploded metaphase originates
when both chromosomal and contintious fibers are destroyed. Such
observations support the concepts that a fidl c-mitosis may in\f)lve an
84 Colchicine
exploded mctaphase and ihat complete spindle inacti\ation is funda-
mental to the unoriented type or lull c-mitosis.
Presence of the pseudospindle"^ or the achromatic sphere'^S' ' (Fig.
3.9) has helped to explain the scattered arrangement in some cases,
notably in Allium root tips (Fig. 3.7). C-pairs are closely appressed
around an achromatic sphere. But comparable cells in regenerating
liver exhibit excellent exploded metaphases without a stainable
sphere. Other scattered types are not comparable to the special case
of All i inn.
The assumption- that a single centrosomic spindle operates in
pushing the chromosomes to the periphery of the cell is hardly ten-
able, for staining has not proved the case, nor have the other tech-
niques subtantiated such mechanisms. It would hardly be consistent to
classify as an unoriented type, one that had such a mechanism as a
central spindle pushing the chromosomes to the edge.
Whatever the final answer will be as to their disposition, they
seem profusely scattered, and seem to lie in the cytoplasm as if each
repulsed the other.
The exploded metaphases are a striking type.^^' ^''> They would
seem to result from the total inactivation of both the continuous and
the chromosomal fibers.
The ball metaphase is more common than the exploded mcta-
phase; it increases in frequency as the concentration increases. A
toxic or poisoning action is logically the basis of a ball metaphase.
The chromosomes are defmitely unoriented and are often massed in
a clump. For that reason the c-mitosis has been called ( linnpcd, a t\pe
related to the loall metaphase.^"' '^~
Prophase-metaphase formations (Fig. 3.2) are more nearly de-
scribed by the term arrested prophase (cf. Chapter 2) , for they re|)resent
leftOAcr prophasic arrangements. AVith no spindle action, chromo-
somes remain stranded in a pre-prophasic arrangement.^^'* In fact there
is complete inactivation. Prophase orientations are not necessarily
disturbed by colchicine, as noted for Dipcadi.''-' Here the chromosomes
are disposed in a pattern determined by the previous telophase. If
the concentration is partially inactivating, a star metaphase results;
total inactivation leads to the prophase-metaphase type.-^- '» The pro-
phase-metajjliase merges into the ball metaphase and clumped meta-
phase depending on the concentration. There may be return by re-
covery to a multinucleate cell. The prophase-metaphase and clumped
c-mitosis seem to be more characteristic of meristematic cells of stems
than of roots. ''^
Distributed c-mitoses have attracted nuich attention because they
were described as a "somatic meiosis" (cf. Chajiter 2) . These are a
subtype of the exploded metaphase. The main diiference between
exploded and distributed metaphase is seen in the disposition of the
Fig. 3.7 — Allium root cells treated with colchicine. A. Cruciform c-pairs associated around
the spindle substance. At bottom of group one pair is completely separated in c-ana-
phase. The timing of separation is upset as well as delayed. B. C-pairs with arms fully
repulsed. A light, unstained area surrounds the chromosome. C. Chromosome reverting
to interphase; dechromatization has occurred. Chromosomal framework associated with
the central substance. D. An amoeboid restitution nucleus around the pseudospindle or
achromatic sphere. The end of at least one c-mitosis. (Photomicrographs furnished by
courtesy of Dr. C. A. Berger, Fordham University, N. Y. After Berger and Witkus, 1943)
86 Colchicine
c-pairs. Polar groupings of c-pairs typily the distributed metaphase.
whereas exploded metaphases are nonpolar. Unquestionably, the
distributed c-metaphase was clearly illustrated in pollen tubes.'^^ The
distributions were equal and unequal. They were not conceived as a
somatic meiosis. In root tips, naphthalene acetic acid and colchicine
increased the number of distributed c-mitoses compared with either
chemical alone. Other chemicals increase this type even more than
colchicine.
3.6: Spindle Disturbance and Cytological Standards
Spindle disturbances in plants may be classified in three cate-
gories:"'^ (1) full inactivation, stathmokinesis,-^ (2) partial inacti-
vation, merostathmokinesis,-^'^ (3) slight disturbance in orientation,
tropokinesis.^^' -^ All these types are produced by colchicine, as al-
ready pointed out. If one wishes to make comparative studies with
other chemicals known to influence mitosis, well-defined cytological
standards of judgment are needed to classify reactions as either dis-
turbed or normal. If the reaction is disturbed, it is important to dis-
tinguish the type according to velocity or strength of reaction. The
most reliable criteria appear to be those based upon tests at telophase,
rather than at earlier stages.'^-^
Abnormal chromosomal distributions may be caused l)y spindle
disturbances in three degrees: first, multipolar; second, ajjolar: and
third, luiipolar. When three or more groujjs of chromosomes join so
as to form discrete groups, partial spindle disturbances are obvious.
These were carefully noted under the general type, mcrostathmoki-
nesis,-^*^ or under the present classification as midtij^olars. Howe\er,
complete destruction or inactivation lea\es one single grouj), or there
may be two groups with no e\idence of spindle function. This is the
apolar distribution. Another specialized distiubance is the close gather-
ing at one focal point described before as the star metaphase; this type
becomes unipolar at telophase.'-^
Colchicine (().0{)5 per cent) a]jplied to Allium root tips for 46 hours,
increases the percentage of trojjokineses. Ihe controls may show as
many as 10.5 per cent, but treated root tips raised the frequency to
21.3 per cent. These disturbances are the first-order changes occurring
at threshold concentration,-^ and are the first signs of spindle dis-
turbance.
3.7: Cytoplasmic Division
Nuclear mitosis and the completed process of cell division are not
synonymous, because the nuclear processes and cytoplasmic processes
taken together make up cell di\ision. Truly, karyokinesis (nuclear
mitosis) and cytokinesis (cytoplasmic processes) are very highly intc-
Spindle and Cytoplasm 87
graied, and are closch coordinated processes/^ One cannoi always
mark the separation between the jjrocesses. For this reason and per-
haps others, biologists use the term mitosis as completely synonymous
with cell division, when mitosis is only one aspect of a dividing cell.*"
A\'hen colchicine acts during a dixision. the significance ol what
has been noted lor mitosis and cell di\ision becomes apparent. The
multijjlication of chromosomes continues in the presence of the drug
at a certain concentration, xvhereas the total absence of spindle fibers
prevents the movement of chromosomes to the respective poles. In-
hibition of fibers has one drastic effect on the cytoplasmic phases of
cell division: the cytokinetic processes are completely eliminated.
Among animal cells the cleavage jirocesses are somewhat specific and
respond to colchicine in a unique fashion. These aspects are discussed
in the next section. In plants no cell plate is formed, and phragmo-
plasts are prevented. For organization purposes these are discussed
separately from animal cells.
5.7— /; Cleavage processes in annuals. Marine eggs have been sub-
jects for studying the mechanism of cell di\ ision since the pioneering
work of Hertwig, Boveri, and \\'ilson. The sea urchin, Arbacia
pnn( tiilata, was therefore a logical selection for Nebel and Ruttle"-
when, in 1937, they wanted to analyze more completely the activity of
colchicine. They established that 10 ^Ai" concentrations block cleavage.
Even a concentration of ().00()2 M inhibits cytoplasmic division'' if
applied 22 minutes after fertilization at 22° to 24.4°C. At this time
eggs are in prophase, metaphase, or early anaphase, and spindle mecha-
nisms are inhibited or destroyed by colchicine (Fig. 3.3) .
If nuclear mitosis passes a certain stage, clea\age is not stoj^jjed
by these concentrations. Therefore, a critical point is reached beyond
which destruction of spindle apjiarently has no effect. These points
emphasize a close integration between nuclear mitosis and cytokinesis.
20. 97, 98
Specific objectives were outlined to determine precisely up to
Avhat stage or stages in the mitotic cvcle treatment was effective in
blocking cleavage and at Avhich stage colchicine Avas no longer effec-
tive. The results showed that suppression of cleavage by colchicine
follows a particular course on the basis of fertilized eggs of Arbacia
pun( tiiJata? The eggs were allowed to stand 10 minutes after fertili-
zation: then different lots were placed in colchicine at 2-mimae inter-
vals dining a 60-minute period. B\ this test, a lapse of 22 minutes be-
tween fertilization (22° to 24.4°C.) and the addition of colchicine was
found as the critical period, because cleavages were not blocked after
that time (Fig. 3.3) . 1 he mitotic stages most generally present at
this time were prophase, metaphase, and possibly early anaphase, each
of which was affected b\ colchicine. 14iese stages regularlv precede the
88 Colchicine
usual furrowing process by about lU to 14 minutes. Therefore, after
the critical mitotic stage, anaphase was passed, the furrowing pro-
cess started, and after that point colchicine did not inhibit cleavage
of the cell into two parts.
Similar results were obtained from tests-" using the starfish.
Asterias forhcsii; the sea urchin, Arbacia punctulata; sea urchins from
Bermuda. Tripneustes esculentus and Lytechinus variegatits: and the
sea slug, Chroinodoris sp. In all cases, the key for inhibiting cleavage
was anaphase. The concentrations varied, but otherwise the general
plan was very similar for all tests. Once the eggs passed metaphase,
cleavage could not be altered by dosages of colchicine that destroyed
the mitotic spindle. If threshold concentrations were used at meta-
phase, furrowing almost divided the egg, and a regression then set in.
This showed that the final closing of cytoplasm is distinctly a process
dependent ujjon the spindle. Cases such as these emphasize the inter-
dependence between karyokinesis and cytokinesis as processes of cell
di\ision that invohe nucleus and cytoplasm.
Cytological evidence for action by colchicine is obtained from the
lakelike bodies appearing where astral rays and spindle fibers nor-
mally should be found'' (Fig. S.?>) . One lake body indicates pro-
phase; two, one on either side of a clumped mass of chromosomes,
point to action at metaj^hasc: and two clusters of chromosomes can
be taken as evidence for disturbed anaphase. All these prevented
cleavage.
Furrowing is dependent upon viscosity changes, and once processes
begin, apjjarently colchicine does not stop cleavage. In an effort to
correlate such changes with the cleavage process, centrifugal exjjeri-
ments were run, but not all results are in agreement." Ihe addi-
tional evidence ''" for viscosity or rigidity relationships and nuclear
mitosis as well as cytoplasmic division are discussed under the mecha-
nisms in the last chajjter.
A demonstrated fact emerges that cleavage is averted if achro-
matic figures are destroyed before a certain mitotic stage has been
reached. Of course, concentration \arial)ilities are important, but
the blocking process appears to be an "all-or-nothing" effect; there-
fore, either nuclei divide and there follows a cytoplasmic di\isi()n, f;r
an arrested mitosis precludes daughter cell formation. For example,
chromosomes, scattered as a result of colchicine, form micronuclei.
and no cytoplasmic di\ ision takes place."*- i*'- ^^ On the other hand, re-
covery among a numljer of star mctaphases may eventually lead to
the cytoplasmic division, because spindle inactivation is not complete.
Depending ujK)n (oiuentration, cleavages may be retarded or
stoj^ped (Fig. 3.3) . The germ cell of Triturus helveticiis L. does
not cleave if a 1:500 colchicine solution is used.''-' Regeneration of
Spindle and Cytoplasm 89
the spindle may determine the course of cytokinesis. These data
have been limited mostly to eggs, where the principles of cytokinesis
in relation to the mitotic mechanism are better observed than among
other animal cells. Further data on the action of colchicine on eggs
are to be found later (cf. Chapter 8) .
In those cases where a lowered \iscosity is related to mitosis, it is
assumed that the gelation-solation phases are influenced.^ If solation
conditions destroy spindles, then lowered viscosity acts accord-
ingly. Sj)indles arc inhibited because colchicine acts upon a mechanism
that changes the solation conditions. But viscosity changes ma\ be
secondary efl^ects \\hile other mechanisms operate before cytoj)lasmic
changes take place. ••"
Birefringence tests show that the normal \ariations of the cortical
layer of eggs of the sea urchin, Psammecbinus tniliaris, presumably
s^'chronized ^\ith sjiindle and monaster expansion, are entirely inde-
pendent.'" The sjjindle and \iscosity changes in the cortical la\ers
may go on simultaneously, yet remain independent. Rhythmical
surface changes of eggs of Tubifex were not modified by arrest with
colchicine. This further substantiates the premise that c\ tojjlasmic
processes are not entirely controlled ^\hen the mitosis is controlled.
In the neuroblastic cell, lowering of c\ toj:)lasmic \iscosity was
visible through the increased activity of mitochondria.-^'-' Brownian
movements were used to indicate the changes. Chromosomes settled
to the lower half of the cell when spindles were completely destroyed.
Disappearance of the spindle and a more rapid Brownian movement
•were correlated. The notable decrease in \iscosit} was suggested as a
consequence of a decrease in the content of ribonucleic acid and
phosjjhorus at the time colchicine acts upon mitosis. •^•*
3-y~-- Cell plate foffiidtion i)! phnit.s. The continuous fibers form
the spindle of c\tokinesis upon which the cell jjlate forms. Between
the spindle and cell wall a phragmoplast completes the fibrous struc-
ture and the cell jjlate across the cell."- ■*■' Since colchicine destroys
or prevents continuous fibers, there is no spindle of cytokinesis or
phragmoplast.
During recovery and regeneration of the sj:)indle, \ arious abnormali-
ties may be seen, but these processes are characteristic only in rela-
tion to recovery and rc\ersible effects of ^vhi(h the cells are capalile
after colchicine.
By the special technic|ues for apphing colchicine at certain stages,
the phragmoplast has been tested specificalh with regard to the role of
the drug acting u]jon such structures already formed. ■'•'* If the
phragmo]jlast is in formation, colchicine can reverse the process,
changing the fibers hack to a fluid stage, a kind of cytoplasmatization.
E\en rudimentary cell plates and the beginnings of septa from each
90 Colchicine
side are arrested. Under these conditions further development is
arrested, and chromosomal bridges extend between the cells.^^
Direct destructive action upon cell plates was recorded also in
wheat root tip cells. Generally, the absence of spindle determines
the formation of a restitution nucleus precluding any form of c)to-
kinesis as well as daughter nuclei/^=^- ^^- ^^' ^^^ ^" The interrelation
between cytokinesis and mitosis is shown by the effects of colchicine.
By centrifuging root tips treated with colchicine, a much greater
displacement of chromosomes against the centrifugal wall was found
among treated cells than among the controls. The action of the drug
was interpreted as an effective lowering of c)toplasmic \iscosity.
Allium root tips treated with colchicine at varying exposures were
centrifuged to determine changes in structural viscosity of the achro-
matic figure. The decrease in \iscosity was indicated. Moreover,
there was a low viscosity at eight hours, when c-mitosis was at a peak.
After return to normal bipolar mitosis the viscosity showed increases
paralleling these recovery processes.
Another view somewhat opposed to that expressed above has been
presented. Since the spindle fibers are inhibited and no achromatic
figure is present to hold the chromosomes in position, greater dis-
placement may take place regardless of \iscosity change. The centri-
fuge tests merely show that the spindle fibers are lacking. Supportmg
this \iew are the obser\ations on cyclosis in Elodea, which does not
seem to be changed by colchicine.
Additional tests showing changes in viscosity among plant cells
are reviewed in Chapter 4.
:}.'j—^: Cytoplasmic (O)istitueuts and cell organUes. The centro-
some, a self-perjietuating Ijody outside the nucleus, becomes involved
with spindle destruction. Its activities are depressed along with
those of the si)indle mechanism. Several centrosomes may accumulate
within a cell treated with colchicine, hence the formation of multiple
stars. Each star probably represents a centrosomic body. These were
carefully demonstrated in Triturus xnndescens.
A confusion arises from the mitochondrial picture and colchicine.
Some say these bodies are affected b\ the drug;i'^^' others report no
change.-^ The concentrations as well as materials vary widely, but it
would seem that some consistent reaction might be obtained. Ho\\-
ever, until now we can only re\ ie^v the j)ro and con. Modifications
involving fragmentation, dispersion, reduction, as well as minor
morphological changes have been seen after colchicine treatments
directed to: (1) Flexner-Jobling carcinoma of rat, (2) liver cells
of rat,^i-^- (3) cells of certain orthoptera, GyrUiis assimilis and
Mflanoplus diffeyentialis.-''' No mitochondrial modifications are re-
ported for neuroblasts in Chortojjiiaga vindijasiata;'^ an observa-
Spindle and Cytoplasm 91
tioii coinciding witli a jjhase contrast observation of Siredon erythro-
blastic prophase-metaphases made by the junior author (un-
published) .
Root meristeniatic mitochondria tended toward constrictions and
fragmentations after exposures to colchicine for more than 25 hours
(0.005 M colchicine) (Fig. 3.9). Shorter exposures, 13 hours, were
less effective. The relation between viscosity and mitochondrial shapes
was believed valid.'^^ The mitochondria were demonstrated in Allium
(Fig. 3.9) in which cases mitochondria did not j^enetrate the
achromatic sphere (Fig. 3.9) (pseudospindle) about which the c-pairs
seemed to collect."^
While the Golgi bodies have not received the attention given other
cvtoplasmic organites,-^'^ fragmentation and scattering of these bodies
were induced in adult mice by 0.1 -mg. colchicine injections.^-^
Metabolic aspects of cytoplasm were demonstrated among tissue
cultures by differential staining with methylene blue (1:10,000).
The arrested mitoses remained colorless \\hile the cytoplasm of
resting cells was diffusely stained. Untreated cells in division are
also colorless because methylene blue is reduced more rapidly when
cells are dividing."'*' This suggests that arrested metaphase reduces
methylene blue like a regularly di\iding cell. This metabolic activity
mav provide an explanation for the e\entual destruction of arrested
mitoses in animal cells'"** (cf. Chapter 2) .
"Bleb" formation occurred at cellular surfaces among grasshopper
neuroblasts-^'^ when mitosis was arrested. Also, notable cytoplasmic
agitations were seen among fibroblasts treated with colchicine and
studied by cinematographic projection. i" These observations call
attention to an unusual activity when cytoplasmic division is pre-
vented by colchicine. This agitation has been described by others
using treated tissue cultures.^^- '^^ Changes at cell surfaces can also
be induced by many other substances, such as mustard gas and ultra-
\iolet radiations. ■"'*•
Some observed cases do not indicate direct action by colchicine.
The marine eggs of Psamynechiuus tniliaris obser\ed for birefringence
characteristics indicated that actions in the cortical layers were inde-
pendent of mitotic arrest."" Tubifex eggs pro\idcd additional cases
for observing the relation lietween changes in c\ toj)lasmic \ iscosity and
mitotic cycles.'''^
3.8: Reversible Characteristics of the Spindle
L.et us summarize what has been detailed from Chapter 2 uj) to
this i>oint. If we compare a colchicine-mitosis (c-mitosis) with a
regular mitosis, our first impressions might well be the foll()^v•ing:
c-mitosis is mitosis without metaphase, anaphase, and telophase;
92 Colchicine
c-niitosis precludes cytokinesis; c-niitosis leads to a restitution nucleus;
c-mitosis prevents daughter nuclear formations; c-mitosis stops the
formations of daughter cells from a mother cell. Oin- sunniiary im-
plies — and similar implications can be found in the literature''^ — that,
whereas during c-mitosis the notable stages of a normal mitosis are
omitted, whereas a single nucleus is formed instead of two, and
whereas one cell begets one cell, the whole c-mitotic process appears to
be a quicker and shorter one. Seemingly, the reason for this is that the
arrested metaphase is a bypass method ultimately short-circuiting, by
the influence of colchicine, true division of a cell. But in reality, these
apparent abbreviations that woidd seem to shorten c-mitosis, re(|uire
more time than a regular mitosis ctnering similar chromosomal trans-
formations. For example, one c-mitosis takes 430 minutes compared
^vith 155 minutes for a normal mitosis. ^'•'* Furthermore, during the
155 minutes, chromosomes become inxohed in metaphase, anaphase,
and telojjhase. During the 155 minutes, two cells each with a nucleus
are deri\ed from a mother cell and one nucleus. In other words, a
c-mitosis (430 minutes) that gives an impression ol a shorter pro-
cedine by omissions, actualh takes 2.8 times longer than the corre-
sponding control (155 minutes) .
These comparative figures are accurate measurements from con-
tinuously recorded cases of individual living cells, passing through the
entire cycles of c-mitosis and mitosis, respectively. Contrary to these
time sequences, Epliedra pollen cells showed no difference between
treated and untreated cells.''^ However, changes may have influ-
enced these time sequences, so that transformations from prophase to
interphase took place without a delayed metaphase.''^
As jiointed out in Chapter 2 and summarily stated abo\e, a time
scale comparison between c-mitosis and normal mitosis is like pro-
jecting a moving picture in slow motion. Action for 155 minutes is
stretched out to 430 minutes. Noav. most of this extra time is taken
up while the chromosomes appear to lie scattered in the cytoplasm,
unoricnted because colchicine inacti\ated the spindle fibers, in con-
trast to the metaphase-anaphase stages that are oriented and activated
by spindle mechanisms. \Vc may refer to this phase as the "intactness
period" of the chromosomes. Chromosomes retain an individuality, an
intactness, ten times longer under colchicine than do those of the con-
trol cultine, because, out of 430 minutes, 249 are relegated to an in-
tactness period, against 23 oiu of the 155 in a contiol cell. Remem-
bering that such data are taken from living cells continuously observed
and recorded, these facts are sii^nificant.
After a c-mitosis is accomplished, the restitution nucleus forms a
single unit that combines the chromosomes which regularly become
distributed equally among two daughter nuclei.'^'' Of coinse, a "pre-
Spindle and Cytoplasm 93
cocioHs reversion" Iroin c-metaphase or earlier arrested stages as well
as a recovery in due course of time, often true for animals''*^' ^^- -^- "•'■
78. !ti,s.>. 1)^ ,^o[ limited to them, creates a restitution nucleus or
daughter nuclei with diploid luimhcrs of chromosomes (centro-
meres) , because in these cases a c-anaphasc does not obtain, under
conditions of rei'ersiou or recovery, from an arrested stage. However,
doubling of chromosomes can and does take place among animal cells.
51, 76, 86, 11. 3, 4, 2, 83. 22. 74. 65. 81. 48 Altliough this piocess of dupUcatiou
is more common to ])Iants treated with colchicine, neither situation
should be regarded as typical for one grouj) or the other. Such gen-
eralizations lead to false conclusions.
rhree statements concisely express the primary concepts: (1)
c-mitosis creates a jjolyploid restitution nucleus via c-metaphase-c-ana-
phase-c-telophase })rocesses; (2) c-mitosis by precocious reversion from
c-metaphase, or earlier arrested stage, may with exceptions, lead to a
nonpolyploid restitution nucleus; (3) c-mitosis may after due time re-
cover from the arrested stage and dexcloj) regular anaphase, instead
of the c-anaphase, thus leading to diploid daughter nuclei.
Greater than all these remarkable features is the underlying bio-
logical principle of reversibility. When the cell, in contact with the
drug for a given time, is removed from the influence of colchicine,
either by actual transfer or by allowing dissipation of chemical dining
a recovery period, the characteristics of reversibility come into locus. •^•''
Cells treated \vith optimal dosages that induce a c-mitosis creat-
ing the polyploid nucleus, recover so that a normal mitosis may fol-
low with a fully finictional bipolar spindle. That is, a restitution
nucleus can regenerate a bipolar sj)indle after the effects of colchi-
cine are remo\ed.-'^
Resieneration amony- the restitution cells is peinianent, and cells
develop spindle mechanisms in each succeeding division with meta-
phase, anaphase, telophase, and, of course, the doubled number of
chromosomes. This new divisional process continues thus, as long
as the cell lineage retains jjower to divide. Polyploidy is thereby main-
tained and continued without attending cvtogenetic changes, except
for those effects related to an increasing numlxr of chromosomes ]jer
cell.-55 No one has demonstrated by careful cytogenetic methods that
colchicine at optimal doses for a c-mitosis leading to polyploidy, also
increases the frequencies of mutations or chromosomal changes.-'-'' '■'-
Caution at this jjoint is advised because miuations and chromosomal
changes mav occur inde])endenily of colchicine but simultanecnish
with a treatment.-'-'
1 he capacity of the cell to recover after a treainunt. to legenerate
a bipolar spindle following a c-mitosis, to reverse the ina(ti\aiing
effects ol colchicine upon spindle: these are, in oin- opinion, the most
94 Colchicine
strikin^• and significant biological characteristics demonstrated when
dividing cells of animals and plants come in contact with optimal
doses of colchicine.
^.8—i: Recovery in j)Iants. Allium root tips transferred to pure
water after specific exposures to colchicine are excellent materials for
tracing recovery of the spindle mechanism. Very slight toxicity, if
any, results from an exjjosure sufficient to inactivate the spindle com-
pletely. Usually 12 to 24 hours in \\ater gi\e adequate time for first
recovery stages. '^^^ "•''• ^^- ''i- ""•■ -i- -^
The regeneration of spindle runs a characteristic course, proba-
bly representative of many plant cells. But most work has been
done with Allium cepa L. specifically, and with root tips rather than
stem tips, generally. By a characteristic course is meant the sequence
of chromosomal groups from full c-mitosis to partial c-mitosis, then to
bipolar spindles. During this course the obvious abnormalities appear
in terms of normal mitosis.-^-^- -^f'- '"■'■ -'■ '■'■ •^■''' "!• ^"'' '• -!• -'^ First, the chro-
mosomes group into what may be called midtijilc star formations
(Figs. 3.6 and 3.8) . There is no connection between the various stars
of a single cell. The chromosomes may be somewhat clumped together.
Shortly thereafter, asynnnetrical and loose spindles appear.
Cells with unusually high numbers are followed in the transition
to normal mitosis. Extremely large cells with high numbers appeared
in tissue cultures of plan.t cells.*'- The first hint that a cell is on the
road to recovery shows in the telophasic stage. Chromosomes are not
condensed into one nucleus when first observed. Later each nucleus
becomes perforated and filled with canals. Next the grouping of
nuclei of a large cell is like a multiple cell.'^i containing as many as
twenty stars. ^^ Perhaps each star represents a regenerating spindle
area. When telophase sets in, fibers running between each grouj) lead
to cell wall formation (Fig. 3.9) . I'hus, the large restitution nucleus
containing many chromosomes, becomes divided into as many as 20
small cells. ''!• ''-
The ob\ ious reduction to many small units means reduced dnomo-
somal numbers. While this is "somatic reducticjn," it does not corre-
sjjond to reduction through meiosis, except in the numerical changes.
Certainly no qualitative genetic reduction takes place such as occurs
in meiotic j^rocesses.-^*'
After 3(i hours most cells have run their normal course. A dia-
gram correlating length of exposure to time for regeneration and com-
pleted reccjvery, has been constructed.^'' Fhe exposures, covering 7 to
30 minutes, rec[uire between 12 to 24 hours for the first spindle regen-
eration, and 36 hoius for regular sj^indle. An increasing exposine, 2
to 72 hoins, retards sjjindle regeneration to 24 hours, and delays com-
plete recovery to 36 and 48 hours. This means that the longer the
exposure, the longer the time for recovery.
Spindle and Cytoplasm 95
Another view is obtained from the 1-hour and 5-hour treatments
A\ith Spitwcia root tij)s. In these cases metaphases were plotted dur-
ing recovery. Complete recovery occurred within 48 hours it" exposure
was 1 hoin-, but (k5 to 66 hours were required for a 5-hour exposure.^
Cytological c()nse(]uences in relation to treatment have been ana-
lyzed. The first teiraploid cell begins a second cvcle after 30 hours.^-^
^•v.•.■^v;^;.;;Srv
Fig. 3.8 — Recovery stages in ceils of roots of Triticum treated with colchicine. A. Multi-
polar groups of chromosomes, unequal numbers. B. Cell with a larger number of
chromosomes showing that several cycles of c-mitosis had been accomplished. Upon re-
covery, cell plates may form between groups. C. A large cell cut into several smaller
ones, a characteristic recovery pattern. D. One cell divided into at least six cells upon
recovery from the efFects of colchicine. These cells do not survive but are replaced by
diploid, tetraploid, or octoploid cells. (Drawings adapted from photomicrographs of Beans
and King, 1938. Their Figures, 31, 32, 34, 35)
octoploids at 72 hours/'^ and after 96 hours, 16-ploid cells, or 128
chromosomes, were in division. ''^^
If one studies the entire root, some new facts come to our atten-
tion that are more meaningful than any absolute ratio between time
and number. Eu|)loid numbers, multiples of 8, predominate so that
usually the count reads 16, 32, 64, 128, etc. There are very few poly-
ploid cells near the root tip; in fact, after 72 horns diploid cells per-
sist a little farther from the tij). Tetraploid and octoploid cells j)er-
sist in e\en larger numbers. At the region farthest fiom the tip. where
lateral root initials are found, giant lobed nuclei were plentiful.''^
1 hese cells were crowded with chromosomes having as high as lOOO
c-pairs. ■"'■"' '•' In these cases no regeneration of the cell took place. As
a rule, the nearer the root tip. the lower the chromosome number. Or
in other words, a greater percentage of cells with high numbers is
found in older portions fjf the root.
96
Colchicine
Just how tar this accumulation can continue with hope for re-
versibility to normal was answered by an elaborate test that required
a series extending over a long time. About 500 chromosomes is the
upper limit beyond \vhich no recovery can be expected, Inii 128 and
(il make the most rajjid rccoverx to bipolar spindle. -^-^
Lethal or toxic effects have been disregarded, but the drug has a
gro^vth-dcpressing influence if shoot gro^vth is the index. Hie effects
Xv- • '.<•■•
^t:-
V vxi -
/
/^■■'
Fig. 3.9 — Allium root cell treated with 0.05 per cent colchicine 32 hours, then fixed and
stained with iron alum haemotoxylin. The lower cells show chromosomes around the
pseudospindle. Shortened mitochondria do not penetrate the area of the pseudospind!e.
Large restitution amoeboid nucleate ceil not in c-mitosis. (Adapted from Mongenot, 1942)
of the poison may be expressed in giowih differences between treated
and control plants. Controls had leaf shoots 34 cm. long on the
se\enth day; .01 per cent of the treated j^lants grew to 15 cm. (about
one-half), and 0.1 per cent of the plants \vere reduced one-lourih, to
8 cm.''-''
j.cV— 2; Rccox'cry in (ininutls. Recovery anahses in animals pre-
sent difficulties not met in jjlant cells because animal cells are not able
to survive as long.-'^' ^'^^ """^ A c-mitotic dose frequently becomes lethal
to the animal, an effect that precludes recovery. Another difficulty is
the \ariation in toxicity between animals as ^vell as the dilierences
when dealing with warm-blooded and cold-blooded animals, and/or
tissue cultures. •^■^' '^^
Spindle and Cytoplasm 97
Among the first experiments at Brussels, 21 hours was considered
a reco\er\ time in manmials, and at 48 horns -'^- ''^' "■' normally divid-
ing cells were in abundance. Many cells degenerated before 24 hours.
Residts with Siredon and Xcn<)j)us ha\e been discussed in Chapter 2.
Generally, 5 to 10 hours represented the duration of arrested mam-
malian mitoses, while in cold-blooded vertebrates mitoses may remain
arrested for several da)s.
Clertain trends are seen not only in the recovery figures with Tn-
turus xiiridcscens,'^ but also in the recovery frequencies in corneal
tissues.i*^' 5" A cornea is treated and then allowed to recover. The
maxinuuii arrested metaphases obser\ed at the first fixation (8 hours)
arc an unoriented type (92 per cent) which means that both con-
tinuotis and chromosomal fibers are inactivated. Only 5 per cent of
the figines are stars and 2 per cent bipolar mitoses. The next fixa-
tion shows a drop in unoriented metaphases and an increase in stars,
69 per cent and 20 per cent, respectively. Bipolar mitoses increase
to 8 per cent. Finally at 72 hours, only 5 per cent of the figures are
unoriented while the stars maintain their niunbers up to 16 per cent,
and most remarkable is the increase in bipolar mitoses to 80 per cent.
The picture at 72 hours is a reversal compared to the 8-h()ur fixation.
Diploid, tetraploid, and octoploid mitoses definitely show that
animal cells can be made to double the number of chromosomes.'^-*'
-^- - A fcAv airaphase bridges, fragments, as well as chromosomes were
found outside the nucleus."^ As late as 168 hours, some bimetaphases,
or the "distributed" c-mitoses, were found in Tn turns, also some tri-
metaphases that present a multipolar picture. ■••*
Conclusions drawn from studies of the recovery pattern are that
(1) chromosomal fibers recover first — otherwise stars ^votdd not be
first to rise and fall; (2) the continuous fibers follow the chromo-
somal in recovery; (3) the interaction between two kinds of spindle
fibers and the centromeres determines the metaphasic type to be ex-
pected; and (4) ;infnial cells may de\elop into polyploid cells ca-
pable of dividing upon recovery.
The nuclear figures were followed during recovery in rats ha\ing
recei\ed single injections following jjartial hepatectomy.^'' The re-
generating liver offered special ad\antages for the tracing ol these
stages; a definite series was noticed."'
At 12 hours, there were t\vo changes; (1) the chromosomes thick-
ened and shortened, while (2) a gradual clumjjing was seen. At 18
hours, the cells were fidl of miniatiue nuclei, the micronuclei. Some
swelling accompanied the clumping.
Between 18 and 48 hours, some amoeboid patterns emerged. These
were obviously a residt of fusing micronuclei.^^' ^^' i*' Perhaps the re-
lated and progressive stages were the binuclear and trinuclear stages.
98 Colchicine
First signs of partial spindles were seen at 48 hours. This is evi-
dence that recovery or reversibility was taking full effect, so that by
72 hours a complete spindle was reformed.
Reversibility is seen in animal cells, but the recovery is complicated
by other effects in addition to arrested mitosis. This is particularly
true in mannnals, where considerable destruction of arrested meta-
jjhases takes place not gi\ing time tor the spindle to recover before
the chromosomes are irreversibly altered.
3.9: Summary
In this chapter and in the preceding one, selected works were cor-
related to describe, first, the action ujjon nuclear mitosis as obser\ed
through chromosomal patterns and, second, the spindle mechanisms
fundamental to arrest by various techniques, ^\^e are aware that little
attention was given to the mechanism of action, theoretical aspects,
and problems of c-mitosis, all of which are suggested by the data.
The action of colchicine involves the cell as a whole and, for ani-
mals, the correlated activity of tissues. Before a discussion of the prob-
lems can be made most effectively, other aspects must be viewed.
Therefore the mechanisms of action as well as the very im])ortant
problem of mitotic poisons are grouped together in Chapter 17. Here
it is hoped that some of the important issues raised by the action of
colchicine on jjlant and animal cells can be brought into a synthesis,
the problcuis of c-mitosis.
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700 Colchicine
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50. Kal'fmann, B., Gav, H., and Hoi.lai.ndi r. A. Distribution of mitoses in the
corneal epithelium of the rabbit and the rat. Anat. Rec. 90:161-78. 1944.
51. Keppel, D., and Dawson. A. Effects of colchicine on the cleavage of the frog's
egg iRana pipiens) . Biol. Bull. 76:153-61. 1939.
52. King, R., and Beams, H. A comparison of the effects of colchicine on division in
protozoa and certain other cells. Jour. Cell, and Comp. Physiol. 15:252. 1940.
53. Lehmann, F., and H adorn, E. (see Ref. No. 55, Chap. 2. 1946) .
54. Lettre, H. Zur Chemie und Biologic der Mitosegifte. Angew. Chemie. 63:421.
1951. Uber Synergisten \on Mitosegiften. \'. Mitt. Versuche zur Aufhebung der
svnergistischen Wirkung durch Phosphagen. Xattnuiss. 38:13. 1951. Uber Mito-
segifte. Ergebn. Physiol. 46:379-452. 1950.
55. Levan, A. (see Ref. No. 56, Chap. 2. 1938, 1940, 1942, 1944, 1949, 1954) .
56. , AND LoTFV. T. (see Ref. No. 57, Chap. 2. 1919) .
57. , AND OSTERGREN, G. (see Rcf. No. 58, Chap. 2. 19^3) .
58. LiTS, F. (see Ref. No. 61, Chap. 2. 1934, 1936) .
59. LuDFORD, R. (see Ref. No. 28, Chap. 1. 1936) .
60. LuscnER. M. (see Ref. No. 63, Chap. 2. 1946a, 1946b, 1916c) .
61. Mangenot, G. (see Ref. No. 65, Chap. 2. 1942) .
62. Martin, G. (see Ref. No. 66, Chap. 2. 1945) .
63. Mascre, M., and Devsson, G. (see Ref. No. 67, Chap. 2. 1951) .
64. Mehra, p. Colchicine effect on the mitotic tli\ision of tlie bodv nucleus in the
pollen grains of some Ephedra Sps. Proc. Nat. Inst. Sci. India. 12:333-40. 1946.
65. Melander, Y. (see Ref. No. 68, Chap. 2. 1950, 1951) .
66. Mills, K. Variations in the rate of mitosis in normal and colcliicine-treatcd tad-
poles of Rana pipiens and Amblystoma jefjersoiiiauuiu. Jour. Morph. 64:89-113.
1939.
67. Miszi'RSKi, B. Effects of colchicine on resting cells in tissue cultures. Exp. Cell
Res. Suppl. 1:450-51. 1949.
68. , AND DoLjANSKi, L. Effect of colchicine on resting cells in tissue culture.
Proc. Soc. Exp. Biol, and Med. 64:334-36. 1947.
69. MoLLENDORFF, W. V. (see Ref. No. 70, Chap. 2. 1939).
70. MoNROY, A.. AND MoNTALENTi, G. Cvclic variations of the submicroscopic struc-
ture of the cortical laver of fertilized and jKirthenogenetic sea urchin eggs.
Nature. 158:239. 1946.'
71. Nebel, B. (see Ref. No. 72, Chap. 2. 1937) .
72. , AND RuTTiE, M. (see Ref. No. 32. Chap. 1. 1938) .
73. OSTERfiREN, G. (see Ref. No. 77, Chap. 2. 1943, 1944) . Cvtological standards lor
the cjuantitative estimation of spindle disturbances. Hereditas. 36:371-82. 1950.
74. Peters, J. (see Ref. No. 79, Chap. 2. 1946) .
75. PiETTRE. L. Action de la colchicine sur les vegetaux. C. R. Soc. Biol. Paris.
131:1095-97. 1939.
76. PiNcus, G., AND Waddington, C. (see Ref. No. 81, Cli;.p. 2. 1939) .
77. Resse, G. (see Ref. No. 83, Chap. 2. 1951) .
78. RiES, E. (see Ref. No. 84, Chap. 2. 1939) .
79. RvLAND, A. A cytological studv of the effects of colchifine, indf)le-3-acetic acid,
potassium cyanide and 2, 4-D on plant cells. Jour. Elisha Mitchell Sci. .Soc.
64:117-25. 1948.
80. Santavv, F. Isolierung ncucr Stoffe aus den Knollen der Herbstzeitlose, Col-
chicine aututnnale L. Pharm. .\cta Helv. 25:248-65. 1950.
81. SciiREiBER, G., AND Pellegrino, J. .\nalise citologica e cariometrica da a^ao da
colchicina sobre a espermatogenese dos Hi'ini])ieri>s. Mem. do Inst. Oswaldo Cruz.
Rio de Janeiro. 49:513-12. 1951.
Spindle and Cytoplasm 101
82. Skdar, a., and ^VILSo^, D. Election microscope studies on the normal and col-
chicin'ized mitotic fignres of the onion root tip, Alliuiii cel>n L. Biol. Bull.
100: 107-1.'). 1951.
83. Sentein, p. Mode d'action da la colchicine sur la carvocmcse de Mnlgc palniaia.
C. R. Soc. Biol. Paris. 137:1.H.V31. 1943. Les eflets mitoclasi(|iies che/ (inelques
Vertcbres. C. R. Soc. Biol. Paris. 139:291-9."). 1915. .\ction dc la colchicine et
de I'hydrate de chloral sur loeuf de Trilurus Heh'eticus L. en developpement.
Acta Anat. 4:256-68. 1947. Les transformations de I'appareil achromatique et
des chromosomes dans les mitoses normales et les mitoses hlotiuces dc lociif en
segmentation. .\rch. .\nat. Hist. Emhrvol. 39:377-94. 1951.
84. Setala, K. Colchicine as carcinogenic agent in skin carcinogenesis in nuce.
Distribution of carcinogenic hydrocarbons in the mouse skin applied during life
and death. Ann. Med! and Biol. Fenniae. 26:126-30. (Index Anahticus Can-
cer. 20:305.) 1948.
85. Shimamura, T. {see Ref. No. S6, Chap. 2. 1939, 1940).
86. SoKOLOW, I. {see Ref. No. 89, Chap. 2. 1939) .
87. Sovano, Y. Physiological and cvtological relations between cokhicuie and
heteroauxine. Bot. Mag. Tokvo. 54:141-48. 1940.
88. Steinegcer, E.. and Lev.-vn. A. (see Ref. No. 42, Chap. 1. 1947, 1948).
89. SuiTA, N. {see Ref. No. 89, Chap. 2. 1939) . . „ „ c
90. Tahmisian, T. Mechanism of cell division. I. 1 he living spindle. 1 roc. Soc.
Exp. Biol, and Med. 78:444-47. 1951.
91. Tennant, R., and Ln liow, A. {see Ref. No. 90. Chap. 2. 1940) .
92. Vaarama, a. {see Ref. No. 91, C.hap. 2. 1947, 1919).
93. Verne. J., and Vilter, \'. £tude de Taction de la colchicine sur les mitoses des
libroblastes cultives in vitro. Concentrations dites fortes. C. R. Soc. Biol. Paris.
133:618-21. 1940a. Mecanisme d'action de la colchicine, employee en concen-
trations faibles, sur revolution de la mitose dans les cultures de fibroblastes
ill vitro. C. R. Soc. Biol. Paris. 133:621-24. 1940b.
94. ViLTER. V. InhiJMtion colchiciniciue de la mitose chez les Mammiferes. C. R.
Soc. Biol. Paris. 138:605-6. 1944.
95. Wada, B. (see Ref. No. 93. Chap. 2. 1940, 1949, 1950) .
96. Walker, R. The effect of cokhicinc on somatic cells of Tradeseanlia {mludosa.
Jour. Arnold Arb. 19:158-^52. 1938.
97. WiLBi'R. K. Ellects of colchicine upon viscosity of the Arbacia egg. I'roc. Soc.
Exp. Biol, and Med. 45:69(5-700. 1940.
98. WoKER, H. (see Ref. No. 95. Chap. 2. 1943, 1944).
CHAPTER 4
Cellular Growth.
The senior author observed unusual "spearlike" tips torniing on
AJliurn roots immersed in a 0.01 per cent solution of colchicine. After
24 hours startling changes in the roots were noted^^ ^^f. Chapter 2) .
Colchicine-titmor,^^ the name given to this growth, is appropriately
descriptive. Similar anomalies were observed earlier by Nemec and
others.35 xhis growth pattern can also be reproduced with chemicals
other than colchicine or by certain physical treatments.'^^- ^-^ Although
the c-tumors were not new to biology, the revival of interest in colchi-
cine brought them to the attention of many experimenters.^*- ^^- ^^' ^^•
44, 37, 59, 55!^13?., 115, 111, OU, SS, 02, 154, 128. IS, 4, 10. S. 21
Roots with c-tumors may have some cells with many chromosomes
within the single cells, l)ecause polyploidy is a consequence of c-
mitosis. The correlation between larger leaves, stems, seeds, and
Howers, and increasing numbers of chromosomes is well established.
135, 152 yi^is concept influenced the first conclusion that c-tumors
were directly correlated with the polyploid cells. On the contrary, an
enlargement of root tijjs is not the result of polyploid cells induced
by the drug, even though polyploid cells may be created at the same
tmie the c-tumor is formed.^^ -fhe c-mitosis and c-tumor are inde-
pendent processes. *-
Now we know that in similar manner, enlarged cells may be in-
duced in Aarious parts of plants.^"'' All these anomalous formations
induced by colchicine are the result of changing the growth pattern.
•5-' 90 Such structures as pollen tubes,'-''' •"'"• i" stylar cells of the flower,
1*^ hair cells of stem and root,-'-^- 55- 1^-^ hypocotyl, and other somatic
cells all show particular enlargements after treatment with colchicine.
They are in contrast to the untreated or normal cells that enlarge by
a cell tension that shows distinct polarity. By a broad interpretation,
all deviations expressed as growth patterns and appearing as a re-
sponse to colchicine will be classified as c-tumors, in spite of the fact
that this name originally designated a specific kind of root tip en-
largement after treatment with colchicine.
[102]
Cellular Growth 103
The processes of meiosis and gametophytic devclojiiiicnt are
changed by colchicine.-' ''• -•'• "''• ^^^- ^-^' ^--- ^^'' Resjionse depcntls iijion
the concentration, stage of tievelopment when colchicine reaches the
cell, length of exposme, and, of comse, concentration. As might be
expected, the spindle is inhibited, but there are also other changes
that accompany the colchicinc-elfect.^ For that reason the problem of
a "colchicine-meiosis" '" is included in this chaj^ter along with the
action upon embryo sac tievelopment^'* and pollen tube studies.^"'
Colchicine acts upon cells dining their differentiation processes.
One noticeable change is foimd in the cell walls. •''^ Their chemical
composition is altered also, and various physical marks show that
action of colchicine is not limited to the mitotic s|)indle or upon
certain cytoplasmic constituents. "•''* Enough data are at hand to prove
that differentiation processes in plants are modified by colchicine. "'•'•
53, 1.56, 151
Among unicellular organisms, processes of division, enlargement,
and differentiation, are closely integrated within one cell. For that
reason one woidd expect to find the results from a colchicine exposure
difficult to interpret. Conceivably, all three processes go on within
one cell at the same time; hence, colchicine may act upon each phase
in a specific manner, yet simultaneously. If this interpretation is cor-
rect, the confusing picture drawn from the literature dealing with
colchicine and microbiological materials may be jjartly explained by
the inability to distinguish the specific process being studied, whether
a cell division, cell enlargement, or differentiation and matmation.
There is general agreement that the actions reported in this research
are contradictory. Under some conditions, however, colchicine is
effective if introduced to specific microbiological cultures within
certain concentrations.
A mechanism for action of colchicine upon jirocesses of gro;\th
and differentiation is difficult to visualize. Nevertheless, there should
be some aspects of metabolism that might help toward the solution
of this problem.if«- 1""'' ^'■'- "- i^s, 142. 180. 90! 5.;, w, 47, 4,s, 45 Generally, the
work with physiology^"^- ^^ has been done with such isolated pro-
cesses as enzyme reactions'-" or respiration^if* imder a restricted set
of conditions for experimental material. At least a start has been
made in this direction, but more can be done in the future.
4.1: Colchicine Tumors in Roots, Hypocotyl, and Stems
1 he root tumor forms at the region of elongation, a section be-
tween the meristematic area and the differentiated cells of a root^"-
35,79,82,02 (Fig. 2.1). Normally cells elongate linearly to the axis of
the root. They seem to show a polarity in this respect. When colchi-
cine is present, an enlargement of the cell takes place in all directions.
That is, an isodiametric expansion occurs, rather than a polarwise
704 Colchicine
elongation. The volumes oi cells Ironi a c-tiinior are about the same
as the volumes of elongated cells in untreated roots. '^- Therefore, the
direction of growth is modified, but not necessarily the total amoimt
of expansion. •'-
Cells of the cortex liccome inllatcd."" This leads to a swelling at
the particular place along the root. Longitudinal and cross sections
of treated and untreated roots within five or six layers of cells show
where the change occurs, and reveal particularly the difference in
the shape of individual cells. These comparative studies confirm the
opinion that direction of gro^vth is altered when colchicine is j^rcs-
ent. The action is not iniiquc for colchicine. Growth-promoting
substances, as naphthaleneacetic acid (NAA) and indolebutyric acid,
induce tumors.^'- ^^' ^^- ■*-• -"■ ''' 34, 44, 79, si, 59, ei Acenaphthene, another
compound that has a c-mitotic potential, may cause tumors on roots. ^^^
Not all compounds that create tumors arrest mitosis. In fact, certain
phytohormones that do not stop mitosis may induce root tip enlarge-
ments. I'he idea of an autonomy of c-mitosis and c-tumors gains sup-
port from these general observations with several chemicals. ^-
Specific thresholds below which no tumors form, are demonstrable
for colchicine. Concentration specificity is shown also by NAA.^^ If
t^vo solutions, colchicine and NAA, are combined, the threshold con-
centration docs not change. *i There is no evidence that two solutions,
each capable of inducing tumors alone, will in combination lower
the threshold value. Thus, the mechanism for creating the tumor
may be different for these particular substances. ^^^ The threshold
changed, however, when sulfonamide (2 per cent prontosil) was
added to colchicine. •• •■'•'
The combined solutions of ???r5o-inositol and colchicine prevented
the usual j^roduction of a c-tumor with roots of AUhim.^^ Apparently
this antagonism by ///^,90-inositol operates at 19°C. since a repetition
at 26°C. did not reveal such antagonism.-*^' The critical role of tem-
perature is seen in pollen tube enlargements, where the maximum
width induced by colchicine occurs only at a sj)ecific temperature. ^-''^
Above or below that optinumi the pollen tubes are close to normal
dimension in spite of the same concentration of colchicine present in
each test.
Venom from bees was demonstrated to have an antagonistic action
upon the formation of root timiors by colchicine."'-' The specific dif-
ferences between kinds of j)lants was also shown. Tomatoes were
more sensitive than wheat seedlings. .\ ()9 per cent reduction of
tumors was obtained for tomatoes and 47 per cent with wheat."^^- ^^
Ethyl alcohol changes the c-mitotic ihreshold for Allium root cells
from 0.006 per cent, when colchicine alone is used, to 0.01 per cent
if alcohol (O.T) per cent) is added. If tlie concentration of alcohol is
Cellular Growth 105
increased to 2 per cent, other poisonous actions occur. Alcohol acts
as an antidote with resjxct to c-mitosis and tlie c-tumor.
When two chemicals work together to accelerate an activity be-
yond the effect obtainable by each chemical independently, the re-
sponse is known as a synergism. Colchicine and numerous other
chemicals have been tried for their synergistic action.'*^ Some give
accelerated response and others do not. Phenylurethane along ^vith
colchicine increases the action of drug upon roots of Allium.^'^
Tissue cultures of HeliunUius tuberosus were handled by com-
bined treatments of heteroauxin (lO-o) and colchicine (10 6) . Small
doses of colchicine enhance the action of heteroauxin because the
tissues seem to divide more actively and huge cells with many chromo-
somes develop as a result. A stimulating action seems evident from
these experiments. Increasing the concentration of colchicine leads to
repetitive c-mitoses and an inhibition of cellular multiplication
among the tissues. ^^
Generally, favorable conditions for growth increase the promotion
of a tumor from a specific treatment.'^^ The range in concentration
is fairly broad, but there are limits marked by minimum and maxi-
mum concentrations, rhe formation of tumors within certain limits
is proportional to concentration. Finally, the thresholds for c-mitosis
and c-tumors are close to each other with some indication that the
threshold for the latter process is lower than that for c-mitosis.s^
As soon as the independence of c-mitosis and c-tumor was sus-
pected, a specific experiment was designed to test autonomy.''' Root
primordia of AlUinn fistulosum were subjected to intense X-ray treat-
ment. Consequendy, the mitotic capacity of meristematic cells was
destroyed. Following X-irradiation, bulbs were placed over colchi-
cine, and typical c-tumors formed with no evidence for several days
thereafter of c-mitoses in these roots. We may conclude, therefore,
that enlargement occurs without a simultaneous division of cells.
Polyploidy following a c-mitosis is not necessary for tumor forma-
tion."'^
Swelling at the hypocotyl when seedlings were soaked in colchi-
cine gave the first evidence that tumors were in no way related to
c-mitosis or induced polyploidy. Although cells in the hypocotyl are
not meristematic, they are capable of elongating or expanding. Colchi-
cine causes an isodiametric expansion of cells much the same as among
cortical cells in roots. •"'-
The tumor formation is proportional to concentration within cer-
tain limits.^^ Different species show different degrees of response to
the same concentration. Another factor is the sj)eciric moment when
seedlings are placed in colchicine. i^'" If the seedling has not yet elong-
ated, there is swelling throughout the entire hypocotyl. But the seed-
706
Colchicine
ling that has already elongated, let us say to 23 mm. before treatment
begins, shows practically no swelling at the hypocotyl.^i'- All these
points fall in line with the proposition that tumor formation is
basically a growth response to colchicine (Fig. 4.1) .
Stems of Tradescaniia cut from the plant and placed in colchicine
show extreme swelling at the node where leaves are attached.i^^ The
nodal enlargements are in every respect comparable to root and hypo-
cotyl tumors. A petiolar swelling also may occur if expanding leaves
are placed in colchicine.
The growth responses observed for roots and stems raised the ques-
tion of a possible hormone action. However, the standard tests for
measuring phytohormone potency gave negative results."'^- ^o- ^^ No
m.
25
/
/
/
20
/
/
15
/
/
y
/
10
5
/
/
/
/
^
^
/
r—
^
^
—
CONTROL
40x10-3
10x10-3
5x 10-3
2xlO-3
1
8 DAYS
Fig. 4.1 — Elongation of hypocotyl of Lepidium seedlings. Reduction in length is pro-
portional to concentration of colchicine. (Adapted from Gremling)
Cellular Growth 107
I
responses were obtained from colchicine applied to the Avena, Heliaii-
thiis, and Pisum tests. ^i"' Colchicine is not a phytohormone, but the
basic relation between gro\\th responses shown by tumors and the
reactions noted lor phytohormones in causing cell enlargement is not
understood. There are numerous cases reported where colchicine
(hanged growth rates.
Resistance to colchicine by cells of Colchiciim was demonstrated
under the secticjn dealing with c-mitoses. A similar resistance can be
proved with colchicine and tumor formation. Enough species of
Colchiciiin were tried to give conclusive proof of a resistance. '^^^ ^^' ^' ^^
Not all plants supposedly containing colchicine are resistant as tested
l)\ the tumor test.**^ The resistance shown by tumor experiments is
not proof of a c-mitotic resistance, and vice versa. This point was
not always apj)rcciatcd because the independence of the two pro-
cesses was not understood until specific tests were finished.
Golden hamsters showed resistance to colchicine under laboratory
conditions.'"' This specific resistance may be explained in the follow-
ing way: Animals inhabiting regions where Colchician is found
Avould come in contact with seeds, fruits, leaves, and corms of the
jjlant and would consume amounts of varying strength. Enough col-
chicine ^\■ould be present to kill suscejjtible individuals, while others
might sinvi\e. Therefore, by selection in nature the hamster may
have acquired this specific resistance.
4.2: Effects of Colchicine on Pollen Tubes, Hair Cells, and Other
Parts of Plants
The number of chromosomes per pollen tube does not increase
after c-mitosis in the generative cell.'^"'' ^^~ An enlarged pollen tube
is independent of the action of colchicine upon the nucleus. When
a pollen grain germinates in artificial media, a tube grows out and
away from the grain (Fig. 4.2) . Such filaments are very narrow and
elongation of the tube is polarwise. Colchicine decreases the length
and increases the width of a tube. An enlargement e\'en greater than
the grain itself may occur (Fig. 4.2) . These are the pollen tube
tumors. .\ stimulation has been reported when hormones are added
to cultures with colchicine. '"^^ i-^*'
A lateral expansion is comparable to the isodiametric extension
of ro(jt oi hyjjocotyl cells. Ihe tubes seem to "bloat" or inHate like
balloons (Fig. 4.2F) . Since there is no bursting, the increase must
take place by an orderly deposition of cell wall material forming the
tube.-^"' Colchicine causes these pollen tube enlargements. AVhen the
concentrations are of low dosage, a stimulation is observed. i''"- ''■
An interaction between concentration and tempcratme condition
\vas expressed in measurements with calculated averages of pollen
v;
tS^i* f!
B
Fig. 4.2 — Pollen tubes of Po!ygonatum pubescens from cultures in sucrose agar, treated
with colchicine and untreated. A. Control culture, poHen tube with generative cell in
metaphase, stained with iron acetocarmine. B. Co.chicine mitosis of a diploid species,
n-10, to be compared with Figure 2.4D of Chapter 2, the tetraploid species, n-20. C, D,
E. Reversion to interphase; c-pairs are not separated completely at centromeric region.
F. Pollen tube c-tumor that is a response to colchicine independen* of any polyploidy.
Tube wall staining shows depositions not commonly observed in .. ol. Stained with
iron alum haemotoxylin. (Eigsti, 1940)
Cellular Growth 109
lube widths.'-' Five-and-oiie-half-hour (ultures at 2o°i'.. luul tiil)es
with a :^() i)er cent increase in width over the control. Xo such sig-
nificant differences in witUli were found at 20°C. or 30°C. Although
the mean ttibe length ^vas less than control for all temperatme levels,
onlv at the optinunn, 25°C., was maximinii width obtained.'-' The
concentration of drug, 0.01 per cent, remained the same for all tests.
No similar increase in width was found upon adding 3-indoleacetic
acid, vitamin B,, or NAA to the culturing medium.
Pollen from ColcJiicuin aiitiiiiiDdle L. was tested for response to
colchicine. Germination was depressed by concentrations ranging
from 1 .0 to 0. 1 per cent."" Tumors were observed comparable to those
in jjollen samples from species not known to jnoduce colchicine, and
thus a resistance such as was shown to c-mitosis and c-tumor has nor
been demonstrated for the case of the pollen tube tumors. The re-
sponse from these tests is of further interest in light of the report that
bees carrving pollen from flowers of Colchicum yield honey that is
poisonous due to a high colchicine content.'^" From this indirect
evidence it woidd thus seem that the pollen contains the drug. The
quantities of colchicine \\hi(h are tarried in tlie flowers are descril)ed
in Chapter 5.
Epidermal jnotuberances on roots, the root hairs, involve no mi-
totic stages. s** These cells are suitable for testing the action of colchi-
cine upon enlargement of root hairs. Eight species of plants were
included in a study to measure differences in root hair develoj^ntent
between control and treated cases. ■''•''
Bulbous tips appeared in contrast to the normal long, thin (da-
mentous root hairs. The polyploid condition is not involved since
the nucleus does not divide. Here again is evidence for an inde-
pendence between the c-tumor and c-mitosis. Sometimes the end of
a particular hair becomes forked. -^-^
Other plant parts, the stem, leaf, and flowers, have hairlike cells.
For Helianthus, a protuberance quite different from the normal is
produced following treatment with colchicine."'
Staminal hair cells of Rhoeo discolor form a chain of cells like
beads.-" Colchicine causes the distal cell to enlarge considerablv be-
yond the normal size. Each cell successively from the tij:) to base is
enlarged, but the size decreases progressively from the tip to the basal
cell. The largest cell, an end cell, is also the youngest. Maxinuun
increase is then projjortional to the age of the cell; yoiuiger cells ex-
pand more than older ones.^^*
The stylar portion of a jnstil is elongate and is composed of elon-
gated cells. Flowers of Tradcscdntid treated with colchicine before the
pistil develops, show modification of these fforal parts.^^** Short,
stubbv prstillate siructmes rejilace the long filamentous styles. The
ntunber of cells does not change, but the manner in which elongation
7 10 Colchicine
proceeds becomes considerably altered. Cross sections as well as longi-
tudinal views are very instructive.!^^
Floral parts from CartJiamus tinctorhis follow similar patterns of
induced changes when treated with colchicine before the flowers
mature. Blunt" wrinkled petals and short, single gynoecia with Avoolly
hairs replace the pointed, elongate petals, double gynoecium, and
stiff, pointed hairs of normal flowers.'^^
Enough data have been collected to confirm the fact that colchi-
cine alters the way in which cells enlarge.i^' Growth by increase in
volume is modified under specific conditions, and this may be related
to changes in viscosity of cytoplasm caused by colchicine.-^' ^-- ^^'^' •^"' ^'^•
126, 88, 98, 10.3
To explain the mechanism for a c-tumor, certain jxirallels were
drawn between viscosity changes in the cytoplasm and dissociation of
the cytoplasmic proteins.i"'^ Colchicine caused a decrease in viscosity
that was correlated with the formation of the c-tumor in Allium. In
this explanation, a dissociation was the primary causal factor. A
similar mechanism was described in connection with the c-mitosis.io3
The idea of a narcosis was also introduced to account for a c-tumor,
but instead of there occurring a narcotized cell division, it is the
growth process by cell enlargement that is infiuenced by colchicine.ios
In regard to this hypothesis and the preceding one. much additional
information is needed for a full explanation of the action of the drug
during cell enlargement.
4.3: Colchicine-Meiosis and Gametophytic Development
In pollen mother cells or megaspore mother cells that are in con-
tact with colchicine at the time of reduction division, the meiotic
stages are converted into a "colchicine-meiosis."'-* Only at this time
can such a process as c-meiosis take place (Fig. 4.3) . Earlier, that is,
during divisions in the archesporium, and in later cycles, when micro-
spores or generative cells divide, the processes become true c-mitoses.'^»
Since the c-meiosis represents a special case, primarily because meiosis
is a particular kind of division, it is discussed in this chapter with
other aspects of growth and reproduction. Obviously the spindle
inhibition is common to both c-mitosis and c-meiosis; so also are the
c-pairing phenomena (Table 4.1) , a secondary action of the suj^pressed
spindle, and the "c-bivalents" accompanying c-meiosis. These and
related characteristics of c-meiosis occur only during a certain time in
the rejjroductive cycle (Figs. 4.3 and 4.4; I"al)lcs 4.1 and 4.2) .'•'• -•'•
124, 148
To help visualize how essential a timing sequence is in producing
the c-meiosis, a survey of the particular cell, treated stage, and ex-
pected results are given in Table 4.2. From this outline one can see
K
^1^
B
1 _ -■■^%:^*' I
W« Vv
•ss
■?it
isil*^'
Fig. 4.3— Pollen mother cells of Tradescantia palludosa. Confrol and treated cultures. A.
Untreated microspore. B. Univalents induced by colchicine. C. Desynaptic metaphases,
four days after treatment was made. D. Diploid microspore from a treatment that be-
came effective at the second meiotic division. E. Octoploid microspore 21 days after
treatment; time of treatment 48 hours, then time allowed for recovery, two meiotic di-
visions inhibited, and one premeiotic c-mitosis. F. Tetraploid microspore, 12 days after
treatment. G. Hexaploid microspore, an unequal division that is similar to a distributed
c-mitosis. (After Walker)
172
Colchicine
that action during division leading up to niciosis creates octoploid
or tetraploid pollen mother cellsJ'^ In contrast, activity dm ing meiotic
divisions I and II creates tetraploid monads, and activity at division
II only, diploid monads. Monadal formation is a special feature of
the c-meiosis. The monads replace the usual tetrads of microspores
forming at the close of a meiosis.-^' ^*^- ^-~- "^^
Since archesporial divisions become regular c -mitoses, these are not
described in great detail here, except to say that one c-mitosis in this
MITOSIS
B
Z
NORMAL
COLCHICINE
C.
MEIOSIS
A N
NORMAL
c
Fig. 4.4 — Comparison of a c-meiosis and c-mitosis. The stage reached when colchicine
becomes effective determines the action in meiosis. (After Levan)
tissue gives rise to tetra})loid j^ollen mother cells, and that two c-
mitoses bring about the octoploid condition. Beyond this degree of
]«)lyjjloidy the meiotic processes are so upset that no finther action
of colchicine can be obtained at meiosis. The premeiotic stages of
Allium ccniiiinn with diploid, tetraploid, and octoploid numbers 7,
14, and 28 pairs, respectively, were observed and followed up to the
first meiosis.'^'' Already at tetraploid stages, the polarities of meiotic
spindles were irregular. The multii>le spindle aspects dining re-
covery from a c-mitosis were noticed at meiosis if the previous c-mitotic
cycles of archesporial cells caused polyploidy.
Pairing of homologous chromosomes and chiasmatal formation
formed during prophase are decisive functions before a regidar meiosis
Cellular Growth
113
or a c-nieiosis begins. Ojlchicine reduces the pairing as shown by the
reduction in diiasniala and increased Irecjuency ol univalents.- The
calculations ironi several independent studies (onfirni the action on
pairing. Allium ceriniinji rarely showed luiivalents in controls, but
among treated cases, 8 cells out of 31 had no bivalents. Moreover, no
cell among 31 jjollen mother cells studied had more than 5 bivalents
^\hen the total with Itdl pairing could have been 7.''* Among Trades-
((Dilld. I-! univalents (Fig. 4.3C') were produced by a lull c-meiosis.
.Similar cases are reported with other species.
The terminali/ation of chiasmata is dirterent when colchicine is
piesent; therefore, there is reduction in chiasmata as well as change
m the kind of chiasmata (Table 4.3).- Whether crossing-over is
changed has not Ijecn tested geneticalh. but the cytological picture
seems to warrant a conclusion that cross-overs would occur in places
they are not generally expected.
If recovery sets in while the univalents are distributed through
the cell, there is no congregation into the equatorial plate. But the
TABLE 4.1
Relation Between Treatment and Stage
(After Levan)
Developmental Stage
Stage Treated
Results Obse ved
Archesporium
Pollen mother cell
Pollen mother cell
Pollen
division I
division II
resting stage
prophase
meiosis I
meiosis II
resting stage
first division
tetraploid pollen
octoploid pollen
no effect
abnormal asynapsis
irregular bivalents
tetraploid monad
diploid monad
no effect
diploid pollen
univalents collect at jjoles where the jjarticidar chromosomes happen
to lie. On the other hand, bivalents, if they have persisted, upon re-
coverv orient in the ecjuator.
Unlike the tendency toward supercontraction at the metaphase of
a c-mitosis, the c-meiotic chromosomes do not show the usual contrac-
tion.''•' In fact, they are less contracted; this is a very striking action
induced by colchicine. Such lack of contraction is correlated with a
decrease in the frequency of chiasmata. These are the major effects
iKjted ^vhen colchicine acts during piemeiotic stages. Full action up-
7 74 Colchicine
sets the meiosis so that abnormal metaphase I and irregularities occur
in subsequent stages.
If prophases have proceeded normally, pairing is regular, but
colchicine introduced at the metaphase stage reduces spindle fibers.
Under these conditions, the bivalents remain scattered in the cyto-
plasm, and the separation of two homologous chromosomes proceeds
TABLE 4.2
Relation Between Time of Treatment and Results
(After Dermen)
Days After Treatment Results
4 meiotic chromosomes in short
broken chains; reduction of
chromosomes not noticed
5 or 6 diploid and tetraploid pollen
mother cells
8 tetraploid and octoploid pollen
mother cells
11 polyploid microspores
12 failure at me'.osis I and 11; hap-
loid, diploid, tetraploid micro-
spores
where each pair happens to lie. Since each homologous chromosome
of the pair is cleft and clearly separated, except at the region of the
centromere, a colchicine-anaphase I is characterized by two cruciform
"c-pairs" lying close to each other. The straight, cruciform anaphase
1 chromosomes are a contrast to normal ones at this stage.
As the first telophase begins, chromosomes lose their staining
capacity, the chromatids remain connected at the centromere, and
the usual transformation to interphase between the meiosis I and II
takes place.ii^ The outlines of chromosomes are difficult to trace at
this stage and can be overlooked, making it appear that division II
begins without an intervening interphase, a prophase II, or a meta-
phase II.
When the second c-meiotic division begins, chromosomes con-
dense and assume a prophase appearance. The contraction of the
chromatid proceeds in a prophase II. During this time the relic spiral
disappears and a chromosome of c-metaphase II comes into the pic-
ture. These chromosomes are held together at the centromere up to
late prophase; then they are straightened, and as fairly long chromo-
somes they separate from each other completely. The second c-meta-
Cellular Growth
115
phase II merges ^vith the second c-anaphase II. All the chromosomes
remain within one cell, so that instead of a tetrad of 4 cells, a monad
results with all 4 sets of chromosomes contained within one cell (Fig.
4.3) . The monad is tetraploid. C-telophase II concludes the c-meiosis
with unraveling and loss of the stainable structure.^^^
The full c-meiosis has been sketched briefly without taking into
consideration deviations and abnormalities caused by different con-
centrations, exposure, and stage at which the drug acts. Abnormal
diploid, tctrajjloid, hexaploid, and octoploid microspores may be
found, as was noticed for Tradescautia and Rlioeo (Fig. 4.3) .-'* Poly-
nucleate cells were produced from certain members of the Aloinae^--
and these cases arose from a treatment that probably began in pro-
phase of mciosis.
Reduction divisions in Carthamus tlnctorius L. were treated by a
special technique in Avhich the entire inflorescence was treated. "^
Under these conditions 10 to 17 pollen grains appeared within a
single pollen mother cell (Fig. 4.5) . Most grains had a nucleus, ex-
cept for the very small grains. In view of the fact that this species is
dicotyledonous, while the major descriptions of c-meiosis were made
from monocotyledonous types, these differences may be in order. The
simultaneous formation of tetrads within a pollen grain of the dicoty-
ledons may accomit for the variations. Carthamus and Allium show
certain fundamental differences.
The aftereffects of colchicine point out a possible influence upon
pairing at meiosis in Antirrhiumn as long as 6 weeks and possibly
TABLE 4.3
Action of Colchicine on Chiasmata in Fritillaria
(After Barber, 1 940)
Treatment
Total
Number
Percentage
Proximal
Locations
Percentage
Medium
Locations
Percentage
Distal
Locations
Control
215
127
80
92
62
70
6.9
25 5
17.5
1.1
0.5%. .
12.5
0.25% .
12.5
up to 15 weeks after treatment ol the plant.'-"' An increase in luii-
valents was 37 per cent among the treated plants compared with con-
trol.^-^ A time lapse of such long duration between treatment and
the colchicine-effect is of particular interest. Whether the colchicine
is retained in the plant or the chromosomal mechanism is specifically
affected was not determined. Similar meiotic irregularities were found
776
Colchicine
in treated plants of Kibes that remained diploid, aiitl thus meiotic ir-
regularities induced by colchicine would seem to be carried along,
not entirely explainable by tetraploidy.^^-^
Colchicuni autitmnale L. is a sterile plant in middle and southern
Japan. Cytological analysis showed many irregularities during meiosis
of these plants. ^-^^ In contrast to these figures, the root tip mitoses
Fig. 4.5^Above. Untreated pollen mother cells and pollen. Below. The large multi-
cellular pollen mother cells and abnormal pollen grains of Corthamus tinctorius. Flowers
treated in an early stage of development. (After Krythe)
were regular. The pollen grains from CoJdiicum were irregular, being
monosporic, disporic, trisporic, or tetrasporic. Many grains carried
fragments. The inter|jretation made from these studies was to the
effect that colchicine contained in the cells of Colchicum created an
autotoxicosis that led to sterility in this species.
Irregular pollen anil jjoor germination were not reported for a
European representative of C. autuninalc L. usetl for pollen tube
germination.*'" In this instance the pollen tubes that formed did not
show a resistance to the {presence of colchicine added to the medium.
There was no evidence that the pollen of Colchicum carried the drug
within the protoplasm of the grains since responses obtained were
reportedly the same as pollen tubes of other species not known to
produce colcliicine, e.g., Polygo)i(il urn'''''' and A)ifnrhiniiiii.^-'
Cellular Growth 117
II the microspore nucleus is treated with colchicine, h typical c-
mitosis appears. Since the haploid numbers prevail, an otherwise
precise picture of the c-mitosis can be obtained. A diploid uninucleate
pollen grain is formed after the c-mitosis (Fig. 4..S) .
When monad microspores with numbeis higher than haploid
divide without colchicine, some interesting cells are formed. 1 hese
may be regarded as an aftereffect of colchicine. Multipolar divisions
are common, and in jxirticular, a tripolar division gives rise to a huge
grain, with two vegetative cells apj)resscd close to the wall, and one
generative cell. On occasion, two generati\e cells are formed."*' These
conditions are similar to the recovery phases described in earlier
chapters.
Pollen grains of Polygonatum with one generative cell, a haploid,
and a tube cell were tested for c-mitotic characteristics (Fig. 4.2) F'
The method of testing is described in detail in Chapter l(i. In Chap-
ters 2 and 3. illustrative material was drawn from pollen tube c-
mitosis, but here it is pertinent to point out that the c-mitosis in this
structure never exceeds the diploid number. Very rarely do the c-
pairs become completely separated, so reversion to the interphase goes
from an arrested metaphase rather than through c-anaphase. Enough
tests have been run to rcj^ort conclusively that there is a termination
to c-mitosis and. unlike the divisions in root tips that continue to
build high numbers, multiple-ploidy has never been found in pollen
tubes with Polygonal inn or reported from other sources. Then the
microgamctophyte never exceeds dijjloidy.
In the case of embryo sac development in Tradescautia, the nuclei
that icgularly divide during the process of gametojihyte formation
seem to build up the amount of chromatin, although as is expected,
no spindle forms with colchicine. Therefore, the chromosomes re-
main together. The si/e of the large nucleus, the size of the embryo
sac, and a tendency toward cell formation lead one to infer that c-
mitoses proceed to but do not go beyond the eight-cell condition, nor-
mal for an embryo sac in Tradescantia (Fig. 4.6) . Aside trom the c-
mitotic aspect, the unusual increase in the embryo sac beyond that
for the control is of interest in light of our discussion about the action
of colchicine on growth ])rocesses involving increase in volume. ^^"^
71ie ovules of Cart ham us tinctorius did not develop into seeds,
and no descriptive cytology accompanied the successive stages that
must have taken place when colchicine acted while the embryo sac
stages were in foiniation. This would be of interest for a comparison
with Tradescantia.'-''- "•*
^.3-1: (Uunetophytcs of mosses, liiu-rn'oyts, and ferns. In n)()8, a
series of experiments with mosses demonstrated that polyploidy could
be induced artificially. Fhe Marchals used regenerative tissues to iso-
late polvj)loid races. Three decades elapsed between the fust work
778
Colchicine
early in the twentieth century and the next significant colchicine ex-
jjeriments.®^ Colchicine has been tried recently for a number of
mosses, using protonemata and propagula, treating the tissues in
special culturing media. Size differences between colchicine-treated
and untreated cells have been used as criteria for the changes in num-
ber of chromosomes (Table 4.4) .
Diploid gametophytes of the male and female thalli from Mar-
chantia polymorpha were made by colchicine.^ Chromosomal check
showed that the numbers were increased. Another hepatic, Palla-
xiacinia spp., was subjected to colchicine. i"^' Again new patterns of
eroAvth showed that chanoes were induced. One mav assume that the
number of chromosomes was increased, although the modification in
cellular form without a corresponding increase in chromosomes makes
Fig. 4.6— Embryo-sac stages of Tradescontio. Untreated stage with cells distributed In
the sac and a smaller cavity. Treated stage with all nuclear material grouped in the
center of sac. The size is not a response to polyploidy. (After Walker)
Cellular Growth 119
TABLE 4.4
Action of Colchicine on Algae and Gametophytes of Mosses,
Liverworts, and Ferns
Species Results Reference
Aulacomnium androf;rnum morphological changes 4-64
Cladophora spp cross wall thickened 4-53
Closteriurn spp temporary inhibition 4-80
Dryopteris fdix-mas morphological changes 4-117
D. subpubescens abnormal sperms 4-94
Gonium spp temporary inhibition 4-80
Goniopteris prolifera abnormal sperms 4-94
Hormidium spp leukophytic isolate 4-1 25
Hydrodictyon spp cellular changes 4-53
Marchantia poh.morpha diploid gametophytes 4-9
Micrasterias thomasianas no c-mitosis 4-67
Nitella mucronata ci.4— oo
Nosloc commune ci.4-o8
Oedogonium spp polyploids 4-140
Oedogonium cellular wall changes 4-53
Pallavacima morphological changes 4-157
Polystoma temporary inhibition 4-80
Spirogyra spp plastid changes 4-1 58
Ulia spp temporary inhibition .
.4-80
it less certain than previously believed possible for chromosomal num-
bers to be increased as cell form changed.
Fern prothalli and sporogenous tissues were tested for the induc-
tion of polyploidy following colchicine."' Evidences of changes in
numbers were obtained for several species of ferns. In another applica-
tion of colchicine to growing prothallia regularly producing sper-
matozoids. some luuisually large sperms were obtained. Also some
changes in the shajx' of cells were noticed along with the increases
in size. Dilute solutions were used for early stages of germination of
the jMothalli.
120 Colchicine
Information at hand shows that the ganiciophyte stages of green
plants can be doubled in manner similar to the sporophytic cells,
notably among the seed plants.
4.4: Microbiological Data
Controlled cultures using unicellular organisms are admirably
suited for experiments \\iih colchicine. A wide concentration range
may be used because the strongest dosages show a minimum toxicity.
Furthermore, the experimental subjects are numerous considering
the bacteria, yeasts, filamentous fungi, algae, and protozoa. Consid-
erable preliminary work has been started, but contradictory conclusions
and no small amount of confusion still exist.
In some cases the methods are not clearly described, nor are they
carefully j^lanned. Modifications such as concentration, media, and
exposure ^voidd prove helpful. The interpretations have been very
narrow, and patterned generally after the known action of colchicine
upon the nucleus of vascular plants and multicellular organisms. As
an illustration, the doubling of chromosomes is a remarkable action
with vascular plants, and it would be helpful to know more about
the hereditary materials in bacteria, but colchicine can hardly resolve
the problem of chromosomes in bacteria when cytologists have had
such great difficulties in demonstrating structures in untreated cul-
tures.
Yeast cells that ha\e an advantage over bacteria in size of internal
structures have been tested with colchicine. The results can not be
considered decisive. Even among the algae where chromosome num-
bers for species have been established, there are no clear cytological
data to pro\c that the number of chromosomes can be doubled by
colchicine. There is discussion of haj^loids, dij)loids, and tetraploids
among fiuigi, but present work with colchicine does not provide
answers either through demonstration of chromosomes or by genetic
evidence.
Changes in the sizes of cells within a culture and direct action
upon the growing organism indicate that the drug has some influence
upon growth processes related to increase in size. Of course, these
changes are not transmitted to succeeding generations. The mechan-
ism of growth by cellular enlargement can not be analyzed from such
tests. Metabolism of bacteria in relation to colchicine represents an
luicxplored field. Preliminary work has been done. In 1907. in-
teresting work was done on temperature and toxicity using cultures
of Paramecinin?^ Otherwise, this field of experimentation has been
overlooked.
Finally the processes of differentiation and cellular structure are
influenced by colchicine. Fungi and algae show evidence that during
Cellular Growth 121
the process of cell wall formation the action of colchicine niodifies
structure.^-"' These aspects are treated in a subsequent section ol this
chapter.
4.^-1: Bacteria. Tests with colchicine have included a range of
species ^'^- ^^'■*- i^- ^^^' ^'' "■^' '^'^' ^^' ^^^' "^' ^^' ^*'^' ^^' "^' ^''' ^'*' ^^' ^^^' ^'^^ Some
report no reaction and others claim that colchicine acts upon gro\\th
bv inhibition. Toxicity was also noted (Table 4.5) .
Certain species of bacteria tolerate high concentrations of colchi-
cine in the mediinn. One source of powdered colchicine had bacteria
present in the material; small quantities of powder added to sterile
solutions of colchicine showed species of Agrobacterium.^^ For a num-
ber of species of microorganisms, colchicine without any additional
nutrient supported bacterial growth. It was a habitat for bacteria.
Undoubtedly these forms were able to use colchicine as a food.
The bacteria gro\\-ing in a medium of strong dosage (1 pei' cent)
]iroduced aberrant cells larger than the initial culture, but no con-
tinuation of these types has been possible. An increase in si/e may
represent a condition similar to the cell enlargements for vascular
plants. These are not hereditary changes. Single cell isolations have
not been reported. It would be of interest to know more about these
types. They should be singled out for subculture, since mass transfer
for isolating the ixuticular deviates has objections. Some morpho-
logical alteration temporary for a specific cidture undoubtedly has
been obtained. Increases amounting to 40 per cent were measured
for Bacillus mesentericus.^'^''-
Polvnuclear cells in Escherichia coli cultures were reported but no
follow-uj) of this work has been discovered.!-^-' Apparently a repetition
has not been accomplished.
In a metabolism test, respiration was inhibited in Micrococcus
aureus. A growth stimulation was obtained for PJiotobacterium phos-
phoreuiu.^"^ No changes were observed in the desoxyribose nucleic
acid and the ribose nucleic acid when cultures of Micrococcus
aureus were used.^' This is a sample of the fragments of information;
more are tabulated elsewhere (Table 4.5) .
4.4-2: Yeasts and oilier fungi. The common brewers' yeast, Sac-
charomyces cerevisiae, has been tested by more independent workers
than any other of the microorganisms. A variety of concentrations of
colchicine Avere used and different techniques for culture, as well as
staining to determine cytological changes were tried. "'^' •^- ^- ■''^' ^!- 1-*'-
54, 39, 144, 75, 9, 6, 119, 52, 132, 145
A wide choice of responses is at hand, ranging from reports of no
action to those citing definite cytological change demonstrated by
special staining methods. Dumbbell-shaped nuclei were seen after a
96-hoin- treatment with 0.1 per cent colchicine. Other workers were
unable to obtain these same residts (Table 4.6) .
122 Colchicine
TABLE 4.5
Action of Colchicine on Bacteria
Species Results Reference
Agrobocterium spp growth not inhibited 4-35
Bacillus mesentericus size increase 40%, growth changes 4-113
Bacterium megatherium negative results 4-149
Bacterium spp no action 4-66
Bacterium spp indecisive results 4-43
"Bacteria" no action 4-1 44
"Coliform bacteria" mutations 4-109
Escherichia coli polynuclear cells 4-134
E. coli phage 4-25
Micrococcus spp inactive 4-19
M. aureus negative results 4-19
Micrococcus spp morphological changes 4~1 49
M. aureus respiration inhibited 4-1 7
Mycobacterium tuberculosis stimulates cells, prevents variants 4-63
Photobacterium phosphorcum growth increases 4-104
Proteus vulgaris inhibition 4-37
Streptococcus catarrhaiis toxic action 4-^^49
S. hernolyticus inhibition 4-37
Camphor induced giantlike cells now called the "camphor forms."
In old cultmes these appear with low frequency. A few were found
after treatment with colchicine, but their frequency was not high
enough to warrant the conclusion that colchicine had the same
capacity as camphor to produce giant forms.^
In light of the known antagonistic action of ethanol as discovered
for cells of Allium, the jjroduction of alcohol by the yeast cell itself
may serve as a kind of antidote or protection against colchicine. ^2
These facts have not been verified with experimental data.
Brewing tests did not bring out specific differences between treated
and control cidtures of Stuc haroinyces cercTlsiae.^- The usual sedi-
mentation, foam head, and other comparative values revealed no
Cellular Growth 123
changes induced by colchicine. Methylene blue was decolorized more
rapidly as e\'iclence of some basic metabolic change.
Tlicre is a possibility that colchicine may serve as a source of
energy. Another conclusion led to the idea that the drug serves as a
buffer against the toxic substances accimiulating in an active cultine.
Filamentous fungi from a variety of families'* have been tested for
j)ossible induction of polyploidy. A polyploid strain of Penicillium
twtatutn was isolated in one laboratory.''- This new strain was sup-
posed to yield more penicillin than the original strain. The poly-
ploids were obtained by another group who rechecked these specific
types. Polyploidy and increased jDenicillin Avas not confirmed (Table
4.6) .11"
TABLE 4.6
Action of Colchicine on Yeasts and Other Fungi
Species Results Reference
Alloniyces javanicus changes induced 4—6
Aspergillus spp mutants 4-1 32
Botrytis cinerea hypertrophy of hyphae 4-145
Cnprinus radians conidia influenced 4-144
Diaporthe pcrniciosa no conidial formation 4-145
Mucoi sp no change 4-9
Penicillium notalum polyploids 4-52
P. notatum no polyploids 4-119
Psilocybe semilanccolata conidia changed A-\AA
Saccharomyces cerevisiac no changes noted 4-4
4-83
4-144
4-75
4-5
^. cerevisiae . .cytological changes 4-126
cells enlarge 4-39
methylene blue decolorized more
rapidly 4-41
stimulation 4-116
inhibition 4-54
Slropharia merderia conidia changed 4-144
Verticillium dahliae no conidial formation 4-145
"Wide range of families" no change 4-9
124 Colchicine
Hypertrophy of the h\phae and faihire to form conidia were
legidarly noted among several species of fungi, but doubling of
chromosomes or evidence of polyploidy was never demonstrated.
Possible mutagenesis^-'^ was reported for Streptomyces griseu.s. Con-
centrations ranging from 0.5 to 1.0 per cent introduce changes in
growth patterns that resemble the tumors previously reviewed. No
better specific information is at hand for the yeasts and fungi than
for bacteria. That mycelial growth may be influenced is probable,
but polyploidy or induction of mutations is extremelv doubtful
(Table 4.6) .
Colchicine increases the frequency with which resistant sporangia
of AUomyces javanicus developed mixed thalli from the sporophytic
generation. When germinating zygotes were treated, some nuclei
were thought to have been converted into polyploids. The cytological
records of chromosomes were not available to confirm the polvploidy."
A series of treatments involved the use of colchicine and sodium
nucleate, so the specific action of colchicine may be in some way re-
lated to the use of the sodium nucleate.
4.4-3: Algae. The first artificially induced polyploid among plants
might well be credited to Gerassimov who treated Spivogyra by tem-
perature shock and apparently succeeded in increasing the volume of
the nucleus. This was done in 1901. A confirmation made some
years later strongly supports the thesis that Spirogyra cells were
doubled. One might hope that colchicine would be useful in repeat-
ing this classical experiment by chemical means, or at least demon-
strate that the drug is not effective, llie results with algae and col-
chicine are not any farther along than those with the other specimens
of fungi. i-*o. 15S. 125, 65, 07, ISO, 9, 88 ^he treatment of Spirogyra with col-
chicine should be tried with a wide range of concentrations and cyto-
logical control.
A polyploid strain of Oedogoniuin was said to be obtained from
treatment with colchicine, but no exact cytological data went with the
report to prove the doubling of chromosomes had taken place. ^^"
Temporary inhibition of mitosis in cells of Micrasterias thoinasi-
anas was recorded in cultures. The general conclusion was reached
that colchicine was ineffective except for some temporary changes in
plastid structure.^" Unfortunately, only limited ranges of concentra-
tions of colchicine were employed for the Micrasterias Avork. Some
dosages may be more effective than others.
Leukophytic variants were isolated from colonies of Hormidium
sp. treated with colchicine.12.3 Several generations of subculture
brought a return to the chlorophyllous type. If a change was in-
duced, the weakness of a non-green variant did not permit a survival
in competition with unchanged chlorophyllous types.
Cellular Growth 125
Plasticl changes are to be expected in the treated generation.
Whether or not changes are retained upon transfer to culture without
colchicine remains unconfirmed. Supposedly the elasticity of plastids
in S/)iyogyra changes inider the infkicnce of colchicine. ^-'^^
Inhibitions at higher concentrations were seciued ^\'ith Gonium
and Polystoma. Upon recovery the cells remained diploid as far as
the in\estigators were able to judge. Some action seems to have been
registered upon the /oospores and zygotes of the green alga Ulva.^'^
Studies dealing ^\ith the cell wall and colchicine are of interest
from the view of diflerentiation. Cell structure and composition of
the wall are modified by colchicine (Table 4.4) .
4.^-^: Protozoa. A number of investigations^- ^i- -"• -^- ^^' ■''- ■'^- "^•
lis, 1.36, 144 oj^ various aspects of colchicine and the protozoa, as well
as regenerative studies^'"'" have been published since 1938. As long
ago as 1907, the action of colchicine on Payamecium was studied in
relation to toxicity and temperature changes.-'^'^ Increasing toxicity
-with raising the temperature was demonstrated by this early work.
No one has repeated these studies in the modern period, but most
have been concerned with cell division and problems of polyploidy.
Undoubtedlv the influence of cytology and genetics preconditioned
much of the experimentation since 1937.
The species of protozoa tried for response to colchicine show tliat
strong solutions can be tolerated at 22° to 24°C. Fission occurs for
a number of species.'^ The microinjections of colchicine gi\e finther
information on the penetrability of the drug that may influence the
reaction. Failine of the drug to penetrate the cell may be one key
in explaining the resistance to colchicine of protozoa as a group. — -
Some retardation in growth and changes in new cells developing
within a culture containing colchicine have been recorded. As a
general ride, the direct action of the chemical upon the cell or nucleus
has not been demonstrated. Some increases in "radio-sensitivity" ac-
companied the prctreatment by colchicine."'' In this case the cells
appearetl to be more sensitive to action of the X-ray after a treat-
ment.^"
Table 4.7 may be used as a reference for a survey of work com-
pleted upon the j^rotozoa as a group.
4.5: Differentiation Processes
Alter a treatment with colchicine the new lea\e->. developing when
growth is resumed, ajjpear wrinkled and distorted. Apparently the
drug has directly or indirectly caused these new types. Some changes
are a residt of chimeras which are discussed in connection with poly-
ploidy. \e\. other very similar anomalies caimot l)e conclated directly
with an increase in the number of chromosomes. These celhilar and
726 Colchicine
TABLE 4.7
Action of Colchicine on Protozoa
Species Results Reference
Amoeba proteits fission not inhibited with 2% solution 4-71
.1. sphaeronucleus microinjection inhibits division of nucleus 4-20
Chilomonas spp fission not inhibited . 4-71
Chlamydomonas spp not effective on division 4-49
4-83
4-144
Chlamrdomonas spp growth retarded 4-24
Euslena spp ineffective 4-71
4-144
4-83
Oxytncha spp no action 4-71
Paramecium spp raising temperature increases toxic action
of colchicine 4-58
P. caudatum fission not retarded 4-71
P. caudatum growth retarded 4-3
P. caudatum radiosensitivity increased 4-57
P. multimicromicleatum no action 4-71
Peranema fission 4-71
Plasmodium relictum no retarding action 4-1 1
P. vivax no action 4-1 1 8
anatomical variations are probably a direct action from the drug by
other means than nuclear changes. ^^•'^ As an example, the c-tumor
response occurs from contact with colchicine. Yet more difficult to
exjjlain are the changes that persist into several generations of propa-
gation."**^' Vegetati^e propagations that continue the anatomical varia-
tions are not as difficult to explain as \ariations that reportedly
persist or occur after several generations of seed propagation.
Not so much attention has been directed to the cell wall and re-
lated problems of differentiation as to nuclear aspects, i.e., c-mitosis.^'^
Colchicine causes modification of cytoplasmic and cellular processes.^-^^
Sufficient evidence is at hand to make this assumption. The actions
of c-mitosis, the c-timior, and differentiation are independent al-
though very closely related to each other. For example, the nearly
Cellular Growth 127
simultaneous action upon division, enlargement, and differentiation
can conceivably take place when unicellulars are subjected to colchi-
cine. At least the processes may merge into each other so closely that
separating the actions becomes difficult or nearly impossible.
Analysis and reports from widely different sources are brought to-
gether in this section that treats the microscopic, microchemical, and
gross anatomical changes in plants.^-Msi, so, 53, i5i, lo.-,, n., 1.35
^.5-/; Microscopic and microcheyiiical data. The cell walls of
treated plants show different types of depositions which form stria-
tions.53 These are regularly observed for pollen tubes growing in
media containing colchicine. When stained, their distinction becomes
more clear. The submicroscopic structure of pollen tube walls has
not been studied. Data are accumulating from other sources that
point up the possibilities in this field. '^
Excellent photomicrographs showed that the cells of algae were
changed after growing in media carrying colchicine. ^'^ The newly
formed portions of cells in Oedogonium showed swelling and local
thickenings inside the cell (Fig. 4.7) . These were scattered without
regular order along the wall. Inner cell walls of Cladophora became
thicker than controls, showing that tmusual depositions had occurred
(Fig. 4.8) . Finally, the regular network characteristic for Hydro-
dictyon became distorted through swelling of the middle parts of
connecting cells (Fig. 4.9) . Also the points of contact were enlarged.
These three cases comparing treated and untreated cells leave no
doubt that colchicine exerts a strong influence during cellular dif-
ferentiation.^'•'*
The root hairs grown in cultures containing colchicine (0.25 to
0.5 )jer cent) offer a comparable source for analysis of cell wall
structure. Earlier we described the tumors that were formed on root
hairs. Now microscopic and microchemical study has correlated the
cell structure with the form taken under treatment. After the cell
walls were stained with chloro-zinc-iodide and these structures viewed
with ])olari/ed light, the irregularly deposited micelles were in dis-
tinct contrast to regular arrangements viewed in untreated root hairs.
Photomicrographs with polarized light are instructive for these com-
parisons.^^
Pollen mother cells develojjing in colchicine (Carthamus tnic-
torius L.) were protoplasmically interconnected at the points where
cells touched each other.'"' Later, as pollen grains formed, one large
cell was composed of nimierous pollen grains within a connnon wall
(Fig. 4.10). Another developmental feature was the wall intrusion
which was essentially an excessive deposition of a callous-like material
on the inner wall (Fig. 1.10). The origin and nature of these de-
vcloiMuents are unknown, l)ul the change is an effect of colchicine.
728
Colchicine
in
B
J)'
D
Fig. 4.7 — Oedogonium cultures, treated and untreated. A. Untreated cell showing the
usual ring and cellular striations. B. Enlargement caused by colchicine, indentation of
cellular layers a result of treatment. C. Inner cell thickening, and depositions. D. En-
largement of the cell from treatment and irregular depositions. (After Gorter)
An interesting vascularization lollowing recovery from colchicine
has been described for the huge cells in Allium roots that form in the
differentiated pericycle at points where lateral roots originate. Scalari-
form vessels developed and a unique tumor was left buried in the
root. 15" Nuclear contents that were estimated to contain over 1000
chromosomes as a result of 6 or more c-mitoses disappeared during
the differentiation process. A complex series of pretreatmtnt with
NAA (0.0002 per cent) and colchicine (0.25 per cent) inters|)crsed
with recovery periods preceded this development. No one can doubt
that an interesting problem of differentiation is presented by this
work.
Stomatal development regularly proceeds from an embryonic
mother cell and eventually forms the guard cells,^'"*' i"*'' ^"^ with as-
Cellular Growth
129
sociated subsidiary components. Independently, several investigations
have shown that colchicine interferes with this differentiating pro-
cess."^ These stomatal anomalies, brought into tocus by reports from
such cases as pollen tube walls, root hairs, algal and fungal cell walls,
as well as other differentiating cells, afford added evidence that colchi-
cine acts in some way upon cells that are differentiating. This is the
first time that so many diverse instances of the action of colchicine
have been brought together under one discussion. These problems
deserve attention. AVc have not exhausted the list of instances that
may ha\e further bearing on this aspect.
7.5-2; Gross anatomical variatiojis. When the outer layer of cells,
the epidermis, has a different number of chromosomes from those of
cells deeper in the leaf, some distortions become evident. These cases
are ^\ell documented and belong to problems in polvploidy. Less
kno^\•n and understood are the cases that cannot be readily exjilained
by chromosomal nmnbers.i'^-^ \ few of these instances are described
liere.
Ne^v shoots of Li gust rum arose after treatment with colchicine.^-
The lea\es ^vere darker green, appeared to be thicker, and answered
the description of an induced polyploid. These characters were trans-
ferred several times by vegetative jMopagation. The chromosomal
numbers did not correlate with these differences.
HK<
B
Fig. 4.8— The end walls of Cladophora with extra depositions in treated cases, B, com-
pared with control, A. (After Gorter)
730
Colchicine
Fig. 4.9 — The network of Hydrodictyon becomes distended and unorganized by treat-
ment with colchicine. A. Control cells. B. Treated cellular network. (After Gorter)
Sugar beets developed alter a treatment showed consistent size
increase for roots, but polyploidy was not found with these particular
cases. Larger roots are regularly developed in known triploid and
tetraploid progenies."-' Barring some error in method, the explanation
for larger beets falls outside the scope of polyploidy. Perplexing
variations appeared in subsequent progenies of sorghum plants that
were treated with colchicine.^*' Chromosomal numbers were diploid,
so polyploidy was not correlated Avith these types. Additional proge-
nies from treated F^ plants were significantly lacking in uniformity
as compared with untreated cases. ^"^ These variants were not classified
with aberrants reported previously and described above, i.e., the
Lio-ustyiun variations, because while the lack of uniformity followed
a segregation pattern, the control material did not show a smiilar
segregation.''- Although no explanation was given, the hereditary
mechanism was not ruled out as a possible cause. The instance is
cited in this discussion primarily to emphasize that results from treat-
ing colchicine are not in every case quickly disposed of as the effect
of a c-mitosis, leading to polyploidy which in turn is the explanation
for new variants. That colchiciire has caused a more basic deviation
not correlated with a doubling of chromosomes seems quite rea-
sonable even though the full explanation remains in question.
Cellular Growth
131
A survey of the literature^''*'' on colchicine hints that moie examples
could be obtained in which colchicine induces changes not directly
correlated with a change in the number ot chromosomes. Obviously
hundreds of polyploids have been induced by colchicine. Yet, along-
side these majority reports come the difficult cases that appear as
anomalous anatomical and morphological deviations. These are cer-
tainly problems for futme study.
4.6: Metabolism and Colchicine
Physiological studies with colchicine that had some relation to
c-mitosis were touched upon briefly in Chapter 3. At the basis of
cellular changes such as c-tumors and cell differentiation there must
also be phvsiological processes invohing action of colchicine. These
are difficult to e\aluate. Howc\er, tests ha\e been run that show
colchicine has a capacity to influence certain metabolic processes as
iniderstood by special tests. i^^- ^*'-' ^-
Enzymatic reactions performed /// iiitro proved that the trans-
formation of starch by malt diastase was accelerated. The basis for
stimulation of this order was not explained, although as a constituent
of the reaction medium, colchicine favored the rate of enzymatic
action. Increasing the concentration of colchicine increased the rate
of reaction correspondingly.^-'
Diastase activity was scored by quantitative measurements of the
increase in sugar (Benedict's solution) . Control \ alues were given
at 100.0, and if the reaction time was accelerated, the value accord-
ingly fell below 100.0. \Vith each tenfold increase in concentration
the rate was increased. V'alues of 84.0 ± 2.5. 78.9 ± 2.5, and 70.3 ±
1.7 were obtained for three concentrations, 10 p. p.m., 100 p.ji.m., and
Fig. 4.10 — Cellular intrusions among the pollen mother cells of Corthamus tinctorios
caused by treatment with colchicine. {After Krythe)
732 Colchicine
1000 p.]).ni., respectively. In other words, a control solution that
reduced 25 cc. of Benedict's solution in a certain time was equal to
100 and the solution (1:1000) with colchicine showed a value of 70.3
± 1.7 because the time taken to reduce the standard amount was
shortened, as expressed by these values.^-'
These data are interesting when correlated with reports of stimula-
tion in growth through seed and shoot treatments.'^" Colchicine may
act upon enzymes in such a way as to accelerate the transfer of starch
to sugar, which processes may in turn stimulate growth.
Excised roots of maize treated with colchicine showed lowered
rates of respiration and dipeptidase response. Also, the elongation
of individual roots was retarded. Since conditions vary from test to
test the comparisons may not be wholly alike. ^^^
Virus tumor tissues (Black's original R, strain from Rumex acetosa
L.) were treated with a wide range of concentrations (0.00001 to
100.0 p. p.m.) of colchicine. i*^! Growth was stimulated with concen-
trations of 0.02 to 0.2 }).|xm. with maximum acceleration at 0.1 p. p.m.
Increasing the concentrations beyond a point of stimulation brought
inhibition. The maxinuuu uptake of oxygen occurred at 0.1 p.]).m.
This value was estimated at 25 per cent above the control. Growth
was measured over a period of .'5 weeks and respiration tests ran for
3 hours. Curves were plotted to show the similarities and differences. ^"^^
Decreases in structural viscosity paralleled the formation of c-
tumors in root tips of Alii inn; the decreases were most pronounced
at 24 hoias.io-^ Changes in cyto])lasmic jiroteins were correlated ^\'ith
changes that led to formation of tumors.
Rates of plasmolysis among Elodca were changed by a pretreat-
ment with colchicine. '^'^ Not only the time for changing the form of
cytoplasm but the sha}je of structures formed after plasmolysis was dif-
ferent in controls and treated cells.
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sulfate, indoleacetic acid ancl colchicine media. Bot. Bull. .\cad. Sinica. 1:9-21.
1947.
King, R., and Beams^ H. Comparison of the effects of colchicine on di\ision
in protozoa and other cells. Jour. Cell, and Comp. Phvsiol. 15:252-54. 1940.
Kostoff, D. Induction of polvploidv by pulp and disintegrating tissues from
Colchicum sp. Nature. 143:287-88. ' 1939.
Krvthe, J. The effect of colchicine on the anthers of (Un llnnnits llinloiiiis
L. Proc. Acad. Sci. Amsterdam. 45:283-87. 1912.
Lang, K., et at. tJber die Hemmuug von Desoxvribonucleotidc spaltenden
Perincntcn (lurch Colchicin. Experientia. 5:449. 1949.
736 Colchicine
75. Laur. C. Experimental study of the action of colchicine on certain phases
of cellular development. Ann. Anat. Path. 15:792-99. 1938.
76. Lee, T., and H\\'ANf;, 1". Growth stimulation by manganese sulfate, indole-
3-acetic acid and colchicine in pollen tube gro^\th. Acta Brev. Sinensia.
8:21-22. 1944.
77. Lefevre, J. Actions similaires sur les mitoses vegetales de I'anethol et des
substances du groupe de la colchicine. C. R. Soc. Biol. Paris. 133:616-18.
1940.
78. Lettue, H.tTber Mitosegifte. Ergel)n. Phvsiol. 46:379-452. 1950. t'ber
Synergisten von Mitosegiften V. Mitt. Versuche zur Aufhebung der synergisti-
schen Wirkung dtirch I'hosijhagen. Xaturuiss. 38:13. 1951.
79. Levan, a. The effect of colchicine on meiosis in Allium. Hereditas. 25:9-26.
1939. The effect of acenaphthene and colchicine on mitosis of Allium and
Colchicum. Hereditas. 26:262-76. 1940. The macroscopic colchicine effect —
^ a hormonic action? Hereditas. 28:244—15. 1942.
V^ 80. , AND Levrino, T. Some experiments on c-mitotic reactions with
Chlorophyceae and Phaeophyceae. Hereditas. 28:400-408. 1942.
81. . AND LoTFV. T. Naphthalene acetic acid in the Allium test. Hereditas.
35:337-74. 1949.
82. , AND Osterc.rex, G. The mechanism of c-mitotic action. Obser-
_ vations on the naphthalene series. Hereditas. 29:381-443. 1943.
\ 83. , AND Sandwall, C. Quantitati\e in\estigations on the reaction of
V yeast to certain biologically active substances. Hereditas. 29:164-78. 1943.
84. . AND Steineggar. E. The resistance of Colchicum and Bulhacndium
to the c-mitotic action of colchicine. Hereditas. 33:552-66. 1947.
85. Lemne, M. The effect of colchicine and acenaphthene in combination with
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86. , AND Lein, J. The effect of various growth substances on the
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1941.
87. Loo, T., AND Tang, Y. Growth stimulation bv manganese sulphate, indole-
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■""Tft 88. Mairoij;, F. Studicn an colchiciniericn Pflanzen. Protoplasma. 37:445-521.
1943.
89. , AND Weber, F. Matricaria cliamnmilla durch Colcliizinierung ohne
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90. Mangenot, G. Substances mitoclasiques et cellules vegetales. £tat actuel
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91. Mariin, G. Action de la colchicine sur les tissus de topinamliour culti\e /;;
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93. Mascre, M., and Devsson, G. Les poisons mitotiques. Biol. Med. 40:1-54.
1951.
94. Mehra, p. Colchicine effect on the mitotic division of the body nucleus in
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Roxb. Ann. Bot. 16:49-56. 1952.
95. MoL, W. DE. Colchicine treatment of Scilla to produce polvploids. Papers
Mich. Acul. Sci. 35:3-7. 1951.
96. MuNTziNG, A., AND RuNQUisT, E. Note on some colchicine-induced polyploids.
Hereditas. 25:491-95. 1939.
97. Naundorf, G., and Haase, E. The auxin metabolism of polyploid plants
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Cellular Growth 137
98. Xebel, B. Cvtological observations on colchicine. Collecting Xct. 12:130-31.
1937.
99. . ANn Ri TTEF. M. Aciion of colchicine on mitosis. Genetics.
23:161-62. 1938.
100. Newcomer, E. Colchicine as a orowth stiniiilalor. .Science. 101:677-78. 194.5.
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and respiration of \irus tumor tissue in Rtimcx acctosii. ,\mer. Jour. Bot.
37:829-35. 1950.
102. XiHOUS, M. Toxic effect of aqueous solutions of colchicine on the germi-
nation of Fisum sativum. C. R. Soc. Biol. Paris. 138:128. 1944.
103. XoRTHEN. H. Alterations in the structural \iscositv of protoplasm b\ colchicine
and their relationship to c-mitosis and c-tumor formation. Amer. Jour.
Bot. 37:705-11. 1950.
104. Obaton, F. Influence de la colchicine sur le developpement de Phnto-
hactcrium phosplwreinu. C. R. .\cad. Sci. Paris. 208:1536-38. 1939.
105. Ollimfr. H. £tude cvto-toxicologique de rinfluence de divers agents physiques
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106. OMara. J. Observations on the immediate effects of colchicine. Jour. Hered.
.30:35-37. 1939.
107. Orsim. .\I.. and Panskv. B. The natural resistance of the golden hamster to
colchicine. .Science. 115:88-89. 1952.
108. OSTERGREN, G. Xarcotizcd mitosis and the jjrecipitation hypothesis of nar-
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Scient. 26:77-87.
109. Parr. L. .\ new "mutation" in the coliform group of i)acteria. Jour. Hered.
29:381-84. 1938.
110. Patton. R., a.nd Xebel, B. Preliminary obser\ations on ph\siological and
cxtological effects of certain hvdrocarbons on plant tissues. .\mer. Join. Bot.
27:609-13. 1940.
111. PiEiTTiE, L. .\ction of colchicine on plants. C. R. Soc. Biol. Paris. 131:1095-97.
1939.
112. PosT.M.A, W. Opermerkingen o\er de cvtologie van normale en \an met col-
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113. Porrz. G. Effects of colchicine on bacteria. Proc. Okla. .\cad. Sci. 22:139-41.
1941.
111. Pr.\aken. R., and Lev.Wj A. Xotes on the colchicine meiosis of Alliiiin cernnum.
Hereditas. 32:123-26.
115. Reise. G. Beitrage /ur A\'irkung des Colchicins bei der Samenbehandlung.
Planta. 38:324-76.' 1950.
IK). Richards. O. Colchicine stinudation of \east growth fails to re\eal mitosis.
Jour. Bact. 36:187-95. 1938.
117. Rosendahl, G. \'ersuche /ur Er/eugimg \c)n Pohploidie he\ Farnen durch
Colchicin-behundlung sowie Beobachtungen an pohploiden Farnprothallien.
Planta. 31:597-637. 1941.
lis. Ri HE. D., et al. Studies in luunan malaria. XI\'. The inelfecti\eness of col-
chicine, S. X. 12,080, S. X. 7266 and S. X. 8557 as curative agents against St.
Elisabeth strain \i\ax malaria. Amer. Jour. Hvg. 49:361. 1949.
119. Sanso.mEj E., and Bannon, L. Colchicine ineflecti\e in inducing pol\pl()id\ in
Fenicillium notatum. Lancet. 251:828-29. 1946.
120. Santaw, F. Polarografie a spektrografic kolchicinu a jcho dcri\atu. Piibl.
Fac. -Med. Brno. 19:149-72. 1945.
121. Sass^ J., .\nd Green^ J. C^tohistologv of the reaction of maize seedlings to col-
chicine. Bot. Gaz. 106:483-88. 1945.
122. Sato. D. Ihe effect of colchicine on meiosis in Aloiiiac. Bot. Mag. Tokvo.
53:200-7. 1939.
123. ScHi'LDT, E., AND GtniEu:!!, I). Colchicine as a mutagenic agent for Strepto-
myces griseus. 111. .Acad. .Sci. Trans. 43:51-52. 1950.
124. Shemamura, T. Effect of acenaphthene and colchicine on the pollen mother
cells of Fritillaria wild var. Thunbergie Baker. Jap. Jour. Genei. 15:179-80.
1939. Studies on the effect of centrifugal force upon nuclear division. CMo-
logia. 10:186-216. 1940.
738 Colchicine
125. SiEBENTHAL, R. A Icucophytic clone of Hormidiiini derived from a culture
treated with colchicine. C. R. Soc. Phys. et Hist. Nat. Geneve. 58:187-92.
1941.
126. SiNOTO, v., AND YuASA. A. Karvological studies in Saccharomxces cerevisiae.
Cytologia. 11:464-72. 1941.
127. Smith, P. Studies of the influence of colchicine and 3-indole acetic acid upon
some enzvmatic reactions. Proc. Okla. Acad. Sci. 21:105-8. 1941. Studies of
the growth of pollen with respect to temperature, auxins, colchicine and vita-
min'b,. Amer. Jour. Bot. 29:56-66. 1942.
128. SovANO. V. The hypertrophv in roots induced h\ several chemicals. Bot. Mag.
Tokyo. 34:185-95. 1940.
129. Sparrow. A. Colchicine-induced univalents in diploid Antirrhinum niajits L.
Science. 96:363-64. 1942.
130. Sreenivasan, A., and Wandrekar, S. Biosynthesis of vitamin C duruig germi-
nation. I. Effect of various environmental and cidtural factors. Proc. Indian
Acad. Sci. 32B:143-63. 1950.
131. Stalffi.t, M. Effect of heteroauxin and colchicine on protoplasmic viscosity.
Proc. 6th Internat. Congress Exp. Cvtologv (1947). Exp. Cell Res. Suppl.
1:63-78. 1949.
132. Steinberg, R., and Thom, C. Mutations and reversions in reproductivity of
Aspergilli and nitrite, colchicine and d-lysine. Proc. Nat. Acad. Sci. 26 (6) :
363-66. 1940.
133. Steineggar, E., and Levan, A. The cytological effect of diloroform and col-
chicine on Aliiujyi. Hereditas. 33:515-25. 1947. The c-mitotic qualities of col-
chicine, trimethvl colchicine acid and two phcnanthrene derivatives. Hereditas.
34:193-203. 1948.
134. Sterzl. J. Morphological variahility of the nuclear substance and genetic
changes induced by colchicine in "Escherichia coli." Nature. 163:28. 1949.
135. Straub, J. Quantitative und qualitative Verschiedenheiten innerhalb von poly-
ploiden Pflanzenreihen. Biol. Zentralbl. 60:659-69. 1910.
136. Sturtevant, F., et al. Effect of colchicine on regeneration in Pelmatohydra
oligactis. Science. 114:241-42. 1951.
137. SuiTA, N. Studies on the male gametophyte in angiospcrms. V. Colchicine
treatment as a proof of the essential function of the spindle mechanism in
karvokinesis in the pollen tube. Jap. Jour. Genet. 15:91-95. 1939.
138. Takenaka, Y. Notes on cytological observations in Colchicum, with reference
to autotoxicosis and sterility. Cytologia. 16:95-99. 1950.
139. Tonzig, S., and Ott-Candela, A. L'a/ione della colchicina suUo s\iluppo
degli apparati stomatici. Nuovo Gior. Bot. Ital. 53:535-47. 1946.
140. Ts'cHERMAK, E. Durch Colchicinbehandhnig ausgeloste Polvploidie bei der
Griinalge Oedogoniuni. Naturwiss. 30:638-84. 1942.
141. Ubatuba, F. Inhibition of growth of oat rootlets. Rev. Brasil Biol. 5:263-74.
1945.
142. Umrath, K., and Weber, F. Elektrische Potentiale an durch Colchicni oder
Heteroauxin hervorgerufenen Keulenwiuveln. Protoplasma. 37:522-26. 1943.
143. Vaarama, a. Permanent effect of colchicine on Ribcs nigrum. Hereditas.
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144. Vandendries, R., and Gavaudan, P. Action de la colchicine sur quelqucs orga-
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146. Vietez, E. Palynological observations on some Spanish honeys. Torrey Bot.
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. 147. Wada, B. Lebendbeobachtungen iiber die Einuirkung des Colchicins auf die
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1940.
.148. Walker^ R. The effect of colchicine on microspore mother cells and micro-
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Cellular Growth 139
effect of colchicine on somatic cells of Tradcscnntia paludosa. Jour. Arnold
All). 19:158-62. 1938. The effect of colchicine on the developing cnihiyo
sac of Tradescantia {mludosa. Jour. .Arnold .\rh. 19:442-45. 19.SS.
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150. Wang. F. Effects of auxin, colchicine and certain amino acids on the germi-
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151. Weber. F. Spaltotlnungsapparat-anomalien colchicinierter TnidcscautiaAAat-
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152. Weichsee, G. Polyploidie, veranlasst durch chemische Mittel, insl)Csondere
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154. Werner, G. Untersuchungen fiber die Moglichkeit der Erzeugung polyploider
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156. WiTKUS, E., AND Berger, C. Induced vascular ditlerentiation. Torrey Bot.
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157. W()Ec;oiT, G. The effect of colchicine on a hepatic. Jour. Hered. 32:67-70.
1941.
158. Yamaha, G., and Ueda, R. Uber die Wirkiuig des Kolchizins auf Spirogyra.
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34:55-57. 1946.
CHAPTER 5
Sources of the Drug
5.1: Scope of Study
In this chapter we shall discuss the pharmacognosy of Colchuuin
and other plants that produce colchicine. Origins, geography, history,
commerce, cultivation, preparation, and applications to biology are
explained in greater detail for Colchicum than is usual in standard
works for pharmacists.
The Greek words pharmakon, meaning drug or medicine, and
gnosis, a knowing, are combined to form the term pharmacognosy.
Literally, the meaning is a knowledge of drugs. This word is iTot so
old as the study of drugs since it was introduced in 1815 by Seydler
through his work, Analecta Pliarmacognostica. A much older name
for this subject is materia medica. and while this is still preferred in
medicine to pharmacognosy, pharmacists prefer the latter word. The
two are not entirely synonymous, for the newer term has a more
limited meaning. Biologies, such as vaccines, sera, and similar com-
pounds, do not fall within the scope of pharmacognosy but are a part
of materia medica. On the other hand, compounds such as waxes,
gums, oils, resins, sjiices, and fibers are included with drugs.
There was much disctission in centuries past as to whether CohJii-
cum should be an official drug in the standard formularies of various
nations. At certain times Colchicinn Avas made official, then dropped,
only to be taken up again in a later issue of the formiUary. Its ex-
tremely poisonous natiae and the lack of proper methods to assay the
drug caused much of the trouble. It was realized that Colchicinn was
a good cure for gout. Medical men also realized the danger associated
with administering the drug. The expressions official or nonojjicial.
acceptance or rejection, are based on the inclusion of a drug in
standard ])harmacopeias of a particular government. The drug may
be official for one country and not another. Today, the standardiza-
tion of colchicine is accinate, and the drug is official in every national
work on pharmacy.'^" Because of its availability, Colchicum luteiim
[140]
Sources of the Drug 141
is pcriniticd as a substitute for C. aiitumualc in India.i^ The stand-
ards of the British Pharmacopoeia do not permit the use of C. luteum,
because the amount of colchicine in raw material is not high enough.
5./-/; Geographical distribution. Figure 5.1 gives the location of
the im])ortant" species of the genus Colcliicum, outlining the main
areas where species are native. Taxonomists recognize 65 species in
this genus,"'* but during the earlier centuries all autumn-flowering
species were grouped in the C. autumnaJe type. Actually, the official
species is distributed over Europe; line 55 outlines this area on the
map (Fig. 5.1) . The majority of species described on the maj) flower
in the fall and produce seed in the spring. Another species known to
antiquity is C. variegatum, number 61. The distribution of C. luteum.
number 1, is the easternmost representative. All are limited to the
Northern Hemisphere and none are reported in the Americas.
5.2: Problems in Pharmacognosy
Maintaining quality, protecting the consumer, preventing fraud,
and regulating traffic become the responsibility of trained pharma-
cognosists.16. 19 During earlier centuries, physicians had to use Colchi-
cum according to their judgment. At times this duty was a heavy
responsibility (cf. Chapter 1). Even today the problem is not com-
pletely solved, for it has been discovered that U.S. P. colchicine may
contain another compound, desmethylcolchicine.-^ The substance has
biological activity; therefore, purification of so-called pure colchicine
is recommended if carefully controlled experiments are to be under-
taken.
The preparation of the drug from the fresh state before drying,
or through processes of drying, must be correct in order to avoid
changes in these complex conq^ounds. Colchicine in solution must
not be exposed to sunlight. Slicing, washing, and exposure to insects
or bacteria can also introduce changes.
Four principal techniques are used to evaluate drugs. These are
(1) organoleptic, (2) microscopic and microchemical, (3) physico-
chemical, and (4) biological methods. Each particular test is de-
scribed in the formularies or standard works on assay of drugs. Many
of the methods have been applied to colchicine.
5.3: Plants Containing Colchicine
One species is famous in every pharmacist's handbook for the
jModuction of colchicine. There are )nany other species that have a
capacity for synthesizing the conqjound in parts of plants. All species
of the genus Colclncum analyzed to date yield colchicine.'''-"'^ An
extensive list of ihcm has been collected (Table 5.1). Two genera,
Merendera and Cohhicum, have been used interchangeably. Species
-D
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Sources of the Drug
143
of each are found in the northwestern Himalayan area. Both drugs
are on sale in the bazaars of the Orient. i"
Isolated substances from Colchicuin nutiimuale and related species
have been studied extensively by Professor F. Santavy and his
colleagues at the Medical-Chemical Institute of the Polacky University
of Olomouc, Czechoslovakia. An tip-to-date summary was prepared
by Professor Santavy exclusively for this book. Accordingly Tables
5.2 and 5.3 combine the significant details from their numerous
published and unpublished works.
The chemical structure of substance F as listed has been de-
termined as desacetyl-N-methyl-colchicine, and differs from colchicine
bv the loss of the carboxy-group attached to the nitrogen ring as can
be seen in the structural diagrams of Chapter 6. Since this compound
F has strong c-mitotic properties and is less toxic than the parent
alkaloid when used with animals, the further examination of related
substances would apj^ear to be worth considerable exploration. A
compound "Demecolcin," marketed by Ciba of Basel, Switzerland, has
been studied extensively and a preliminary survey shows useful appli-
cations to some types of malignant growth. These data are found in
references to papers by Bock and Gross (1954) , Meier, Schar, and
TABLE 5.1
Principal Pla.nt Sources of Colchicine
Colchicum autumnale L.
C. montanttm L.
C. arenarium VValdst. and K.
C. neapoliianum Ten.
C. alpimim DC.
C. luteum Baker
C. multiflornm Brot.
Merendera bulbocodium Ram.
M . caucasica Biel.
M. persica Bois and Kotsch.
Gloriosa superba L.
Merendera sobolifera Fisch.
R'.dbocodium ruthenicum Bung.
Tojieldia glacialis Gaud.
T. calyculala Whlnd.
I'eratrum album L.
r. nigrum L.
Anthericum ramosum L.
Hemerocallis fulva L.
Ornithogalum umbellatum L.
O. comosum L.
Tulipa silvestris L.
Asphodelus albus VVilld.
Fritillaria montana Hoppe.
Lloydia serotina Salib.
Muscari tenuiflorium Tausch
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748 Colchicine
Neipp (1954) , Moeschlin, Meyer, and Lichtman (1954) , and Santavy,
Winkler, and Reichtstein (1953) .*
Probably the best method of detecting colchicine is the polarogra-
phic techniqne used to great advantage by Santavy and his col-
leagues."^ By these newer methods, other compounds have been
identified in the seed, corm, and flowers. A section is devoted to this
problem.
5.5-/; Colchicuni autumnale L. We mentioned earlier the un-
usual character of this autumn-flowering crocus. Not many plants
bloom in the fall and mature seeds the following spring. Since the
flowering and fruiting cycle is directly correlated with development
of corm and seed, and since colchicine production is related to these
processes, knowledge of development is important. The content of
colchicine will vary from season to season, and with different en-
vironmental conditions. Seeds are a rich source of colchicine after
maturation. The corms reach a peak of colchicine about June or
July. A vast amount of information has been reported over a period
of 20 centuries, yet it is surprising to learn how few textbooks bring
together a complete report on comparative morphology, anatomy, and
physiology in relation to drug production. More than passing atten-
tion will be given to such details in this chapter. ^^
The corm has two coverings when dug in early summer, the outer
brown membranous and an inner reddish-yellow layer. Beneath these
coats lies a yellow body that composes the bulk of the corm and most
of the tissues that yield colchicine. The corm is conical, somewhat
rounded on the surface, and flattened on one side. At the base of
the flattened area a smaller corm, or bud, fits into a groove or de-
pression. When this young bud begins development, the larger,
parental corm usually carries the maximum colchicine per dry weight
of body.
A bud develops in Jvdy, and during August or September stalks
of flowers appear. Floral activity is the first index that the young
corm has been active. Violet and reddish flowers in a cluster ranging
from two to six break through the membranes of the corm just de-
scribed and appear above ground. Corms that are not placed in the
* H. Bock and R. Gross, "Leukamie unci Tumorbehandlung mit einem Nelienal-
ca\oid aus, Colchicuin autuninnlc (Demecolcin) ." Acta Hoeinatol. 11:280-300. 1954.
R. Meier, B. Schar, and L. Neipp. "Die W'irkimg von Demecolceinaniiden an
Zellen hi iiilro." Expericntia. 10:74-76 . 1954.
S. Moeschlin, H. Me)ei, and A. Lichtman, "Ein nciies Colchicuni-Xehcnakaloid
(Demecolcin Ciha) als cytostaticum myeloischer Leukamien." Schweiz. Med.
VVschr. 83:990. 1953.
F. Santavy, R. Winkler, and T. Rcichstein. "Ziir Konsiilution von Demecolcin
(Substance F) aus Colchiciuu uutumualc L." Hehetica Chim. Acta. 36:1319-24.
1953.
Sources of f he Drug 149
soil develop liowers when the time is right. They do so without
attention as to water or nutrition. For this reason unusual attention
is given to the corm for ornamental purposes.
Each flower measines 10 to 20 cm. from base to tip of petal. The
six stamens and six floral parts are united in a tube from the top
to the carpels below. Three carpels of an ovulary show the relation
to the liliaceous group. At tlic base of the long tube is the superior,
syncarpous ovulary. Regularh', the corm is deep enough in the soil
so that about one-half of the flower is above the surface; thus, the
ovulary is well beneath the soil surface. Following fertilization, the
ovules begin a development that proceeds during the entire winter."
A progression of development and colchicine content was noted over
the long period of time that elapses from fertilization to maturation.
Pollination development begins soon after, but the content of colchi-
cine is low. There is not much increase during the early stages. In
other words, the increase in the winter is very small compared to
the gain that occurs in content of colchicine as seeds mature. The
total time studied extended from August of one year to April of the
next.'- '^^ •'^-
In early spring the fruit capsule rises out of the soil. Expanding
leaves accompany the fruit development. In the vicinity of Olomouc,
Czechoslovakia, the green capsules contain small, watery ovules until
about the middle of May. From May to July the content of colchi-
cine increases from 0.2 to 0.5 per cent. As capsules mature, the walls
split and seeds fall oiu.'
5.5-2.- CoJchic.um liiteum Baker. Because of its availability in
India, the Indian pharmacopeia accepts this spring-flowering species
as a source for colchicine. ^i' ^■'*- ^■^
The product called colchicine is Surinjan-i-talkh. Undoubtedly
this drua: has been used for manv vears, certainlv before the present
studies of pharmacognosy were conceived in their present level. Col-
lection of the corm for colchicine must be coordinated with the flower-
ing and fruiting cycles. Each corm is inclosed in membranous layers,
under which lies a hard, white bud. The daughter corm that pro-
duces the next season's plant is found in a groove at the base of the
parent corm.
At altitudes of 7000 ft., the buds develop early in March or late
February. Flowers aj^pear when the snow melts; the })lant is one of
the earliest to flower in the area. The conmion name for the species
is Kashmir hermodactyl.
A scape bearing golden flowers, two or three per cluster, emerges
from the corm. Fruiting stalks develop soon after pollination. The
capsules mature, and leaves form. Finally the seeds mature, and a
cvcle is thus completed Avitliin one season, from March to May.
750 Coichicine
5.^-5; Other sources for colchicine. Numerous sources of colchi-
cine exist in nature (Table 5.1), and undoubtedly more will be dis-
covered. A notable case is Gloriosa superba producing 0.3 per cent
colchicine compared with 0.5 per cent for C. autumnale. The un-
usual demand for colchicine made by plant breeders should stimulate
search for other sources.""^ These are the problems that pharma-
cognosists are surveying, particularly in areas where plants have not
been thoroughly studied.
When colchicine is extracted from Colchicum, other compounds
aj^pear in the residue, some of which have proved to be valuable. New
products of biological interest might well be revealed through ex-
amination of the species that yield colchicine. By new methods of
analysis a large amount of important work has been done in recent
years with compounds of colchicine and its derivatives.^^
5.4: Cultivation, Collection, and Preparation
An important source of raw material has come from the plants
growing in natural habitats.^ A large area in southeastern Europe
supplied much raw material that was purified into colchicine and
distributed throughout the world. About 1939 the sudden demand
for large portions to be used by geneticists in creating j)olyploids
created a shortage in the market. Almost simultaneously, the war
interrupted production and trade in Colcliiciiin. The prices in-
creased and colchicine was difficult to obtain.
There are standard practices for cultivating most drug plants,
and similar work has been done with Colchicum.-^ A general pro-
cedure is as follows: Seeds are sown in September, in moist, shady
locations and are covered with a thin layer of soil. After germination
the next spring, seedlings are set out 60 cm. apart. Cultivation prac-
tices are continued for three years. Corms are dug and prepared for
the market.
If seed supplies are to be made from cultivated plants, four years
of propagation are necessary. Actually a five-year cycle is required.
A common practice involves the use of seeds produced in natural
habitats. Seeds are collected by bagging the ripening capsules.
Another method for producing raw material under cultivation is
to set out the corms that come through the regular corm and bidb
markets. Or the corms may be dug in the wild state and transferred
to a field for intensive cultivation. Production of colchicine is in-
fluenced by environment. A survey from 1 1 1 localities in Moravia
showed that colchicine produced by seed \aried from 0.6 to 1.23 per
cent. An average of 0.8 per cent colchicine was obtained. "• *• ^
Sources of the Drug 151
Drug production can be increased by the application of fertilizer.
Increases in colchicine per corm were made when PoO-, was added.^*'
The methods for adding the fertilizer to soil and details of these
tests have not been rcj^eated or confirmed. These data are correlated
with a variability in jjroduction of colchicine found for different
localities.
Variation in production of colchicine appeared to be a function
of size of seed (Fig. 5.2) . The number of seeds per gram varied from
183 to 406. As the number of seeds increased, there was an increase
in the percentage of colchicine per 100 grams of raw material. The
size of seed is a response to en^•ironmental condition, and in turn the
production of colchicine is changed by the seed form. Standards set
for content of colchicine must account for variation in raw samples
of Colchicuiu. Not enough attention has been paid to the relation
between en\ironmental conditions and production of colchicine. ""^^
Colchicum hiteum is collected from natural sites exclusively. The
corms, rather than the seeds, serve as a sovirce of colchicine. There
are large areas of the northwestern Himalayas, notably in the grass-
lands, where the plants are abundant. Their locations are at levels
from 4000 to 7000 ft. AV^hile the total content of colchicine is not as
high for C. Juteum as the officially recognized species, enough can be
gathered to make this a valuable drug plant.
The dried whole corms are collected from March to May. By
this time the fruits have matmed and leaves have dried down. The
corms are dug and prepared for market according to practices estab-
lished by collectors who have been working at this trade for many
years.
Altitude influences the production of colchicine in the seed more
than in the corm, according to a study made in the European Alps
for C. aututnnale. Collections were made beginning at 50 m. and
continuing in locations up to 2200 m. The content of colchicine in
the seed sample was found to diminish with increasing altitude. The
difi^erences were not so great for the corm.'^
5.5: The Crude Drug
Dried corms and seeds of ColcJiicion are official in standard
pharmacopeias. ^1 Since 1946, C. luteum has been accepted in the
Indian standards. Dried corms are bitter and have a disagreeable
odor. There are two drugs in the Himalayan collections known as
the bitter and the sweet surinjan; the former is C. luteum.
Collections are made and corms sliced 2 to 5 mm. thick after
drying. Each piece should be about 3 cm. wide. A black layer along
the side becomes prominent. In transverse section the ground tissues
J 52
Colchicine
350
5
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COLCHICINE °/o
15
Fig. 5.2 — Size of seed can be correlated with percentage of colchicine per gram. The
smaller seeds yield more colchicine per gram of raw material. Environmental conditions
influence the size of seeds. Larger yields occur when number of seeds per gram exceed
300. (Adapted from Buchnicek)
appear grayish at certain points; these mark the vascular bundles ot
the corm and are distinct features. In the apical and basal regions
the pieces are subconical and plano-convex, respectively. The use of
specific marks of identification help to prevent the substitution of
material not genuine.
Sources of f/ie Drug 153
Histologically, the crude chug can be identified by the presence
of typical cells. Epidermal cells are rectangular and polygonal, meas-
uring 60 microns on the average. The walls are brown and thickened.
Ground tissues are full of starch grains, usually simple; if compound,
the comjjonents are from two to three parts. Vascular bundles run
longitudinally through the corm and are of the collateral type. Xylem
vessels are narrow, spiral, or annular, and about 30 mm. in diameter.
Seeds of Colchicum are subspherical, 2 to 3 mm. in diameter, hav-
ing a dark brov^n and rough seed coat. A large, hard, yellow endo-
sperm surrounding a small embryo is embedded near the surface of
the seed. Strong HCl colors the endosperm yellow, indicating the
presence of oils.i'- ^^ The seeds are bitter, but they do not have the
same disagreeable odor found with corms. Large enough amounts of
colchicine are contained in seeds that poisonous effects can be pro-
duced if warm-blooded animals eat a certain (juantity.
5.6: Compounds Isolated From Colchicum
From 1901 to 1949, many reports have been made to establish the
amount of pure substance to be expected from a given amount of
dried raw material. The corm, seed, fruit, and flowers have been
studied, and variations recorded. ^s. o<i. f'- Some of the basic reasons for
variation have been mentioned. 1 here are sources of variation that
occur because different methods of extraction and assay have been
used.^' ^0 A survey of some of the literature shows the variety of
methods that have been advocated and used.-- •='• •''• ^^- "• ^^^ i**- 1'-*- --•
.SI, 33, 3.-., 37, 41, 42, 43, 52, 66, 73 Improvements in methods have come
through the use of polarography and chromatography. •^-- ^'i- *^^ A
large field of chemistry of plant products has been opened by the
application of these new technics to drug plants. The idea that
Colchicmn produces only cokhicine must be changed in light of the
important compoinids that ap[)car with pure drug.^^
The treatment of corms with boiling water during preparation
for market causes water-soluble portions to leach out. Difterent solu-
bilities and physical properties show that even the so-called pure
drug is not a single compound. These impurities have been detected
in pollen germination studies. Obviously very few biological experi-
ments have been jjerlormed ^vith jjinc colchicine. There are dif-
ficulties in making absolutely pine colchicine in large quantity.
In addition to the comj^ounds obtained from the raw material,
there are derivatives made in the laboratory by degradation w^ork
from the drug. Enough has been done to prove that specific chemical
substances related to colchicine are obtainable. The details of such
work are extended in the cha])ter dealing with chemistry of colchi-
cine.
154 Colchicine
Santavy and his colleagues have isolated compounds from the
corm, seed, truit, and flowers. Their general method involves the
extraction from dried powder of particular portions of the plant.
Fats are extracted by petrol ether, followed by alcoholic extraction.
The use of water, then ether, and finally chloroform brings out an
extract demonstrated to have reducible substances when subjected
to polarographic analysis. By chromatographic differentiation, specific
and identifiable compounds have been reported. Details of the pro-
cedures are given in papers written by Santavy and liis associates. "^^
Isolated substances, the chemical and physical properties of which
have been observed, are tabulated in Table 5.3. The work by F. San-
tavy and liis group extends greatly our knowledge of the specific chem-
ical components that may be obtained from tlie Colcliiciim plant.
Classification is made by grouping substances as neutral and phenolics,
basic and glucosidic compounds. The particular part of the plant
used is listed so that others may repeat the isolation of similar com-
pounds.
Substances A, B, C, D, E, F, G, J. and I have been derived from
the corm, seed, fruit, and flowers. In some cases the substances liave
been found only in certain parts. Pure colchicine is identified as
compound A. Desmetliylcolchicine appears to be similar to compoinid
C. Another material, colchicerin 3, corresponds to compound G. Bio-
logically, these compounds liave different toxicities and produce dif-
ferent effects upon mitosis. Compound F is less toxic than colchicine
yet more active in blocking mitosis.
Sunlight induces changes in a solution of colchicine. •'■^ Irradiation
changes the structure of colchicine to a product known as lumicolchi-
cine. At present two kinds of lumicolchicine, I and II, are obtain-
able. Lumicolchicine I is identified with substances obtained from
the seed and flower. Lumicolchicine II is similar to compound J. By
irradiation and also through chemical treatment, compounds may be
converted from one structure to another. These tests show that the
stability of pure colchicine must be regarded as a possible source of
variation in biological experimentation.
Only a small portion of this important development in pharma-
cognosy has been given here. The possibilities of undiscovered identi-
fiable and active compounds open new fields for experimental work.
Colchicine has j^rovcd to be a very imique substance. The discovery
of related compounds synthesized by the plant is even of greater
interest.
REFERENCES
1. Albo, G. Sur la signification physiologique de la colchicine dans les diflcientes
especes de Colchicum et de Merendera. Arch. Sci. Phys. Nat. 12:227-36. 1901.
2. Anderson, A., et al. Modified assay methods for crude drugs involving the re-
moval of interfering substances by enzymic digestion. I. Modified assav method
Sources of the Drug 155
for Colchicum corm and seed. Jour. Amer. Pharm. Assoc. Sci. Ed. ,^7:319-21.
1948.
3. Beer, .\., et al. Chemical study of Colchicum sljeciosuni Stev. C. R. Dokl. Acad.
Sci. URSS. 67:883-84. 1949.
4. Belleau, B. The biogenesis of colchicine. Experientia. 9:178. 1953.
5. Blazer, Z., and Slouf, A. The examination of the Colchicum seeds, fruits
and leaves of the domestic origin. Hortus Sanitatis. 2:68-74. 1949.
6. Bryan, J., and Lauter, W. A note on the alkaloid content of Gloriosa roth-
childifnia O'Brien. Jour. Amer. Pharm. Assoc. Sci. Ed. 40:253. 1951.
7. BucuMCEK, J. Coichicin in reifenden Herbstzeitlosensamen. Pharm. Acta
Helv. 25:389-401. 1950.
8. , and Santavv, F. Mnozstvi kolchicinu v semenech ocunu zeme Mora-
vskoslezske (Content of colchicine in tlie seeds of meadow saffron from Moravia
and Silesia). Acta Acad. Sci. Nat. Moravo-Silesicae. 20:1-16. 1948.
9. , and Hejtmanek, M. Toxicita kolchicinu studovana na lebistes
reticulatus. Zvlast. Otisk z Casopisu Biologicke Listy. 31:122-29. 1950.
10. Cattelain, E. La colchicine alcaloide du Colchicum auiumnatc. extraction,
proprictes, constitution. Jour. Pharm. et Chim. 3:162. 1926.
11. Chopra, R. (see Ref. No. 7, Chap. 1. 1933).
12. Clewer, H., et al. The constituents of Gloriosa superba. Jour. Chem. Soc.
107:835. 1915.
13. Cook, J., and Loudon, J. (see Ref. No. 9, Chap. 1. 1951) .
14. Davies, E. The assay of Colchicum by the phosphotungstic method. Pharm.
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15. , AND Grier, J. Colchicine, its assay, isolation and special properties.
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3
CHAPTER 6
ChenriLstry
by James D. Loudon*
6.1: Extraction and General Properties
Colchicine is commonly extracted from the seeds and corms ot the
autumn crocus, Colclucum autumnale, Linn., but it is also present in
numerous species of Colchicum (Alboi) as well as in other Liliaceae
(Klein and Pollauf-) . Extraction is effected by alcohol (Zeiself
Chemnitius-*) and the concentrates after dilution with water are
freed from insoluble fats or resins. The aqueous solution is then
repeatedly extracted with chloroform and the colchicine is recovered
in the form of a crystalline addition complex with the solvent. From
this the chloroform is distilled off in steam or alcohol and evapora-
tion of the residual solution yields amorphous colchicine which may
be crystallized from ethyl acetate as pale yellow needles (Clewer,
Green, and Tutin'') . Chromatographic purification of the chloroform
solution on alumina greatly facilitates the procedure (Ashley and
Harris'") .
Pure colchicine, CooHo-.O^X, forms fine, practically colorless needles,
m.p. 155°; [ajo^ — 119.9° (c — 0.878 in chloroform), as determined
by Mr. T. Y. Johnston at Glasgow. It is readily soluble in alcohol,
chloroform, or in cold water, but is less soluble in hot water or in
cold benzene and is almost insoluble in ether. From these solvents
there is a tendency to crystallize with solvent of crystallization which
may markedly affect the melting point. Concentrated aqueous solu-
tions dc})osit crystals of the sesquihydrate which, despite its relatively
sparing solubility in water, does not crystallize from more dilute
solution unless induced to do so by seeding (Loudon and Speak-
man") . Dilute mineral acids and alkalis color colchicine an intense
yellow, while nitric acid (d,1.4) produces a violet color which slowly
changes to yellow and finally to green: other color-reactions are de-
Lectuier in Chemistry, University of Glasgow, Scotland.
[159]
760 Colchicine
scribed by Zeisel.^ Although under suitable conditions colchicine
forms precipitates with many ot the usual alkaloidal reagents,^ its
classification as an alkaloid is questionable. It is essentially a neutral
substance with a honiocyclic ring-structure: on the other hand, it is
associated in the plant with compounds of allied structure, some seven
crystalline and kindred alkaloids being known (Santavy and Reich-
stein**) .
6.2: The Functional Groups
Hydrolysis of colchicine by boiling with very dilute hydrochloric
acid yields methyl alcohol and colchiceinc, C^iH^.^OuN, which is
acidic, gives a deep olive-green color with aqueous ferric chloride
(distinction from colchicine) , and on further hydrolysis with more
concentrated acid yields equivalent amounts of acetic acid and tn-
mcthylcokhicinic acid, Ci.,HoiOr,N (ZeiseP) . This last compound is
amphoteric and contains a primary amino-group (Johanny and
Zeiseli") ; hence the two-stage hydrolysis may be represented as follows:
C10H1SO4 (OMe) (NH.COMe)
-^ MeOH + Cic,Hi,04 (OH) (NH.COMe)
-> MeCOoH + CigHisO, (OH) (NH,) .
Trimethykolchicinic acid contains three methoxyl groups which, by
prolonged hydrolysis, are demethylated and colchicinic acid, CifiHig
O5N, is produced. Correspondingly in colchicine itself the presence
of four methoxyl groups is shown by the usual Zeisel estimation.^
The four methoxyl groups and the acetylamido-group together
account for five of the six oxygen atoms of colchicine. Since the sixth
oxygen is unresponsive to carbonyl reagents, it was at one time
thought to be part of a carbomethoxy group (-CO.OMe) or of an
oxygen ring system. The former view is in harmony with the ready
hydrolysis to colchiceinc which has acidic character but which, on
the other hand, also shows definite enolic properties and when methyl-
ated by diazomethanc, yields two readily hydroly/able O-methyl
ethers, namely colchicine and iaocolchicine (Meyer and Reichstein;ii
Sorkini-) . Similarly trimethykolchicinic acid reacts with benzenesul-
phonyl chloride to give two di (benzenesulphonyl) derivatives (W^in-
daus^^^) , in each of which one of the acyl gioups is attached to nitro-
gen while the second ajjpears to be attached to oxygen since fairly
mild hydrolysis converts both compounds into the same A^-benzenesul-
phonyl trimethylcolchicinic acid. This duplication of O-derivatives
strongly suggests that in colchiceinc and in trimethylcolchicinic acid
there is a tautomeric enol system capable of giving rise to paired O-
derivatives which are either steric or structural isomers. Accordingly
the sixth oxygen atom is considered to reside in the carbonyl group
Chemistry 161
oi an enolonc system in colchiceine and of a corresponding enolone-
niethvl-ether system in colchicine.
Although neither colchicine nor colchiceine reacts with the usual
carbonyl reagents, hydrogenation results provide evidence ot the
presence oi a carbonyl group in each. Bursian^* found that with a
platinum catalyst both compoinids absorbed three moles of hydrogen
and that thereby colchicine gave a mono-alcohol while colchiceine gave
a diol. In each case therefore a new hydroxylic function has been pro-
duced and may well arise from reduction of a carbonyl group by one
mole of hydrogen. The absorption of two further moles of hydrogen
shows the presence of two olefniic groups, while the presence of yet
a third olefinic group, which resists hydrogenation, was indicated by
the interaction of liexahydrorolchicine, C^jHyiOoN, with perbenzoic
acidic or with monoperphthalic acid (Tarbell et al.^^) to form an
oxide, CjoH:„07N.
Summing up: The evidence suggests that colchicine is the methyl
ether of an enolone which contains three additional methoxyl groups,
an acetylated primary amino-group. and three non-benzenoid dotd:)le
bonds:
Ci,H, (OMe) 4 (NH.COMe) (:0) (=) 3-
6.3: The Structural Problem
The saturated hydrocarbon, Ci.jHoo, which corresponds to this
assemblage of groups, fall short of the j)araftin, Ci,;H34, by six hydro-
gen molecules each of which in default indicates the presence of either
a carbon ring or a benzenoid type of double bond. Four of the miss-
ing hydrogen molecules are at cjijce accounted for by the demon-
strable presence of a benzenoid ring; the remaining two must there-
fore denote two further ring systems. Colchicine is accordingly tri-
cyclic and the respective rings, both in the alkaloid and in its
degradation products, are designated by the letters A, B, and C.
6.5-/; Ring A. The presence of the benzenoid ring (A) is shown
by the formation of •5:4:5-trimethoxyphthalic acid (I), or its anhy-
dride, from colchicine and many of its derivati\'es on oxidation with
hot alkaline permanganate (Windaus^''- i") .
6.5-2; Ri)ig B. The most penetrating insight into the molecular
structure of colchicine is obtained through a series of degradation
products (Windaus^^' ^^) derived from N-aceiyliodocolchinol. C20H22
O5NI. This compound is formed from colchiceine by the action of
iodine in the presence of alkali. It is definitely phenolic and is re-
duced by zinc and acetic acid to ^i-acetylcocJilnol, C^qH^^O.-.N, which
on methylation ailords N-acetylcolchinol methyl ether. The latter still
contains the acetylated primary amino-group and may be deaminated
162 Colchicine
in se\eral ways: (1) directly, by heating with phosphoric oxide in
xylene (Cook and Graham;i^ Barton, Cook, and Loudon^o) whereby
two isomeric compounds, Cif,H2i04. are formed and are named de-
amjuocolchinol methyl ether and hodeammocolchinol methyl ether, re-
spectively; (2) by hydrolysis to the primary amine, colchinol methyl
ether, followed by reaction with nitrous acid to form a carbinol
CO2H
COoH
(Cohen, Cook, and Roe-^) which on dehydration^^ yields the same
pair of isomeric products; (3) by Hofmann degradation of colchinol
methyl ether whereby only deaminocolchinol methyl ether has been
isolated (Windaus--) .
Barton, Cook, and Loudon-'^^' established the structure (II) for
deaminocolchinol methyl ether and the structure (III) for the iso-
compound on the following grounds. Both isomers afforded the same
dihydride when hydrogenated in acetic acid with a palladium cata-
lyst; they must therefore differ only in the location of a double Ijond
which must be ethylenic in type. Deaminocolchinol methyl ether was
oxidized with sodium dichromate in acetic acid to 2:3:4:7-tetrametho-
xyphenanthraquinone (VIII) , together with a by-product which was
recognized as an unsaturated ketone, CioHisOr,.
Formation of the quinone, which was identified by synthesis,
establishes the presence of a (bridged) diphenyl system and fixes the
methoxylation pattern. The nature of the three-carbon bridge in
deaminocolchinol methyl ether (II) was next determined by oxida-
tion with osmium tetroxide to a glycol (IV) which, by scission with
lead tetra-acetate, yielded not the normally expected di-aldehyde (V)
but a mono-aldehyde (VI) formed from (V) by internal condensation.
This mono-aldehyde — later synthesized —was identified by oxidation
to 2:3:4:7-tetramethoxyphenanthrene-10-carboxylic acid which was
also synthesized. Similar stepwise oxidation of /.vodeaminocolchinol
methyl ether (III) gave 2:3: 4:7-tetramethoxy-9-phenanthraldehyde
(VII) , identical with a synthetic specimen.
These results leave little room for doubt that deaminocolchinol
methyl ether and its wo-compound are correctly formulated. More-
over, Cook, Dickson, and Loudon--' Irdxe shown that the synthesized
o
SG
o a-
s
u
X
o
o
X.
o
X
u
X
o
X
o-
"V
«
"V
164
Colchicine
parent hydrocarbon corresponding to (II; H for OMe) reproduces in
all essentials the behavior just described and, iurthcr, that this hydro-
carbon is isomerized to 9-methylphenanthrene by successive heating
with liydriodic acid and zinc dust. Such isonierization accounts lor
the isolation of 9-inethylphenanthrene by AVindaus-- during an at-
tempt to dcmethoxylatc dcaminocolchiuol methyl ether, and it con-
OH
Me
OH
(IX)
Me
c:o
CHO
OMe
(X)
tributed to his formulating the latter compound as either 2:3:4:6- or
2:3:4:7-tetramethoxy-9-methylphenanthrene, each of which, when
synthesized by Buchanan, Cook, and Loudon."-^ proved to be distinct
from the degradation product. Tarbell, Frank, and Fanta,-^' who pre-
pared deamino-iodocolchinol methyl ether from A^-acetyliodocolchinol
and oxidized it to a derivative of homodiphenic acid, likewise con-
clude in favor of a 7-membered ring B as in (II) .
The first synthesis of a significant deri\ative of (II) was effected
by Buchanan. Cook, Loudon, and MacMillan.-" The sequence of re-
actions used lor the ring-contraction (II) -^(IV) was applied in the
Chemistry 165
opposite direction to expand the central ring of 2:3: l:7-tctramethoxy-
10-niethvlj)hcnanthrenc (IX). Hiis took ad\antage of the known
reactivity of the 9:10-double bond in phenanthicnes and hvdroxyla-
tion, scission, and renewed cyclization led to an unsatinated ketone
(X) identical with the one produced, as already mentioned, by oxida-
tion of deaminocolchinol methyl ether. Moreover, by applying the
same series of reactions to 2:3:4: 7-tetra-methoxy-9-methylphen-
anthrene (XI) Cook, Jack, and Loudon-' obtained an isomeric tm-
saturated ketone (XII) . This was reduced to the saturated ketone
(XIII) and thence by oxiniation and rcneAsed reduction was con-
verted to tlie (rt) -amine (X\' I) . Optical resolution of this amine,
through its salts Avith (-]-) -6:6'-dinitrodiphenic acid, afforded the
( — ) -base and hence the ( — ) -acetyle derivati\e and these resj^ectively
were identical with colchinol methyl ether and its A'-acetvl derivative
^^o
/\
OMe
(XI)
(XII)
MeO
MeO
\U'
(XIII)
OMe
(XIV)
as obtained by degradation of colchicine. -"^ By a different loute start-
ing from the 9-monoxime of 2:3:4:7-tetramethoxyphenanthraquinone
Rapojjoii, Williams, and Cisney also synthesized the (h=) -amine
(XIV) and showed it to be identical witli i acemized colchinol mcthvl
ether.2»
A second series of degradati(;n prcxiucts has a bearing on the struc-
ture of ring B. \\^indausi'' found that A"-benzoyltrimethylcolchicinic
166
Colchicine
acid (prepared by di-bcnzoylation of triinethylcolchiciuic acid and
preferential hydrolysis of the O-ben/.oyI group) was oxidized by cold
alkaline permanganate to two products, namely N-benzoylcolchinic
anhydride, C^aH^iOjN, and a corresponding lactone, N-benzoylcol-
chide, CjcjH^mOijN, which he formulated--' as derivatives of 1:2-
dihydro-2-methylnaphthalene. With the recognition of ring B as 7-
MeO
MeO
NHBz
MeO
UcO\
MeO
X
y CO
I !
CO o
( XVI )
MeO
MeO
N
MeO
CH2.CH2
\
CHo
CH.COoH
CO
CO2H
( XVIII )
membered in the colchinol series, it was at once evident that A-
benzoylcolchinic anhydride might be better represented by formula
(XV) and A^-benzoylcolchide by a corresponding lactone structure.
To test this view. Cook, Johnston, and London-^*' deaminated the
anhydride and showed that the lesidtant deaminocolchinic anhydride
was not identical with ():7:8-triniethoxy--^methylnaphtlialene-l:2-di-
carboxylic anhydride — as it would be on the Windaus formulation —
nor indeed could it be a naphthalene derivative since it showed
ethylenic behavior towards reduction. From the reduction products.
Horning, Ullyot, and their colleagues''^ isolated a dihydride and
established its structure as (XVI 1) in synthesis and cyclization of the
oxaloacetic acid (XVIIl) . Thereby the 7-membered rings in A^-
benzoylcolchinic anhydride (XV) and its deaminati(jn product
(XVI) are unequivocally proved.
Accordingly both lines of degradation — the first, through A^-
acetylcolchinol, involving a process which makes ring C benzenoid;
the second producing A'-benzoylcolchinic anhydride ai)parently by
Chemistry 167
direct oxidation ol ring C - consistently lead to the conclusion that
ring B ol colchicine is T-nienibered.
6.9-9; ^'"i( ^'- ^t will now be evident that the enolone projjerties
ol colchiceine derive trom the third ring, namely ring C, and that the
structure to be assigned to this ring must also interpret the conversion
ol colchiceine into A^-acetyliodocolchinol. This transformation is
empirically expressed by
C,,,H.,;A;N + I ^ C:.OH,,0,NI + [CHO]
and die colchinol derivative so produced may be formulated as (XIX)
which is in harmony with the observation that its methyl ether yields
4-iodo-5-methoxyj)hthalic acid on oxidation.'^ •'- Two further links
between the structure of the alkaloid and that ol colchinol are known.
Cecil and Santaxy-^-^ obtained iV-acetylcolchinol directly by oxidizing
colchiceine with alkaline hydrogen peroxide. Again, colchicine (but
MeO
Mao's
/
NHAc
MeO
')on
( XIX)
NH.Vc
MeO
MeO
C02Me
(XX)
not colchiceine) is isomeri/cd when heated with sodium methoxide
in methanol (Santavy;-^^ Fernholz'''') forming the methyl ester {(lUo-
colchicine) of a carboxylic acid (c///ocolchiceine) ; and Fcrnholz^-^
conxerted this acid into A'-acetylcolchinol l)y the standard procedure:
RCOTi-^RNH. -^ ROH. The structure of aJJocoUhlnuc is there-
fore sec urely fixed as (XX) .
768
Colchicine
Even before all of these facts were available, Dewar^^ suggested
that ring C of colchiceine was trojiolonoid and on this basis the struc-
ture of colchiceine is represented by the tautomeric system (XXI) ^
(XXII) . The validity of this formulation is now generally accepted
and an earlier formida, proposed by Windaus,-- need not be dis-
cussed here.
6.4: Comparison With Tropolones
It is necessary, however, to refer briefly at this stage to some of
the more general featines of tropolone chemistry (for more compre-
MeO
MeO
NHAc
MeO
MeO
MeO
O
OH
NHAc
(XXI)
( XXII )
OH
hensive treatment, see Cook and Loudon-''") . Tropolone (2-hydro-
xycyc/oheptatrienone) and its derivatives have aromatic properties,
the reactivity of the ethylenic and carbonyl functions being sup-
pressed. Thus the compoimds are substituted by electrophilic reagents
but do not react with carbonyl reagents. The hydroxyl group is
markedly acidic. Salt formation is accompanied by development or
intensification of color, and coordination complexes are produced
with ferric or cupric ions. Tropolone itself exhibits feebly basic
properties and yields a hydrochloride and a picrate. Tropolone
ethers resemble esters in their ready hydrolysis. With varying ease
individual tropolones (or their ethers) are isomerized by hot alkali,
the 7-membered ring luidergoing contraction to the benzenoid struc-
ture of an appropriately substituted benzoic acid (or ester) . Catalytic
hydrogenation of tropolones is seldom simple. When complete, it
yields octahydrides which are l:2-diols, but it may involve loss of
oxygen, and ketonic intermediates are frequently detectable.
The general analogy with colchiceine, implicit in this account of
tropolone behavior, is borne out by more specihc comparison. Like
unsymmetrically substituted tropolones, colchiceine is known only as
a single substance which yields two isomeric methyl ethers, colchicine
and wocolchicine, corresponding to the tautomerides (XXI) and
Chemistry 169
(XXII) . The ester-like properties of these ethers are revealed in their
rapid hydrolysis to colchiceine and in their reactions with ammonia
and amines wherebv colchicamides are formed,-^'^ the rea(ii\e methoxyl
group being replaced by an amine residue. Hydrogenation of colchi-
ceine, or of colchicine, is complex, i^- ^■'- ^^- ^f- ^'^ but there is evidence
that hexahydrocolchiceine is a 1 :2-diol,i'' ^- and less fully hydrogen-
ated material shows ketonic properties.-'* Polarographic measure-
ments made by Santavy and by Brdicka,^"'' and infrared absorption
studies by Scott and TarbelH^ confirm the similarity between colchi-
ceine and tropolones. Moreover, r/Z/ocolchicine (XX) is at once seen
to be the benzenoid isomerization product of a methyl ether derived
from either (XXI) or (XXII) . Its production corresponds to that
of methyl benzoate from trojjolone methyl ether (Doering and
Knox-*'') and explains the origin of the trimellitic acid (benzene-l:2:4-
tricarboxylic acid) which ^\'indaus obtained from colchicine by suc-
cessive alkali fusion and oxidation. ^'^
6.5: Structure of Colchicine
The tautomeric nature of colchiceine allows two possible formula-
tions of colchicine, its methyl ether. It is not easy by chemical means
to distinguish between these alternatives but the distinction can be
made by X-ray crystallographic analysis. King, De Vries, and Pepin-
sky-**' in this way examined an addition complex of colchicine and
methylene di-ioclide and not only confirmed the tricyclic structure
with its two fused 7-membered rings but also showed that colchicine
is the particular methyl ether (XXIII) . It follows that /.vocolchicine
has the methyl ether structure corresponding to (XXII) .
6.6: Miscellany
So far in this chapter discussion has been directed primarily to
the evidence on which the structural formula of colchicine rests.
There remain to be noted several reactions and items of chemical
interest, which are either at {^resent incompletely evaluated or only
indirectly related to the alkaloid's structure. For instance it is known
that nitration of colchicine yields a mononitro-colchicine, reducible
to an aminocolchicine, but the seat of substitution in these derivatives
is not yet definitely ascertained (Nicholls and larbelH') . Bromina-
tion of colchicine yields mono-, di-, and triljromo deri\aii\es (Zeisel
and Stockert^') . Bromination of colchiceine yields a tribromo acid
which Lettre, Fernholz, and Hartwig^^ formulate as (XXIV) by
analogy with the bromination of tropolones^" and because the com-
pound is readily decarboxylated to a tribromo derivative of A'-acetyl-
colchinol. Oxidation of colchicine ^vith chromic acid in aqueous solu-
tioti yields a ketone, namely oxycolchicine, C:,2H280-N, in \vhich a
170
Colchicine
methylene group of tlie alkaloid has been oxidized to carbonyl.22, 5o
Molecular rearrangement is almost connnonplace in colchicine's
chemistry. It is inherent in the changes, already described, by which
the 7-membered rings ot the alkaloid or its derivatives become con-
tracted to 6-membered rings. It is also encountered in formation of
the carbinol (().8) by the action of nitrous acid on colchinol methyl
MeO
MeO
NHAc
( XXIII )
NHAc
CO2H
ether and is again found in dehydration of this carbinol whereby
deaminocolchinol methyl ether (and its isomeride) is produced. Both
of these reactions are known to involve Demjanow-type rearrange-
ments (Cook, Jack, and Loudon"'^) and through them ring B, initially
7-membered. is contracted and re-ex|jandcd in successive steps. More-
over, colchicine itself is sensitive to ultraviolet light and is isomeri/ed
in aqueous solution by simlight. 1 hereby three isomerides, namely
U-, IS-, and y-liunicolchicine are formed (Grewe and Wulf;'''- Santavy^-^)
but their molecular structures remain undetermined.
Synthesis — the ultimate challenge of a natural product to the
organic chemist — has still 10 be achieved for colchicine although, at
Chemistry 171
the liiuf ol writing', preliminar\ \v()rk in ihis diicdion is cnoaoing
nuuh attention.''^ "•'•' The colchicine striictme is novel chielly in re-
spect ol the two fused 7-nienibeie(.l rings ol its tricyclic svstcni. These
lings are retained in a coni]jound, C,.|Hj,;0;., which Rapoporl and
W^illianis-''^ prepared Ironi colchicine by a series ot hydrogenation
reactions. In this jjrodiict ring A ol colchicine is unaltered, but rings
B and C are fidly reduced and devoid ol substituent grouj^s. Syn-
thesis ol this conipoimd is potentially more simple, although also less
significant, than that ot colchicine itself. But even total synthesis of
the alkaloid, when achieved, is unlikelv to have more than academic
importance: synthetic colchicine will not soon pro\ ide an economic
replacement of the natmal product. Here another issue is joined,
for it may be possible from a study ot the alkaloid and its immediate
derivati\es to discern some pattern of atoms or groups, ^\•hich is as-
sociated ^vith colchicine's elfeci on mitosis. By incorporating this
molecidar pattern in simpler and more accessible compotuids it
would then be possible to search on a rational basis for synthetic
substitutes. Already several attemjits have been made to achieve this
end and some success has been claimed for compoimds modeled on
the earlier, partly erroneous formida of AV^indaus (see work by Lettre
discussed in Clhapter 1 7) . As woidd be exj^ected, tropolone deriva-
tives have been investigated for their effect on cell mitosis. For in-
stance, p-acetamidotropolone (XXV) — a compound possessing obvi-
ous structmal similarities to colchiceine — was examined, in Trades-
cnntia cells //; t'/t'c;, bv "\\\ida''" 'who records a strong^ radiomimetic
NH.Ac
v
o
OH
( XXV )
acticju and regards the compound as a possible mtuagenic sul^stance.
Its effect, however, does not appear to be identical with that oi colchi-
cine.
As an aid to biologital studies Raffauf, Farren, and UlKot''^ ha\e
jjrejjared C^^-labeled deiivatives of colchicine by metliylation ot col-
chiceine with labeled dia/omethane and by acet\lation of desacetyl-
colchicine with labeled acetyl chloride.
172 Colchicine
Mention was earlier made of congeners of colchicine (6.1) . These
include a demediylcolchicine —or "substance C" — in which one of the
three methoxyl groups of ring A is demethylated. Horowitz and
Ullyot*'- find what is probably the same compound present in U.S. P.
colchicine to an extent of some 4 per cent. It is also interesting that
Bellef's-'^'^ has isolated a glucoside, namely colchicoside, C27H0.0OUN,
from C. autuinnalc and that this glucoside may be hydrolyzed to. and
synthesized from, "substance C" and glucose. The glucosidic link
probably involves the oxygen atom which in ring A is adjacent to
ring B. Santavy and his colleagues have improved the technicpie of
isolating colchicine from C. (lutiniDuiJe and have examined its sea-
sonal variation in the plant. ''"^ They also surveyed various Colchicuin
species for alkaloid content and found C. arennrium W.K. to be par-
ticularly rich in colchicine. Finally they have made considerable
progress towards elucidating the structures of colchicine's co-alkaloids
^- ^^ and it is already apparent that at least several of tliese are simple
modifications of the structural pattern of colchicine.
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Chemistry 173
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CHAPTER 7
Pharmacology
7.1: Colchicine in Medical Therapeutics and Forensic Practice
The ninetCL'iith (cntui y medical literature contains many references
to Colchicion prc-jjarations.^^ 1 hese were widely used in the treatment
of gcjut. a disease in which se\ere jjain is associated ^\ith the deposition
of uric acid crystals near the joints. It was logical to attempt to cure
other ]xiinful joint ailments with the same drug, and references may
be found dealing with the treatment of various types of "rheuma-
tism." Ihe medical interest in the drug had two very different conse-
c|uences. Scientists took tip jirecise pharmacodynamic experiments in
order to reach a better luiderstanding of the therapeutic effects of col-
chicine. Various animals and organs were treated with the drug, and
important new facts wvve proclaimed in learned papers. .\ typical
paper of this t\ pe is that of Jacob], which suuniiari/es all that was
known of the drug in the 189()'s.-^-^' Frecjtient reference will be made to
it, and to a chapter contributed by Fuhner-' in Hetfter's textbook
of pharmacology. Most of the contributions of the last centiny are now
onlv of historical interest and will not be reviewed in this chajjter.
Today interest in colchicine pharmacology has been re\ ived,'--' and it
is apjiarent that man\ conclusions will ha\e lo be changed in the
light of modern work. In 1952. it was stated that the mechanism of
action of colchicine, from a j^harmacological ])oint of view, was "largely
unknown."--^
Another and more redoubtable consecpience ol ihe use ol the drug
against gout in the nineteenth centin\ was the increasing number ol
cases of fatal human poisoning.'^- '^ While one author is claimed to
ha\e taken as much as 20 mg. of colchicine in an experiment lo study
the toxic reactions,''' there are reports of severe physiological dis-
turbances and even death in jjatients that had absorbed only a few
milligrams of the diug.^"' It is cpiite dilluuh to compare all these
findings, for the j:)reparations of C.oU liicutn may have been different.
E\en after the crystallization of the alkaloid b\ Houde, preparations
[175]
176 Colchicine
were not standardized. Recent Avork re^ icwed in otlicr chapters indi-
cates the complexity of the alkaloidal content of Caleb innu and the
great differences in loxicitx of substances cheniicalh \er\ close to
colchicine.
Forensic medicine cjuite natmally was often interested in the prob-
lem of htmian poisoning, accidental or criminal. A vast amount of
literature on this subject exists. ])ut it has not been found necessary to
include it in this book. HowcAcr, one most imjjortant fact made clear
in this field is the long jjersistence of the alkaloid in the body after
death.-' The jiroblcms of the metalDolism of colchicine will be taken
up further in this chajner.
.\11 ^vork on colchicine before 1934, excepting onh iliat on blood-
forming tissues and Ijlood cells, which will l)e discussed later, was
confined to pharmacological methods and chemical testing. No study
of the morj)h()logical changes was made, and these remained unsus-
pected for a long time. 1 he aim of this chapter is not to give a detailed
study of the j^harmacology of colchicine, but to place it in a new per-
spective, that of spindle-poisoning. The significance of this in a field
apparently so distant from cytology can be illustrated b\ modern
descriptions of death from colchicine poisoning. These will sho^v some
of the comj)lexities of the jjharmacology of that ver\ ancient drug,
Colcliuinn.
7.2: Colchicine Poisoning in Man
The junior author happened to make the first detailed post-mortem
study after the disco\ery of the action of colchicine on cell division. ^^
In 1941, a woman of 42, attempting suicide, swallowed 60 1-mg.
pills of colchicine "f4oude." She lived eight days after this very
high dose; delayed letliality is nearly always found in colchicine poi-
soning. Vomiting and diarrhea were |jrominent, the I)lood mea in-
creased to \.5 gm. per thousand, and there were nervous troubles which
were considered to be e\ idence of polynetuitis. An important decrease
in the number of white blood cells and of platelets was noticeable.
A bone-marrow study was performed only two hours before death, that
is to say, eight days after colchicine had started to act. The abnormal
percentage of metaphases, mainly of the star type, illustrated that
sjjindle activity had not yet entirely recovered (Fig. 7.1) .
Microscopic evidence of this was found at the post-mortem exami-
nation.-- Arrested metajjhases coidcl be seen in lymph glands, in the
spleen, and in the Lieberkiihn glands of the intestine. 1 he histological
changes in the liver were remarkable. Here, 4 per cent of all li\er cells
were in a condition of arrested metaphase. .\bout 15 per cent of these
mitoses were ball metaj^hases, while the others showed scattered
chromosomes. Other findings interesting from the ]:)oint of \ icAV ol the
Pharmacology 177
general action ol the alkaloid were hypertrophy ot the adrenal cor-
tex, Avhere no mitoses ^\■ere to be seen, hypertrophy of the Langerhans'
islets, and hvijerbasojihilia of the anterior lobe of the pitnitar\. These
weie considered to bring e\idence of an "alarni-reaciion,"' that is to say,
a nonspecific j^itnitarx -adrenal stininlation. Ihe kidneys did not
shoAV an\ particular chani^es. \viih the exce))tion of a \ery small
^^?^
^^
^
M A+T
M A+T
Fig. 7.1 — Colchicine poisoning in man. Metaphasic arrest in the bone marrow, left,
granulocytes; Right, erythroblasts. The shaded areas indicate the normal repartition
and variation in the percentage of each stage. (After P. Dustin" )
number of mitoses. Mitoses arrested by colchicine could be iound
both in exocrine and endocrine tissues of the pancreatic gland.
The principal findings were (I) the persistence of mitotic changes
long after the ingestion of colchicine, indicating that this substance
is only slo^vh metabolized, (2) evidence of a general toxic reaction,
and (3) considerable changes in the li\er, where the proliferation of
hepatic cells was made c\ident b\ ihe nn"lotic "stasis" ])r()duced by
778 Colchicine
spindle destruction. These changes ^vere considered at the time as
evidence ot mitotic stimulation by colchicine (ci. Chapter 9) ; they
are probably only an indirect effect, the alkaloid having destroyed
hepatic cells and later arrested the mitoses needed lor regeneration.
One other similar pathological description has recently been
published. ^^ This was a case of acute poisoning. A five-year-old girl
swallowed an inikn()\vn number of seeds. These were later identified
as belonging to the genus Colchi( inn. Repeated ^omiting and ab-
dominal pain were the first signs of toxicity. The central temjierature
rose and the pulse became fast. Death followed in 38 hours. Cerebral
edema was conspicuous. Small hemorrhagic dots were seen on the peri-
cardium and the peritoneal serosa. The duodenal mucosa was swollen
and dotted with man\ hemorrhagic zones.
Evidence of mitotic poisoning was visible in the li\er, where some
cells were in a condition of arrested metaphase. Others showed evi-
dence of degenerati\e alterations. Arrested metaphases were con-
spicuous in the bone marrow; a small number could be foimd in the
duodenal mucosa. Pycnotic destruction of lymphocytes in lymph
glands, Peyers patches, and the thymic cortex was probably the result
of the combined action of the mitotic poison and of the general alarm-
reaction.""
Colchicine was detected b\ a biological method, while chemical re-
actions remained negative. Large quantities were found in se\eral
organs, in particular the liver, the kidney, and the brain. Extracts
from these tissues displaced a typical spindle-poisoning effect when
brought into contact with chick fibroblast cultures.
In the complex changes which take place when a large dose of
colchicine is absorbed in man, it is evident that some are related to
the poisoning of cell division, for instance bone-marrow inhibition. '■'■ ^^
while others, such as the destruction and regeneration of liver cells,
and the evidence of stress, are of a more complex nature. Vomiting,
which may appear shortly after the drug is taken, is one major sign
of a series of disturbances which clearly have nothing to do with the
cytological effects which have been studied so far. These will now be
described from data on various mammals and \crtebrates, before
analyzing the changes possibly related to spindle inhibition. The
important problem of the metabolism of colchicine in the bodv will
be discussed in a later paragrajjh.
7.3: Disturbances Unrelated to Mitotic Poisoning
Vomiting, diarrhea, bloody stools, and a progressive paralysis of
the central nervous system are the most evident signs of toxicity. Death
occurs within several hours in warm-blooded animals, or several davs in
cold-blooded \ertebrates. after injections of the largest doses. In 1906,
Pharmacology 179
colchicine was called "this most remarkable sUnv i)()ison."-" Progres-
sive nervous paralvsis leading to respiration arrest, appears to be the
main cause of death, whatever the animal tested. Recent research has
brought new emphasis on this nervous action ol colchicine.--'
y.^^-i: Nerx'ous system, central and JMni j^lioal. An experiment
jjerlormed nearh' 50 years ago gives a remarkable demonstration ot
the sensitivitv ot the nervous system towards colchicine. While the in-
jection ot even the largest doses killed a cat only alter several hours,
the intracerebral injection ot the drug had a spectacular and rapid
action. Very soon the blood pressure was found to increase, and the
respiration became rapid and deeper. After 35 minutes, a sharj) fall in
the blood pressure indicated vasomotor paralysis. One hour alter the
injection, the animal died of respiratory paralysis.-"
.\n important series of findings in rats and cats points to the
ner\()us s\stem as one of the principal causes of the various etlects of
colchicine poisoning. This work can only be summarized here. 2=^
Some of the most significant obser\ations are listed. Vomiting cannot
be, as was sometimes thought, the consequence of pathological modi-
fications of the gastrointestinal tract brought about by mitotic arrest.
The same is true for diarrhea, a frequent synijitom, which would
appear to be a consequence of intestinal congestion and ulcerations. -«
No diarrhea and almost no vomiting is found in animals injected with
barbiturates, even when the dose of colchicine is lethal.
The central temperature falls sharply after colchicine. This may
be pardy a result of stress and nonspecific toxicity'^ «•• (Fig. 7.2) . but
the cur\es indicate that the decrease taking place in the first ten hours
has another cause. This is now believed to be a central nervous effect.--^
Another fact points in the same direction: Animals treated with
colchicine display an increased sensitivity. While unanesthetized cats
die only after eight to ten hours, the same dose of colchicine brought
death in less than two hours when the animals had received barbi-
turates jM'eA-iously.--^ Barl)iturate or ether anesthesia also proved to
be abnormally dangerous in animals which had received the alkaloid
first.
Arterial constriction leading to high blood pressure has been men-
tioned. Experiments of brain transsection in the cat demonstrated thai
this also was a consccjuence of a central nervous stimulation.-"-
Howe\er. other territories of the ner\ous system are attectcd In
colchicine. The neuromuscular apparatus appears to be the most
sensitive, though only after repeated administration of the alkaloid
can the modifications be detected. An atrophy of the hind (piarters
of cats injected daily with 0.05 mg. per kg. of body weight was ()l)ser\ed
after two weeks. The leg muscles were converted into thin strands.
There was no e\idence of muscular damage. .Abnormal responses U)
780
Colchicine
acetylcholine were ob,ser\ed. There was no true neuromuscular block.
Anesthetic properties have also been descril:)ed; these are probably
of central origin. Death often follows a period resemblino narcosis.
In the dog, this apj^ears before the muscle paralysis. In cold-l)looded
animals, the nervous changes may be very slow to appear. In frogs
RECTAL TEMP.
98° F_
COLCHICINE : 2 MGM. PER KGM.
36°C.-
35°-
34°_
97'
96<
95=
94°
93<
92'
10
20
SO
CORTIN
NaCl+NaCit
40HRS. 50
Fig. 7.2 — Action of cortin and sodium on the temperature fall of rats after colchicine
intoxication. (After Clark and Barnes ')
kept at low temperature, reflexes disa|)pear progressi\eh, the corneal
being the last, and this not until sexeral weeks after an injection of
colchicine.-"
7.5-2; Striated tnusdr. Recent studies of the frog's sartorius muscle
have brought ne^\^ evidence of a muscidar action of colchicine. In 187.5,
irreversible changes in striated muscles of frogs injected with a large
dose were first reported.-" Later "owcolchic ine" was showu to be
Pharmacology 181
e\u cinch toxic in I'rogs.^^ II the injected animals leapt within a few
minutes after tlic cliuo- took effect, their legs remained stretched and
exhibited fibriliarx twitchings. The rectus abdominis muscle of the
frog was also modified by colchicine, and contracture appeared after
repeated stimulation. ^'^ This was considered to be a "i.undsgaard
effect," identical whh thai induced by many sul)stances iniei lering with
glycolysis.
A detailed analysis of the sartorius muscle of frog treated with
especially purified preparations of colchicine has brought to light many
facts, which will be summarized here and which are illustrated by
Figure 7.3. The curarized muscle preparation was subjected to supra-
maximum electrical stimulation. Colchicine concentrations above 10"^
M produced a sustained increase in contractile force, which reached
more than 60 per cent with 1.6 X 1^^ " '^^- Larger doses resulted in
contracture and failure to respond to stinuilation. The increased con-
tractility was paralleled by an increased demand for oxygen, which
may be the double of the controls after two hours. Cafieine ajjpeared
to act synergically on this increase in oxidative processes, while meta-
bolic inhibitors such as azide, fluoroacetate, and malonate jMevented
this action of colchicine. The rate of glycolysis was increased two to
three times with colchicine concentrations of 6.4 X 1*^ " ^^^' ^s evi-
denced by the amount of lactate produced. Hydrolyzable, but not in-
organic, phosphorus was also increased. These facts do not ajjpear
to point towards a change in ATP utilization. They resemble closelv
those of caflfeine. The action of colchicine in increasing the available
energy is called "relative rarity," and thus one more curiotis effect of
the alkaloid appears to have been discovered. 2=*
7.5-5.- Smooth inii.scic (Did intestine. Conflicting re}>()its have
been puljlished on this subject. Ihe discovery tliai diarrhea is of cen-
tral origin may be the explanation. A strong increase in the intestinal
movements has been described in animals under ether anesthesia. •'■'
A similar effect has been found in frogs.-*' It was abolished by atropin.
Increased tonus and atitomatic movements have also been described
in sj)leen, uterus, and bronchioli. In the dog, the action on smooth
muscle has been said to l)e innnediate, resembling that of pilocarpin,
and to be antagonized by atropin.-' Quite different results have been
reached by other workers on isolated intestine.-'' ^^ The innnediate
effect was one of depression. The reactions towards adrenalin and
atropin were not altered.
The local action on the intestine is paralytic, and was found to be
related to the changes taking place in the mucosa, especially hemor-
rhage.-*^ In a cat, injections of colchicine (1 mg. in saline) were made
in liuated seements of the small intestine. A strong congestion and
hemorrhages arc to be seen locallv within 21 hours. With larger doses.
200
Q^
'2
lOO
o
500
400
300
Qco2
200
100
O
1.8 OO
OXIDATIVE ACTIVITY
lO-^M
4x10-3m
1.6x10-2m
6.4x10-^M
ANAEROBIC GLYCOLYSIS
4x10-^M
1.6x10-2m
64x10-2XM
1.200
Total
lactate
600
ACCUMULATION OF LACTATE /
.
/
/'
/
10-3 M
— 1. 1 1 1
/t
4x10-^M
n
l.6x10-^M
i 1 • 1
0123401 23401 23401 234
TIME (hrs.)
Pharmacology 183
up to 5 nig. colchicine, the hcnion hagcs arc apjiarcni after 8 hours.
This docs not appear lo be in any wax rehited to a release ol his-
tamine.'* which is one ol the toxic actions ol colchicine locally ajjplicd
on the skin.'"'
Recent work-'' indicates that colcliicine has no direct action on
the smooth muscle ol tlie intestine.
-.5-7; Hfuirl iind ciniilatioti. The heart is apparently in-
sensitive to colchicine, either in irogs or in manunals. The isolated
heart ol the frog may beat in a 1 per cent solution ol colchicine.-"
in mammals, the heart may go on contracting regularly for as long as
two hours after death by colchicine poisoning."^ As a consecpience,
blood pressine is onh depressed immediately before death.
There is no "cneral agreement about action on xasomotor nerves.
1 1 - ■
A\'hile having no action on the heart's sympathetic fibers, •''= colchicnie
has been found to increase the hypertensive action of epinephrine
in the rabbit under urethane anesthesia. i- In a dog under chloralose
anesthesia, a similar potentiating effect could be measuied 1)\ changes
in blood pressure and intestinal contraction.-^*^ This latter observation
has not been confirmed, and only the excitatory actions of ej)inephrine
on the \ascidar bed aj)ijear to be well pro\ed.-''
7.4: Disturbances Possibly Related to Mitotic Poisoning
Several remarkable effects of the alkaloid will be gathered under
this heading. Our purpose is, when possible, to relate pharmacological
effects to the histological changes resulting from spindle destruction.
However, this is ob\ iously far from being simple, and this paragraph
should only be considered as a tentative grouping of cellular reactions.
It will i)e noticed that the leukocytosis-promoting effect of colchicine,
which nearh led to tlie discovery of its action on mitosis,-"- -^ is
probablv only remotely linked to mitotic arrest. Its origin may be the
action of the drug on the central ner\ ous system. Howe\er, it is associ-
ated with some of the first descriptions of tissues altered by colchicine,
and has often been tjuoled as the origin of modern cytological work
in this field. For this reason, the problem will receive more attention
here.
j.^-i: A(ti())i on the blood. A substance that arrests h)r some
hours the mitoses taking place in the bone marrow and destro\s many
of them, would be expected to dej^ress blood lormation. Kxiensive
celhdar destruction has been lound in the bone marrow ol nmc.'"
Considerable congestion and a decrease in the number of nucleated
cells are the consequence of this destruction, in some expeiiments. 20
Fig. 7.3 — Action of colchicine on the isolated Sartorius muscle of the frog. Broken
lines: controls. The oxidative activity and anaerobic glycolysis are measured on cof-
feinafed muscle (1.9 x 10 'M). The lactate concentration is expressed in microgm gm
of muscle. (After Ferguson,"^ slightly modified)
784
Colchicine
per cent of all the nucleated cells of the marrow were arrested at
metaphase."" That this actually decreases the output of young red
blood cells was made clear by reticulocyte counts in the blood of
rabbits. Normal animals and rabbits with phenylhydrazine-induced
hemolytic anemia were utilized (Fig. 7.4 and 7.5) . A sharp but
transient iall in the percentage of reticulocytes is a convincing demon-
stration ol the inhibition of blood formation. --
35.000 7
I
30.000 6
I
25.000 5
20.000 _^
15.000
10.000
I
5.000
2.500
colchicine i
jr. 4-
,r.b.c. xlO^
euk.
^r«tic.%o r.b.c.
SO
45
40
35
30
25
20
15
10
5
1
1
•,\
-•— s
^ I
I I
I I
_
1
9 DAYS 10
F!g. 7.4 — Blood changes in the adult rabbit. Colchicine-leukocytosis and sharp fall of
the numbers of reticulocytes (immature red-blood cells). The importance of the mitotic
disturbances of the erythroblasts is evidenced by the slow return of the reticulocyte
number to normal, and by a slight anemia. (Unpublished, after P. Dustin"^)
On the other hand, Dixon and Maiden-' disco\ered that in rabbits
and dogs an injection of colchicine was followed by a considerable in-
crease in the number of circulating white blood cells (Figs. 7.6 and
7.7) . These authors, while reporting this curious effect, mentioned
that 12 hours after the injection, tlie bone marrow of rabbits apjK-ars
empty of most of its nucleated cells. Fhis is in agreement with
observations of bone-marrow aplasia, sometimes fatal, which have
since been recorded in the medical literature (cf. Chapter 10) .
The British authors-^ expressed their conclusions in a rather mis-
leading way, to cjuote: "evidence is conclusive that colchicine is a pow-
erfid stimulant to the bone-marrow, since it tmns out into the circu-
lation all the elements including the erythroblasts, and leaves the
"D
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786 Colchicine
25.000
r leukocytes /mm»
. . leukocytes (total)
/
* lymphocytes
/
/
20.000
^^ • • granuiocyies
\
/
^
\
/
\
\
/
\
^
/
\
\
/
\
\
/
15.000
/
■^
\
i
\
\
/ A
\
/
N
/
" .
/
^ ^
/
10.000
/
/ '
~~~^
/
~"-^
^ / /
<
/ / /^
\ ' : /
^
\ / / /
^
5.000
\\ / //
\\ ''■•'''/
•^ 1 1
1 1 1
hours: O
10
15
20
25
Fig. 7.6 — Modifications of the leukocyte count in the blooci of a rabbit injected 7.8
mg'kg colchicine, after 5.2 mg kg atropine sulfate. (After Dixon and Maiden'')
marrow relati\cl\ dcnuticd ol corpuscles."* 1 his is no true stimu-
lation, and the authors are more precise when in the same j^aper
they mention that the cells "are swept out . . . oi the bone-marrow . . .
into the circulation" (see Table 7.1) .f
It appears evident, however, that these authors did obser\e some
of the facts of mitotic arrest. But not being histologists, they tailed
to appreciate the exact significance of the facts. In !*)()(). Dixon-"
wrote:
A further effect of (okliicine is to excite karyokinesis. This action on the
mairow cannot be adequately determined at present, but it slioidd not l)e
regarded as specific to the leukocytes, but rather a type of the action wliich
goes on to a greater or less degree in other tissues of the body, but is
necessarily more easily in\estigated in the wanderin.s^ cells ol the blood. J
*W. Dixoii anci W. Maiden. "Colchitine, With .Special Reference to Its Mode of
.'^.ctlon and Effect on Bone- Mai row," Jour. Physiol., 37 (HK)S) . p. 7.'i.
fibid., p. 62.
J W. Dixon, .1 Minutiil of I'liin iiuKology (London: Arnold, UKKi) , \y. 9().
Pharmacology 187
30000 r
.leukocytes ( total )
-.lymphocytes
. granulocytes
25.000 _
20.000 _
15.000^
10.000
5.000
hours: o 10 20 30 40 50 60 70 80 90
Fig. 7.7— Modifications of the leukocyte count of a dog injected 0.34 mg kg colchicine.
(After Dixon and Maiden"')
In a later paper,-i j, j^ nRiitioncd tliai after repeated injections ol
colcliicine in rabbits, "sections of smears ol the bone-niarroAv . . .
exhibit proliferation . . . : j^lrtitifiil mitotic forms (tni orcasionaUy be
obsen'cd" [our italics].*
There can be no doubt toda\ that the sionificance ol these his-
tological changes was not grasped. These pui)li( ations on colchicine
pharniacologv were widely quoted, and Un 2t) vears text books
* ]our. Piiysiol.. ;?7(1908), p. 7(i.
788
Colchicine
mentioned that colchicine increased the numbers of Ieukoc\tcs. No-
body appears to have been interested enough to study more precisely
the bone-marrow changes, and it is only in 1934 that this T\as done.^'''
Colchicine-mitosis -was then disco\ered at once, lor in the laljoratory of
A. P. Dustin, Sr., problems of mitosis and mitotic stinudation had been
studied for many years, and the proper technicjues had been de\ eloped.
TABLE 7.1
Effect of Colchicine on Blood Count in Rabbit*
(Injection with 0.02 gm. colchicine made at 1 :05 p.m.)
(After Dixon and Maiden)
Cellular Types
Time of Blood Count
1 P.M.
Total leukocytes per cmm.
Granulocytes (%)
(pseudoeosinophils)
Eosinophils.
Mast cells .
Myelocytes . .
Monocytes . .
Lymphocytes .
Erythroblasts
(per cent leukocytes)
8850
37
1
10
45
1:30
3:00
4600
16
1
4
4
75
6700
50
1
7
3
7
32
5:00
9:15
9650
36
0.5
6.5
7.5
4.5
45
20,000
16
1
1
7
4
71
41
* Weight of rabbit, 1800 gm.
\\'hile the changes occurring in the blood-forming tissues were
then described, first in mannnals,^' then in amphiljia,!' the Dixon
and Maiden experiments were repeated in rabbits by another author,
unaware of the problems of mitotic regulation and poisoning. i'' The
effect of repeated small (from 1 to 5 mg.) daily injections was studied.
Immature white and red blood cells were foiuid in the blood stream.
The percentage of hemoglobin and the number of red blood cells
progressi\ely decreased. The marrow was ver)' cellular, ^vith leukoblas-
tic areas far in excess of the erythroblastic ones. The following con-
clusion was reached, to cjuote: "Colchicine, undoubtedly, stimulates
the formation of new cells in the marrow, and induces immature
cells ... to apj^ear in the peripheral blood, but . . .its destructi\e po^vers
outweigh its stimidant effect. "* Here again, the action on the mitotic
spindle was missed.^"
*C. R. Das Gupta, '" Ihe Action of Leiicopoietic Driii;s," Indian Jour. Med. Res.,
26 (1939) p. 997.
Pharmacology 189
Ai i^iesciit. no dear relation can be discovered between the in-
hil)ition ot mitotic growth and tlie colchicine-leukocytosis, and clearly
ncAV Avork is badlv needed in this field. Some facts are of interest
ho^\e\ er.
It has Ixen disco\ered that in leukemic patients and in normal
men a single dose of colchicine (2 mg.) may increase considerably the
ntunbei- of platelets. The bone-marrow megakaryocytes do not change
in number, but there is evidence of a greater |)latelet-building activity
by their c\ toplasm.-^"' ^^ In essential thrombopenia, where megakaryo-
cvtes are present but appear to be unable to produce platelets, this
effect of colchicine was not found. It is evidently not related to
mitosis. 1)11 1 may be similar to some other membrane changes induced
by the alkaloid (Chapter 4) .
Some recent work attempts to relate the bone-marrow changes and
leukoc\ tosis. This is often preceded by a transient period of leuko-
penia. Avhich appears to ha\e no causal influence on the leukocytosis.""
Bone-marroA\' studies in mice and rabbits all {(Mifirm the increase
of arrested metaphases, which is about 15-fold in the rabbit after 15
hours. The erythroblastic cells become progressively more numerous
than the granuloblastic; the increase is from 10-15 per cent to more
than 60 jjer cent in mice. The immature cells increase in proportion,
because the adult cells leave the marrow. There is no visible relation
between this phenomenon and the mitotic changes.'" However, re-
peated daih injections of 12 /^g. of colchicine increase considerably
the number of leukocytes in the blood of mice (more than 250,000 per
cmm.) . It has been suggested"" that these changes may be the con-
secjuence of a central nervous stinudation of the bone marrow. This
is in line with more recent pharmacological data (see above) and
merits close attention.
rhe following changes of blood cells after colchicine may be
mentioned here, though an explanation is not evident. Young rats,
aged 1 and 3 days, de\elop anemia, and a single injection decreases
the red blood cell diameter." ^ 1 hese two facts may bear some relation
to the decrease in the numbers of reticulocytes, which ha\e a larger
diameter than average red blood cells. An increase of "monocytoid"
leukocvtes in a case of fatal human poisoning'^-' parallels the ob-
servation of abnormally great nmnbers of histiocytes in guinea-pig
tissues after repeated injections.''" Several imjjortant data on blood
cells studied by culiine //? I'ltro with the hel}) of colchicine will be re-
ported in Chapter 9.
j-^-2: Ski)i. Iidir. <nui frtit/icrs. Colchicine arrests the mitoses in
the hair follicles in mannnals. Inhibition of haii' growth tan be seen
in rats in the \ icinit\ of colchicine injections, and loss of hair has been
found in human intoxication.^' In birds, similar changes may be ex-
190 Colchicine
pected to exist. l)ui tlic lollowino results are not necessarih the con-
sequence ot mitotic poisoning.
In hens, 1.5 nig/kg of colchicine causes death in 36 to 48 hours.
The symptoms are those already described: diarrhea, vasomotor dis-
tmbances, and nervous paralysis. Injections of 1.2 mg/kg are not fatal.
They cause a shedding of the feather buds in j^laces where the feathers
were remo\ed 15 days previously.- The feathers which grow next have
a white extiemity. Two similar injections, 7 and 14 days later, give
to these feathers a deejj black barring. The other feathers of the
animals darken. An analysis of the rate of growth of the feathers
demonstrates that colchicine acts immediately and that it modifies the
feather gro^\•th for 4(S hours. It was demonstrated lateri'^ that the
section of the spinal ner\es could bring about similar changes of color.
The authors are led to the conclusion that colchicine may act b\ affect-
ing the nerxous sxstem. a conclusion remarkably in line with later
research.--^
7.5: Nonspecific Toxic Changes
In considering the modifications of an organism \vhich lias been
injected or which has received by any route a substance as toxic as
colchicine, nonspecific changes must be taken into account.'''' These
may be difficult to sejjarate from effects of the drug itself, and only
future work will enable this aspect of the subject to become clearer.
For instance, while the influence of the pituitary-adrenal sxstem is
known to be great in all types of "stress," there are only txvc^ jjapers
on the action of colchicine in adrenalectomized animals. -^^- It was
demonstrated that an important ninnl)er of the nuclear pycnoses of
thymus and lymphoid tissue are only indirectly the consecjuencc of
mitotic jjoisoning. Pycnosis is much less ajij^arent in adrenalectomized
animals. ^- Xo work has been reported on the general effects of the
alkaloid after hypophysectomy. This should be important, consider-
ing the possibility of the jiituitary gland taking part in some central
nerxous stimulation of leukocytosis.
The facts assembled here may only have a distant relation to stress
and the alarm-reaction. It is known, howexer, from experimental
work-^* and from human pathology-"' that this reaction can appear
after colchicine. Also, sexeral of the changes reported have also been
obserxed after other mitotic poisons, chemicallx unrelated to col-
chicine.■•"■ It is logical to believe that they belong to the vast groiij) ol
nonspecific tissue changes."'*
7.5-/.- The "Jionnonr-mhnetic" actions of colchicine. The idea
of colchicine haxing some direct hormonal action xvas put lorxvard b)
botanical work."'- It led to some curious experiments which are im-
Pharmacology 191
nortiiiit lo consider ulun one knows how olten the alkaloid has been
nsed lor the detection ot hormone-stimulated growth (Clhaj^ter 9) .
Durini; the brecdino season, the fish Rhodeus itinanis displays
biilliant red "niijjtial colors," which are related to the expansion ol
chromatophores and to local hyperemia, 1 hese colors appear in
animals treated with male hormones. Colchicine alone has the same
effects. •^-- ■^•^ Nuptial colors are displayed bv fish subjected tor 10
minutes to a 1,5/1000 solution, or tor 35 minutes to a concentration of
0.75/1000, Colchicine and hormones add their effects, and the tidl
skin changes could l)c produced in 2 instead ot 20 hoins with hormone
alone. The oxygen consumption of the animals ^\■as also increased."'"
Howe\er. the "endocrine" mechanisms ot this action of colchicine may
be ciuestioned. In females of the same species, no increase in the size
of the o\ipositor was noted.'' The changes of the male fishes, where
\asomotor mechanisms play a great part, may have been either the
consecjuence of a nervous action, or of the general toxicity of colchicine.
The possibility of stimulating the action ot ijituitary hormones ])y
the alkaloid was strongly suggested by experiments on the ovulation of
isolated ovaries of Rana pifjicns. This was considerably accelerated,
both in \\hole animals and on isolated ovaries (Fig. 7.S) . The eggs
were tcrtili/aljle, biu none e\er dixided. Colchicine was believed to
bring a "true j:)otentiation" of the pituitary hormones controlling
ovulation.'- In the rai)i)it, however, no jjotentiation of the action of
pregnant mare's serum, containing gonadotropic hormones, on the
rate of ovulation could be detected.'*- Colchicine had no action on the
weight of oxaries of mice similarly injected, or on the seminal vesicles
of rats injected with testosterone,"'- Neither do results of experiments
on silk-worms-^-' justify the conclusion that colchicine is "hormone-
mimetic," 1 he onh ]K)ssil)ilit\ is that through nonspecific action,
this toxic drug could stimulate the secretion of hormones b\ endocrine
glands, in ]:)articidar the j)ituitary.
7.5-2; Liver and kidney damage. The mechanism ot these changes
is not clearly tmderstood, but it certainly plays an important part
in the general toxicity of the drug. I hough bile secretion has Ijcen
supposed to be increased, se\ere degenerative changes and necrosis
have been described in the livers of mice,""' especially after repeated
injections.^" In mice, the LD-,„ dose induces li\er cell steatosis in one
hour.''- Steatosis ot heart muscle cells and kidney tubules xvas also
noted. Female mice appear U) be more resistant to this damage than
males.
Mitoses ot li\cr cells ha\e Ijeen described in hiinian poisoning bv col-
chicine. 1 here are often arrested metaphases. c\en long after the drug
has been administered, a fact xvhich is explained in its slow excretion.^'
Three days after injection ot colchicine in mice, normal mitoses also
192
Colchicine
ZOO -
CD
0
O
LJJ
LL
O
a:
UJ
D
Z
150
100
— •— COLCHICINE
— O- CONTROL
HRS
Fig. 7.8 — Action of colchicine on the release of eggs from the ovary of the frog,
treated in vitro with pituitary powder. (After McPhail and Wilbur")
have been observed in liver cells. These will be discussed in the next
paragraph. After se\eral injections of colchicine, man\ arrested
mitoses are to be seen. The stages of recovery lead often to bizarre
nuclei which may resemble those of megakaryocytes. Cellular damage
may not be evident at all, and the cause of these divisions is not clear.
A hormonal stimulation related to stress and the adaptation svndrome
is possible. •''■'■
Pharmacology 193
In (hronic intoxication ol mice, after daily injections ol 12 to 15
fxg. lor 20 to 30 days a great niniiber ol liver nuclei are irregularly
shaped. More than 40 per cent ol these contain spherical bodies re-
sembling huge nucleoli. These are diffusely stained by acid dyes. They
persist 13 days after the end of the injections. No mitoses were seen,
a lailur surprising fact.-*^ It may be suggested that these intranuclear
bodies result from arrested mitoses, and represent sj)indle material,
similar to the hyaline globules and ))seudospindles (Chapter 3) .
Kidnev damage has been mentioned repeatedly, ^'*- •'- but has never
been described in detail. It should be borne in mind while considering
in Chapter 9 the use of colchicine in studies on the mitotic growth ol
kidney tubules.
-.5-5.- The "l/itc" mitoses. In many experiments on mitotic
])oisons. and in jKUticular after the injection of trypaflavine (acri-
Haxine) , normal mitoses coidd be found in unusual locations several
davs after the mitotic poisoning itself."' Colchicine is also effective,
and this i^ one of the observations that led to the belief that a true
mitotic stimulation existed. Actually, things are probably iar more
comi:)licated.
In adult mice,^' divisions could be observed in many locations:
liver cells and Kujiffer cells, endothelial and epithelial cells of the pan-
creas, sali\ar\ cells, histiocytes, and renal epithelial cells. Some of these
mav be abnormal, but normal mitoses are usually found in liver,
pancreas, kidney, and adrenals, from one to two days after an injection.
\vhile some of the divisions may be of a regenerative character, for
instance in liver and kidney, the important fact is that this is not a
phenomenon observed with colchicine alcjue. It obviously needs
further investigation, because very few authors appear to have taken
notice of it. In the light of all recent work on stress, the hypothesis
that pituitary-adrenal stimulation of cellular division has taken place
as a consec|uence of the general toxicity of colchicine, deserves notice.
7.5-7; ChemicaJ changes of the blood, llie idea of the alkaloid
producing a stress effect may liel[-) to explain some unrelated facts
mentioned in the ]jharmacological literature. The hyperglycemia
following the intra\enous injection of 1 gm/kg of glucose in the dog
is increased 10 to 12 hours after colchicine.^^ The lethal dose of the
drug in this species is 1 mg/kg. It decreases the blood sugar and also
the body temperatine.''^ The action on the glycemia does not appear
to be related to j)ancreatic islet activity. The LD-,,, dose has the same
effeci. In j^ancreatectomized dogs, (;n the contiary, the glycemia again
reaches its normal level within (i to 11 hours.*'"' The influence of the
adrenal cortical hormones has not been studied in these experiments.
Evidence has been presented that the adrenal j^lays an imjiortant part
in controlling the temperature fall observed after colchicine poisoning
(Fig. 7.2) .
194 Colchicine
Considerable changes oi blood-clotting time have also been re-
ported in rabbits injected with large doses of colchicine. This mav be
five times too long.-**^ It will be mentioned elsewhere that hemorrhage
has been considered an important factor in the action of the drug on
neoplastic growth.^ One author has found that the direct action of
colchicine, added /// 7'itr<j to oxalated blood plasma containing
thrombin, was to decrease the clotting time from 20 to 15 seconds.
Much remains to be learned about what happens when a complex
organism is imder the influence of such a poisonous chemical. It is
e\ident that much of the re\iewecl work is incomplete, that even the
exact chemical structure of the "colchicine" that is injected is not al-
ways known, and that we are confronted with a puzzle in which speci-
fic effects of colchicine are intermingled with general toxic reactions
involving hormonal stinudation and metal^olic changes. The im-
portance of all these ajjparently innelated facts emerges when one
considers colchicine's action in gout, which will be discussed later. It
is first necessary to ha\e some idea of the metabolic changes, if any, of
colchicine within the body. The study of this problem has recently
received some new light-
7.6: Metabolism of Colchicine
Forensic medicine demonstrated long ago that colchicine could Ijc
detected, apparently unchanged, in the bodies of patients who had
died of an overdose.-" Experiments on cold-blooded animals, which
can withstand considerable amounts of the alkaloid (Table 7.2) , dem-
onstrated that this remained unchanged. They also brought atten-
tion to the considerable \ariations in toxicity depending on body
temperatiue.-"- "^' ''^ For instance, a frog is able to withstand an in-
jection of 50 mg. of colchicine. For several days the chemical may be
detected unchanged in the urine. If such an animal, two to three
weeks after the injection, is warmed to 32°C., a temperature in itself
harmless, death super\enes in a few days. Progressive nervous paraly-
sis is evident, a typical manifestation of colchicine poisoning. Similar
facts are to be found in hibernating bats, which do not appear to be
affected by colchicine.''^ Once the animals are warmed and awake,
the characteristic nervous poisoning becomes \'isible.''^
After injection in dogs and cats, colchicine is chemically detected
in the feces and urine. Similarly in man, it is excreted unchanged in
the urine. However, only a fraction of the initial dose can be re-
covered.-^ This suggested to early workers that the alkaloid was
modified and metabolized in the animal and human bod\. The
striking effect of temperatme suggested that some of these changes
may only be possible in warm-blooded animals, or in artificially
warmed amphibians. Table 7.2 shows that the toxicity of colchicine is
Pharmacology 195
about I he same in mammals and frogs when the latter are kept at
30-32 °C.
It was also known that solutions of colchicine that had l)een left
standing- and haAC become brownish, probably as a result of oxidation,
become far more toxic to frogs, even at low temperatures.-^-^ In 1890, an
attempt was made to separate the toxic fraction of these oxidized
TABLE 7.2
Relative Toxicity of Colchicine
(After Fuehner^*)
Lethal Doses. After
Subcutaneous Injection
Species (gm /kg of body weight)
Rana esculenta, 15-20°C L200-2.000
Rana esculenta, 30-32=0 0 . 002-0 . 004
White mouse 0.003-0.010
Rabbit 0.003-0.005
Dog 0.001
Cat 0.0005-0.001
preparations, and a substance tentatively named "oxydicolchicine"
^\•as isolated. 1 his was believed to be made of iwo molecules of col-
chicine linked by an oxygen atom.^^ Artificial oxidation of colchicine
with ozone yielded a similar substance. A ftnther experiment at-
tempted to prove that the kidney was the organ in which colchicine
was oxidized to a more toxic product. About 330 mg. of amorphous
colchicine -were added to defibrinized hog's blood, and this was slowly
perfused through the hog's kidney. From this organ 42 mg. of a
brown substance were recovered. This, like "oxydicolchicine," dis-
played a rapid toxic action in the frog, where the symptoms were vis-
ible about one hoin- after the injection of 30 mg.
These experiments do not appear to ha\e been checked by modern
methods. This would be interesting now that the chemistry of the
alkaloid has made such great progress (cf. Chapter 6) . No std^stance
of the structure assigned to '"oxydicolchicine" has been described. On
the other hand, experiments ^viih mitotic poisoning are conflicting.
In mice, solutions of colchicine lose about 20 per cent of their cytologi-
cal activity after fi\e weeks of standing.^"
The fate of colchicine in the animal b(Kly has been si tidied by
modern methods, chemical, biological, and physical. A colorimetric
196 Colchicine
method ol titration was checked by measuring the mitosis-arresting
properties ot solutions either by injecting them in mice or by study-
ing their action on tissue cultures. ^^ Alter a single injection the blood
level in the adult rat decreased rapidh, and remained stable alter
a few minutes. The tissues contained less alkaloid than the blood.
Elimination was by the bile and intestine, and within a few hours,
10 to 25 per cent of the dose injected was to be loimd in the intestine
and its contents. Elimination by the urine only lasted a short time,
wdiile the blood concentration was at its highest. Within If) hours,
50 per cent appeared to have been eliminated. There was neither
evidence of a change into a more toxic substance, nor of any selective
tissular fixation. The cumulative toxicity of repeated injections is a
simple consequence of the slow excretion.
By growing Colchicujn in an atmosphere containing radioactive
carbon, C'^, in the form of CO,, a biolooical svnthesis of radio-
— o /
active colchicine has been made possible.'^*' lire fate of this in
the body of mice has been tested. One fact of imjjortance is that four
hours after the injection, no more colchicine coidd be detected in the
central nervous system, muscle, heart, or blood. Most of the radio-
acti\e alkaloid ^vas detected in the kidney, the sjjleen, and the intestine.
Neoplastic tissue (sarcoma 180) did not contain more colchicine than
the liver. An unexplained fact is that while the spleens of control
animals were a site of active fixation, no more colchicine could be
found in this location in tumor-bearing mice.^ These observations
appear to demonstrate that the alkaloid brings about quite rapidly
some change in the brain without becoming fixed in this tissue.^
Evidence will be presented elsewhere (Cliapter 9) that colchicine may
be retained for some time in tissues of cold-blooded animals {Xenopus
tadpoles) .
Finthei" research is also necessary in this field, for there appears
to be some contradiction between the stability of colchicine as evi-
denced from old and modern work, and the l)iological activity and
specificity of this molecule. These problems will be discussed in the last
chapter of this book.
7.7: The Treatment of Gout
Logically, colchicine pharmacology should be an introduction to
its use in medicine and should enable us to imderstand why this
plant alkaloid is elfective in treating a disease of inic-acid metabolism.
However, as will be noticed, actual data on ]jharmacology are of
small helj) in understanding the curative properties of Colchicum.
Many complicated side-effects have been described, many strange
properties investigated, but modern medicine is ajjparently not much
closer th:in the Ebers Papyrus in explaining the medical use of this
plant.
Pharmacology 197
Gout, which was still called a forgotten disease in 191()/''^ has re-
gained nnich medical attention. New methods of treatment and
neA\- methods of study have brought this change. Also, the frequency
of cases of gout ma\ liaAC increased in some coimtries. The principal
and painful lesion that affects the joints of gouty patients results
from dcjiosits of uric acid. This chemical was believed to be mainly
related to nucleoprotein metabolism. Studies ^\■ith radioactive uric
acid, marked with N^^ have helped to understand the origin of the
so-called "miscible pool" of uric acid, which is considerably increased
in some cases of gout. This has been demonstrated to originate from
many pathways of metabolism. All proteins, carbon dioxide, anmionia,
glycine, serine, and carbohydrates may be used as building blocks
for uric acid. Methods for studying the changes of the "miscible pool"
of uric acid have been developed.-''- -■'■ '^-
This has been mainly the consequence of the discovery that
steroid hormones like cortisone,'' and the adrenotropic hormone of
the jMtuitary (ACTH) may play an important part in gout and may
possibly be used for its treatment.-^, ^s. 29 Now, the nonspecific toxic
reactions of colchicine poisonings have been described. These would
result in an increased secretion of ACTH and cortisone.'"'-'- ■''^ Could
colchicine possibly act in a nonsjjecific way in this disease?
The considerable amount of work, mainly clinical, which has
been published these last years on this subject can only be rapidly
reviewed here.-^^- •*-^- ^'•- *'''• ^*' '^^- '"' Current practice of handling gouty
patients with colchicine has recently been summarized.-^
The doses which elicit in animals the alarm-reaction and ACTH
secretion are far larger than those effective in human therapeutics. The
Thorn test of adrenal stimulation demostrates effectivel) that in
patients with diseases other than gout, therapeutic doses of colchicine
do not stimulate the pituitary and the adrenal. The urinary elimi-
nation of 17-cetosteroids is not modified either.^''- ^•' A positive 1 horn
test is demonstrated by a rapid fall in the numbers of eosinophil
leukocytes in the blood. In one case only was this positive, the eosino-
phils falling to 53/cmm. and later rising to the normal number of
269. This, however, was in a man Avho had taken 24 mg. of colchicine
in 24 hours, that is to say more than six times the usual dose.
On the other hand, while ACTH and cortisone may be effective in
the treatment of gout, they have by no means taken the place of col-
chicine. This is now used either at the same time or after the injections
of hormones, and it is recognized that its action is unrelated to the
alarm-reaction, and to ])ut it shortly, "entirch unknown." -•''
Some workers believe that the acute crisis of gout, the origin of
which is by no means clear, is related to allergy. Colchicine has been
found to decrease the intensity of the anaphylactic shock in guinea
pigs injected with ovalbuniine.' In j)atients suffering from diverse types
798 Colchicine
of allergy, such as serum sickness, Quinke's edema, or urticaria, col-
chicine has been used with results comparable to those of the anti-
histamine drugs. •^^- '^''^ Colchicine, however, does not antagonize his-
tamine, and this new use in thcrajicutics now presents finthcr im-
sohed problems.
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200 Colchicine
48. LoicQ, R. Recherches sur les effets de la colcliicine sui la coagulabilitc du
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Pharmacology 201
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CHAPTER 8
Embryonic Growth, in Animals
8.1: Action on Gonads and Early Development
Eggs have often proved to be an excellent material for colchicine
research, and in previous chapters results of work on various types
of eggs have been mentioned. Nuclear structure is modified in Tiibi-
jex,--- -3 the nuclear sap becomes granular in the Anodonta e^g,^^
spindle changes are most evident in Arbaria^- '^'^ disturbances of
cleavage are noted in Spliaerechinus}' while curious surface changes
have been described in both Tiibifex-^^ and Arbacia.-*^ The size of egg
cells, their conspicuous spindle, and the possible induction of poly-
ploidy were factors making them useful in some of the early colchicine
research. It is remarkable, howe\er, that the first paper on this sub-
ject was written by two botanists.-'''
We shall consider here only facts which have not been observed
in ordinary cells, and which are related to the special physiology and
cytology of eggs. Since there are few papers on modifications of
spermatogenesis, it was thought natural to describe some of the re-
sults which may prove important for the possible induction of poly-
ploidy in animals. This last problem will be discussed more
thoroughly in (Chapter 16. On the other hand, the disturbances of
embryonic growth related to mitotic poisoning result in some quite
peculiar malformations which will be considered later in this chapter.
S.i-i: The cleavao^e of eggs. All work in this field points towards
the complexity of colchicine actions, which are not only related to
the stage of maturation or growth reached Ijy the eggs or the young
embryo, but also to the concentrations of alkaloid used. For instance,
in some of the early work on the egg of Rana pipiens the classification
of cellular changes proved to be very difficult because of great dif-
ferences of sensitivity. 1^ A 1:1000 solution suppressed all cleavage and
led to cellular disintegration; at 1:10,000, colchicine did not disturb
the first cleavage, but the next ones were irregular and the grooves
between the cells were only shallow; at 1:100,000, three cleavages
[ 202 ]
Embryonic Growth in Animals 203
proceed normally, but in many eggs the grooves faded away later.
Even when the concentration was only 1:1,000,000 and when some
ajijKirently normal embryos grew, abnormal cleavages were visible,
and on the third day all the embryos were found dead. It was evi-
dent that even when nuclear mitosis proceeded normally, cleavage
could be inhibited. Gastrulation was made impossible, the eggs as-
suming a meroblastic type of growth.
It was soon discovered that in Arbacia the sensitivity of the eggs
decreased rapidly after fecundation;^ 40 minutes later, from 90 to
100 per cent of normal cleavages could be observed. In the sea
urchin Paracentrotus, before fecundation, the eggs may live only in
a 1:200,000 solution. Later, cleavage is quite abnormal. If colchicine
is apjjlied at fecundation, a 1:60,000 solution does no more than dis-
tiab gastrulation. A temperature effect was also observed. Inhibition
of growth was nearly complete if colchicine had been allowed to act
at 25°C., even if the eggs were kept at lower temperatures later. On
the contrary, colchicine at 15°C. permitted growth to the morula
stage, or, if the eggs were placed at 25°C. after colchicine, as far as
the 16-celled stage. This temperature effect was tentatively related
to permeability changes. •'^o
1 he peculiar behavior of egg cells and the first stages of develop-
ment of amphibia have been the subject of a thorough analysis, re-
lated in many papers of the French author, Sentein.^*, 35 i^[]^q other
workers, he founcl that cleavage disturbances were not closely related
to mitotic disturbances; precocious cleavage could, in some eggs, lead
to anucleate blastomeres. The complexities of the action of colchicine
are revealed by the various cytological anomalies described: poly-
ploidy, plurinucleation, asymmetrical development, chromatin bridges
between nuclei, pycnosis, and pluricentric mitoses. The last were
found during recovery and are comparable to the multiple stars de-
scribed in Chapter 3.
The variable reactions during development were analyzed in
Tritunis, Pleurodeles, Bnfo, Rcuia, and Anihlysloma.^^ After gastru-
lation, typical arrested mitoses of the star type are the rule, Avith
clumped chromosomes that are progressively destroyed. In the earlier
stages, however, nuclear changes are quite different. Rather concen-
trated, 1:500 and 1:1000, solutions of colchicine were used. How-
ever, the cytological changes were always delayed, as observed by the
other authors mentioned above. ^' i"^' ^^ First of all, cleavage is in-
hibited, the nucleus completing its division. The result of this is the
frequent observation of binucleate blastomeres. The spindle may be
completely destroyed; large, probably j)olyj)loid nuclei are found
later. However, the normal niunber of chromosomes is most often
maintained because the spindle, even in these high concentrations of
204 Colchicine
colchicine, recovers. This leads often to pluripolar spindles, which
are considered to be an important factor counteracting the poly-
ploidizing action of the alkaloid. Recovery is incomplete, and chromo-
some coiuits demonstrated a great variability from cell to cell.^*^
Another peculiarity of the spindle of amphibian eggs is its asym-
metrical reactions towards the depolarization effects of colchicine.
The hypothesis has been put forward that this may be related to a
differential sensitivity of the centrosomes, whether of paternal or
maternal origin. ^^
Similar disturbances of development have been described in Rana
agilis^ and Bii^o vulgaris, where an apparent decrease of cellular res-
piration was observed.^" The exact relation between mitotic changes
and the abnormalities of later development, which will be related in
the next section, are most difficult to understand. A detailed de-
scription of the action of colchicine on the cleavage and early de-
velopment stages of the fish Oryzias latipes cannot possibly be svmi-
marized here, but should be consulted by embryologists interested in
chemically induced abnormal growth. ^9
The changes described in the egg of Tiibifex, an invertebrate, are
remarkably similar to those reported in vertebrates. In 1:30,000
solutions of colchicine some eggs are able to divide twice. One of the
main effects is on cytoplasmic limits, which may disappear after hav-
ing been normally formed at telophase.^^
A relative resistance towards colchicine, changes in sensitivity re-
lated to developmental stages, the absence of polyploidy in the em-
bryos, and peculiar actions on cleavage are the main facts which at
this time emerge from a great amount of observations.-^- ^^ There is
no doubt that cytologists and embryologists have many more prob-
lems to solve and probably new types of colchicine effects to discover.
8.1-2: Male gametes. There are surprisingly few data available
on the action of colchicine on spermatogenesis. In mice, aged 22
days, some arrested mitoses (or meioses?) have been reported in early
work.-^ In adult animals, colchicine brought evidence of nuclear and
cytological destruction. Arrested mitoses of spermatogonia in rats in-
jected with inore than 1.4 mg/kg of the drug have been described.
The spermatocytes did not appear to be altered, akhough 24 hours
after the injection the nixmber of metaphases Avas somewhat in-
creased.•''-
Personal observations of the junior author (unpublished) are that
in the testes of mice injected 1.25 mg/kg, most of the spermatocytes
have no more spindle 24 hours later. Spermatogonia appear to be
unaltered, and the stages of meiosis are normal, as long as no spindle
activity is required. Many spermatids with vacuolated nuclei may be
observed, but this ]:)henomenon is a consequence of the general
Embryonic Growth in Animals 205
toxicity of colchicine, and has been described under various experi-
mental conditions and with other mitotic poisons. "^^ With less toxic
colchicine derivatives, spindle inactivation is apparent in a few horns.
Depending on the doses injected, recovery is possible, or considerable
cellular damage may be found. Binucleated spermatids may result
from the spermatogonia! mitoses during recovery assuming the "dis-
tributed" type with two nearly equal groups of chromosomes (cf.
Chapter 2) .
In fowls also, colchicine may induce severe degenerative changes
in testicular cells. These are followed by regeneration seven days
later.17
No polyploid spermatozoa have been reported in vertebrates. On
the contrary, in the insect Triatomn infestans (order: Hemiptera) ,
colchicine not only inhibits the spindle function, but as a consequence,
modifies considerably the size of the spermatids (Fig. 8.1) . This is
observed after nine days, wlien all spermatogenetic cells have dis-
appeared. The simple numerical relations between nuclear sizes are
a strong evidence in favor of polyploidy, although the exact inter-
pretation of these facts awaits further research. ^^
Control
Colchicine 9 days
420 842 1677
3073
Fig. 8.1 — Action of a prolonged treatment by colchicine on the nuclear diameters of
the spermatids, expressed in conventional units, in Triatoma infestans. Several categories
of polyploid nuclei with diameters in the relation 2,4,8,16. (After Schreiber and Pelle-
grino'"')
206 Colchicine
In Chapter 16, a technique of inducing polyploidy in vertebrates
will be discussed. This involves using sperm treated with colchicine.
It should be mentioned here that the alkaloid has not been reported
to affect adult spermatozoa. i-- 1''
8.2: Colchicine-induced Malformations
The artificial production of embryonic monstrosities has received
a great impetus from the work of Ancel and Lallemand.^' -• -^ This
was initiated around 1937, and, together with the use of other chemi-
cals, has opened a new field in developmental research. A detailed
survey of this is to be found in Ancel's recent book. La Chimiotera-
togenese.^
Through a small opening in a chick's egg, a minute quantity of
a solution of colchicine in saline is introduced. The embryo is ob-
served, to make sure that no abnormalities exist at the start of the
experiment. The opening is closed and the egg hatched in an incu-
bator.
One of the most striking results was the production of a malforma-
tion which had been described in calves by Gurtl (1832) and called
schistosomus reflexus. This is a peculiar type of celosomy, that is, a
total hernia of all the abdominal and thoracic viscera, residting from
an absence of the anterior body wall. Lesbre, in 1927, used the term
stropliosomy , or body-turned-inside-out, for the rachis and tail are
strongly bent backwards, the hind limbs located close to the back of
the head (Fig. 8.2) . Such a malformation had never been seen in
chicks, and naturally aroused great interest in colchicine. Further
testing of more than fifty substances, several of which induced various
abnormalities of development, demonstrated that only ricine and
abrine could initiate stropliosomy.
Figure 8.3 shows the difference between the formation of celosomy,
which is much more frequent, and stropliosomy; the posterior bend-
ing of the caudal part of the spine plays a great part in the second
tyj)e of anomaly. The colchicine treatment of the eggs must be done
within a quite definite period. The optimal period is after 48 hours
of incubation; before this time, or after 68 hours, it is ineffective.
Only 5 hours after the introduction of colchicine into the shell,
the embryo demonstrates an exaggerated forward flexion of the infra-
cardiac region. Many of the embryos die at this moment. Some also
display a dorsal flexion of the caudal extremity of the rachis; these
are the ones which will eventually become strophosomic. This mal-
formation does not distmb the formation of the embryonic organs,
and the chicks are capable of living nearly until hatching, the longest
observed duration being 19 days. A similar condition had been
>-^\\\N-,
Fig. 8.2 — Strophosomy induced by colchicine in the chick. A. Normal chick at 12 days
of incubation. B. Strophosome at the same age. There is a total hernia of all viscera,
no abdominal wall, and a backwards flexion of the hind limbs. C. Another stropho-
somic chick, after 13 days incubation. The animal is seen from the rear, the herniated
viscera hang underneath, the legs here folded on the back. (After Lallemand"')
208
Colchicine
Fig. 8.3 — Origin of strophosomy in chicks. Injection of colchicine in the eggs at 48 hours
of incubation. A. Control at the time of injection. B. Control, incubated 72 hours. C,
D. Colchicine-treated embryos, incubated 72 hours. These are future strophosomes, as
indicated by the backward flexion of the tail. E, F. These chicks, similarly treated, will
only develop celosomy. The tail is bent forward. (After Lallemond'')
known to exist in calves, which may be born strophosomic after an
intra-uterine growth of normal duration.
The caudal bending of the embryo appears quite important, and
it is to be noted that pycnotic nuclei arising from arrested meta-
phases are to be found in this region, mainly in the nervous system
and the smroiuiding tissues. Neither the chorda nor tlie intestinal
epithelium shows evidence of cellular destruction.
Embryonic Growth in Animals 209
The problem of the determination of strophosomy has been fur-
ther studied by local applications of colchicine in agar strips.^^^ In
embryos with 25-28 somites, the region between the omphalomesen-
teric vessels and the hind limb is the most sensitive in regard to this
malformation. Absence of tail and hypophalangism and absence of
tail were also observed; these phenomena led to a study of colchicine
on the expression of the anomaly, polydactyly.^'^ In other animals,
colchicine is also a teratogenic agent,* but the changes mentioned are
of very different types, ranging from exogastrulation" to variations in
pigmentation, cyclopean eyes, abnormal blood formation, and dis-
turbances of body flexures.^'* In the frog, many of the reported
anomalies^-- ^'^ could also be initiated by X-rays, a fact strongly
suggesting their relation to mitotic disturbances.
One other result is worth mentioning. Local application of a
1:7000 solution of colchicine on the posterior limb of Xenopus larvae
resulted in a decrease in the number of toes.^ With increasing effects
all but the fourth toe disappeared during development. This is
paralleled by no other type of regressive evolution of toes in verte-
brates.
8.3: A Tool for the Study of Embryonic Growth
The use of colchicine for the detection of zones of maximal
growth and of growth stimulation or inhibition will be discussed at
length in Chapter 9. The "colchicine method" is fundamentally
based on the observed increase in metaphases, arrested because of
the absence of spindle, in growing tissues. Mitotic multiplication of
cells is made more visible. Some of the difficulties of this method in
adult animals will be discussed in Chapter 9. It is evident from all
that has been written in this chapter, that in embryonic growth the
complexity of the changes brought about by colchicine is consider-
able. Not only does the alkaloid inhibit mitoses, it may also com-
pletely alter the normal course of growth. Only a few experiments
yield facts that are simple to interpret.
For instance, in chick embryos treated at the forty-second hour
of development with dilute solutions of colchicine, there could be
observed, 24 hours later, an "overjiroduction of cells." -'^ The amount
of neural tissue appeared to be increased, and several neural folds
were to be seen, even in animals where the number of arrested mitoses
did not appear to be great. These facts were considered as good
evidence of mitotic stimulation and increased neuralization, that is
to say, a colchicine-induced malformation. Chicks with spina bifida
have been found in some experiments.^ The number of mitoses
seemed considerable to the author who observed for the first time
these neural changes, but no accurate quantitative counting w^as done,
nor, in fact, could have been properly done because of the malforma-
270 Colchicine
tion itself. It has also been suggested that the apparent increase in
neural tissue was merely the consequence of abnormal cellular migra-
tions, not of modified mitotic activity. ^'^
Analysis of patterns of embryonic growth is made difficult by
many facts. One is the varying sensitivity of tissues and stages of
development. In Molge palmata Schneid., the zones of highest mitotic
activity are the most sensitive to colchicine;^° in other regions, the
same concentration may yet enable mitosis to recover and to proceed
to telophase through star and incomj^lete star metaphase. In Dis-
coglossus pictus Orth., some periods of growth are very sensitive to
the mitotic arresting activity of colchicine. The fifth day, correspond-
ing to the "primary metamorphosis," when swimming is initiated, is
one of these periods. In Discoglossis, Rcma, and Xenopus. the meta-
morphosis is a period of increased sensitivity. The regions of the
embryos where the mitoses are the most numerous are, rather natu-
rally, the most rapidly altered by colchicine. Instances are the nervous
system, the olfactory bud, and the germinative region of the eyes.^^
These carefully studied facts do not leave much to say about
papers which attempted to detect zones of growth by colchicine,
especially in amphibia, i"^- -^ for the complexities of the problem were
not properly understood at the time of their publication. Some facts
emerge, however, from the literature on this subject and are worth
mentioning, for they may be starting points for further work. In
young mice, colchicine demonstrated that liver and pancreatic cells
cease to divide at about 20 days after birth;^! the mechanism which
prevents any further division, except in regeneration (Chapter 9) ,
is unknown. In mice also, ganglionic nerve cells have been found, by
the use of colchicine, to divide until three weeks after birth.-" Colchi-
cine has also been used to bring about the death of the litter of preg-
nant mice,!^ and to induce the formation of tetra- and octoploid cells
in embryos of the fish Coregonus when the eggs had been treated
three hours with a 0.5 per cent solution. Hastening of the meta-
morphosis of Rana fiisca tadpoles is also reported. '^^
The publications which have been reviewed in the last paragraph
would seem to indicate that colchicine is of little, if any, use in the
study of embryonic growth. However, it must be recalled that most
of these results have been published during the early phases of colchi-
cine research, before the proper techniques could have been designed.
Two recent papers show that important facts can be made clear by
using colchicine as a tool in embryos.^
In the first one, the jiroblem was to assess the comparative mitotic
activities of the embryonic megaloblasts (young red blood cells) of
the chick embryo, and of the megaloblasts of human Addison-Biermer
anemia (cf. C^hapter 9) . These cells resemble closely the embryonic
ones, though their existence is an evidence of pathological growth
Embryonic Growth in Animals 217
related to vitamin B^o, or folic acid, deficiency. A dose of 0.015 mg.
of colchicine in saline solution ^vas found to arrest all mitoses in the
voung chick embryo. The number of mitoses found after four and
eight hours was counted. This gives a precise idea of the proliferative
activity of these cells. In chicks at the sixtieth hour of growth, eight
liours after colchicine, the number of megaloblastic mitoses is in-
creased more than tenfold; while in controls. 38.6 cells per thousand
are in division; in treated chicks, eight hours after colchicine, the fig-
ure reaches 457.9. This increase is markedly greater than that found in
the bone marrow of Biermer anemia patients. However, the technique
being different, the comparison is not quite valid. What is more in-
teresting from the viewpoint of embryological growth, is that the
mcgaloblasts are demonstrated to divide more than the undiffer-
entiated connective cells from which they originate.
A detailed study of the relation between differentiation of the
red blood cells and cell division in the chick embryo at different
stages of growth has clearly indicated a decrease in mitotic activity as
soon as hemoglobin is synthesized. Colchicine has been a remarkable
tool for the precise study of this problem. ^ No doubt, it will not be
the last contribution in a field open to many types of investigation
(cf. Chapter 9) .
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9. CoEOMBO, G. L'azione della colchicina sulla sviluppo embrionalc di Rana
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212 Colchicine
applications of colchicine on Leghorn and pohdactylous chick eml)iyos. Jour.
Exp. Zool. 101:339-50. 1946.
11. Haas. H. T. A. Uber die Beeinflussung des Zellkcrns durch Pharmaka. Arch.
Exp. Path. 197:284-91. 1941.
12. Haggqvist, G., and Bane, A. Chemical induction of polyploid breeds of mam-
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Handl. IV. Ser. 3:1-14. 1951.
13. Hall. T. S. Abnormalities of amphibian development following exposure of
sperm to colchicine. Proc. Soc. E\p. Biol, and Med. 62:193-95. 1946.
14. Havas, L. J. L'action de la colchicine administrce seule ou en combinaison
avec des hormones sur la croissance et sur le dc\eloppement des embryons de la
grenouille. Magyar Biol. Inst. Kozl. 1942-43.
15 Hutchinson, C. The earlv development of the nervous system of Amblystoma
studied by the colchicine techniciuc. I. Medullaiv plate changes. Anat. Rec.
70:Suppl. 3:39. 1938.
16 Tahn, U. Induktion verschiedener Polyploidiegiade bei Rami iniilxnaria mit
Hilfe von Kolchizin und Sulfanilamid. Z. Mikr.-anat. Forsch. 58:36-99. 1952.
17 Jenkins W R., and Bohren, B. B. The effect of colchicine on the seminiferous
tubules of fowl testis. Poultry Sci. 28:650-52. 1949.
18. Keppel, D., and Dawson, A. Effects of colchicine on the cleavage of the frogs
egg {Rana pipiens) . Biol. Bull. 76:153-61. 1939.
19. Kerr, T. Mitotic activity in the female mouse pituitary. Jour. Exp. Biol.
20:74-78. 1943.
20. Kjellgren, K. Studien-iiber die Entuicklung dcr Neuronen nach der Geburt.
Acta Psych, et Neurol. Suppl. 29. 1944.
'^1 Lallemand, S. Realisation experimentale, a laide de la colchicine de poulets
strophosomes. C. R. Acad. Sci. Paris. 207:1446-47. 1938. La strophosomie
chez I'ambrvon de poulet, reaction teratogene de la colchicine. Arch. Anat.
Hist. Embryol. 28:217-53. 1939. Action de la colchicine sur lembryon de
poulet a divers stades du developpement. C. R. Acad. Sci. Paris. 208:1048-49.
1939.
22. Lehmann, F. E. Der Kernapparat tierischer Zellen und seine Erforschung mit
Hilfe von Antimitolica. Schweiz. Zentralbl. .\llg. Path. 14:487-508. 1951.
23. , AND Hadorn, H. Vergleichende Wirkungsanalyse von zwei antimito-
tischen Stoffen, Colchicin und Benzoquinon, am lubifex-Ei. Helv. Physiol,
et Pharm. Acta. 4:11-42. 1946.
24. LiTS, F. {see Ref. No. 61, Chap. 2) .
25. Mills, K. O. Variations in the rate of mitosis in nomial and colchicine-
treated tadpoles of Rana pipiens and Ainblystowa jefjcrsoniamim. Jour.
Morph. 64:89-113. 1939.
26. Monrov, a., and Montalent, G. Cvclic variations of the submicroscopic struc-
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Nature. 158:239. 1946.
27. Nebel, B. R., and Ruitle, M. L. The cvtological and genetical significance of
colchicine. Jour. Hered. 29:3-9. 1938.
28. Paff. G. The action of colchicine upon 48-hour chick embryo. Amer. Jour.
Anat. 64:331-10. 1939.
29. Pincus. G., and Waddington, C. H. Tlie effect of mitosis-inhibitmg treatments
on normally fertilized pre-cleavage rabbit eggs. The comparative behaviour of
mammalian eggs in t'ii'o and in vitro. Jour. Hered. 30:514-18. 1939.
30. PoussEL, H. Influence dc la colchicine sur le developpement de I'oeuf doursin:
remarques sur quelques conditions daction. C. R. .Soc. Biol. Paris. 136:240-42.
1942.
31. RiES, E. Wann erlischt die mitotische Vermehrungsfahigkeit der Gewebe.''
Z. Mikr.-anat. Forsch. 43:558-66. 1938.
32. Roosen-Rlnge, E. C. Quantitative studies on spermatogenesis in the albino
rat. II. The duration of spermatogenesis and some ertects of colchicine. Amer.
Jour. Anat. 88:163-76. 1951.
Embryonic Growth in Animals 213
33. .SfiiRFiBFR, G., ANr) PiLLEGRiNO, J. Aiiulisc citologica e carionictrica da acao da
colchicina sobre a espermatogenese dos Hemipteros. Mem. Inst. Oswaldo Cruz.
Rio de Janeiro. 49:513-42. 1951.
34. Sentein. p. Mode d'action de la colchicine sur la carvocinece de Molge pahnata
Schneid. C. R. Soc. Biol. Paris. 137:13.3-34. 1943. .Action de la colchicine sur
les mitoses de maturation chez le triton. C. R. .Soc. Biol. Paris. 137:132-33.
1943. Relation entre la mito-inhibition et les troui)les de I'ontogenese chez
les oeufs et les larves d'anoures et d"inodeles. Bull. Acad. Sci. Montpellier.
7(i:51-53. 1946. Action de la colchicine et de I'hydrate de chloral sur I'oeuf de
Trilitriis helvetirus L. en de\elo])penient. Acta .\nat. 4:256-67. 1947. Action
de substances mitoinhibitrices sur la segmentation et la gastrulation de I'oeuf
de triton. C. R. .Soc. Biol. Paris. 142:208-10. 1948. Action comparee des sub-
stances antimitoticjues sur la segmentation et la gastrulation chez les anoures.
C. R. Soc. Biol. Paris. 142:206-8. 1948. .Analyse du mccanisme de la caryo-
cincse par Taction de substances antimitoticjues sur I'oeuf en segmentation,
[our. Ph\siol. Paris. 41:269-70. 1949. Xomelles obscr\ations sur Taction des
substances antimitotiques: effets de la colchicine, du chloral et du carbamate
d'ethyle (urethane) sur la segmentation de I'oeuf d'amphibien. C. R. .Assoc,
des Anat. 35:355-63. 1949. Structiuc des no\au\ geants polvmorphes obtenus
par transformation telophasicjue des chromosomes dans les cineses bloquees de
i'oeuf. Rapports entre polyploidic, amitose et pluripolaritc. C. R. Assoc, des
.Anat. 36:613-20. 1950. Sur les deviations de Taxe mitotique au cours de la
segmentation de Toeuf traite par la colchicine, et leur signification. C. R. Soc.
Biol. Paris. 145:87-89. 1951. Les transformations de Tappareil achromatique
et des chromosomes dans les mitoses normales et les mitoses blocjuces de Toeuf
en segmentation. Arch. Anat. Strasbourg. 34:377-94. 1952.
35. . Mise en e\idence des zones germinatives de Toeil par le blocage des
mitoses chez les larves d'amphibiens. C. R. Soc. Biol. Paris. 140:185-87. 1945.
Action experiinentale de la colchicine sur la mitose chez quelques batraciens
anoures a Tctat adulte et au cours du developpement. Mont|)ellier Med.
21-22:494-95. 1942. Les differences de sensibilite a Taction de la colchicine
chez les larves de batraciens. Bull. .Acad. Sci. Montpellier. 76:61-62. 1946.
Sur Taction comparee de la colchicine et du chloral sur les cellules epitheliales
et nerveuses des lar\es d'amphibiens. C. R. .Assoc, des .Anat. 34:440-51. 1947.
36. S\ARnsoN. ... Chromosomes studies on Salmonidae. L Haeggstroms (Stock-
holm) . 1945.
37. I'rbam. E. Lassunzione di ossigeno in uova di anfibi trattate con colchicina.
Boll. ,Soc. Ital. Biol. Sper. 23:637. 1947.
38. \'ax Ros. G. Recherches experimentales sin^ la \acuolisation nucleaiie des
spermatides de la souris. C. R. Soc. Biol. Paris. 147:547. 1953.
39. A>'aterman', a. J. Effect of colchicine on the de\elopment of the fish embrvo,
Oryzias latipes. Biol. Bull. 78:29-34. 1940.
40. Welds, C. M., and Wimsatt, W. A. The effect of colchicine on earlv cleavage
oi: mouse ova. Anat. Rec. 93:363-76. 1945.
41. W'u.BUR. K. M. Effect of colchicine on the viscositv of the Arbacia egg. Proc.
•Soc. Exp. Biol. 45:696-700. 1940.
42. WoLSKY, A. Untersuchungen iiber die Wirkung des Colchicins bei .Amphibien.
-Arb. Ung. Biol. Forsch. Inst. (Tihanv) . 12:352-58. 1940.
43. . .AND .Aleodeatoris, L IL Histologische Befunde an Colchicinbe-
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1941.
44. AVoKER, H. Phasenspezifische AV'irkung des Colchicins auf die ersten Furchung-
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Eies. Rev. Suis.se Zool. 51:109-71. 1944.
45. Woodward, T. M., and Estes, S. B. The mitotic index in the neural tube of
the 48-hoiu- chick as determined bv the use of colchicine. Anat. Rec. 84:501.
1942. Effect of colchicine on mitosis in the neural tube of the 48-hour chick
embrvo. .Anat. Rec. 90:51-54. 1944.
CHAPTER 9
Experimental Growth, in Animals
9.1: Endocrinological Research
One ot the most striking features of colciiicine, wliettier injected
into animais or acting upon tissue cultures,--^ is ttie accumuiation of
mitoses arrested at metaphase (Fig. 9.1) . Tiiis is a consequence of tlie
absence of spindle (cf. Cliapter 3) . The increase in the number of
mitotic cells was soon understood to be most useful for the analysis
of growth by cellular multiplication. Several lines of research were
started in the years 1934-36. At this time, the isolation and the
synthesis of hormones were proceeding rapidly, in particular, the
steroid hormones of the sexual glands. These substances have most
powerful physiological effects, the principal being to stimulate cells
to increase the rate of appearance of new mitoses. Now, ordinary
histological technicjues give only an instantaneous picture of the
state of the tissues at one given moment. If the cell divisions proceed
very .rapidly, there will be small chance of observing them in a micro-
scopic slide. Colchicine, by arresting all these rapid cellular changes,
would be able to let the mitoses progressively accumulate in a given
tissue. Counting would be easier, and easier also the localization of
regions of maximal growth.
While several authors understood the uscfidness of colchicine as
a tool for the study of growth, the largest amotnit of work was done
in the field of endocrinology. Allen, Smith, and Gardner- are to be
credited with the publication, in 1937, of an excellent paper with
splendid photomicrographs that gave added impetus to research with
this new technique. They were studying the action of estrogens in
the mouse. After injecting the still chemically impure hormone of
that type at their disposal, "theelin," they observed that colchicine in-
creased tremendously the visible mitotic action in tissue sections. In
the vaginal epithelium, they mention "a most incredible number of
mitoses." * In a single transverse section of the vagina, controls in-
* E. Allen, M. Smith, and W. V. Gardner, "Accentuation of the Growth Effect
of Theelin on Genital Tissues of the Ovariectomized Mouse by Arrest of Mitosis
With Colchicine," Amer. Jour. Anat., 61 (1937) , p. 324.
[214]
Experimental Growth in Animals 215
jected with "theelin" alone showed 20 to 30 dividing cells. After
colchicine, this was increased to more than 1500 in about 10 hours.
In one experiment in which "theelin" and colchicine were injected
simultaneously, the authors wrote that "the general impression is
that approximately every other cell is in mitosis."* I'hese results
aroused great interest, and marked one of the starting points for
Fig. 9.1 — Graphical representation of the course of cell division in a fibroblast culture
treated by colchicine (1/20,000,000). During the two first hours, no notable changes.
Later, progressive accumulation of arrested mitoses. Each horizontal line represents
one mitosis; it is interrupted at the end of metaphase. Any vertical line indicates th-
number of visible mitoses at one moment, that is to say, the mitoses which should b
seen in a fixed preparation. This number progressively increases under the influenc
of colchicine. The rate of apparition of new prophases is not disturbed with this con-
centration. There is no mitotic stimulation. (From a cine-micrographic recording. After
Bucher, 1939)
e
le
ce
colchicine research outside of the Brussels laboratory. Together with
the discovery of colchicine polyjjloidy in 1937, this study initiated
the publication of a great number of papers in which colchicine was
mainly considered as a tool for making mitotic growth more visible
and easier to analyze.
However, any tool has its advantages and its shortcomings. Many
workers do not appear to have considered carefully the fundamental
problems involved in what Allen called the "freezing" of mitoses.
* Ihld.. p. 325.
276 Colchicine
Some of the complexities have aheady been scrutinized in the first
chapters of this book. A few more considerations about this particular
problem of multiplying the numbers of mitoses by destroying their
spindle will be useful for future workers in this field. While the
number of papers published about the colchicine method appears
to be on the decrease, so far as can be assessed, for colchicine is not
always mentioned in the titles, much work remains to be done. This
chapter will point out several unexplored fields.
9.2: Theoretical Considerations
Most of the American authors, following the first papers of Allen,
those of Brues^^- -**•-!• -- on liver regeneration, and the tissue culture
work of Bucher--^ and Ludford,*''- considered colchicine simply as a
means of stojjping any mitosis at metaphase. The complexities of
colchicine pharmacology (Chapter 7) should alone call for more cau-
tion.
A. P. Dustin, Sr., in a paper published in 1936, but which could
not have received much publicity, demonstrated the utility of colchi-
cine as a tool.^i He had noticed the increased number of divisions in
the wall of a parasitic cyst in a mouse, a fact which was the starting
point for experiments rjn the healing of ^vounds, revie^ved further on
in this chapter. In his own words, "colchicine enables the detection
of the otherwise invisible state of preparedness to mitosis." * It
throws into an abortive division all the cells which are ready to
divide, or had been prepared for mitosis, for instance, under the in-
fluence of endocrine or other stimidi. This was in agreement with
the line of thought which had led to the discovery of colchicine's
action in 1934, and which was the study of the regulation of mitotic
growth.
The theories of "mitotic arrest" or "arrest after mitotic stimula-
tion" are conflicting. In work where tlie location of mitoses is the
main purpose and where no quantitative data are required, colchi-
cine is useful whatever the opinion one has about a possible stimula-
tion of mitosis. This problem, however, should not be overlooked.
For instance, several authors have thought it possible to calculate
from the number of mitoses found after colchicine, the average dina-
tion of these mitoses, had they not been arrested. This dmation is,
of course, an indication of the rapidity of cellular growth in the
tissues studied. It should be clearly realized that such calculations
imply several unknown factors, and they have a precise signification
only if the following conditions are fulfilled:
1. Colchicine arrests all mitoses, shortly after it has been injected
and until the end of the experimental period.
* A. P. Dustin, "La Colchicine, Reactif de llniniinence Caryocinetique," Arch.
Portugaises Sci. Biol., 5(1936), p. 41.
Experimental Growth in Animals 217
2. The intermitotic period is much longer than the duration of the
experiment, and is not modified by the cxj^eriment.
3. The arrested mitoses are not destroyed before the moment the
tissues are fixed and examined.
4. The tissue is homogeneous from the point of vie\v of mitosis, that
is to say, mitotic rates and intermitotic periods do not vary from
one region of the tissue to another.
5. The mitotic rate does not \ary chning the experimental period,
in control animals.
Such conditions are not often fulfilled. One type of experiment in
which they are is liver regeneration; this will be considered further.
In mammals, cellular destruction is a factor which cannot be ignored.
If, however, the above-mentioned causes of error do not exist, the
average duration of mitosis can be found by the formula A = Mt/X,
in which M is the mitotic index before colchicine, and X the index
found t hours after the injection of the alkaloid.
If this formula is applied to the resvdts obtained in the experi-
ments referred to in the previous paragraph,- it is found that after
"theelin" stimulation, the average duration of mitoses would be 10
minutes. This is a remarkably short period, and it may be questioned
whether mitoses can be completed so rapidly. However, results ob-
tained by A. P. Dustin, Sr., in the uterus of the rabbit after stimula-
tion by chorionic gonadotropic hormones, are rather similar.^^ The
increase in the ninnber of mitoses was observed in repeated biopsies.
Figure 9.2 shows that it was considerable, and that in one animal,
the calculated duration of each mitosis, had it not been arrested by
colchicine, would be 12 minutes. These results bring some evidence
for mitotic stimulation, for the prophase mitotic index increased also.
This indicates that more cells were undergoing prophase than ex-
pected; that is to say, a true stimulation took place. This index rose
from 7.56 to 14.8 in 2 hoins, and from 4.8 to 24.4 in 7 hours. It must,
of course, be supposed here that the duration of each prophase was
not affected by colchicine.
Such results are rather complex, for the mitotic index could have
been modified by the traumatisms of the biopsies themselves, and also
by the continued action of the hormone. The possibility of a synergic
action of hormones and colchicine cannot be rided out'^'^ (cf. Chapter
The following results-^' are all the more interesting, for while
they apparently could demonstrate such a synergism, a much simpler
explanation is possible. Table 9.1 gives the results of mitotic counts
in the seminal vesicles, after stimulation by a single large dose of
testosterone. There appears to be a veritable "explosion" of mitoses,
to use the expression coined by A. P. Dustin, Sr. Does this give evi-
dence of mitotic stimulation by the alkaloid? The counts of the con-
218
Colchicine
trol animals demonstrate that it does not, for it can be seen that be-
tween the thirtieth and thirty-fifth hours after the hormone injection
the mitotic index rises sharply. If colchicine had been injected at
the thirty-first hour, a mitotic increase from 2.92 to 108.60 would have
been observed, and this could not be explained by the theory of meta-
phase arrest. This increase is, however, not only the result of mitotic
X 35 mitotic index
^C'2
CALCULATED DURATION OF
MITOSES = A = '^-^
x30
x25 .
x20
X 15 .
x lO -
x5 -
X I
hours : I
Fig. 9.2 — Progressive increase of the numbers of mitoses, in repeated biopsies from
the rabbit's uterus, after stimulation by chorionic gonadotropins and injection of col-
chicine. Calculated duration of mitoses on the assumption that colchicine does noth-
ing more than arrest them at metaphase. (From original data of A. P. Dustin, 1943 )
stasis, but also of the progressive action of testosterone, demon-
strated by the fact that in untreated animals the mitotic count rises
about threefold. Therefore, colchicine alone has increased the mitoses
only from about 10 (2.92 X 3) to 108.60 within 4 hours, which means
that the average mitotic duration nuist be about 25 minutes or less.
This agrees with knowledge of mitotic duration in mammals.
Such an example demonstrates the intricacies of quantitative
Experimental Growth in Animals 219
work with colchicine. Others will be found in this chapter. Here,
as in other fields of colchicine work, problems must not be over-
simplified, and here especially, the greatest care should be taken in
all quantitative estimations. It is striking that it is when colchicine
is considered as a tool that the need for fundamental knowledge is
the most apparent.
9.3: Cellular Multiplication in Normal Growth
Gro^\•th patterns in the organs of adult animals can be revealed
far better after colchicine than with ordinary tissue sections. The
alkaloid may do more than simply locate the germinative zones of
organs; inider strict experimental conditions, it may solve some
quantitative problems of growth. Another method, which has brought
excellent results, is to study the growth of explanted tissues. This
has been done bv the ordinary methods of tissue culture,-^- ^-' ®* or
TABLE 9.1
Mitotic Activity in the Seminal Vesicles of Cas-
tr.JlTed 80-dav-old Rats Treated With 0.3 mg. of
Testosterone Propionate
(Abridged from Burkhart^')
Time Alter Treatment
(hours)
Control
Colchicine
15.
19.
23.
27.
0
0
0.28
5.00
2 .92
10.68
0.04
0.24
0.20
2.04
31
7.60
35
108.60
by a modified technique in which cellular multiplication was ob-
served only for a fe\\' hours after explantation.^s, 24-27 Some of the
results demonstrating how useful colchicine may be as a tool in such
work will be summari/ed here.
9-3-1 : Studies in vivo. Some of the early work in this field was
done on the ovary. Colchicine, l)y increasing from 11 to ,H5 times
the number of mitoses that could be observed in the germinal epi-
thelium of the ovary of mice, demonstrated that this Avas a region of
active growth.^- ■^'*' ^^' ^" Similar facts were observed in guinea pigs.
76, 77 Yhe relation between the mitotic activity in the ovarian follicles
220
Colchicine
and the estrus cycle were carefully analyzed (Fig. 9.3) . In the endo-
thelial cells oi the theca interna of the ovarian follicles, immediately
before ovulation, the karyokineses were found to increase about sixty-
fold. Arrested mitoses of follicular cells in the rat can be found
around eggs after they have reached the uterus (Fig. 9.4) ^ Some
follicles are found to be growing rapidly while others are quiescent.
I40
I30
I20
no
lOO
90
80
12 70
I/)
g 60
u. 50
O
d 40
z
30
20
lO
O
16
MITOTIC PROLIFERATION IN THE DEVELOPING AND
RETROGRESSING CORPUS LUTEUM
endothelium
luteal cells
connective tissue
theca externa
6 7 6 9 lO II 12 13 14 15 16
DAY OF ESTROUS CYCLE
Fig. 9.3— Mitoses in the corpus luteum of the ovary of a normal mature guinea pig,
studied by the colchicine method. (After Schmidt")
This fact is not evident in central animals, because the number of
mitoses is too small.
In the pituitary glands of mice, colchicine increases the number
of mitoses about threefold. This is an indication that these mitoses
are normally of long duration. Many data have been gathered about
the mitotic activity in this organ in various physiological conditions.
'•• 5- Table 9.2 shows how evident is the action of age on mitotic
activity when the number of metaphases has been artificially in-
creased by spindle poisoning. ^'-
A quantitative study of cell regeneration in the mucosa of the
intestine in rats has been made possible by colchicine. It was known
that the intestinal cells are continuously shed, but how long it took
for the whole epithelial lining to be replaced was not known. Table
Experimental Growth in Animals 221
9.3 gives the results, with the percentages of dividing cells and of
mitotic stages in control and colchicinized aninials.'^'^ From these
results, it is apparent that mitotic arrest at metaphase has increased
in six hours the number of cell divisions by 17.63/3.32. The mitotic
duration, calculated as indicated in Section 9.2, is 3.32 X 6-0/17.63 =
1.13 = 1 hr. 8 min. It can be calculated from this result that in
37.7 hours (1.57 days), 100 per cent of the cells will have divided;
that is to say, a complete renewal of the e])ithelium will have taken
place. This is, of course, only statistically correct, for there must re-
main a certain number of stem cells so that growth may persist.
These cells will divide into one differentiating cell and one stem cell
identical to the first. A great discrepancy between results obtained
with radio-phosphorus on the nucleic acid turnover and the figures
given bv the colchicine method as used by the same authors has been
discovered.'^! This may throw more light on the complex problems
of growth in differentiating tissues.
The skin of small rodents has been excellent testing material for
the study of growth as analyzed by colchicine. A very extensive series
.^cSt
o°^~,ooo
oor.'^rf OoOOO w-o,a
<^
>Oo
Fig. 9.4— Colchicine-mitoses (black dots) in an ovarian follicle (left), ancJ in Follicular
cells surrounding an egg found in the uterus in the rat. (After Allen et al. )
of experiments has been carried on, especially by Bullotigh.-^ -^ This
has provided ample material for a precise analysis of growth and the
fundamental mechanisms of mitosis. Further reference Avill be made
to some of these jiapers in the section on hormonal stimulation of
mitosis. Diurnal variations, the action of sleep, the efiects of blood-
sugar level and ol injections of starch, have led to the most im-
222
Colchicine
portant conclusion that carbohydrate metabolism is indispensable
for mitosis in epidermal cells, and that it provides the energy neces-
sary for a cell to initiate division. Once prophase has started, no
further energy requirements are apparent, and mitosis proceeds as
if it were an all-or-none reaction.-^- -^ These experiments have also
shown that the mitotic increase after colchicine corresponds to a
TABLE 9.2
Effect of Age on Mitotic Actimtv in the Pituitary Glands of Female Rats
(After Hunt^^)
Age
(days)
Pituitary Mitoses
(per sq. mm.)
96 77.5
148 45
188 32
220 15
300 5
normal duration of about three hours. This is very long compared
to that of ten minutes mentioned in Section 9.1. The difference may
be partly explained by the action of hormonal stimulation, which not
only increases the number of new cells starting to divide but also
apparently shortens the duration of mitosis. This will be considered
in a subsequent paragraph. Some other complexities of the study
of epidermal growth and of the action of colchicine can be under-
stood by the tact that the alkaloid may decrease the number of ne^v
mitoses,-^ and that unless observations are made within six hours
after the injection of the alkaloid, some arrested metaphases may
proceed to telophase.
TABLE 9.3
Dividing Cells (per cent) in the Ileal Epithelium of Male Rats
(After Leblond and Stevens^")
Per Cent Nuclei Un-
dergoing Mitosis
(Normal and
Abnormal)
Stages (per cent)
Pro-
phase
Meta-
phase
(Nor-
mal)
Meta-
phase
(Degener-
ating)
Ana-
phase
Telo-
phase
Controls. . . .
Colchicine. .
3.32 ± 0.35
17.63 ± 0.82
24
2
36
57
0
41
5
0
35
0
Experimenfal Growth in Animals 223
These studies of the epithelial growth in mice lead to a most
interesting development which will now be considered: the study of
groAvth in cxplantcd tissues.
y.5-2; Grou'tJi in vitro. Many of the fundamental discoveries
related to colchicine-mitosis were made on tissue cultures.^- -^' '^^- '''-•
84, 88, 90 ^hc importance of metaphase arrest in increasing the num-
ber of visible mitoses without modifying the mitotic rate has been
illustrated bv Figme 9.1. Other results on the action of colchicine on
neoplastic cells in tissue culture, and on the mitosis-arresting proper-
ties of colchicine derivatives and other mitotic poisons will be related
in Chapters 10 and 17. Tissue culture work offers definite potentiali-
ties for further investigation. The utilization of synthetic or semi-
svnthetic media and the roller-tube technique are some of the modern
aspects of tissue culture Avhich could benefit from colchicine.
On the other hand, most important results have been obtained
by simplified methods in which surviving tissues are utilized. Within
the short duration of the experiments, mitoses proceed normally, and
problems of bacterial contamination, transplantation, and dediffer-
entiation do not arise. These methods have been used in the study
of the skin and bone marrow of mammals, including man.
As a consequence of previously mentioned work on the skin of
the ears of mice, Bullough-^ developed a technique of in vitro study
of the mitotic activity. In vivo experiments had demonstrated that
glucose-*' and oxygen-' were indispensable for providing the energy
required for cell di\'ision. Glutamate was further demonstrated to
increase the rate of cell division. The /?? vitro method should eventu-
ally bring forth important new data on the metabolic requirement
of epidermal cells. Colchicine increases the amount of visible mitoses
and makes counts simpler. However, because of the long duration
of cell division in this type of tissue, colchicine does not produce any
of the spectacular increases \\'hich have been seen in other organs.
An important residt was to establish that a linear relation existed
between the number of arrested mitoses and the oxygen tension.
While only 0.4 mitoses could be seen in pure nitrogen, the figures
were 3.9 for 60 per cent nitrogen and 40 per cent oxygen, and 8.3 in
pure oxygen.-" The general significance of these results is made clear
bv nearly identical findings with bone marrow cells.'' This work has
been done mainly in Ital)'. Astaldi and a group of collaborators first
studied the colchicine response of human bone marrow.*' This is
readily available by sternal puncture, and colchicine has provided
a new insight on the growth of this tissue. This growth is far more
rapid than that of skin; in mammals, bone marrow and intestinal
mucosa are the tissues which ha\e the highest mitotic index. After
explantation, small fragments were kept at 37°C. in human serum,
224 Colchicine
and their growth could be studied for as long as 36 hours. The
number of mitoses was considerably increased by colchicine, and
the authors have indicated that this "stathmokinetic index," as it has
been called, may throw considerable light on many problems of
normal and neoplastic celhdar division. Some of these will be men-
tioned in Chapter 10.
Very small amounts of colchicine are effective; dilutions of
1:1,000,000 were used. The alkaloid may disturb slightly the normal
maturation of cells of the erythroblastic series. This is only visible
after 12 hoius hi vitro, and for most experiments, important data can
be recorded from 4 to 8 hours after colchicine. The action of em-
bryonic extracts" and that of irradiation with X-rays^ have been
studied on normal marrow. This has also been compared with
marrow from patients suffering from Addison-Biermer anemia (cf.
Chapter 8) , polycythemia and leukemia (Chapter 10) , and thalas-
semia (Cooley's anemia) ."
Figure 9.5 demonstrates that the mitotic activity of erythroblasts
(young red blood cells) is depressed by absence of oxygen. This ex-
periment was carried on in a vessel in which a partial vaciuuu could
be maintained. It is made clear by colchicine that the younger cells,
the basophil erythroblasts, are more depressed than the more
differentiated ones, which have already some hemoglobin. These
important results are to be compared to those mentioned above, on
the importance of oxygen for mitosis in the epithelial cells of the
mouse's ear.-" This might have passed entirely imnoticed if a tool
had not existed to increase the number of visible mitoses and make
counting a simple proposition. It must, however, always be kept in
mind that control experiments shotdd be made, for it remains to be
proved that colchicine, which has such a wide variety of pharma-
cological effects (Chapter 7) , does not disturb some mitoses more
than others. These experiments are, of comse, entirely based on
the assumption that the alkaloid does no more than "frce/e" the
mitoses at metaphase.-' -^' ^^
9,4: Hormone-stimulated Growth
A considerable number of papers have been published following
the contributions of Allen, Smith, and Gardner.^ It is not con-
templated to review them all here, even if such a task were possible,
for many papers of endocrinological interest do not mention in their
titles that colchicine has been used, and it has become impossible to
keep up a complete set of references. Table 9.4 gives a summary of
some of the work which has been jHiblished. It is evident that the
sex hormones have been the most studied, partly because their iso-
lation and chemical identification took place in the period im-
MITOSES
<J\J ■
■ \
•
•
•
•
go-
t \
•
•
•
•
•
so.
0
0
H
•
70.
o
o
8
•
\
60-
O
o
o
8
o
o
0
•
• \
• \
• \
0
\&
0
0
• >
V
so-
0
0 ^v
0
V 8
•
\
0
\^,
<k !
•\
40-
30-
o
o
o
o
-x
t
o
o
0
X
•
\-
o
o \
V
20-
o
0
0
0
o
0
o N
8
•
•
0
^ 0
lO
r-
1
1
1
0
PRESSION:
760mm. 660
Hg.
560 460
360
260 160
Fig. 9.5 — Linear relation between pressure of atmospheric air and mitoses in bone-marrow
erythroblasts studied by culture In vitro. The results are expressed as percentages of the
maximum mitotic rate, i.e., that of basophil erythroblasts at atmospheric pressure. (After
Astaldi et al.")
TABLE 9.4
Experiments on Hormone-influenced Growth
Hormones
1. Pituitary hormones
gonadotropins
chorionic gonadotropins
prolactin
adrenocorticotropic
hormone (ACTH) *
thyrotropic hormone
anterior lobe extract
2. Ovarian hormcnes
a. estrogens ("theelin,"
estradiol, estrone, etc.)
b. progesterone
Animal
niouse
rabbit
guinea pig
Alolee marmoraia
mouse
gumca pig
rabbit
pigeon
rat
rat
guinea pig
rat
mouse
rat
guinea pig
woman
Rhodeus amariis
rabbit
rat
Receptor Tissue and
References
uterus (muscle)'
uterus'**
hypophysis''*
parathyroid^*
adrenals^'^
testis (interstitial cells)'*
seminal vesicles; uterus^-
prostate'*
cloacal epithelium'*
uterus (glands and muscle)'
testis (interstitial cells)^'
uterus'"'^"^
crop-sac^*'^^'"''
adrenal corte.x"
thyroid*"
thyroidi2iif"
Langerhans' islets*"'-'^
vagina^'''!
uterus: glands,^' muscle^
mammary gland''^
hypophysis J^'*^
rectum^'
ovary'^'
skin2^
seminal vesicles^"
various tissues-^
uterus (muscle)'
vagina"'-
hypophysis*^
parathyroid^''^
seminal vesicles'*^ '^'
ventral prostate"''^'
nipple*"'
uterus (muscle)'*
vagina'^''-
ovipositor'*
uterus (glands)^*
(muscle)^*
parathyroid§'-
vagina'-
prostate"
Experimental Growth m Animals 227
TABLE 9.4 (continued)
Hormones
c. pregnane
3. Testicular hormones (androgens')
testosterone, androsterone,
androstenediol
androstane
4. Adrenal cortical hormones
a. desoxycorticosterone
b. cortisone
c. corticosterone, total ex-
tract of cortex
5. Other hormones
thyroxine
tachysterinc
insulin
Animal
woman
Rhodeus amarus
mouse
rat
guinea pig
Rhodeus amarus
Molge marmorata
Receptor Tissue and
References
* Non-purified extract of pituitary.
t Extracted from human urine.
t Also experiments with stilbestrol.
§ With estradiol benzoate.
seminal vesicles''
vagina'*''^
ovipositor^*
seminal vesicles*"''*^''"''^'^"
Id. transplanted to females-
prostate*^
seminal vesicles'^'**'^^'^'''^^
prostate*^-'
thyroid (in female)^'
parathyroid {id.Y^
adrenal*^
skin
cpoophoron^''
ovipositor'*
prostate''*
cloacal epithelium'*'
rabbit
uterus*'
mouse
skin2'
Rhodeus amarus
ovipositor'*
mouse
rat
adrenal^'
regenerating kidney^'
rat
parathyroid'^
rat
Langerhans' islets''
111
ediately following \\)'i~ , when colchicine was taken up as a "fad."
Endocrinologists appear to have lost some of their interest in this tool.
and this may explain how such important substances as cortisone
and ACTH have hardly been tested by colchicine methods. Most
of the W'ork was on hormones which stinuilated mitosis; cortisone,
on the contrary, appears to have an inhibitory action.-'^ llie useful-
ness of colchicine in the study of mitotic inhibitors has not yet been
fully understood, and further work will undoubtedly demonstrate
228
Colchicine
that this is a tool for the study of mitotic activity, whether stimulated
or depressed. Results reported in Chapter 10 support this opinion.
9-4-i: Pituitary hormones. Prolactins the hormone stimulating
secretion of the mammary gland in mammals, was used in one of
the first and most spectacular experiments of this type. In birds, this
hormone stimulates ilip (rop-sac. This organ secretes "milk" by a
TABLE 9.5
MiTosii IN THE Crop-Sac of the Pigeon After Prolactin Stimulation
(Colchicine is injected 9 to 1 1 hours before the animal is killed.)
(After Leblond and AUen^s)
Number
of
Animals
Colchicine
Pro-
lactin
{bird
units)
Mitoses per 2000
Cells (Average,
Smallest, and
Greatest Figures)
Average
Mitotic
Index
Colchicine controls . .
Prol;irlin controls
2
6
9
6
0.10-0.25
0 . 40-0 . 50
40
40
40
12 (9-15)
46 (8-173)
15 (9-21)
27 (11-48)
534 (210-1075)
1
2
■*
1
Prolactin-colchicine. .
0.10-0.35
0.50
1
26
process which has no relation to that observed in mammals. The
bird's "milk" is made of fat-laden cells desquamating from the thick-
ened epithelium of the crop-sac. Table 9.5 shows the increase of the
mitotic index for this epithelium in pigeons injected with prolactine
and colchicine. •'**• -^^ In one animal, 5-^ per cent of all epithelial crop-
sac nuclei were found to be in a condition of arrested mitosis. The
average increase of the mitotic index is 37-fold, and calculation based
on the assumption of arrest only, leads to the result that the pro-
lactine-stinuilated cells must divide in about 16 minutes. It is not
certain that such a calculation is correct, because many factors, for
instance cellular differentiation, are involved. Also, from the pub-
lished photomicrographs-^"' it is not evident that the thickness of the
control and the colchicinized epithclia are comparable. Whatever the
significance of these quantitative estimations may be, colchicine
demonstrated clearly that connective tissue cells, and muscular cells
of the crop-sac wall also divided under the influence of prolactine.
This fact had never been observed.^*'- 5"
The thyroid-stimulating hormone, thyrotropin, also increases the
cell divisions in the thyroid. 1 his is made much more evident by
spindle poisoning. In controls 6.3 mitoses were found per 100 thyroid
Experimental Growth in Animals 229
vesicles in the guinea pig. This figure Avas increased to 16.8 by the
hormone alone, and to 119 by hormone -j- colchicine.^- A method
tor the detection of increased amounts of this hormone in the urine
ol patients has been proposed^i (Table 9.6) . A response is positive
when more than 4 mitoses per 100 vesicles are detected." Other
authors have confirmed these results, but some abnormal resjjonses
were attributed to a rhvthmic growth response of the thyroid. "^^
The gonadotropic hormones stimulate mitotic growth in many
tissues, and this was studied by means of colchicine as early as 1937.^-
In the uterine glands of guinea pigs, colchicine made clear the
location of the zones of maximal growth. Action of pituitary hor-
mones on endocrine glands will be considered later. Results of work
on pregnant guinea pigs may be mentioned however, because they
bring e\idence of many, often unsuspected stinudations of mitosis
by the increased amount of gonadotropic and other steroid sex
hormones during pregnancy. -^^ Especially notable is the stimulation
TABLE 9.6
Mitoses in One Microscopic Field in the Thyroid of the Guinea Pig
(After Bastenie")
Number
Substance Injected
of Cases
Mitoses
Colchicine alone
0.5-1
Anterior lobe extract + colchicine
35
Extracts of urine + colchicine:
m^rxedema
5
7-7
myxedema after treatment
3
0.1-0.5
hyperthyroidy
3
0.15-0.25
hypothyroidy of pituitary origin
3
0 . 05-0 . 4
Froelich's syndrome
2
0.05-0.1
Acromegaly
1
0.2
Other diseases, without thyroid disturbances
3
0.2-0.4
of the paratltNroids, exocrine glands of the pancreas, and kidney
tubules — changes which would have been unnoticed -without col-
chicine. This important work does not seem to ]ia\e been pursued so
far as colchicine is cone erned (Table 9.7) .
The absence of pul)lications on the adrenocorticotropic hormone
(ACTH) and colchicine has already been mentioned.^* It is still
230
Colchicine
TABLE 9.7
Mitotic Index in Organs of Pregnant Guinea Pig
I: without colchicine
II: 9 hours after 0.625 mg./lOO g. colchicine
A: embryos less than 5 mm. long
B: embryos from 5 to 15 mm.
C: embryos longer than 15 mm.
(After Cavallero^^)
Hypophysis
(anterior lobej . .
Thyroid
Parathyroids
Adrenal cortex ....
Adrenal medulla . .
Langerhans' islets. .
Corpus luteum ....
Kidney
Pancreas (exocrine)
Liver
Controls
I
0
0.2
0
0
0
0
0
0
0
0
II
2
0.3
1
0
0
1
2
0.5
0
A
I
5
2
1
2
0
0.3
1
2
1
0
II
17
7
14
4
0
B
I
1
1
0
0
0
0
6
9.5
0
0
II
4
0
0
6
5
10
0
0
0
0
0
0
0
*
0.5
0
0
II
6
3
2
1
0
0
5
18
5
0
* The figure given in the original paper has been omitted because of a typographi-
cal error which it has not been possible to correct (Cavallcro, personal communication).
more remarkable that the growth hormone, somatotropin (STH) ,
has only been studied ^vith the colchicine method in a single paper,
which pointed to stinudation ot hemopoiesis."* This shows that many
pathways remain open. The results obtained with other hormones
are good evidence that important and inisuspected findings still re-
main before us.
().4-2: Sex hormones. These are poAverful stimidants of mitotic
growth. Some of the results with estrogens have been reported in the
first paragraph of this chapter.-- ^^ It was not always realized that
estrogens may stimidate growth in other epithelia than those of the
genital tract. In his observations on mice, Bidlough, using colchicine
to detect the increased mitotic activity, demonstrated stimulation in
most tissues, including connective tissue.-* In further experiments,
this author has called attention to a remarkable effect of estrogens.
Figure 9.6 shows that colchicine increases the mitotic index of the
40t
o o
Diestrus \ ^,thout Colchicine
Esirus f
10.00 11.00 12.00 13.00 14.00 15.00 16.00
Time of day
Fig 9 6— Mitotic activity, as demonstrated by colchicine, in the epidermis of the ear
of female mice, in estrus and diestrus. Controls: dotted lines. The far greater increase
observed after colchicine during estrus is considered to be an indication that normally
epidermal mitoses last longer in diestrus. (Modified, after Bullough, 1950" )
232 Colchicine
epidermis of the ear considerably more during estrus than during
diestrus. The mitoses were counted hour by hour by cHpping small
Iragments of the ear. This difference can be explained by a shorten-
ing of the time taken for one division, from about 2 hours in diestrus
to 34 hour in estrus. This significant result is not discussed; other
possible hypotheses are, for instance, synergic action of colchicine
and hormone, or changes in the duration of interphase. The alkaloid
is simply considered to stop metaphases.-- -"'• "-
Androgenic hormones, also, stinudatc mitotic growth, and the
use of colchicine was advocated in 1937 for the study of the changes
in the seminal vesicles'*'*' *'•''• ^- (Fig. 9.7) . The accumulation of arrested
mitoses in the prostate or seminal vesicles of castrated mice or rats
has been projjosed as a test for androgens.'''' In mice, colchicine
helped to jjrove that the prostate is a more sensitive reactor than
the seminal vesicles to testosterone.^^ Data about the "explosive"
aspect of mitotic stimulation when studied with colchicine in these
tissues has been discussed already and presented in Table 9.1.
The quantitative aspects of the seminal vesicle reaction to various
androgens and related hormones have been carefully investigated.^*^' *^
Figure 9.8 demonstrates how the increased number of mitoses heljjs
to establish the linear relations between the doses of androgen in-
jected and the intensity of the reaction. With other hormones, such
as progesterone and estrogens, though the mitotic index may increase,
no such relation is foiuuH" (Fig. 9.9) .
Colchicine also brought further evidence that in the female
guinea pig, the epoophoron reacted to colchicine like the male epi-
didymis, of which it is the anatomical homolog.*^
g.^-^: Mitotic sti/nuldllon o^ endocrine glands. Though pituitary
hormones play a great jxirt in mitotic stimulation in various organs,
the cells of the pituitary may also undergo mitosis under the in-
fluence of hormonal stimuli. '^^ ■^''' "^ Colchicine helped to demonstrate
that in virgin female rats, ovariectomy did not promote pituitary
mitoses. ^'-^ On the contrary, injections of estrogens, natural or syn-
thetic, enlarge the pituitary as a consequence of mitotic growth made
evident with colchicine.''"* It has, however, been shown that castration
could influence the numbers of c-mitoses of the basophil cells of the
anterior lobe of the i)ituitary.'^i There are no data about the posterior
lobe of the organ, which may be an interesting object for future col-
chicine work.
Several papers deal with mitotic stimulation in the cortical region
of the adrenals.'-*-' ^'^^ «''' ^••'' *' In inmiature female rats, colchicine re-
veals a stimulation which reaches its maximum 96 hours after an
injection of testosterone. At the same time, however, mitotic activity
is increased in thyroid, parathyroid, and ovary. This may be evi-
^B
i»i
M
Fig. 9.7 — Mitotic stimulation by testosterone propionate in the seminal vesicles. Above.
Hormone alone. Below. Hormone — colchicine. (Original photomicrographs from
Bastenie and Zylberszac ")
1 doses
3 4 5 6 7 8 9 10 12,515 175 20 Y30 40
Fig. 9.8— Seminal vesicle test with testosterone propionate. The line (below), without
colchicine, does not make clear the correlation between number of mitoses and dose.
With colchicine, a linear relation is evident (above). (After Dirschel et al. ")
Experimental Growth in Animals 235
100
200
500 1000)f 2000
10 20 doses SO
pjg 9.9— Seminal vesicle test with various androgens. Amplification of the number
of visible mitoses by colchicine. {After Dirschel et a\.*")
dence ot an indirect action via pitiiiiaiy stimulation."^ The same ap-
plies jMobably for the increased mitotic activity detected in the thy-
roid of female rats injected with testosterone.*'"'
The mitotic activity of the parathyroid glands of mammals is
usually very low, and is difficult to study; hence, the utility of colchi-
cine. FoUiculin (estradiol) and progesterone injections in the rat
result in the appearance of many mitoses.^- This eflfect may be the
consequence of hypocalcemia. The contrary, hypercalcemia, proba-
bly explains why irradiated tachysterin (Holtz's A.T.IO) decreases
the parathyroid mitotic activity. Testosterone injections increase mi-
toses in this organ; this may be an indirect effect mediated by the
pituitary.^^
236 Colchicine
In the Langerhans' islets of the pancreas, pituitary stimulation
9-' '>" and pregnancy increase the number ot mitoses, as detected by
colchicine.
It is surprising. to find no paper dealing ^vith mitotic stimulation
in the interstitial (Leydig) cells ot the testes. In guinea pigs injected
with chorionic gonadotropins, these cells increase in number, but
colchicine failed to detect mitoses. It was concluded that the hor-
mone-secreting cells originated from ordinary connective cells.^^
Further work on this tissue is obviously needed. ^'^
9.5: Regeneration and Hypertrophy
The problem which was under study in the laboratory of A. P.
Dustin, Sr., since about 1920 and which led to the discovery of the
properties of colchicine was that of the regulation of growth and
mitotic activity in pluricellular animals. In vertebrates, for instance,
cell division takes place only in some tissues, and then in an orderly
way. While in the adult, nerve cells become incapable of any mitosis,
other organs, such as the liver and the kidney, while nearly devoid
of any mitotic activity in normal conditions, may grow rapidly by
cellular multiplication after surgical excision. In the rodents, and
in ]jarticular the rat, large portions of the liver may be removed
surgically. The remaining cells start to divide at once, and regenera-
tion of the normal liver mass is remarkably rapid. -^ The exact de-
terminism of this cellular growth is unknown. This was one of the
first subjects to be studied with the help of colchicine as a tool for
a better analysis of mitotic activity. i»- -•'• -i- -- Hence, the work which
had been initiated in order to understand better such problems as
regenerative growth led indirectly to the discovery of a new tool,
colchicine, which was rapidly put to use in several countries. ^^^ ^^' ^^' *^
The problems of cellular division in wound healing, which is closely
related to regeneration, will be considered in the next section of this
chapter. This work deserves special attention, for important results
aj)pear to have been often overlooked. Once again, colchicine was
taken up with enthusiasm as a new tool; new discoveries were made
possible, but only in a few instances w\as the study pursued long
enough to come near a solution of the problems.^i This field ap-
pears today as one of the most promising for futvne research.
9.5-7.- Liver. In the rat, as much as 68 per cent of the liver
parenchyma may be removed surgically. After an initial period of
edematous swelling lasting about 24 hours, cell division takes place.
This type of growth has been extensively studied, for it lends itself
to quantitative estimations of the numbers of new cells formed each
day.'-^ The duration of mitosis was found to be between 48 and 53
minutes. After colchicine, many arrested mitoses are visible. Their
Experimental Growth in Animals 237
luimlxi can be ex]jlainecl on the basis of niitoiic arrest. i"- -"• -i Some
show only slight abnormalities, but most are of the exploded tvpe
(Fig. 2.5) . A\nien u|) to one-fifth of all the li\er cells are in this
condition, swollen and their chromosomes dispersed, the liver be-
comes extremely friable.-- The various stages of restitution after the
injection of colchicine have been descriljcd and illustrated in C>hapter
2. It is surprising that the regeneration is only slightly slowed down
by several injections of the sublethal dose of 50 mg. This has been
explained by the fact that the exploded metaphases, after building
cells ^vith many micronuclei, regained normal nuclei by the fusion
of the micronuclei (Figs. 2.7. 2.8, 2.9) . These facts remain rather
difficult to understand from a quantitati\e point of view.
Apart from this work, liver regeneration studied \vith colciiicine
has pro\ided some material for counting the chromosomes. This is
done readih in the exploded metaphases. Diploid, tetraploid, and
octojjloitl nuclei were observed, a fact which agrees with karyometric
chita.'"' About the analysis of the differential growth of various liver
constituents — liver cells, Kupfter cells, bile canaliculi, blood vessels
— hardly anything is known, and there remain ample opportunities
for fin ther colchicine research.^-^- '''^- "^^ The biochemical stimulus to
mitotic growth after hepatectomy is also unknown; some unpub-
lished results obtained at Brussels indicate that the ligature of bile
ducts ma\ increase mitoses, as observed in the liver b\ the colchicine
method.
p-y-::: Kidney. The increase of the \olimie of one kidney after
removal of the other is closely related to regeneration. It proceeds
by mitotic growth. This is particularly difhcult to analyze in such
a complex organ as the kichiey, and any tool increasing the niunber
of visible mitoses is most helpful.^'' ^'■^- -^^ The great ninnber of mitoses
obser\ed in rats injected with 2.5 mg/kg after tniilateral nephrectomy
and killed 10 hours later is apparent from Table 9.8.
The jjroblems of kidney mitoses in this condition and in other
experiments carried on to throw light on the causal factors have
been the object of several jniblications from the Brussels school. After
tmihiteral nephrectomy, the maximal niunljer of mitoses is found
during the first four days in the convoliued tidjules, then in the
glomeruli, and on the seventh day in Henle's loops and the Schweig-
ger-Seidel tubules.^i- *'■'' No mitoses are to be foimd in the epithelium
of the renal }jelvis. Exploded c-mitoses are the most frequent in
the con^oluted tubes. If a partial nephrectomy is added to the abla-
tion of the other kidne), the remaining tissue shows mitoses in all
locations, including the pelvis. Ligation of the ureter, without ne-
phrectomy, also stimulates kidney cells to divide, a fact ^\hich may
prove of great experimental importance"'^ (Fig. 9.10). .Another re-
238
Colchicine
markablc result is found when colchicine is injected into animals
after one renal artery has been ligated.^^ Yhe ischemic kidney shows
a considerable number of mitoses, mainly in the excretory (Schweig-
ger-Seidel) tubules and the pelvis (Fig. 9.11). Similar facts have
been observed in kidneys made partly ischemic by the endocrine
kidney operation of Selye.^^ The following experiments were aimed
TABLE 9.8
Mitotic Index in the Remaining Kidney of Adult Rats Injected With Colchicine
(After Carnot and May^^
Cortex
Med
ulla
Days After Unilateral
Nephrectomy
External
Zone
Internal
Zone
Total
Controls
3
1.5
0
4.5
3
43.5
13.5
0.5
57.5
8
43
9
1.5
53.5
14
18
18
4
0
0
0
79
21
18
at finding the possible nature of the mitotic stimulus.^! The number
of renal mitoses after nephrectomy was decreased by injections of
thiouracil, a drug which depresses thyroid function. Thyroidectomy,
however, did not prevent or retard the increase of size of the re-
maining kidney in the rat.^^ Thyroxin was nevertheless found to
stimulate renal mitoses as much as woidd a nephrectomy. When
this was carried on and thyroxin injected afterwards, the mitotic
increase was greater than expected, lliis may indicate a truly syner-
gic action of the two stimuli. Ihe differences in body weight be-
tween controls (nephrectomy alone) and the other rats, and the
fact that the mitotic counts were corrected for 100 g. of body weight,
make these results difficult to interpret and suggests the need for
further research (Table 9.9) .
The hypothesis which was put forward following these data was
that thyroxin did not act directly on renal tissue, but that the in-
creased jMotein catabolism resulting from the action of the hormone
provided the factor responsible for mitosis.^^ Some substance present
in the urine may be suspected since, as mentioned above, ligature of
the ureter promotes cell division (Fig. 9.10). However, such mitotic
activity is mainly located in the connective tissue of the kidney. An
important fact is that unilateral ureter ligation promotes mitosis in
1900-r mitoses
1800.
« Connective cells
^ , Convoluted tubules
^ , Henle's loops
♦ Glomerul
, , Medullar zone
Fig. 9.10 — Mitotic activity in the kidney of the rat after ligature of the ureter, studied
with the colchicine-technique. (After Herlant )
4«'
^^^
♦ sJ'-lI
W
Fig. 9.11 — Colchicine-mitoses in the kidney of the rat, 72 hours after ligature of the
renal artery. Above. Star and ball metaphases with clumped chromosomes in the renal
pelvis. Belovi?. Exploded metaphases in the tubuli contort!. (A. P. Dustin and Zylberszac ' )
Experimental Growth in Animals 241
the other kidney also; tliis resembles closely the changes of com-
pensator\ hypertrophy (Fig. 9.12) . Substances reabsorbed from the
mine ma\ promote division first in the ligated kidney and later
in the other one. Research by other workers has suggested that
xanthopterin or substances ol that chemical constitution may initiate
the kidnex hypertroi:)hy. The problems are far from being solved,
but the utility of colchicine for the observation of mitotic growth
has been amply demonstrated.
9-^-^: Other organs. The folloAving results give an indication
of the multiple uses of colchicine as a tool. In the l.angerhans' islets
of the j:)ancreas, alloxan brings about a selectixe destruction of the
so-called |5-cells, which secrete insulin. Regeneration and mitoses of
these cells are j^revented if the animals receive insidin. This proba-
bly acts through a pituitary mechanism, for extracts of the pituitary
gland increase considerably the number of cell divisions in islet re-
generation. Colchicine-mitoses are also observed in the anterior lobe
of the pituitary-^''" (Table 9.10) . The regeneration of the adrenal
cortex after unilateral adrenalectomy in rats has also benefited from
the use of mitosis arrest. ^^ In rats also, colchicine helped to demon-
strate that compensatory hypertrophy of parathyroids after partial
parathyroidectomy does not take place in hyj:)ophysectomi/ed ani-
mals-"' and that testosterone inhibited the epithelial mitoses in thymic
regeneration following X-irradiation.-"'
TABLE 9.9
Action of Thyroxin on Renal Hypertrophy After Unilateral Nephrectomy:
Number of Mitoses in a Median Section of the Whole Kidney,
9 Hours After Colchicine
(Abridged from Herlant^')
Mitoses
Experiment
Convo-
luted
Tubules
Henle's
Loops
Glo-
meruli
Me-
dulla
Connec-
tive
Tissue
Total
L Unilateral
nephrectomy
(4 rats;*. . .'
2. Thyroxin alone
(4 rats) t
3. Unilateral
nephrectomy
-f thyroxin
(7 rats^t
61-125
173-252
315-589
8-19
3-5
35-65
0 3
0-2
2-15
26-87
2-5
25-152
44-62
3-7
31-132
163-210
186-250
523-722
* Animals weighing 260-360 gm.
t Six daily doses of 0.25 mg. thyroxin; killed the seventh day after 2 mg/kg col-
chicine. Animals weighing 120-220 gm.
242
Colchicine
9.5-7; Regeneration in developing animals. The complex actions
of the Colchicum alkaloid in embryonic development and larval
groAvth have already been reviewed. It is not surprising that in some
conditions colchicine may actually inhibit regenerative growth; thus,
it could not properly be used as a tool. In AmbJystoma opacum and
A. punctatinu, 18 to 25 mm. long, limb regeneration was studied
IIOOt mitoses
1000
900
800
700
600-
500-
400
300
200
100
T-
I \ A
I \ / \
. Ligated kidney
, Non- ligated kidney
I i
\l ^
\
\
\
\
\
\
\
\
\
_] I I L.
_] I ] 1_
days: 2 4
8
12 14 16 IB 20 22 24 26 28 30
Fig. 9.12 — Unilateral ligature of the ureter in a rat. Mitoses in ligated and non-ligated
kidney, detected by the colchicine-method. (After Herlanf^)
when the larvae were placed in 1 : lOOO or 1:5000 solutions of colchi-
cine. If this was done at the moment of amputation, all regeneration
was suppressed. \^arious degrees of inhibition of the limb-blastema
formation and of further differentiation, according to the length of
the colchicine treatment, were described.®*^
The regenerating tail of tadpoles of Xenopus laevis reacts simi-
larly.*^'^ In very dilute solutions of colchicine, this material provided
some results which apjjeared to indicate not only that mitoses ^vere
arrested at metaphasc but that a true mitotic stimulation existed.
Figure 9.13 shows that in control animals the number of mitoses is
quite small. It colchicine is assumed to have only a metaphasic arrest-
ing action, it is possible to calculate the number of mitoses which
should be observed at various intervals, for the duration of mitosis
has been observed and calculated in Xenopus (Chapter 3; . Figure
9.13 indicates that many more mitoses are found than expected, and
01 >. -
u ^ T3
C O C
O IV 3
-r, ~ 4*
O C 0)
3 0
Q.-Z
E i;
O o
M- ■= —
J3
0
>
■ - OJ O
-^ w
4, 0) O
^ c
O m
"0
c
lU
c
3
a
o
c
V
•5 2
*- o
-a
05
c c
'■Z o
a
i"2
c S
0) O
D) _Q
<ll
o
c -z
0
o
D
■J .
£
O O 0)
*- ■- ^
u (U o
ill
■D
o
3
o
c
<
I
o
c
o
-o
244
Colchicine
that instead of a gradual rise, there is a steep increase on the fifth
day. However, the experimental conditions are complex and stimuli
from other growth-promoting substances cannot be exclnded. These
data with those given in Section 9.2 comprise the best evidence to
date of possible mitotic stimulation of animal cells by colchicine.
In Xenopus, a short treatment, one hour in a 1:2000 solution, may
comj)letelv inhibit growth. However, regeneration often proceeds
normally during the first three days after this "colchicine shock" be-
cause cellular migration is not disturbed. On the fifth day, on the
contrary, when divisions should be taking place, regeneration was
completely inhibited (Fig. 9.14). Some pharmacological conclusions
are important to mention; they are the results of an extensive series
of experiments on this favorable material. Colchicine was demon-
TABLE9.10
Influence of Alloxan Diabetes on Pancreatic, Pituitary, ancI Suprarenal
Mitoses; Inhibition by Insulin; Stimulation by Pituitary Extracts
I: rats injected with 150 mg/kg alloxan
II: ?W. -f 10 to 20 units insulin per day
III: id. + pituitary extract (about 32 mg. dry powder per day)
(After Cavallero'^)
Mitoses
Langerhans' I
slets
Anterior Lobe
of Hypophysis
Adrenal Med
ilia
Days
I
II
III
I
II
III
I
II
in
1 . . . .
8
0
3
15
15
24
2
4
1
2 . . . .
7
1
24
24
19
16
9
7
3
3. . . .
44
2
132
54
2
64
1
0
0
4. . .
81
0
185
81
9
164
'-1
1
1
0
5. . . .
31
1
86
15
14
12
0
9
0
9
2
0
8
27
8
10
0
0
0
12. . . .
7
1
7
27
22
8
0
7
2
strated to act locally, for no inhibition was observed when only the
anterior part of the larva was immersed in the solution. This is also
evidenced by the absence of inhibition if colchicine is applied to
another wound close to the amputation. Experiments in which the
tail blastema was amputated and growth resumed, demonstrated that
colchicine did not penetrate more than 2 mm. from the wound. These
also showed that colchicine was fixed in the tissues of the wound for
Experimental Growth in Animals 245
at least three days. Such a fixation of the alkaloid in tissues has not
been described in j)]iarniacological work (Chapter 7) . The inhibition
ot regeneration Avas clearly the consequence of a great number of
the mitoses, sometimes up to 70 per cent, being destroyed after a
prolonged period of metaphase arrest (cf. Chapter 3) S'^ Similar re-
sults have been re]M)rted in Rtnia tempornria tadpoles. The local
days
Fig. 9.14 — Inhibition of the regeneration in the tail of Xenopus laevis after a short
treatment with colchicine. Dotted line: normal growth curve. I. Inhibition of regener-
ation for more than 5 days, then resumed growth. II, III. Strong and persistent inhi-
bition of growth. (After Lehmann et al. 1945, and Lijscher" )
application of a 1:500 M solution of colchicine for only 20 minutes
inhibits the regenerative growth of the tail, but has no influence on
the gro^\•th of the tadpole.^''
These facts, ajiart from the conclusion that colchicine is not
always a harmless "tool," indicate a remarkable property of the alka-
loid of becoming fixed in some tissues. This is surprising for a sub-
stance soluble both in water and in lipids. Pharmacologists should
pay attention to this possibility, for instance in the analysis of the
action of colchicine on muscle and brain. Nearly all data available
on colchicine metabolism in warm-blooded animals contradict this
246
Colchicine
idea of a fixation of the alkaloid. One of the purposes of this book
is being fulfilled whenever similar contradictions between work done
in widely separated fields of research are brought to light.
9.6: Wound Healing
The histological changes found in wounds after injections of col-
chicine were some of the most surprising observed by A. P. Dustin,
Sr.^- They appeared to give good support to the theory that a true
mitotic excitation followed the injection of the alkaloid. Experiments
were performed in rats. Two parallel incisions were made in the
dorsal skin, and alcurone grains inserted as an irritant in the wounds
before suturing. One of the scars was removed as a control at the
time colchicine was injected. The dose was 1.25 mg/kg and the ani-
mals were killed 9 hours later. This method made available some new
facts about w^ound healing and the formation of granulation tissue
near the alcurone grains. The endothelial cells are the first to divide.
Extraordinary pictures of capillaries with up to 10 c-mitoses in a
single section were observed. These cells appeared swollen. The
rapid mitotic growth was not noticeable without the use of the colchi-
cine tool.^-
In nerve regeneration, the alkaloid, by increasing the numbers
of mitoses, makes clear that their repartition is different on both
sides of a section. This may result from the influence of the disintegra-
tion products of myelin on the division of the Schwann cells (Fig.
9.15) .36
16 17
Fig. 9.15 — Colchicine-mitoses in a regenerating nerve of the rat. The shaded zone is
that of cicatrisation following sectioning. There are more mitoses in the Schwann cells
in the peripheric end, at left, than in the central part of the nerve. (After Delcourt )
Experimental Growth in Anin^als 247
Bone repair has been studied in rabbits. i" The tibia was cut
transversely, without damaging the periosteum otherwise than locally.
Mitoses were coimted from day to day, the animals being killed 9 to
10 hours alter 0.625 mg/kg of colchicine. The amplification of the
mitotic changes made estimations of relative growth far easier than
in control animals (Fig. 9.16).
PERIOSTEUM
. , ENDOSTEUM
. ^ ENDOTHELIAL CELLS UPERlOSTEUM
HIST?OCYTES J I ENDOSTEUM
days:
Fig. 9.16 — Repartition of mitoses during bone repair, studied after injection of colchicine.
(After Borghetti and Parini')
These few papers have studied only some limited aspects of heal-
ing and inflammatory reactions. Here again, large fields remain open
for investigation, and it is surprising that more work has not been
completed.
9.7: The Action of Chemicals on Mitotic Growth
Few papers have been published in this section, a surprising fact,
for colchicine could no doubt help in the study of many substances
affecting growth. In work on vitamins, for instance, many experi-
ments could be imagined. Some results with folic acid antagonists
will be mentioned in Chapter 10.
The possibilities of finding new facts is illustrated by the follow-
ing experiments: Young rats were intoxicated with carbon tetrachlo-
ride and studied at various intervals by a routine (olchicine technique
(Fig. 9.17). Arrested mitoses were observed in the liver cells and in
248
Colchicine
Kupffer cells
. , Liver cells
, .Adrenal cortax
, Hypophysis,anter. lobe
O 49 21 23 57
hours
Fig. 9.17 — Mitoses in liver and endocrine glands during experimental carbon tetrachlo-
ride poisoning, detected by the colchicine-method. (After Cavallero' )
the Kuptter cells in relation with the progressive cirrhotic changes
on the liver. No mitoses were observed in bile ducts, though the
number of these apparently increased.''"^ After 15 inhalations of car-
bon tetrachloride, an increased number of reticuloendothelial mitoses
could be observed in the spleen. A systematic study of the endocrine
glands revealed evidence of mitotic stimulation in the adrenal cortex,
the pituitary, and later, the adrenal medullary zone. These divisions
do not appear to be related to local damage, and may be an evidence
of a pituitary stimulus arising from "stress" (cf. Chapter 7) .
Some work on the mitotic stinudation in the thyroid of rats in-
jected with thiouracil may be mentioned here."*^-^^ -phe stimulus
lor cell division is not, however, the chemical itself, but the secretion
of the thyrotropic hormone by the pituitary, as mentioned in Sub-
section 9.4-1. Colchicine has also helped to study, in experiments
of this type, the mitotic changes which take jjlace in the pituitary. i''
Results obtained in young rabbits on the influence of thyroidec-
tomy and thiouracil on healing of cornea wounds are important to
consider under this heading, for they throw light on some difficulties
of interpretation.45 Doses of 5 mg/kg of colchicine were injected 4
hours before killing the animals. The results are summarized in
Experimental Growth in Animals 249
Table 9.11. It is evident that the mitotic index is more depressed
by thiouracil than by thyroidectomy, but it seems surprising that
this fact is not at all noticeable without colchicine, thiouracil-injected
animals having a slightly higher mitotic count than the controls. The
authors think that the count alter thiouracil results from a double
cllect, i.e., a decrease of the mitotic rate, which would decrease the
mitotic index, and a simultaneous lengthening of the duration of
mitosis, which would have the opposite effect.
4.
TABLE 9.11
Corneal Mitotic Counts in a Rabbit
(After Fleischmann and Ereckler^^)
Without
Colchicine
With
Colchicine
Controls
92 ± 35
100 ± 17
393 =fc 59
Thiouracil-treated
Xhvroidectomizcd
168 ± 42
228 ± 41
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Experimental Growth in Animals 251
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Doctoral Dissertation. Universitv of Chicago Library. 1940. .\ study of the
carlv effects of androgenous substances in the rat bv the aid of colchicine.
Jour. Exp. Zool. 89:133-66. 1942.
30 BiRRiLL. M. W., AND GREENE, R. R. Androgen production during jnegnancv
and lactation in the rat. Anat. Rec. 83:209-28. 1942.
31 C\RNOT. P., AND May, R. M. La regeneration du rein chez le rat etudiee an
nun en de la colchicine. C. R. Soc. Biol. Paris. 128:641-43. 1938.
32. Castelnuovo, G., and Freud. J. 'Mitogenese dans IV-pithclium \aginal des rats.
Arch. Int. Pharm. Ther. 61:491-93. 1939.
33. Ca\allero. C. £tude de la cirrhose experimentale par le tetrachlorure de car-
lione a I'aide de la reaction stathmocinetique icolchicinique) de Dustin.
Arch. Int. Med. Exp. 14:1-14. 1939. Reactions hormonales an cours de I'intoxi-
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par la niethode stathmocinetique (colchicinique) de Dustin. .Arch. Int. Med.
Exp. 14:15-22. 1939. Les glandes endocrines an cours de la grossesse. £tade
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de Dustin. .\rch. Int. Med. Exp. 11:123-35. 1939. .Application de la methode
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34. , and Pellegrini. G. F. L'effetto colchicinico nel "rene endocrino" di
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nelle cariocinesi provocate sperimcntalmente. .\rcli. Ital. .\nat. Emlirvol. 47:
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36. Delcoirt. R. fitude de la regeneration iles nerfs peripheriques par la reaction
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37. Desclin. L. .\ propos de l'action androgenique de la progesterone. C. R. .Soc.
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38. Dornfeld, E. J.. AND Berriav, J. H. Stimulation of mitoses in the germinal
cpiiliclium of rat o\aries b\ intr;icapsular injections. Anat. Rec. 109:129-38.
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39. Dirschel, W., -and Kropp, K. \'itamine und Hormone. 5:280. 1944.
40. . ZiLLiKEN. F. W., and Kropp, K. Colchicin-Mitosen Test an den A'esi-
culardriisen der kastrierte Maus. 11. Die Spe/ifitat des Testes. Biochem. Z.
318:4.34-61. 1948.
41. DisTiN. A. P. La colchicine, reaciit tie limminence caryocinetique. .\rch.
Portugaises Sci. Biol. 5:38-43. 1936. Etude de I'hypertrophie compensatrice du
rein par la reaction stathmocinetique. Acta Unio Internal. Cancrum. 4:679-
83. 1939. Rechciches sur le mode d'action des poisons stathmociuctiques.
.Action de la colchicine sur rutcrus de lapine impubcie sensibilise par injection
prealable d'urine de femme enceinte. .Arch. Biol. 5 1: 1 1 1-87. 1943.
42. . and Chodkowski, K. Etude de la cicatrisation par la reaction colchi
cinique. Arch. Int. Med. Exp. 13:641-62. 1938.
43. , AND ZvLBERSZAc. S. Etude de Ihypertropiiie compensatrice du rein par
la reaction stathmocinetique. Bull. Acad. Med. Belg. Vie. Ser. 4:313-20. 1939.
41. Flkisciimann. W., and Kahn, S. Uber das Colchicin als Hilfsmittel beim
Studiiun hormonal bedingter Wachstumsvorgange. Biochem. Z. 296:374-82.
19.38. The use of colchicine in the assav of androgens. Endocrinology. 25:
798-800. 1939.
252 Colchicine
45. Fleischmann, W., and Brfckler, I. A. Mitotic and wound-healing activities
of the corneal epithelium in thiouiacil treated and thyroidectomized rats.
Endocrinology. 41:266-68. 1947.
46. Gatz, a. J. The cellular changes induced in the testes of the alhino rat by
artificial cryptorchidism aided by the arrest of mitosis with colchicin. Anat.
Rec. 70:Suppl. 1:87. 1937.
47. GiNESTE^ D. J. Recherches sur la regeneration des elements de la glande cor-
tico-surrenale par la methode colchicini([ue. Action de di\ers facteins. C. R.
Soc. Biol. Paris. 140:221-22. 1946.
48. Granel. F. La sensibilite de Tepoophore a la testosterone. Reaction coklii-
cinique. C. R. Soc. Biol. Paris. 131:1255-56. 1939.
49. Gregoirf^ C. Recherches sur les relations entre thymus et surrenales. II. Les
reactions des celhdes du reticuliun epithelial thvmique a I'ablation des sur-
renales. Arch. Int. Pharmacodyn. 67:446-63. 1942. Sur le mecanisme de
I'atrophie thvmitjue declanchee par des hormones sexuelles. Arch. Int. Pharma-
cod)n. 70:45-77. 1945.
50. GiJTHFRT, H. Der Einfluss \on Hypophysenvorderlappenextracten und Col-
chicin auf Kerngrosse und Kernteilung in der Schilddriise. \'irchous .\rch.
307:37-70. 1940. Die Einfluss von Hvpophvsenvorderlappenextracten und
Colchicin auf die Langeihanschen Inseln des P;inkrcas. \'ircho\\s Arch. 307:
175-99. 1940.
51. Herlant, M. Influence du thiouracyl sur I'hypertrophie compensa trice du
rein. Bull. Acad. Rov. Belg. Classe. Sci. 5e Ser. 33:567-76. 1947. Activite
mitotique des celhdes icnales au coins de I'hvdronephrose unilaterale. Bull.
Acad. Roy. Med. Belg. 6e Ser. 13:315-30. 1948. Experimental hydronephrosis
studied by the colchicine method. Nature. 162:251-52. 1948.
52. Hunt, T. E. Mitotic activity in the anterior hypophysis of female rats of difler-
ent age groups and at different periods of the dav. Endocrinology. 32:334-39.
1943.
53. Jau.fr, J. W. Mitotic index of hyperplastic interstitial cells of the guinea-pig.
Proc. Soc. Exp. Biol, and Med. 39:281-83. 1938.
54. Kerr, T. Mitotic acti\ity in the female mouse pituitary. Jour. Exp. Biol.
20:74-78. 1943.
55. KuzELL, W. C, AND CuTTExn. W. C. Pituitary muotic changes after the ad-
ministration of oestrogen antl after o\ariectomy. Entlocrinolog\. 2(5:537-38.
1940.
56. Lahr, E. L., and Riddle. O. Proliferation of crop-sac epithelium in incid)ating
and in prolactin-injected pigeons stiulicd wiili tlic coUhicinc-method. .\mer.
Jour. Physiol. 123:611-19. 1938.
57. ^ , Alwell, L. H., and Riddle, O. Mitosis oi)ser\ed under coUhicine in
crop-sac tissue after subcutaneous and intramuscular injection of prolactin.
Arch. Int. Pharmacodyn. 65:278-82. 1941.
58. Leblond. C. P. Action de la prolactine sur le jaljot du pigeon mise en evidence
par I'arret des mitoses a I'aide de la colchicine. C. R. Assoc, des Anat. 32:241-
47. 1937.
59. , and Allen, E. Emphasis of the growth effect of prolactin on tiic crop
gland of the pigeon bv arrest of mitoses with colchicin. Endocrinology. 21:
455-60. 1937. '
60. , AND Stevens. C. E. The constant renewal of the intestinal epithelium
in the albino rat. Anat. Rec. 100:357-78. 1948.
61. Lettre, H. tJber Mitosegifte. Ergebn. Physiol. 46:379-452. 1950.
62. LuDFORD, R. }. The action of toxic substances upon the division of normal and
malignant cells in vitro and in vivo. ,\rch. Exp. Zcllforsch. 18:411-41. 1936.
63. LiJscMFR, M. Hemmt oder fordert Colchicin die Zellteilung im regenerierenden
Schwanz der Xenopus-Larve? Rev. Suisse Zool. 53:481-86. 1946. Die Heni-
mung den Regeneration durch Colchicin beim .Sch\vanz der Xenopus-Larve und
ihre entwicklungsphysiologische Wirkungsanalvse. Helv. Phvsiol. et Pharm.
Acta. 4:465-94. 1946.
64. Mai.in.skv, J., AND Lang, B. Hyperplasie du foie de rat apres hepatectomic
])arlicllc et influence des corps cohhicines sur celled. ('.. R. Soc. Biol. Paris.
145:609-12. 1951.
Experimental Growth in Animals 253
65. Manus, M. B. C. Zaadblaastest met l)ehulp van colchicine. Xederl. 1 ijdschr.
Gcneesk. 81:4128-29. Samenblasentest mit colchicin. Acta Biev. Neeil. i'nysiol.
7:173. 1937.
(i(). M< ruAii.. M. K., AM) W ii.BiR. K. M. Absence of potentiation of gonadotropin
and steroid function in mairmials by colchicine. Endocrinology. .'?'): 196-97.
1944.
67. Morato-.Manaro, J. Accion del acetato de deso\\corticosterone sobrc el utcro
de la coneja infantil estiidiato por el metodo colchicinico. Arch. .Soc. Biol.
Montevideo. 10:110-14. 1940. Accion de los androgenos sobre la vesicula
seminal de la rata, estudiata por el metodo colchicinico. .\rch. Soc. Biol.
Montevideo. 10:193-201. 1941.
68. Xathanson. I. T., Brlks, A. M., and Rawson. R. \V. Effect of testosterone
iMoiMonate upon thvroid and parathvroid glands in intact immature female
rat. Proc. Soc. Exp. Biol, and Med. 43:737-40. 1910.
(39. AND Effect of testosterone propionate upon the mitotic activity
of the adrenals in tiie intact immature female rat. Endocrinology. 29:397-401.
1941.
70. Paschkis. K. E., Cantarow. \.. Rakoff. A. E., and Rothenberg, M. S. Mitoses
stinudation in the tlnroitl gland induced by thiouracil. Endocrinology. 37:
133-35. 1945.
71. PoMFRAT. G. R. Mitotic actisitv in the piluitar\ of the whue rat follounig
castration. .\mer. Jour. Anat. 69:89-121. 1941.
72. PiNDEi.. M. P. Etude des reactions vaginales hormonales chez la femmc par la
nuthode colchicinique. .Ann. Endocrin. 2:659-64. 1950.
73. Roc.ERS, P. \'., AND Allen, E. Epithelial growth caused by stinuilation with
various smear methods as demonstrated bv mitotic stasis with colchicine. Endo-
crinology. 21:629-32. 1937.
71. SAcciHETL C, AND Blanchini, E. .Actiou directe de la S. T. H. sur les activites
de la moelle osseuse himiaine normale. Le Sang. 21:344-54. 1953.
75. ScHEiBLEV. C. H., AND HiGGiNs, G. M. Effect of administration of colchicine
after partial removal of the liver. Proc. Mayo Clin. 15:536. 1940.
76. S(HMn)T. I. G., and Hoffman, F. G. Proliferation and ovogenesis in the germi-
nal epitheliimi of the normal mature guinea-pig o\ary, as shown bv the col-
chicine technique. Amer. Jour. Anat. 68:263-72. 1941.
77. Schmidt. I. G. Mitotic proliferation in the ovarv of the normal mature guinea-
pig treated with colchicine. .\mer. Jour. .\nat. 71:24.5-70. 1942.
7S. Sfntein. p., and rucH.NrANN-Di'i'i.Essis, H. Mise en c\idence de mitoses dans
Ihypophvse du cobaye par Paction de la colchicine. \'ariation de lactivitc
divisionnelle a letat normal et apres injections d'hormone gonadotrope. Mont-
pellier Med. 23-24:16,3-64. 1943. Sur la presence des mitoses colchiciniques
dans le cloacjue et la prostate du Triton marbre (Molge mannorata Latr.)
soumis a Faction des hormones scxuelles et Inpojjlnsaires. Montpellier Med.
23-24:240-42. 1943. Sur quelques particularites d'action de la colchicine sur les
glandes endocrines du cobave injecte d'hormone gonadotrope. Montpellier
Med. 29-30:133-35. 1945.
79. Shorr. E., and Cohen, E. I'se of colchicine in detecting hormonal effects on
vaginal epitheliinn of menstriuiting and castrate women. Proc. Soc. E\p. Biol,
and Med. 46:330-35. 1941.
80. SrEiN, K. F., AND Foreman, I^. Effect of th\roid substances in the ovarian caj)-
side upon mitosis in the germinal epithelium. Anat. Rec. 105:643-56. 1949.
81. Stevens, C. E., DAoisr. R., and Leblond. C. P. Rate of synthesis of desow ri-
bonucleic acid and mitotic rate in li\er and iniestiue. Jour. Biol. Cliem. 202:
177-86. 1953.
82. Takfavaki, K. .Mitotic aclivitv in seminal vcside cells transplanted to female
mice. Jour. Fac. Sci. Tokyo Iniv. 5:291. 1941.
83. Teir. H. Cokhicine-tests for the purpose of ascertaining cell division regen-
erative conditions in the liver of the rat. Acat. Path. Microb. Scand. 25:45-51.
194S.
81. I tNNANT. R., AND I.iEBOW, A. A. I hc' .Ktioiis of coUliicinc and etli\l(arl)\ ianiine
on livsuc cultures. ^ ale Jour. Biol, and Med. 13:39-19. 1910.
254 Colchicine
So. Thales-Marhns. Test rapido i);iia o hormonio masculino: mitoses na
•genitalia accessoria. Biasil Med. 51:717-19. 1937. Test rapide de Ihornione
masculine: mitoses dans les genitalia accessoires de males castics. C. S. Soc.
Biol. Paris. 126:131-34. 1937^
86. Thornton, C. S. Ihe ettect of colchicine cm lind) legenciation in larval
Amblystoma. Jour. Exp. Zool. 92:281-93. 1943. Colchicine and limb regenera-
tion in lar\al Amblystoma. Anat. Rec. 84:512. 1942.
87. TisLOWiTZ, R. Uber die Latenzperiode von Testosterone luul Testosterone-
propionat. Kongressber. 16. Internat. Physiol. Kongr. 1938. The colchicine
test as a method for determining the lime of onset and the duration of action
of male substances. Endocrinologv. 25:749-53. 1939. The action of estrogens in
inducing mitoses in the muscle, connective tissue, and epithelium of the pros-
tate and seminal vesicle as determined by the colchicine techni(]uc. Anat. Rec.
75:265-74. 1939.
88. ToRO. E., AND Vadasz, J. Untersuchiuigen iiber die Wirkung \on Colchicin und
Corhormon in Geuebekulturen mit Hilfe von Filmaufnahmcn. Arch. Exp.
Zellforsch. 23:277-98. 1939.
89. Uelinger, E., Jadassohn, W., and Fierz, H. E. Mitoses occurring in the acan-
thosis produced by hormones. Jour. Invest. Derm. 4:331-35. 1941.
Verne. J., and Vilter, V. Etude de Taction de la colchicine sur les mitoses des
hinoblastes cultives in I'itro. Concentrations dites fortes. C. R. Soc. Biol. Paris.
133:618-21. 1940. Mccanisme d'action de la colchicine, employee en concentra-
tions faibles, sur revolution de la mitose dans les cultures de fibroblastes
in vitro. C. R. Soc. Biol. Paris. 133:621-24. 1940.
91. Williams, W. L., Stein, K. F., and Allen, E. Reaction of genital tissues of the
female mouse to the local application of colchicine. Yale Jour. Biol, and Med.
13:841-46. 19H.
92. Wolf, O. .Mitotic acti\itv of stinudalccl rat adrenals and spleen measured l)v
colchicin technic. Anat. Rec. 70:Suppl. 1:86. 1937. Mitotic activity of the
islands of Langerhans and paratlivroids of rats following piiuitarv extract and
colchicine injections. Biol. Bull. 75:377-78. 1938.
93. Worthington, R. \.. and Allen, E. Growth of genital tissues in response to
estrone as studied by the colchicine technicpie. Vale Jour. Biol, and Med.
12:137-53. 1939.
90
CHAPTER 10
Neoplastic Growths
— In Animals and Plants
10.1: Colchicine in Cancer Research
Mitotic changes iiidiiccd ])y colchicine in a Crocker sarcoma of
the mouse were described by Proiessor A. P. Uustin, Sr., in 1934-^
(Fig. 10.1) . This now recognized classic research marked a new trend
in the study of cancer. At that time, the toll of life from bacterial
diseases ^\■as declining as a result of the use of the sulfa drugs, and
the relative incidence of cancer was gaining the impressive figure it
has reached today in civilized countries. It is not surprising that the
discovery of a specific action upon mitosis, the metaphase arrest, at-
tracted Avide attention. This research made clear for the first time
the possibility of arresting cell division with chemicals acting specifi-
cally. Such a relation had, it is true, been demonstrated several years
earlier in the Brussels laboratory,-^' -■' but colchicine, being such a
unique chemical, helped greatly in convincing research men of the
possibility of cancer chemotherajn'. A. P. Dustin, Sr., grasped im-
mediately the potentiality of this new approach.-^ His 1934 publica-
tion anti the demonstration given by his school at the Second Inter-
national Cancer Congress, held in Brussels in 1936, markctl a turning
point and led many people to woik on neoplastic gro^vth.
It is quite remarkable that colchicine, like other plant substances
used in popular medicine, such as chelidonine,-*^ may have been uti-
lized in cancer treatment long before that date. At least two French
textbooks of pharmacology^*^' •''" mention that Dominici, the great
French hematologist and radiotherapist who died in 1919, had ob-
served favorable effects of colchicine in cancerous patients who had
received X-ray Avhile under treatment for gout. \We have been unable
so far to discover the original text of Dominici's observation and his
publication. 1 he idea of some interrelation between gout and cancer
was mentioned in 1920 in Belgiiun by A. P. Dustin, Sr.-^ Again,
[255]
« *'
•
. ^ • , . , * • . ♦ • ^
■.» •• •• • » ■ -
• •-•• ■*... •
• >
." • ' ' ^*^' ■• •-' • •
• * . . - . • • , • • • v.- .•- ' • ,
• •. ■•'• • -; A ••:„..-... • ■...•••.
■ * - _ _ ■
• ".••,• ".-•'• '"»■■* ' * ■.,♦.»
• * . . . • , .
. . -- ♦ . » • -
Fig. 10.1 — Action of colchicine on the Crocker sarcoma in a mouse. All the nuclei which
appear as black dots are in a condition of arrested metaphase of the "ball type, with
clumping and progressive fusion of chromosomes. There is no hemorrhagic effect m
this area. Nuclear staining: iron-hematoxylin. (From an original preparation from the
collections of the Department of Pathology, Brussels University. A. P. Dustin, 1934"')
•. ' •■• •'
*
■•- ' J- • .
•'
.* .*!
♦ t - \
• •
*
Neoplastic Growths 257
in liie fii-,t report ol Iav()ra1)lc effects of colchicine on tninors in mice
and in one epithelial cancer in a dog,^ made in 19:^5, the author,
E. C Amoroso, did noi nuntion anv of the work done in Brussels,
hut -writes:
Follo\\ing on some earlier ol)scr\ations (unpul)lislied. 1927) \\h\d\ I made
with the'late Prof. M. R. J. Hayes on the beneficial ettects of deep X-rav
thera])v on neoplasms in patients suffering from acute attacks of gout, wliuh
u-ere ix-ing treated with cokhicum. a series of experiments was . . . jihmned. *
These results are only known in a preliminary lorm, and no detailed
paper appeared later. They may have influenced one re}Jort on favor-
ahle restdts of the treatment by colchicine of a malignant growth in
a mare.''' I'he fiist report in English on the action of colchicine on
normal and malignant cells in tissue cultures, which was pid)Hshed
in 19o().^" ackno^\•ledges these references and claims not to have been
infltienced by the work done in Brussels.-^' ^^ Jt is. however, surpris-
ing that this paper also describes the effects of arsenical derivatives
on the spindle, for this was discovered in Bclgiiun in 1929 and had
onlv received scant attention. '''"• -''
Manv experiments and also j^ractical applications of colchicine in
experimental and lunnan tumors weie made; this subject has been
reviewed recently.^' The concltrsion was reached that colchicine is
no cure ior cancer. However, nuich work is now in progress^"' -- m
the search for chemicals, more or less related to colchicine, with a
lower general toxicity and a more specific action against malignant
cells. The study of these will be described in the last chapter of this
book.
The disco\ery of colchicine heralded a greater search for mitotic
poisons, i.e., substances specifically harndul to dividing cells. This
subject has become so extensive that is more and more diilicuk. even
for specialized workers, to review it all.
It has been shown in previous chapters what a unic[ue substance
colchicine is as a tool for detecting cellular proliferation. It could
be used as such for the study of carcinogenesis, on the one hand, and
malignant groAvth on the other. A surprisingly limited amount of
research has been conducted in this direction.-'^' •"'-■ ♦'^ However, in-
teresting results have been obtained recently with the use of colchi-
cine in vitro. This work demonstrates the quite unexpected fact that,
apparently, cells from acute leukemia, a disease in which cellular
proliferation was always believed to be extremely rapid, grow much
more slowlv than the normal constituents of the human bone mar-
row.-
A section related to the j^roblem of plant overgrowths and tumors
is included in this chapter because some carefid work has been done
* E. C. Amoroso, -(.oldiicine and I imioui C.routli." Xnlinc, 135(1935) . \). lili<>.
258 Colchicine
in this field. The basic relationship bel^veen the action of colchicine
and abnormally proliferating plant cells remains unsolved. An in-
duced vascularization similar to that referred to in Chapter 4 may
be related to this problem, and would provide a promising new
approach.
The combined action of colchicine and X-irradiation on animal
and plant materials has been studied in several laboratories. No
decisive results appear to have been obtained. Ho^vever, some re-
cent research indicating the action of irradiation on metaphasic
chromosomes, shows that this work is ^vell A\orth reviewing.
All the studies on neoplastic cells point towards the same inescap-
able fact: Whereas colchicine, as a treatment for gout, may well have
been observed prior to 1934 to have some favorable action against
cancer, all the papers ^vhich connect both have been published since
1934. This clearly indicates the significance of the cytological work
published at that time by A. P. Dustin-^ and demonstrated at the
1936 Cancer Congress.
10.2: Experimental Study of Neoplastic Cells
Malignant cells, especially in animal tumors, often display "spon-
taneous" mitotic abnormalities. These have been compared to those
induced by colchicine, and it has been suggested that the cells were
under the influence of some mitotic poison acting like colchicine. ^9
It has been suggested that this may be lactic acid.'-* However, these
spindle disturbances often appear to be the consequence of more
deep-seated nuclear changes, closely related to the cause of malignancy
itself, and leading to chromosome breakages and rearrangements. In
early human carcinomas, however, it has been pointed out that the
spindle changes appeared first.^^ xhe behavior of such cells when
brought under the influence of colchicine is of great importance, for
it would be of value to determine whether a specific destruction of
malignant cells by a spindle poison is possible.
The effect of colchicine on cancerous growths has been studied
either by injecting the animals with the drug, or by explanting the
abnormal cells in vitro and using the methods of tissue culture. This
last procedure has been followed with a mammary carcinoma''- and
a sarcomai* of the mouse, and with Ehrlich mouse carcinoma grooving
as an "ascites tumor" in the abdominal cavity.^"- ''^ Concentrations of
100 X iO'''M to 1.25 X 10-*^ M inhibit outgrowth from the explants
and arrest cell divisions. This efTect is still evident on carcinoma
cells at a concentration of 0.5 X lO'^A/. In culture containing ex-
plants of both tumor and embryonic kidney, the latter showed the
greatest cellular destruction following the mitotic arrest. Differences
of sensitivity between various strains of carcinomas were found, \vhile
the Crocker sarcoma showed fewer arrested metaphases.^'
Neoplastic Growths 259
The ascites tumor enables colchicine to be brought in direct con-
tact with the malignant cells in vivo. The tumor cells float freely in
the fluid which gradually fdls the abdominal cavity. It is possible,
simply bv pipetting cells hom the abdomen, to examine all the
changes brought about by the injection of colchicine. i^- ^"' ''•■ Growth
curves of the tumor indicate that on the average each cell divides
every 2 to 2i/o days. After an injection of colchicine, the jjcrcentage
of mitotic cells rises in 9i/o hours from 1.2 to 14.2. Thirteen hours
after injection, it reaches 18.2, and falls to 2.0 after 48 hours. From
these figures, the normal average duration of mitosis can be calcu-
lated as follows: 1.2x9.5/14.2^1.2x13/18.2 = 0.8 hours, or 48
minutes.
Scattered groups of chromosomes and micronuclei are observed
in the colchicine-treated tumor cells. ^i- •'' Resting (intermitotic)
nuclei are also affected; their chromatin network becomes coarser. ^^
In sarcoma-bearing mice, a series of experiments was carried out to
determine whether administration of colchicine had any effect upon
subsecjuent growth of the tumor cultivated in vitro.^^ Clolchicine
(().()()4 to 0.06 mg.) was administered by subcutaneous or intravenous
injection, and fragments of sarcoma were removed for cultivation at
various intervals after treatment. The growth of tumor tissue in vitro,
obtained from an animal treated Avith colchicine, was inhibited to a
large extent. Colchicine arrested mitoses, both normal and neoplastic.
In human malignant growth, colchicine has been found useful
for the study of cellular multiplication. In 1 1 patients injected with
1.5 to 4 mg. subcutaneously or intramuscularly, modification of tumor
mitoses Avere observed. ^^ Four other patients did not show any re-
sponse, a fact which is not surprising, the dose being kept relatively
small by comparison with doses administered in animal work, because
of the great toxicity of colchicine in man. In one case of adenocarci-
noma of the bowel, the progressive increase of the mitotic index
could be followed by repeated biopsies. The control specimens had
an index of 2.6, which rose to 7.-^ Aac hours after colchicine and
reached 19.6 after 12 hours. This last biopsy demonstrated a con-
siderable increase of arrested mitoses. It is regrettable that, owing
partly to the too great danger of colchicine poisoning (cf. Chapter 7) ,
no further research of this type has been conducted. Now that new
and less toxic colchicine derivatives are available^o (Chapter 17), a
more thorough study of the rate of growth of human neoplasms may
be possible. This could then be compared with data on normal tis-
sues obtained by the same method.
Colchicine may yet be used on explanted human tissues, and it
is surprising that only iwo papers on tliat sul^ject can be recorded
up to now. In polycythemia vera, a disease in which the abnormal
number of red blood cells has often been considered closely related
260 Colchicine
to malignant growth, and which may end in leukemia, the increase
of metaphases of bone-marrow cells explanted /?? vitro in a solution
of colchicine was found not to differ from normal.'' The striking re-
sults obtained with marrow of patients with acute leukemia have
been mentioned in Section 10.1.^
10.3: Cancer Chemotherapy
It is evident that the data on the growth of neoplastic cells treated
with colchicine are meagre. Workers were quickly attracted by the
false idea of finding a cancer cure, and they injected colchicine into
animals bearing various timiors. Botanists, also, painted plant timiors
with colchicine. Neither were much interested in the fundamental
changes taking place. As a result, the cytological data are often in-
complete and only mention "cellular destruction," "nuclear frag-
mentation," or "tumor necrosis and hemorrhage." This emphasis on
the gross changes in animal tumors has led to a neglect of the funda-
mental problem which is at the base of any cancer chemotherapy:
Are malignant cells more severely damaged than normal ones? This
is of great importance with a chemical like colchicine which affects
all types of mitoses. The appearance of large zones of hemorrhage
in tumors treated with colchicine has led some workers^' ■*'^' '^'^ to the
conclusion that this is the main action of the drug and the only
possibility of obtaining a destruction of the neoplastic growth. This
problem will be discussed first, though it is quite evident to all en-
gaged in cancer chemotherapy that a drug the main action of which
would be hemorrhagic destruction, is of no use in medicine.
lo.^-i: The hemorrhagic effect and metabolic changes. Many re-
ports on experimental tumors in mammals, whether induced by car-
cinogens or grafted, showed that colchicine was unable to prevent the
neoplastic giowth.^^. c6. is, os j^ the sarcoma 180 of the rat even the
largest tolerated doses were unable to arrest all mitoses at meta-
phase.i^ From the unaffected ana- and telophases the malignant
growth resumed its activity once colchicine was discontinued.
On the other hand, the metabolic changes in tumors treated by
colchicine were being investigated. In grafted tumors in rats the
metabolism, measured /'// vitro, was found to decrease. At the same
time, the ascorbic acid content of the tumors was considerably lowered,
and large zones of hemorrhage were seen.^ This last change was be-
lieved to play a great part in the regression of the tumors. Similar
changes could be observed after the injection of Bacillus typhosus
extracts. It was not reported that these bacterial products induced
any nuclear or mitotic change.^ Similar hemorrhages were also noticed
in other grafted carcinomas, in spontaneous mammary tumors, and
in methylcholanthrene-induced tumors of mice. They were most ap-
parent 18 to 20 hours after colchicine. The spontaneous tumors ap-
Neoplastic Growths 261
peared the most resistant towards this new "colchicine-efTcct." A
parallel decrease in ascorbic acid content, respiration, and glycolysis
was obscr\'ed.^
The significance of these hemorrhages, which appear only with
sublethal doses,- is not clear. It has been suggested that mitotic
poisoning of the endothelial cells of the tumor capillary bed (cf.
Chapter 9) may play an important part.**" Escherichia coli filtrates
have similar hemorrhagic proj^erties, and add their eftect to those of
colchicine, but the over-all toxicity is also increased. The polysac-
charide extracted from Serrdtia inarcescens is interesting, for it also
produces hemorrhages in timiors and has been shown to interfere
with cell division.""
Tumors treated with colchicine become quite fragile. In the Flex-
ner-Jobling carcinoma of rats the injection of distilled water in the
tumor has a destructive action 15 hours after colchicine. These ex-
periments, which were done on a great number of animals, have been
reported only in a short note.'^*'
In a recent review,'*' the effects of colchicine on 17 different strains
of tumors and 49 spontaneous mammary carcinomas in mice have
been sunnnarized. AVliile the effects vary according to age, genetic
constitution, rate of tumor growth, toxicity of colchicine, and histo-
logical structure, the hemorrhagic effect was considered to be the main
factor in tumor regression. In highly cellular and soft tumors grow-
ing on RIII mice, complete cures were reported. Regression is ob-
tained only by doses very close to the lethal one and far above those
that simply arrest mitosis. Soft and rapidly growing tumors respond
well, while slowly growing and fibrous tumors are resistant.
This conclusion applies only to the experience of one group of
authors, and instances can be found of malignant growths which re-
spond to colchicine without any hemorrhage. Such is the case of a
benzopyrene-induced sarcoma (HL tumor) in albino rats." The re-
gression appeared here to bear some relation to a decrease in the
pyrophosphatase of the neoplasm, while liver and kidney pyrophos-
phatase were not affected.
Further exam])les will be given of favorable effects unrelated to
hemorrhage, which is clearly related to verv toxic doses and is of no
practical interest in chemotherapy. The hemorrhagic effect is one
more of the riddles of colchicine, but to insist too much on it as the
main mode of action of the drug on tumors is to discourage any
further work on nontoxic derivati\es Avith mitosis-arresting jiroper-
ties.
70.5-2.- Auinitil tinnors. One of the most striking effects of colchi-
cine noticed in the first experiments on animals44 was the destruction
of lymphoid and thymic cells following the metaphase arrest of their
mitoses. This action is certainly related to the general toxicitv of
262 Colchicine
colchicine and to a "stress" releasing cortisone and other lymphocyte-
damaging hormones from the adrenals (Chapter 7) . It led to the idea
of treating lymphoid iimiors in C3H strain mice with colchicine. ^-^
The malignant lynijjhocytes, like those of thymus and spleen, under-
went a pycnotic destruction after injections of 0.025 mg. repeated
every third day. The average duration of life of the animals after
the tumors had been grafted was significantly prolonged. In controls
it was 31.5 days; in those injected with colchicine, 50.5 days. Histo-
logical study sho^ved that the reticidum cells and some of the neo-
plastic hiiiphocytes escaped destruction, and resumed growth when
the injections were interrupted. In another series of experiments'^ a
permanent regression of the 6C3HED lymphosarcoma (in C3H mice)
was obtained by daily injections of 0.5 to 0.75 mg/kg after the tumor
had reached a diameter of about 1 cm. The animals cured from the
grafted neoplasm became immune to further graftings of the same
tumor. No similar effects were observed after cortisone. This ap-
pears to rule out the jjossibility of colchicine acting on tumor growth
by the indirect pathway of the pituitary-adrenal system. In these
lymphoid tumors, colchicine destroyed the cells and their mitoses,
and no mention is made of hemorrhage playing any part in the
chemotherapeutic action.^''' ^
In epithelial tumors the results vary considerably. For instance,
the Brown-Pearce carcinoma of the rabbit showed some increase in
the percentage of metaphases after 1 mg/kg of colchicine. The re-
sponse was, however, so unpredictable as not to warrant further
study.-"'' Some authors have reported an important prolongation of
life in mice bearing the Ehrlich carcinoma,^" ^^'hile in various other
timiors of mice and rats no such jjrolongation could be claimed. "'-• *'^- '^^
Studies on virus-induced malignant growths in fowl are of in-
terest. In animals grafted with the Rous sarcoma, doses capable of
arresting the testicular mitoses did not modify the tumor growth.
Larger doses killed the lairds. ^*'' In avian erythroblastosis, a dose of 1
mg/kg injected over a jjeriod of five days did not alter the evolution
of the malignant growth of blood cells.''- Some inhibition of the
growth of the Rous viius has, however, been observed, ^^ especially
when this is cultivated on the chorioallantoic membrane of eggs.
It appears that considerable variations in sensitivity towards colchi-
cine exist from one tumor to another,^^' '*'^ and that the toxicity of
the drug has often limited its use. Further work should clearly be
aimed at many different tiunors and at the use of the new colchicine
derivatives, which are discussed in Chapter 17.
/0.3-5; The Sliope pajjilhjjna in rahhits. This is a virus-induced
tumor, which is very widespread in this species. A closely related
virus, myxomatosis, has even been advocated as a tool for the ex-
termination of rabbits in Australia and other countries. This tumor
Neoplastic Growths 263
is benignant, but under the influence of carcinogens it may become
malignant. A series of papers has been devoted to its possible cure
bv colchicine."^'- ^^- •'^■' This may be obtained after injections of colchi-
cine in the animal."'' \\ hile one is always limited by the toxicity re-
actions, it was found that the local application of a colchicine oint-
ment to the skin tumors could definitly cure a great number of
animals. A remarkable and rather perturbing fact was noticed.''-' If
both ears of a rabbit are inoculated with the Shope virus, and a cure
is obtained on one side with the colchicine ointment, the tumors of
the other ear become more liable to undergo a malignant change into
carcinomas. The conclusions of these papers are most important for
they opened a new pathAvay for the use of colchicine in human
patiiology.'^^ To quote: ". . . these experimental data suggest the
possibility of using colchicine in human therapeutics . . . by local
applications, to precancerous lesions or benignant skin tumors." *
The results obtained in tumor-bearing patients will now be discussed.
10.4: Chemotherapy of Human Neoplasms
The suggestion of a local application of colchicine, enabling a
strong concentration to act upon abnormal cells without general
toxicity symptoms, was taken up in 1941. Colchicine, either in a
paste or an injection as an oily solution, was applied to metastatic
nodules of epithelial cancers.*"'" The volume of the treated metastases
clearly decreased.
However, it appeared more logical to begin Avith benign growths
of the skin. Some of these, such as the venereal papillomas or warts,
may be very extensive, and their treatment by usual methods involves
large surgical excisions. These are virus-induced growths, compar-
able to the papillomas of the rabbit. A colchicine-lanoline paste
(0.05 per cent) was applied twice daily to six of such cases.^i Re-
markable regressions were observed after several weeks of treatment.
The tumor became more and more resistant to colchicine, and ni
the last stages, had to be removed surgically. 1 his was facilitated con-
siderably by the regression of the size and extension of the tumor.
Colchicine-mitoses can be found in great numbers in biopsies of
treated papillomas.*^ It is quite evident that the regression of the
neoplastic growth is a simple consequence of the arrest of its cell
divisions. No hemorrhage is to be seen. It appears also that the mito-
ses of normal skin are less modified by the treatment, for there is no
.skin ulceration, and after the tumor has disappeared, the skin has a
normal aspect. ^^- ^
* A. Pevron. G. Poumeaii-Delillc, ;m<l R. LaFay. I.a tiimeur de Shope du
lapin et sa sterilisation par la cokliiciiif. Hull. Assoc. Franc, tlude Cancer 26:633.
1<)37.
264 Colchicine
Colchicine has now been replaced in the treatment of such warts
and papillomas by another substance of plant origin, podophyllin, a
resin extracted from Podophyllum sp.^''' This substance is a complex
mixtine of chemicals, the most active being podophyllotoxin and the
peltatins. 1 hese are, quite like colchicine, mitotic poisons, and they
interfere mainly with the spindle formation. ""^ The use of the resin
of podophyll was known in the United States as a popular medical
remedy; it is remarkable that another plant, known in Europe to
have good effect on warts, Chelidonium ma jus, contains an alkaloid,
chelidonine, which has also been demonstrated to inhibit spindle
formation in tissue cultures.^" Chelidonine was advocated for the
treatment of cancer at the end of the nineteenth century. 20
These empirical remedies, probably centuries old, are most in-
teresting, and it may be recalled that Dioscorides recommended the
use of Ephemeron, a species containing colchicine, in the treatment
of some tumors. Colchicine-paste has also jiroved to be successfid in
the treatment of some skin cancers of the basal-cell type.^^- i" In
ulcerating mammary tumors, interesting results have been obtained.
A striking fact is that here again the growth of normal skin appears
to be less altered than that of the neoplasm. ^^
In hiunan malignant tmnors, the effect of colchicine has so far
proved quite disappointing, and from the reports available, it is
difficult to understand how it cotdd have been observed to be of any
benefit to cancerous patients. ^ It may arrest tumor mitoses in man,^-^
but this effect is never powerful enough to stop the malignant growth.
The toxicity of colchicine is redoubtable. Even in a series of four
patients, where some favorable eftects were noticed, one case of severe
leukopenia was noted, and another patient lost almost all his hair.*^^
In another series, two out of three patients died of agranidocytosis,
which was probably the consequence of mitotic inhibition in the
bone marrow. 1-
In severe neoplastic blood diseases, colchicine has also been tried
by a few investigators. In lymphoid tumors the results were of no
practical interest,^'^ and intramedullary injections did not change the
fatal course of acute leukemia.-*^ In chronic myeloid leukemia, a
disease which is known to respond favorably to many mitotic poisons,
more promising results have been recorded. In one patient, who
received 0.5 mg. of colchicine three times and later twice daily, the
leukocyte count was found to fall from 110,000 to 2400. This im-
provement was only of short diuation.^'^' ^^
These data, which are very sketchy, may seem to ride out colchi-
cine for the treatment of cancer in man. However, recent develop-
ments are more promising, though still in an experimental stage. In
Hodgkin's disease, a neoplastic condition affecting mainly the lymph-
Neoplastic Growths 265
oid tissue, excellent effects have been described. Colchicine ad-
ministered intravenously produced a sharp tall in temperature, which
in these patients is oltcn very high."-'' Substances chemically close to
colchicine but less toxic arc being tested; "methyl-colchicine" has
tjuite recenth j)i(ned to be ol \aluc in the management of cases of
chronic myeloid leukemia/'^ It is quite evident that it is too early to
draw a conclusion about the future o£ colchicine in cancer therapy,
and that far more Avork remains to be done.
10.5: A Tool for the Study of Cancer Chemotherapy
The mitotic stasis resulting from spindle destruction can make
visible small changes in the mitotic rate which would pass unnoticed
in microscopic sections (cf. Chapter 9) . Some promising work has
been initiated in this field. Urethane, at a dose of 0.5 gm/day, has
been demonstrated not to modify the number of mitoses, studied with
the colchicine method, in the Walker rat carcinoma 256.2' Azagua-
nine,**^- ''' on the other liand, has been proved to be one of the most
remarkable chemotherapeutic substances. This antagonist of guanine
and adenine can be demonstrated not to affect normal mitoses, while
strongly decreasing those of the BroAsn-Pearce carcinoma. This tumor
Avas studied Avhile grafted in the anterior chamber of the guinea pig's
eye.*"' This type of mitotic depression is made more evident by the use
of colchicine.
Another type of experiment was planned for the study of an anti-
folic drug, aminoj^terine. Ihis substance is widelv used in the treat-
ment of acute leukemia, \\4ien large doses are injected into mice,
the cell divisions in the intestine do not take place any more for about
48 hours. During this period of mitotic inhibition, cellular and
nuclear groAvth are not impaired, and very large nuclei are formed.
When these divide again, the mitoses are of excejnional size. Colchi-
cine Avas used as a tool to arrest these mitoses and to provide a greater
number for study, as a consequence of the mitotic stasis. Also, the
shortening of the chromosomes made their counting easier, and ball
metaphases provided excellent material for photometric measure-
ments. These experiments indicated that the increase in nuclear size
was neither the result of polyploidv nor of polyteny.-''
10.6: Plant Tumors
Whatever may be the exact relation between tumors in animals —
and. ill particular, cancerous growths — and the Aarious types of gall
formations induced in plants by Bacillus tumefacieus, insects, etc., it
is interesting to compare the effects obtained with colchicine with
those described for animal neoplasms. In a series of experiments on
Lycopersiciim escxdenlum inoculated with B. tumejaciens, a 1:10,000
266 Colchicine
solution of colchicine, locally applied, decreased the number and the
volume of the induced tumors without disturbing the growth of the
plant itself. '^^ An extensive series of experiments was started shortly
after on seven species.^" By injecting colchicine in plants at the time
of infection by B. (urnefaciens, tumor growth was only prevented in
9 out of 61 plants. On the contrary, to arrest the growth of tumors
and to destroy them later were possible in most cases by several tech-
niques of application of the alkaloid. In Tagetes patula, these tumors,
after daily paintings with a 1 per cent colchicine solution, stop grow-
ing after 7 days and then progressively decrease and die. The princi-
pal microscopic effect is a great enlargement of the tumor cells, four
or five of the colchicinized ones occupying the area of 30 normal
ones. This enlargement is the most visible with rather concentrated
solutions of colchicine (up to 0.1 per cent) . The smallest cells are
64-ploid (1536 chromosomes), the larger 1014-ploid (24,500 chromo-
somes) . Some nuclei have irregular shapes and some cells are multi-
nucleated. Cellular death is a direct consequence of the extreme de-
gree of polyploidy which is reached, the giant cells becoming at some
stage quite unable to divide any further. There is no effect on the bac-
terial growth. !•' Similar results have been obtained in Pelargonium
and Riciiius.'- It was supposed that the death of the tumor was the
consequence of its isolation by a layer of cork."-
Though animal cells, through failure of centromere division, can-
not usually go through repeated colchicine mitoses, it is thought-
provoking, however, to compare these effects with those of X-rays in
animal tumors. Cellular proliferation after X-ray therapy is also
stopped when cells become gigantic and highly polyploid through
repeated abnormal mitoses.
10.7: Colchicine and X-rays Associated
When the first work on colchicine and tumors was done in 1934,
ionizing radiations were supposed to have the most harmful effects on
mitotic chromosomes, and it was expected that accunudating such a
great number of divisions, as seen in sarcomas for instance, would
increase the radiosensitivity of the tumors (Fig. 10.1). Most recent
work, however, shows that the sensitive period of the mitotic cycle
is before prophase, and thus, accunudating metaphases could not be
expected to increase radiosensitivity since the rate of prophases is not
disturbed.^'' This is confirmed by most work on colchicine and tumors,
whether in animals or in plants.
lo.-j-i: Animal tumors. X-rays were observed to be considerably
more efficient in killing in vitro tumor cells when these had been
previously treated by colchicine (Flexner-Jobling grafted carcinoma
of the rat) .^'^ Here the test used was the grafting of fragments of
Neoplastic Growths 267
imiioi, the number of "takes" being decreased. Colchicine (1 nig/kg)
administered 15 hours before irradiation (188 r. twice weekly) in-
creased also the effects of X-rays as measiued by the size of tumors in
surviving animals. No similar increase in mice and rats, even with
large doses of colchicine, was found.^- In the Yale carcinoma of the
mouse, 2 mg/kg produced extensive necrosis and hemorrhage, but a
border of viable tissue was always seen to persist.^^ The addition of
2500 r. produced only a slightly higher rate of curability "not signifi-
cant to warrant further investigation." •^- In the Ehrlich carcinoma,
colchicine was injected every day (5 mg.) and 260 to .^00 r. delivered. i'
Some results seemed to indicate an improvement of the colchicine
action by X-rays, which alone are not effective. However, if the dose
of irradiation was increased, the life span of the colchicinized mice
became shorter than the nontreated controls. From Table 9.2, it is
clear that no significant improvement is obtained by combining the
two treatments. It must, however, be pointed out that this is a radio-
resistant tumor, not well suited for such studies.
One paper mentions that in a case of gastric carcinoma, two metas-
tases were irradiated with the same dose of X-rays, while one was
injected with colchicine; the post-mortem disclosed that the latter was
severely necrotic, a fact which is not surprising in view of a large
local injection of colchicine and which does not demonstrate a true
synergism between the two agents. ^'^
The action of colchicine on human tumors has been followed by
nndtiple biopsies.^-'' The patients were injected intramuscidarly with
2 mg. of colchicine. An increase of the metaphase percentage was
noted, as well as some hemorrhage and cells with highly polyploid
mulei. These data, which are supposed to open the way towards a
treatment with colchicine and X-ray combined, were not examined
critically, and the variations observed may be entirely fortuitous.
A series of clinical rej)orts have been published-^^, 49, 43 about
colchicine increasing the effectiveness of X-rays, but these results are
not statistically valid and cannot be accepted without finther re-
search. Colchicine was used for some time as a routine in irradiated
cancerous patients at the Cancer Hospital, Brussels, with no convincing
results (unpublished) .
/0.7-2; Plant overgroivtlts. In plants, experimental work'^^ brings
some significant detailed cytological data on the action of irradiation
on mitoses previously arrested by colchicine, which ai:)pear to be ab-
normally fragile. Root tips of Fisiim satimnn and Allium cejxi were
dipped into a 1:2000 sohuion of the alkaloid, and irradiated (3500 r.
in one minute) at various intervals later. Prophases were observed
to be quite resistant, but the c-metaphases were very rapidly modified,
the chromosomes clumping together and later undergoing katachro-
268 Colchicine
matic changes into apparently normal restitution nuclei (6 hours
after irradiation) . The nuclear membrane may give some protection
to the prophasic chromosomes.
The results of these changes on the growth of the root tips and
of the leaves of bvdbs of Allium cepa have been studied.^- Exposure
to 0.01 per cent solutions of colchicine induces the well-known root
tip swelling, the so-called c-tumors, and when the plants are replaced
in water, growth is resumed. If the root tips are irradiated with 900
or 1500 r. after 48 hours of colchicine, growth is arrested and leaf
development is strongly impaired. These effects are greater than those
obtained by irradiation alone. The action of X-rays appears to be
independent of the nuclear division stage. After 48 hours of colchi-
cine, "some non-recognizable toxic effects in the cell . . . sensitize it to
irradiation." * The same author has published detailed results of
investigations on the combined action of colchicine and X-irradiation
on onion root tips.^'^ It appears evident that the two actions add
their effects, but the mechanism is not clear, and does not seem to
be related to an increase of mitotic cells at the time of irradiation.
For instance, the 48-hour colchicine bulbs are more vulnerable to X-
treatment, "even though the time of exposme occurred when the
number of dividing cells had passed the peak of metaphase arrest. "f
Irradiation by 900 r., which has only a temporary retarding effect on
growth, inhibits completely cellular multiplication and growth with-
out any immediate death of the tissues when the roots have been pre-
viously treated for 48 hours with a 0.01 per cent solution of colchi-
cine. A long exposiue to the alkaloid seems necessary, for, "while
colchicine causes analogous cytological changes at 6, 12, 18, 24 and
48 hours, the larger exposures induce some microscopically unrecog-
nizable alterations. This . . . arrests growth permanently and com-
pletely [with 1500 r.]"t The oiJiimum growth-inhibition effects
were observed after 1500 r. and a more than .^6 hours' exposure to
colchicine.
On the other hand, onion bulbs treated for 45 minutes in a 0.05
per cent solution of colchicine, then irradiated with 300 r. and re-
placed in the solution, showed less chromosome rearrangements than
controls, while the number of breakages was not appreciably altered.
It is supposed that the short colchicine treatment could not have in-
creased the metaphases. but impairment of the sjMudle function may
slow the movements of chromosomes. This would leave less oppor-
tunity for the broken ends to reunite into abnormal structures. ^^
* M. Levine, "The Action of Colchicine on Cell Division in Human Cancer,
Animal and Plant Tissues." Ann. N. Y. Acad. Sci., 51 (1951) , p. 1400.
j-Ibid., p. 1397.
% n>i(l.. p. 1399.
Neoplastic Growths 269
It is evident that work in this field is particularly difficult, because
the interpretation ol the results depends on the action of two agents,
each having a (oniplex nature. It has recently been shown that nieta-
jihase chromosomes could be singled out and destroyed in a beam of
neutrons^'' Modern cytological and radiobiological methods should
enable similar experiments to be jjerformcd with arrested metaphases.
1 he exploded type would be an excellent test object for a study of
the action of irradiation on isolated chromosomes.
10.8: The Study of Carcinogenesis
Chapter 9 has shown how useful colchicine could be in the analysis
of growth. It is regrettable that more studies have not been done
on the first stages of malignant change under the effect of various
carcinogens. For instance, the action of azo-dyes on the liver, and the
various factors which are known to influence the origin of liver car-
cinomas have never been subjected to the colchicine method. From
the few instances which will be quoted here, there is little doubt that
the early changes in mitotic activity in the liver would be fascinating
to study with the colchicine tool.
In one of the first modern papers on colchicine, this was de-
scribed as a tool for the detection of the increased mitotic rate in
the skin of animals painted with the methylcholanthrene.-^ Shortly
after, in the 39th Annual Report of the Imperial Cancer Research
Fund, similar findings were described in mice painted with benzo-
pyrene. This British work does not appear to have ever been pub-
lished in extenso. These early results, demonstrating for the first
time that mitotic activity is increased shortly after the application of
carcinogens, is in agreement with later findings." These confirm the
idea that some subtle cellular change takes place soon after the first
painting with a carcinogen even when no malignant growth will
develop for several weeks. Colchicine could evidently be used for
studying all the intermediate stages between benignancy and cancer-
ous growth.
Another observation published in 1934 is remarkable.-^ In methyl-
cholanthrene-treated mice a great increase in the numbers of mitoses,
as detected by colchicine, was found in the thyroid, in the salivary
glands, and in histiocytes. The meaning of this remains unknown.
A single paper gives a detailed cytological study of the hair follicles
of mice,^4 [^ normal skin, in embryos, and in skin painted Avith
methylcholanthrene. Ultracentrifugation studies were carried out to
study the cellular viscosity. This was not found to be modified, even
in arrested mitoses.
rhere is also a possiljihty that colchicine may act as an anti-
carcinogen. In mice im]jlanted with methylcholanthrene and in-
270 Colchicine
jected with colchicine, no skin tumors appeared."^- This result is
contradicted by experiments demonstrating that methylcholanthrene
tumors appeared in 30 days in mice injected with colchicine.*'^ The
time for the controls was 100 days. There is no evidence trom the data
of the literature that colchicine may be itself a carcinogen.
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Studies on tissue metabolism. XII. The action of colchicine on transplanted,
induced and spontaneous mouse tumors. Biochem. Jour. 34:280-84. 1940.
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13. BRiicKE, E. T. v., and Hijber, E. v. tiber die erfolgreiche Behandhmg emer
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1160-61. 1939.
14. Brues, a. M., Marble. B. B.. and Jackson, E. B. Effects of colchicine on growth
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190-93. 1943.
16 Carr J. G. The effect of some substances influencing cell activity upon the
growth of the Rous n° 1 sarcoma. Brit. Jour. Exp. Path. 23:221-28. 1942.
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mice. Jour. Path. Bact. 44:469. 1937.
19. Dermen, H., and Brown, N. A. A cytological study of the effect of colchicine
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21. Dickinson, L., and Thompson, M. J. Chemotherapeutic investigations with
Rous sarcoma virus. Brit. Jour. Piiarmacol. 7:277-86. 1952.
22. Downing, V., Hartwell, J. L., Leitlr, J., and Shear, M. J. Effect of a single
injection of colchicine, colchicine deri\atives and rchited compounds on mouse
tumors. Cancer. Res. 9:598. 1949.
Neoplastic Growths 271
23. Dii Bii.iiR, H.. AND AVarrln. S. I.. The cfTcct of cokhid'nc on the mitotic
activity of the Brown-Pearce rabbit epithelioma. Cancer Res. l:9()(>-fi9. 1941.
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27. Gri,f.n, W. J., Jr.. and Lushbaugh, C. C. Histopathologic stndv of the mode of
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28. GuicHARD, A., Brette, R., and Philii^pe. L. P. Essai de traiiemeni dc tlenx
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1946.
29. Garrigues, R. Snr ceitaines anomalies de la mitose obser\ces ilans dn cancer
hnmain. C. R. Acad. Sci. Paris. 216:822-24. 1943.
30. Guver, M. F., and Claus, P. E. Irradiation of cancer following colchicine.
Proc. See. Exp. Biol, and Med. 42:565-68. 1939. Destructive effects on carci-
noma of colchicine followed 1)\ distilled water. Proc. Soc. Exp. Biol, and Med.
43:272-74. 1940.
31. Ha\as, L. J. L'action de la colchicine snr le de\eloppement tin "'phvtocar-
cinomc" de la tomate. Bull. Assoc. Franc. Cancer. 26:635-()2. 1937. Colchi-
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32. HiRsciiEELD. J. W., Tennant. R... and Ot'GHTERSON, A. W . The effect of col-
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Biol, and Med. 13:51-59. 1940.
33. HuANT, E. Action de la colchicine siu- la radiosensibilite des tnmenrs malignes.
Ga?. Hop. Paris. 15. 1911. \on\elles considerations cjuant a Taction de la
colchicine sur la radiosensibilite des tnmems. (.a/. Hop. Paris. 15:2.30. 1944.
34. . Action de la colchicine associce a la radiotherapie dans le traitement
des tumeurs malignes. Acta Unio Internat. Cancrum. 9:83-93. 1953.
35. Isch-Wale. p. Quatre cas de maladie dc Hodgkin traites par la colchicine.
Le .Sang. 23:689-93. 1952.
36. King, L. S., and Sullivan, M. Effects of podoph)llin and colchicine on normal
skin, on cond\loma aciuirinatum and on verruca vidgaris. Arch. Path. 43:374-
86. 1947.
37. Klein. G., E., and E. The viabilitN and the average desoxvpcntose-nncleic a( id
content of micronuclei-containing cells proiluced by colchicine treatment in the
Ehrlich ascites tumor. Cancer Res. 12:484-89. 1952.
38. Kneedler, W. H. Colchicine in acute myelogenous leukemia. ]our. .\mer.
Med. Assoc. 129:272-73. 1945.
39. Lenegre, J.. AND Soi'LiER, J. P. De Taction de la colchicine sur certaines
tmneurs ganglionnaires. Bidl. Mem. Soc. Med. Hop. Paris. 58:402-4. 1942.
10. Lei-tre, H. Einige Beobachtnngen iiber das VVachstnm des Mause-.\sciles-
Tumors imd seine Beeinflnssnng. Hoppe-Seyl. Z. 268:59-75. 1941. Ergebnisse
und Probleme der Mitosegiftforsdinng. Xaturwiss. 3:75-86. 1946. Uber .Milo-
scgiftc. Ergebn. Plnsiol.' 46:379-152! 1950.
41. , AM) Kramer, W. Fine gegen C;oUhicin resisieiue .\bait des Miiuse-
Ascitestumors. Naturwiss. 39:117. 1952.
42. Levine, M. Colchicine and X-ravs in the trealmcnt of pl;nu and animal over-
growths. Bot. Rev. 11:145-80. 1945.
272 Colchicine
43. Levine, M. The action of colchicine on cell division in human cancer, animal
and plant tissues. .\nn. N. Y. Acad. Sci. 51:1365-1408. 1951.
44. LiTS, F. Contribution a I'ctude des reactions cellulaires provocjuees par la
colchicine. C. R. Soc. Biol. Paris. 115:1421-23. 1934. Recherches sur les reac-
tions et lesions cellulaires provoquees par la colchicine. Arch. Int. Med. Exp.
11:811-901. 1936.
45. , KiRSCHBAi^r. A., and Strono. L. C. Action of colchicine on a trans-
planted malignant lymphoid neoplasm in mice of the C3H strain. Amer. Jour.
Cancer. 34:196-213.' 1938.
46. LoEPER, M., et al. Therapeuticpie mcdicale. V. Peau; s)philis, cancer. Masson
et Cie. Paris. P. 358. 1932.
47. LuDFORn, R. J. Colchicine in the experinrental chemotherapy of cancer. Jour.
Nat. Cancer Inst. 6:89-101. 1945.
48. Factors determining the action of colchicine on tumour growth. Brit.
Jour. Cancer. 2:75-86. 1948.
49. Mallet, L., and Le Camus, H. Poisons caryoclasiques et radiotherapie dans le
traitement du cancer. Presse Med. 52:230-31. 1944.
50. Menetrier, p. Cancer. Formes et varietes des cancers et leur traitement. In:
Nouveau Traite de Medecine et de Therapeuticiue (P. Carnot et P. Lereboul-
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51. Moeschlin. Personal communications. 1953.
52. XicoD, J. L. La colchicine dans le traitement du cancer de la souris. Schweiz.
Med. Wschr. 72:1074-77. 1942.
53. OuGHTERSON, A. W., Tennant, R., and Hirschfei.d, J. W. Effect of colchicuie
on human tumors. Proc. Soc. Exp. Biol. 36:661-64. 1937.
54. Paletta, F. X., and Cowdry, E. V. Influence of colchicine during methylcholan-
threne epidermal carcinogenesis in mice. Amer. jour. Path. 18:291-311. 1942.
55. PARNtENTiER, R., AND I)usTiN, P., Jr. Reproduction experimentale d'une
anomalie particuliere de la metaphase des cellules malignes (mctaphase 'a
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mitotic abnormalities induced by hvdroquinone in aniirral tissues. Rev.
Beige Path. 23:1-11. 1953.
56. Paul^ J. T., Brown, W. O., and Limarzi, L. C. Effect of colchicine on m\eloid
leukemia. Amer. Jour. Clin. Med. 11:210. 1941.
57. Pevron, a., Lafav, B., and Kobozieff, N. Sur la regression de la tumeur de
Shope du lapin sous Taction de la colchicine. Bull. Assoc. Fran^. Cancer. 25:
874-75. 1936. Sur la regression du papillo-cpithclioma du lapin sous Taction
de la colchicine. C. R. Acad. Sci. Paris. 205:378-80. 1937.
58. , Poumeau-Delille, G., and Lafay, B. La tumeur de Shope du lapin
et sa sterilisation par la colchicine. Bull. Assoc. Franc. Cancer. 26:625-34. 1937.
59. , AND Sur revolution maligne du papillo-epithelioma du lapin
et son mode de regression sous Faction de la colchicine. C. R. Soc. Biol. Paris.
126:625-28. 1937. L'histopathologie et les modalites eyolutives de la tumeui
cutanee de Shope chez le lapin. Bull. Assoc. Franc:. Cancer. 28:180-94. 1939.
60. PiTON, R. Recherches sur les actions caryoclasiques et caryocinetiques des com-
poses arsenicaux. Arch. Int. Med. Exp. 5:355-411. 1929.
(H. PoiLssoN, K. T. Colchicinbehnadling av maligne soulster hosmus. Norsk. Mad.
Laegevidensk. 96:735-36. 1935.
62. Rliffilli, D. Azione di un veleno statmocinetico sulleritroblastosis dei polli.
Boll. Soc. Ital. Biol. Sper. 16:140-41. 1941. Azione della colchicina sulla can-
cerogenesi da metilcolantrene. Xota preventiya. Boll. Soc. Ital. Biol. Sper.
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63. Schairer, E. Der Einfiuss des Cokhicius auf den Mausasciteskrebs. Z. Krebs-
forsch. 50:143-54. 1940.
64. ScHjEiDE, O. A., AND .Ai.LEN. B. M. The relation of mitosis to the manifestation
of X-ray damage in hematopoietic cells of tad-poles. Jour. Cell Comp. Phvsiol.
38:51-67. 195l':
65. Seed. L., Slaughter. P. P., and Llmarzi, L. R. Effect of colchicine on hiunan
carcinoma. Surgery. 7:696-709. 1940.
Neoplastic Growths 273
(56. Slldam, B. E. J., AND SoETARSo, B. Dc werking van colchicine of cnkele c\-
peiimenteele Ratteiisarcome. Geneesk. Tijdschr. Xed.-Ind. 78:3187-96. 1938.
67. Sfntein, p. Laction des toxiques sur la cellule en divison. Effets de la colchi-
cine et du chloral sur les mitoses et tissus norniauv et sur quekjucs tunieurs
malignes. These. Montpellier. 1911.
68. Setala, K. Colchicine as carcinogenic agent in skin carcinogenesis in mice.
Ann. Med. Biol. Fenniae. 26:126^30. 1948.
69. Shai'iro, D. M., Weiss, R., and Gellhorn, A. The effect of azaguanine on
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primary subcutaneous tumors to injection of a hemorrhage-producing bacterial
polysaccharide. Jour. Nat. Cancer Inst. 4:461-76. 1944.
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1951.
72. SoiACOLU, T., AND CoNSTANTiNESCO, M., AND D. Actiou de la colchicuie sur les
tiuneurs yegetales provoquees par le Bacillus tiunefacieiis. C. R. Soc. Biol. Paris.
130:1148-50. 1939.
73. Tennant, R., and Liebow. A. Actions of colchicine and ethylcarbvlamme on
tissue-cultures. Yale Jour. Biol. Med. 13:39-49. 1940.
74. Thomas, P. T. Experimental imitation of tinnour conditions. Nature. 156:
738-40. 1945.
75. \'illars^ R. £tude cytologique de Taction des rayons X siu- les racines colchi-
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76. \Villiamson, G. The treatment of tumours by the injection of colchicine.
Jour. Rov. Army Vet. Corps. 8:23-25. 1936.
77. WooDSiDE, G. L.; Kidder, G. W'., Devvev, W C, and Parks, F. E., Jr. The influ-
ence of 8-azaguanine on the mitotic rate and histological appearance of certani
normal and neoplastic tissues. Cancer. Res. 13:289-91. 1953.
78. Zirkle, R. E.. and Bloom. \V. Irradiation of parts of individual cells. Science.
117:487-93. 1953.
CHAPTER 11
The Experimental Polyploids
11.1: 1937 — Beginning of a New Era in Polyploidy
Colchicine replaced practically all the techniques used to double
the number of chromosomes in plants. The procedure was new and
could easily be fitted to many different kinds of plants. Within a
short time geneticists became convinced that a very useful tool had
been discovered, because colchicine methods were more effective and
more suitable for making polyploids, plants with additional sets of
chromosomes, than any formerly used.
Immediate and wide universal interest in colchicine developed
among botanists, as shown by the rapid rise in popularity that fol-
lowed closely upon the announcements of chemical induction of
chromosomal doubling.'^' 12.52,53.62 \ ^ew era in polyploidy investi-
gations began in 1937, the year the colchicine method was discov-
ered.36. 72
Soon the advantages of colchicine became clear. One out of 600
cotton plants treated by "heat-shock" became polyploid (1:600), but
colchicine procedures applied to a comparable group yielded 50 poly-
ploids from among 100 (1:2) of the cotton plants surviving the
chemical treatment. ^ Similarly the superiority of colchicine was dis-
covered by workers at the chromosome laboratory, Svalof, Sweden,
where up to the time colchicine was introduced, elaborate heat-
shock machinery, with refrigeration controls, had been used to double
the number of chromosomes.^^ Swedish botanists soon discovered that
such complicated equipment was no longer necessary.^*' A rapid
change-over to colchicine took place.-**. 3. 8-'i4, 16, 20, 21. 23, 25, 20. 3... 32, 4i.
43, 46, 51, 50, 54, .50, 57, 58, 59, 63. 64, 05, 60, 69, 70, 73, 74 TllC Switcll tO Colchi-
cine in Sweden and elsewhere was so fast that it appeared that the
colchicine "fad" in research had arrived.'-' -^
As we mentioned in Chapter 2, colchicine was not the first chemi-
cal to be tried and used for doubling of chromosomes. Other chemi-
cals, heat-shock methods,!^ production of callus tissue,'**^ and other
[274]
The Experimental Polyploids 275
techniques yielded polyploid types/'" The reason these methods were
replaced is found in the two specific advantages demonstrated by col-
chicine: First, colchicine was very effective for making polyploids
Avith many different species; and second, the drug was applied easily
to young growing plants Avith very little damage being done to them.
There are several noteworthy features of colchicine that account
for its effectiveness as a polyploidizing agent. Brieflv, colchicine is
highlv soluble in water; colchicine is not toxic to plant cells even in
strong dosages; colchicine is effective in concentrations ranging from
1.0 to 0.01 per cent (1:100 to 1:10,000) ; and finally, it is soluble in
lipoids. Furthermore, the effect obtained during a treatment is wholly
reversible. Thus the drug is almost "made to order" for changing
diploids into polyploids.
After recovery from treatment the new tissue from treated genera-
tions (Co = generation) and the progeny of succeeding generations
(Ci = first, Co r= second, etc.) do not show damage of a hereditary
nature. The usual changes associated with multiplication of chromo-
somes, gigantic characters in leaf, flower, fruit, and seed, are trans-
mitted to the next generations; there is no evidence that "deteriora-
tion" ^" sets in after colchicine reaches the protoplasm. While the
treated plants may perhaps have wrinkled leaves, distorted stems, and
various anatomical malformations, such temporary changes disappear
in Cj, Co, and later cycles.
Gene changes or chromosome repatterning have not been proAed.
s'^- "1 although preliminary tests led to these suggestions. This much
is certain: Changes comparable to those produced by X-ray have not
been found, and if we choose to use the word mutation, it must be
clearly stated that colchicine does not cause gene mutations. Only in
the broad sense of mutation, which includes chromosomal doubling,
may we use the term in connection with colchicine as a producer of
mutations.--^ If the definition is limited to gene changes and chromo-
some repatterning (inversions and translocations) , colchicine does not
cause mutations" Hence it is incorrect to classify colchicine with
mutagens, such as p-acetamidotropolone, a 7-carbon compound which
appears to cause chromosomal breakage.'^
More knowledge about the meaning and use of chromosome num-
bers in relation to species relationship formation is desirable. Every
experimenter before commencing a project Asith colchicine should
know the drug is not a chemical fertilizer; it is not a phytohormone;
it is not a weed killer; it is not a vitamin; it is not a mutagen; and
finally, colchicine is not merely one more organic substance on the
present long list now at the disposal of many persons interested in
plants.2» The drug has specific and limited uses; therefore, reports
giving directions to spray a field with colchicine or to soak the soil
as one would witli fertilizing agents, are completely erroneous.
276 Colchicine
In this chapter and the next iour chapters the future possibilities,^^
limitations, and accomplishments are given. Miracles were predicted
in the numerous writings in praise of colchicine, but there often
followed a serious disillusionment for those not informed in poly-
ploidy and cytogenetics.^^ A wave of great enthusiasm for colchicine
in some quarters was succeeded by a loss of interest. Totally dis-
counting colchicine, however, is quite wrong.
n.2: Terminology
In the rapidly expanding field of cytogenetics, new terms are con-
stantly being added, while others are modified as more information
is acquired. The two terms, auto-syndesis and allo-syndesis, have been
used with exactly opposite meanings by two groups. Now each time
the terms are used, an explanation must accompany the usage. When
autopolyploidy and allopolyploidy were first pointed out by Kihara
and Ono in 1926,^=^ the distinctions were based on materials at hand.
When many more examples came into consideration, the differences
were not as specific as one might desire for a classification. Terms and
their meanings often introduce added confusion. The terminology
and definitions used here have in large part been adapted from Clau-
sen, Keck, and Heisey.^^ Extensive work on terminology has been
done by Stebbins.*"^
Ploidy, in recent usage, means /o/r/ (from the Greek pJoos) and
a combining form like (oid) . Thus the prefixed word polyploidy
means many-jold. This refers to the number of sets of chromosomes
for a particular plant or animal. Monoploid refers to those cells or
individuals with one set; diploid, twofold; triploid, threefold; tetra-
ploid, fourfold. Then autoploid means self-fold; ainphiploid, both-
fold.
Polyploidy describes a serial relation of numbers in multiples
starting from some basic number. If the number is 7, then the poly-
ploid series would read 21, 28, 42, for triploid, tetraploid, and hexa-
ploid, respectively.
Autoploidy is an abbreviated form of the term autopolyploidy and
will be used for those polyploids formed by nudti plication of sets of
chromosomes within the limits of a species. Admittedly, the range is
wide, and complications arise in classification because the autoploid
with four homologous sets will differ from the one derived from two
subspecies, that is, the doubled intraspecific hybrid.
Amphiploidy embraces the polyploids derived from the additions
of two distinct species. A sterile hybrid AB upon doubling becomes
the amphijiloid AABB. If the number of species included increases
beyond two, a polyploid-amphiploid condition obtains.
The Experimental Polyploids 277
Segmental allopolyploid is an amphiploid which shows character-
istics of autoploids with respect to pairing of chromosomes, resem-
blance to parents, and fertility; yet the amphij^loid exhibits enough
tlilference between the genomes contributed by the parents to fall
within the scope of amphiploids. Segmental types are important for
practical and theoretical reasons. Our discussion of the segmental
allopolyploid will be included in Chapter 12 (The Amphiploids) .
Genome designates the set of chromosomes derived from a species;
the term may be used to express a relationship between species. Ex-
tensive use has been made of genomes since many intersi>ccific hy-
brids have been made and doubled with colchicine. Among species
of Gossypium the genome concept is related to geographical distribu-
tion of species. The genomes of Trituum refer to generic contribu-
tions. The original term was introduced by Winkler in 1920.
Dysploidy refers to a series of polyploids in nature whose basic
numbers are not nuiltiples. A dysploidy is superimposed upon an
amphiploid series. A good example is found among the Cruciferae,
where basic numbers 5^ 6. 7, 9. 11 fall at levels of diploid, tetraploid,
and hexaploid status.
Aneuploidy is a condition in which chromosomes are added or
lost from the diploid set of chromosomes. Aneuj:>loids may or may not
represent balanced genotypes. 1 he loss or addition may be found at
polyploid levels. For example, the nullisomic is essentially aneuploid.
Cryptic structural hybridity*'*'' designates a chromosomal differentia-
tion in very small segments that does not readily find expression in
configuration at metaphase of meiosis. Pairing of chromosomes may
be bivalent and apj^arently normal, for the segments that are differ-
entiated are so small that no opportunity is afforded for abnormal
configurations during synapsis. For these reasons a structural hy-
bridity of this nature may be indistinguishable from the genetic
hybridity.
11.3: Cataclysmic Origin of Species
The origin of a new species by gene mutation or chromosomal
repatterning (inversions or translocations) is a slow process and re-
quires a long time. Surprisingly, there exists in nature, alongside
these slower processes, a very rapid method that can catajndt a new
species into existence within a generation or two.''' This sudden
origin is called "cataclysmic evolution." -^ By this process a new plant
is separated at once from its immediate jjarents and is destined to
occiipv new environments different from either, or both, of its pro-
genitors, (Fig. 11.1) .'"'
m
Colchicine
SPECIES A
A, A,
II
SPECIES B
B, B,
X
DIPLOID 2n
DIPL0ID2n
A, B
DIPLOID HYBRID
Jl
CHROMOSOME DOUBLING byCOLCHICINEl
I
T
f\i Ml Ml A I
nil
AUTOTETRAPLOID
A, A, B, B,
4n
A, A,
II
AMPHIPLOID
4n
T
X
B, B, B, B,
AUTOTETRAPLOID
4n
B, B,
GAMETE 2n
GAMETE 2n
Fig. 11.1 — Use of colchicine to make autotetraploids. Doubling the chromosomes of in-
terspecific diploid hybrid. Amphiploids made by hybridizing two autotetraploid species.
(After Wexelsen)
Thi.s kind of evolution was loinuilated as the A 'X B hypothesis
by VVinge in 1917 before any examples were well known, although
the doubling of Primula keivensis was on record. •'•' According to the
A \ B hypothesis, a polyploid series with a basic number of 7 would
read 21, 28, and 42; or triploid, tetraploid, and hexaploid, respec-
tively. These can originate as follows: A triploid, sterile hybrid
arises from the hybridization ])ctwcen the diploid, 2u =i 14, and a
The Experimental Polyploids 279
tetraploid, 4;? = 28; upon doubling of the 21 -chromosome triploid, a
hexa})loid (42-chromosome) species originates.^'' In this way species
h\bridization, followed by doubling of the chromosomes, fulfils the
principle of the Winge hypothesis. Among the wheats (Triticinae)
there is an excellent chance to show how this mode of evolution
accounts for speciation as well as the production of mankind's most
\aluable economic crop species, hexaploid wheat, (42-chromosome
Triticum aesthnim L.) .•*^ However, on a purely numerical basis and
without a knowledge of the only known case to support his assumption,
the A X B hypothesis was outlined to explain the origin of species with
high chromosomal numbers. The data which Winge needed were
published by Digby for Primula keioensis.^^
The facts of cataclysmic evolution became clearer, for new tetra-
ploids Avere discovered"'^ or synthesized continuously from 1926.
These include Miint/ing's synthetic Galeopsis tetraliil;''^ Primula
kewensis, arising under culture at Kew Gardens;*"^*^ Karpechenko's
Raphanobrassica,-^ a doubled intergeneric hybrid between radish
and cabbage. Finally Spartina fownsendii}^ a new polyploid of recent
historic times, is a new species which invaded a habitat not previously
occupied. The mud flats along the channel coastline of England
abound with this new species, but records show that prior to 1870 no
plants were present in this area.^^
Two important conclusions emerge from the numerous studies
dealing with polyploidy and evolution. (1) Polyploid species are
abundant in nature: by one estimate as many as 50 per cent of the
flo\\'ering plants are in some dui^licated form. (2) Valuable economic
crop species (food, fiber, and others) are polyploid, e.g., bread wheat,
cotton, oats, sugar cane, tobacco, grapes, berries, nuts, and many other
horticultural and floricultural species. In the first instance our
problem may be called cataclysmic evolution in nature; in the second,
evolution under domestication.^"^
Polyploid agricultural species originated through the years in
nature without man's guidance, but under his hand and through his
selection they may have become quite different species than if left
to natural processes of selection. When man eliminates certain types
and nurtures the environment for his choice plants, the situation is
not com))arable to nature's elimination process and selection that goes
on conijjetitively without cultivation. Nevertheless, the problems of
evolution in nature and imder domestication'*'* are very closely inter-
related. That is why closer integration of theoretical and j^ractical
work seems advisable in j^olyploidy research. Increasing the in-
formation about the origin of jjolyploids in nature improves our posi-
tion in the planning (jf a ne^v hybridization program.'-'' Furthermore,
the data from countless selections by the practical breeder could be
valuable for analysis with purely theoretical objectives in mind.^^
280 Colchicine
When colchicine was discovered as a tool for doubling the chromo-
somes, it was believed by many that evolution was about to be
speeded up out of proportion to anything known. The tool, col-
chicine, did in fact remove a serious bottleneck^'? in permitting a
doubling of the species hybrid by a new and more efficient method
than ever before available. Many newcomers to the ranks of new
species have been produced; this is evident if we compare our list
of amphiploids produced since 1937 with the list made before that
date. There is no doubt of a speeded-up tempo, but unless one
possesses a broad and deep knowledge of cytogenetics, he will fail to
see that the expected "miracles" have been forthcoming. The intro-
duction of a new variety of wheat by ordinary standards requires
about 15 years.«« To produce a new polyploid variety is as difficult,
if not more so.
11.4: Classification of Polyploids
The two principal classes of polyploids are (1) autoploids derived
from homozygous diploids, e.g., tetraploid maize,''o and (2) amphi-
ploids, like Raphanobrassica,-'^ resulting from hybridization. These
two types are not difficult to distinguish. They are extremes with the
autoploid carrying four sets of homologous chromosomes AAA A, and
the amphiploid. two diploid sets AA and BB. The difficulties in
classifying polyploids arises when dealing with examples between the
different types, that is, polyploids with both autoploid and amphi-
ploid characteristics.'''^ There are many cases - and more are being
made continuously — that are intcrgrading types and, as such, are not
easily classified into the autoploid or the amphiploid category.
Problems of classification in polyploidy are similar to those in
other systematic studies. For example, everyone agrees on which
individuals of the species belong to the Mammalia and the Sperma-
tophyta; however, among the microorganisms a classification problem
has new difficulties. Since the bacteria are so widely studied in re-
lation to human disease, the medical bacteriologists find it illogical
to group them with the fission fungi, or Schizomycetes, of the plant
kingdom. As a matter of fact, some bacteria do have plant and animal
characteristics, and so present a distinct problem in classification.
Likewise in polyploidy, the borderline cases have characteristics that
are both autoploid and amphiijloid. As colchicine increases the
number of polyploids, the intergrading types are increasing at the same
lime.
The artificially induced hexaploid Phleurn nodosum, created by
colchicine,-^^ ^ad^y be used as an example of the disagreement on clas-
sification because the true nature of its autoploidy is in disinite.
When all the evidence is carefully reviewed in this case, the complex-
The Experimental Polyploids 281
ities of classification become very real. These are problems requiring
iurther study which cannot be resolved entirely in this review. There
are other cases. In fact, the gioup between the autoploid and amphi-
ploid provides the most interest and perhaps the greatest opportunity
lor practical and theoretical work in polyploidy. E\en though one
cannot decide definitely on the classification, there is no need for
concern, for he may utilize the opportunities presented by these
intergrading polyploids without classifying them.
One way to explore this group has been oj^cned by an inquiry into
the special kind of polyploid called the "segmental allopolyploid."*''^
Good reasons were given to justify the establishment of this special
group. Some types of polyploids have segments of chromosomes so
closely associated that pairing is between the two parental genomes,
and therefore they cannot be considered as strictly amphiploid; but
in other segments, there is enough differentiation to prevent pairing
of the chromosomes that originate from the different parents. View-
ing the chromosomes segment by segment, instead of as whole chromo-
somes or even whole genomes, gives one a more critical picture of the
basis for borderline types between the autoploid and the amj^hijiloid.
Theoretical and practical aspects are greatest among the ])olyploids
that fall between the unquestionable autoploid and amphiploid.
Pairing of chromosomes is of limited value in classifying the
polyploids e\'en though this cytological method is one way to point
out the difference between the autoploid and the amphiploid. Some
diploid species hybrids may show pairing at the diploid level, but
this does not necessarilv happen. On the other hand, complete lack
of pairing at the diploid level does not insure total bivalents at the
polyploid stage.^- Less and less reliability is being placed on pairing
of chromosomes as a measure of homology and a means of distinguish-
ing the autoploid from the amphiploid. As more examples come into
view, the case for pairing is increasingly complicated. Other factors
must be considered.
Sterility and fertility characteristics may separate the amphiploid
from the autoploid. The latter is invariably less fertile than the
diploid, and the amphiploid changes from a sterile condition to a
fertile one upon doubling of the chromosomes. In reviewing many
cases, one can find wide variation in degree of sterility among the
autoploid and the amjjhiploid cases. Actually, the causes of sterility
are so complex that this relationship is of little help in trying to
classify the two types. Yet basically, sterility may be closely related
to some basic cytogenetic mechanism.
The best solution to the classification problem appears to he the
chart developed bv Cilauscn and his colleagues^'' on which they place
the amphiploids in a relative position depending upon a series of
282 Colchicine
characteristics that place the tyjie closer or farther from one of the
two classes. Table 12 of their work is worth considerable attention
for those interested in the classification of polyploids. As would be
expected, the known polyploids form an intergrading series from the
extreme autoploid to the amphiploid, which is a completely diploid-
ized type. Colchicinc-induced polyploids cause increasing inter-
gradation as more and more examples appear.
For purposes of reviewing the colchicine-induced polyploids, re-
sorting to taxonomic authority has served a very useful purpose. If
the polyploid has been a product of doubling a species hybrid in-
volving accepted species, then the type is considered amphiploid,
while the diploids made tetraploid are autoploid. Admittedly the
system is artificial and does not delve into the real problem that
makes a polyploid what it is. However, with the view of handling
large amounts of data and many polyploids, this method of classifica-
tion is simpler. At no time has the basic feature of the segmental
allopolyploid or its significance been overlooked. Those character-
istics that are peculiar to the segmental allopolyploid are important
practically and in certain evolutionary aspects.
11,5: Principles of Polyploid Breeding
Within five years, from 1938 to 1942, examples of all the major
agriculture species of Sweden were converted into polyploids.^fi. «9. i
In other places throughout the world vast numbers of polyploids
were created at about this same time. Colchicine accounted for many
of the new polyploids, but few of these could be used in agriculture.
7.3, 65, 49, .54, 56, 57, 63, 35, 62, 44, 19, 21, 22. 30, 32, 3, 5, 8,9.15, 16 Xllis may COmC aS
a shock to i)ractical agronomists. A re-examination of the principles
basic to polyploid breeding was needed. Since so much material was
at hand, polyploids were used to test a number of points about chro-
mosome doubling as a method of plant breeding. The principles enu-
merated below have been stated directly as such or indirectly through
the work of a number of investigators.
The application of colchicine permitted the production of large
numbers of polyploids from diploids. One would expect these new
polyploids to replace the standard diploid varieties.''^ However,
artificially induced polyploids are, at the beginning, "raw" polyploids
without exccption.^*^ Such types are generally unselected, so the task
of jjlant breeding has only begun after the polyploid has been made.'**'
Too many investigations disregarded the principle of raw polyploids
and tested the tetraploids against the selected diploids. Naturally,
the tetraploids failed to measure up to diploids in all-around per-
formance. What is even more surprising is the condemnation of
colchicine when tetraploids, apparently as raw polyploids, failed to
The Experimental Polyploids 283
outperform the l)est diploids. Statements that colchicine causes
"harm"^" to the plants are also difficult to iniderstand.
A second principle well known to practical breeders is the use of
!ar2;e populations. If one starts with a few plants, his project is
doonietl Ijciore a start has been made. Two qualifications should be
stated in this respect. The self-fertilized species should be used with
more strains and fewer plants from each, while the cross-fertilized
types demand many plants, but these can be taken from fewer strains.
In both instances, large numbers of tetraploid genotypes must be
made as the material for future selection work.^'' Naturally, a few
jilants cannot serve as a substitute for mass production.
Each successful tetraploid nuist eventually have genotypical bal-
ance. Through selection the relation between plant and its environ-
ment must be brought into an adjustment. i'' Practical breeders are
accjuainted -with the need for the all-around performance of more
than one characteristic. It is not enough to acquire disease resistance,
or some other quality, to the exclusion of those equally as impor-
tant.*^^ The new tetraploids are no exception in this respect. The
transfer of a specific gene for disease resistance must not be per-
mitted at the expense of the whole genotype which may be thrown
out of balance — that is, if success in a practical way is anticipated.
Therefore, the opportimities for selection begin with the polyploid,
and the difficulties are also started as we shall learn in subsecjuent
sections.
The genetic traits of the polyploid are an accumulation of those
contributed by the diploid. It does not follow that a very good diploid
\vill always give rise to the best polyploids. But there is this rule
to be observed that a polyploid, like the diploid, is a plant with
genetic traits that segregate and respond in selection according to the
same rules as the diploid.
In judging the chromosomal ninnbers of natural species, there is
a law of optimal numbers above or below which the maxinunn per-
formance or adaptation cannot be expected. The polyploid series of
Phleum is a good example.'**'> Those types with best characteristics as
polyploids were found in the ninnbers 6 X '- an<^l 1 1 X 7. One cannot
expect to achieve success by doubling a tetraj)loid, so the di|)loid species
are needed for a start. Chromosomal doubling of natural tetrajiloids
in cotton from 52 to 104 chromosomes creates very weak and poor
plants; obviously this exceeds the optimum nimiber.^ There is, how-
ever, another point to be remembered: If the number of diverse
genotypes can be increased during the process of doubling high num-
bers with plants having good fertility, vigor and growth are possible.
Merely stating that the numbers cannot be above a certain value is
too limiting. In nature the natural polyploids are combinations of
two or more genomes that can I)e recognized. For example, the hexa-
284 Colchicine
ploid wheat combines three genomes, and after this process the optimal
number of 42 seems to be attained.
Cross-fertilizing, or allogamous, species are more promising as a
group than the self-fertili/ing types. This general rule seems to hold
for a large number of plants included in the Svalof experiments.
Some qualification needs to be made, for the sampling was not as
extensive as might be desired. The changes from incompatability to
compatibility upon doubling the number of chromosomes is an in-
volved genetic problem, not merely a result of the tetraploid nature,
but consisting of a combination of events that create the changes.^''
1 he autoploids are almost without exception less fertile than the
diploids.***^ Therefore, seed and fruit yields, if dependent upon seed
production, will at once suffer in the polyploid stage, at least before
selection can be done to rectify the situation. The sterility barrier is
by-passed when a hybridization is included with the doubling; then
the degree of fertility generally improves, but not always. The prin-
ciple of reduced fertility after polyploidy from the diploid should
always be considered by every one starting a new project. Then the
changes that might be induced by selection in the later generations
can be considered along with the sterility-fertility relations. Granted
that fertility levels can be raised by selection, the danger of introduc-
ing other changes constantly attends the selection processes.
The part of the plant to be used for economic production becomes
a first consideration, for the root and shoot yields will not be in-
fluenced by sterility. Vegetatively propagated plants are a new prob-
lem. They need not pass through the reproductive cycle that is so
critical to a polyploid at many levels. Perennial plants are favored,
and plants that produce propagating shoots like the grasses are im-
mediately more favorable than the strictly seed-producing annuals.
A principle of transfer of characteristics from one species to
another has been mentioned frequently in polyploidy work. Among
many species the favorable traits are jnomincnt in the wild species.
There is at once a desire to introduce this character into the valuable
commercial species. A notable case is the mosaic resistance transfer
in tobacco. 1" This problem is discussed in greater detail later, but
it should be noted that the transfer of such a trait is in effect a prob-
lem of polyploidy l)reeding. On a plan in blueprint stage, the idea
appears relatively simple, but now it is well known that accomplish-
ment is quite difficult. One of the greatest obstacles in transfer is
the introduction of undesirable traits along with the desirable ones
being sought.
Combining the good features of two diploid species into the amphi-
ploid is another aspect of how hybridization and the doubling of
chromosomes offer opportunity for future programs of selection. A
The Experimental Polyploids 285
new s[)ecies such as the Cucurhild inosdiata X C. maxima amphiploid
combines good traits from two diploids. A new species of economic
potential is apj:)arent. However, intersj^ecific segregations in tlie fifth
and sixth generations show that a lack of Lniiformity can be expected
(cf. Chapter 12) . Such variation is not what the breeder hopes for
in a true l^reeding variety. By transfer of whole genomes into a hy-
brid the characters of the polyploid can be influenced. If in later
generations there is pairing between the two genomes that originated
\\\x\\ the two species, the chance for segregation is good. If the segre-
gates are undesirable and if the interchange is so great that the
original type is lost, all the transfer is circumvented by the after-
breeding effects. Transfer in Gossypiimi has presented a very difficult
problem, that of introducing the good characters and maintaining all
the original traits of the cultivated varieties. In spite of the ])roblems,
the principle of transfer is basic in polyploid breeding.*""'
The advantages balanced against the disadvantages are necessary
for a final evaluation. •''i No tetraploid within a certain species may
be expected to surpass the diploid in all respects. Therefore, the
desirable traits balanced against the unfavorable ones should be cal-
culated to see whether the new result is in favoi of the tetraploid or
the diploid. Triploid sugar beets are not perfect, but there is the
important fact that the triploids can be grown to a larger root size
before the percentage of sucrose decreases than is the case for the
diploids."**' In this way the triploid has an advantage over the dip-
loid, Avhile for seed production, germination, and growth problems
the triploid is sometimes at considerable disadvantage beside the
diploid. Tetraploid rye offers another notable example of balancing
two sets of characters. ^^
All plants arising from treated generations may not be totally
tetrajjloid. The diploid cells may be found mixed with the tetra-
ploid, and a mixoploid condition may persist.^" Or the layers of cells
may differ one from the other, so that the shoot apex is stratified with
respect to its ploidy.-'^ These are called periclinal chimeras discussed
in Chapter 14 (The Aneuploids) .i"' From the point of view of poly-
ploid breeding the mixoploids and chimeras are very important prob-
lems. The reversion of jjolyploid to diploid is sometimes explainable
on the basis of a chimera, or sometimes it may arise from cross-breed-
ing.
Stabilizing the polyploid by selection and In preventing the re-
version to the diploid or through segregation, to some inferior type
is a problem that confronts the plant breeder after the polyploid has
been produced. The first and second generations may be quite uni-
form, but later generations less so. Or the first generation may have
defects that yield to selection in later generations. The effectiveness
286 Colchicine
of selection between diploid and aniphiploid is one of degree and
speed rather than absolute difference. Genetic types can be isolated
more quickly in diploids than in polyploids if one can base his evi-
dence on a specific character and extend the idea to a whole set of
characters.* Selection as a result of interspecific segregation creates a
good opportunity for making wholly new lines.""
Regardless of the plant, whether diploid or tetraploid, the testing
methods are important to success in measuring the gains made, in
keeping the good qualities, and in raising the standards if possible.
In tetraploid rye the testing side by side of diploid and tetraploid
is inijjossible, and consequently an adjustment must be made by a
yield factor with another plant.^i This at once complicates evaluation
of the polyploid against the diploid. There are many other prob-
lems of testing peculiar to certain plants, and tetraploids are involved
because the success of the polyploid may depend upon the mode of
testing rather than the qualities of the polyploid itsell.
The list of principles is not comj^lete in the above survey, but
a start has been made. More information is needed before the ad-
ditional principles of polyploidy breeding can be described in gieater
detail.
11.6: The Scope of Research
Colchicine increased the frequency of induced polyploids beyond
that possible with any other method known up to 1937. This dis-
covery had two major effects upon research in the plant sciences all
over the world. (1) Polyploidy, already a subject of study, was in-
creased immediately. (2) New programs were started because greater
reliability could be placed upon this technique and much time could
be saved in converting the diploids into polyploids. The net result
of these two developments has been an unusually great expansion in
research with polyploidy in many nations.^^- ^^ In fact, a detailed re-
view of all work with colchicine goes beyond the jjermissible allot-
ment of space in this review.
One might single out specific cases where certain scientists have
had an exceptional influence upon jjolyploidy and greater than aver-
age progress has been made accordingly. For example, the personal
interest that Vavilov took in polyploidy led to great activity in cyto-
genetics in Russia.'" In Sweden, Nihlsson-Ehle made special efforts
to organize laboratories such as the chromosome laboratory at Svalof
and other institutes in that country.^" These and other special in-
stitutes^-' tluoughout the world were at work on problems in poly-
ploidy before colchicine became known as a tool for creating poly-
*See Reference Xo. 10 -i in C^haplei 12.
The Experimental Polyploids 287
ploids. When colchicine appeared to be usetul, its future possibilities
were expressed in several American papers''' published by Chronica
Botanica in 1940. A broad view was taken at this time.
The progress made in Sweden Irom 1937 to 1947 was rapid. Scien-
tists irom every nation observed the scope of this work as a restilt of
demonstrations made before two international congresses, the genetics
meeting of 1948 and the botanical meeting of 1950. Obviously, the
discovery of colchicine in 1937 appeared at a very favorable time in
the history of plant sciences in Sweden. A large amount of work was
done in Russia from 1937 to 1947, but less attention has been given
to this contribution."^ Already in 1945, Professor Zebrak reported in
a lecture at the University of California that numerous polyploids in
the Triticum group had been made, perhaps not exceeded elsewhere
in the world. "^ The extensive report on the situation in biological
sciences in Russia matle in 1948 gives a general survey of the status
of research with polyploidy before 1947. After 1948 the use of colchi-
cine was apparently not encotnaged in Russia.^' There can be no
tloubt that Vavilov had an important influence on the use of poly-
ploidy as a research method.
Japanese geneticists have made direct and special contributions
to practical and theoretical phases of polyploidy.''^ The trijiloid
watermelon, triploid sugar beet, tetraploid radish, and tetraploid
melon have been \n\\. into agricultural practice since 1937.-^^ Much
progress has been made at the Kihara Biological Institute, Kyoto,
where a number of workers have been able to make their contribu-
tions. Furthermore, the influence of this laboratory ^vas directed to
other institutes in Japan. Polyploidy has been a familiar subject, and
there has been close integration of theoretical and practical problems
under the direction of one group of Avorkers.^"^
Accomplishments in the field of polyploidy by three nations,
Sweden. Russia, and Japan, are cpiite out of proportion to the
relative number of scientists, and particularly of geneticists, in each
country. In this respect, the progress made in the United States is far
behind these others if one compares the total work in plant sciences
in relation to the progress made in the area of polyploidy. There-
fore, one cannot imderstand w4iy colchicine and polyploidy are
thought to be tools owned solely bv America. They are not. In fact,
no nation can claim a priority in the use of colchicine and in progress
made by its application to polyploidy. The records of the Seventh
International Genetics Congress show some unbalance, l)m l)\ the
time the Ninth Congress was held, there was an equalization, so that
no single group has dominated the j^rogram of colchicine and prob-
lems in polyploidy. Historically the situation has been clarified since
the early period of w'ork with colchicine.
288 Colchicine
There is another aspect in the scope of research with colchicine
that tends to be overlooked. Scattered throughout the world, special
institutes were at work on species whose background was recognized
to be polyploid, such as Gassy pi inn, ^- i5- C7, 35 JSHcotiann,^-^ Triiicum^'^-
"^ Sohniuni, and others. Iheoretical problems and the practical im-
portance of polyploidy Avere well known before 1937. One outstand-
ing case is the British Empire Cotton Research Station at Trinidad,
British West Indies, where diploid and tetraploid Gossypiinn was
studied in detail (cf. Chapter 12) . Soon after colchicine became
kno\\'n, it was applied to the sterile hybrids on hand.*'" The drug was
merel)' incidental to the whole jjroject, and many polyploids were
made as a matter of routine in the larger program. For these reasons
research with colchicine did not get prominent notice in their pub-
lications.
The application of polyploidy breeding in Nicotinna began before
colchicine was discovered. After 1937 the number of polyploids for
this genus was increased. i" A transfer of disease-resistant traits from
one species to another is an example of polyploid breeding and a
contribution of experimental genetics. ^''^
Breeding programs with forage species,-* Triticiim^^ fruits, and
flowers are under way in many places. The state and federal stations
in the United States alone represent a large program.-- Polyploidy
is included in many of these programs. Public and private institutions
throughovu the ^vorld have put colchicine to work.
A complete list of research centers and projects using colchicine
would be laige. The bibliography and list of polyploids indicate the
international character of such research.
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36. Havas, L. A colchicine chronology. Join. Hered. 31:115-17. 1940.
37. Hill, H.. and Meyers, W. Isolation of diploid and tetraploid clones from
mixoploid plants of rye grass (Lolium perenne L.) , produced by treatment of
germinating seeds with colchicine. Jour. Hered. 35:359-61. 1944.
38. Hudson, P. Personal communication. Cambridge Univ., England. 1953.
39. HusKiNS, C. Polyploidy and mutations. Amer. Nat. 75:329-44. 1911.
40. Jorgensen, C. The experimental formation of heteroploid plants in the genus
Solanum. Jour. Genet. 19:133-210. 1928.
290 Colchicine
41 Karpichfnko, G. Tetraploid six-rowed barlevs obtained 1)y colchicine treat-
ment. C:. R. Dokl. Acad. Sci. URSS. 27(1) :47-50. 1940.
42. Klhr, a., and SMrrH. H. Multiple genome relationships in Xicoliana. Cor-
nell Univ. Memoir 311. Pp. 19. Agr.' Exp. Sta., Ithaca, New York. 1951.
43. KiHARA, H. History of polyploidy. Monograph on polyploidy. Baisusei Sogen-
slia. Tokvo. March 25, 1947.
44. Krvthe. j., AND Wellensiek, S. Five years of colchicine research. Bibliog.
Genetica. 14:1-132. 1942.
45. KucKucK, H., AND Levan, a. Vergleichende Untersucluingen an dijjloiden
und tetraploiden leinsippen iind tetraploiden Kreuzungsnachkommenschaften
nach vieljahriger Sclektion. Ziichter. 21:195-205. 1951.
46. Levan, A. Polvploidiforadlingens Xu\arande Lage. Sver. Utsadesf. Tidskr.
Pp. 109-43. 1945.
47. LvsENKO, T. The situation in biological science: Verbatim report of the pro-
ceedings of the Lenin Academy of Agricultural Sciences of the U. S. S. R. For-
eign Languages Pulilishing House, Moscow. 631 pp. 1949.
48. Mangelsdorf, P. Evolution under domestication. Amer. Nat. 86:65-77. 1952.
49. McFadden. E., and Sears, E. The genome approach in radical wheat breeding.
Jour. Amer. Soc. Agron. 39:1011-25. 1947.
50. Mendes, a. Observacoes citologicas em Coffea. XI. Metodos de tratamento
pela colchicina. Bragantia. 7:221-30. 1947.
51. MiJNTZiNG, A. New material and cross combination in Galeopsis after colchi-
cine-induced chromosome doubling. Hereditas. 27:193-201. 1941. Cytogenetic
properties and practical value of tetraploid rve. Hereditas. 37:1-84. 1951.
52. Nebel, B. Cytological observations on colchicine. Collecting Net. 12:130-31.
1937.
53. . AND Ruttle, M. Action of colchicine on mitosis. Genetics. 23:161-62.
1937. The cytological and genetical significance of colchicine. Jour. Hered.
29:3-9. 1938.'
54. NiSHiYAMA, I., and Matsubayashi, G. a list of induced polyploids in the plant
(a review). Kihara Inst. Biol. Res. Seiken Ziho. 3:152-71. 1947.
55. NORDENSKIOLD. H. Svuthcsis of PhlciUN jnateiise, L. from P. iKidnsinn. L.
Hereditas. 35:190-214. 1949.
56. Pal. B., and Ramanujan, S. Plant lireeding and genetics at the Imperial .\gri-
cultural Research Institute, New Delhi. Indian Jour. Genet, and Plant Breeding.
4:43-53. 1944.
57. Parthasarathv, N.. and Kedharnath, S. The improvement of the Sesame
crop of India. Indian Jour. Genet, and Plant Breeding. 9:59-71. 1949.
58. Peto, F., and Boyes, J. Comparison of diploid and triploid sugar l)ects. Can.
Jour. Res. Sec. C. Bot. Sci. 18:273-82. 1940.
59. "Ramanhiam, S., and Deshmuk. M. Colchicine-indiued polvploicK in crop
plants. III. Oleiferous Brassicae. Indian Jour. Genet, and Plant Breeding.
5:63-81. 1945.
60. Randolph, L. An evaluation of induced polyploidy as a method oi hieedmg
crop plants. Amer. Nat. 75:347-65. 1941.
61. Richmond, T. Advances in agronomy. Vol. 2:63-74. Academic Press, Inc.,
New York. 1950.
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59:79-87. 1939.
63. Sacharov, v., et al. Autotetraploidv in different varieties of Inickwheat. C. R.
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30:38-43. 1939. Amphidiploids in the seven-chromo.some Triticinae. Mo. Agr.
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Inc., New York. 1948.
The Experimental Polyploids 291
fi"). SiMONKT, M. Production craiiiplii(lii)l()idcs fertiles et stables par intercroise-
nients d'especes lendues aiiioieiraploides aprt-s trailements c:ok'hicini(]Ucs.
C. R. Acad. Aor. France. 33:121-23. 1947.
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Press, New York. (543 pp. 19r)(l.
(57. .Stephens, S. The internal niedianisni of speciation in Cusixpnun. Uui. Rc\.
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7L'. ^\'^I.LE^slEK, S. The ne^vest fad, colchicine and its origin. Chron. Boi. 5:15-17.
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ADDITIONAL REFERENCES FOR LISTS OF POLYPLOIDS INDUCED BY COLCHICINE
Dela^ . C;. Nombres chromosomi(|ues chez les phanerogames. Re\. C\tol. Biol.
Veg. 12:1-368. 1951.
Ti.scHEER, G. .Allgemeine Pfianzenkarxologie. Gebriider Borntraeger. Berlin Nikolas-
see. 1953.
CHAPTER 12
The Amphipioids
12.1: Amphiploldy and Implications
New species can arise suddenly In interspecific hybridization and
doubling of the chromosomes. Such an act in nature separates the
new amphiploid, a potential species, from its parental progenitors.
New amphiploid species are able to invade new habitats, an invasion
not possible by either parent. A new ecological range, as well as re-
productive isolation from all other species, is acquired. More data
are now at hand from amphipioids produced in the laboratory, be-
cause colchicine has provided an effective method for making the poly-
ploids after the interspecific hybridization has been made. Principles
of theoretical and practical value can be developed.
Not all autoploids and am])hiploids separate into clear-cut cate-
gories since certain of their characteristics tend to overlap.^- Many
amphipioids produced by colchicine show autoploid characteristics.^!
The genetic and cytological changes that take place in later genera-
tions of propagation among such amphipioids are difficult to interpret
when there is interchange between the two parental genomes. A
classification designed by Clausen, Keck, and Heisey sought to visual-
ize how a gradual merger between autoploids and amphipioids obtains
if a number of cases \ue compared. Table 12 in their paper places
amphipioids in positions from the upper left-hand corner to the lower
right, in a gradient from autoploid to amphi))loid.-i The conclusions
incorporated in this chart were made after analyzing natural and
experimentally produced amphipioids.
While the limits between some autoploitls and amphipioids are
not clearly defined, the requirements for the success of an amjihiploid
as a new species are extremely sharp, almost to the jjoint of bemg
restrictive. Limits aj^pear to be set that cannot be violated, that is, if
the new plants are to succeed in nature. We should consider whether
the requirements for success in agricultural situations are not equally
restrictive. The requirements may be somewhat different, but new
[ 252 J
The Amphiploids 293
polyploids must meet exacting demands in order to succeed as new
crop species.
The diploid, interspecific hybrid, if it is to become a successftd
pohjjloid, must have good vigor, excellent growth of vegetative
characters, and an all-around vegetative cycle that is in harmony with
its environment.-^ Combined with these characteristics, the two
parental genomes shovdd be incompatible in the diploid hybrid to
the extent that no interchange can occur between them. 1 here should
be no gene exchange betAveen the parental sets of chromosomes,
which means no intergenomal pairing. Briefly, the dijiloid hybrid
according to these requirements should be entirely sterile until a
doubling of the chromosomes occms. Working in almost direct
opposition to these conditions, describing the source of ami)hij:)loid
from dijjloid hybrids between sj^ccies, are biological laws that tend
to j)re\ent achiexing the best-suited sterile hybrid. To acqiure such
genome incompatibility between the parents, one immediately moves
the relationships of the two species farther aj^art. Usually the farther
apart they are, the more difficult the hybridization Avill be. Even
after the hybrid has been made, a more distant relationshijj often
results in plants that are weak, j)oor in vigor, and lacking in good
growth generally. A poorly growing diploid hybrid cannot be ex-
pected to change into a vigorous, successful amphiploid by merely
dotd^ling the number of chromosomes.
If hybrids are made from species too closely related, gene ex-
changes between the parental sets of chromosomes occur. Then after
four or five generations, segregations tend to destroy the individuality
of the amphiploid from the parental type.-^ Of course, by gene ex-
change the transfer of a trait from one species to another at the poly-
ploidy level can occur. The moment gene exchanges take place, the
fiUme of the amjjhiploid as a distinct and isolated individual becomes
entlangered.^ Cytological mechanisms may automatically cause the
plants of later generations to drift to one or the other parental type.
Experimentally produced amjihiploids have been studied for
enough generations to demonstrate that genetic exchanges can take
place between the two parental sets of chromosomes. From a jilant
breeder's point of view this woidd seem to offer opportunity. Other-
wise a strict independence between genomes, like those of Raphnno-
hrassica, permits a true breeding type distinct from either parent, f)tit
further hybridization with either jjarental species to improve the
amphiploid is ineffective. -'^ If the amphiploid is not like the Raphauo-
bra.ssica case and intergenomal pairing does occur, gene exchange
leads to segregation in F^ and later generations. Many segregates may
be weak, sterile, and jjoor. Occasionally, new and xigoious com-
binations may arise. Certainly a scries of new lines can be developed
when there is exchange between genomes."''
294 Colchicine
Suppose that lines are isolated by selection after interspecific
segregation among progenies of aniphiploids. One cannot expect these
lines to compete in nature as successful independent aniphiploids in
the same rank as a distinct and differentiated species. From an agri-
cultural standpoint these lines need not be new species, and they may
or may not be valuable as new i:)olyploids. If the transfer of genetic
traits is made from one parental species to another, and the species
of commercial importance is improved, the result is not a new poly-
jiloid.-" For example, mosaic resistance was transferred from N.
ghiti)iosa to the A^ (ahacitrn genome. "^ The characteristics of com-
mercial tobacco plants were not changed, but the disease resistant
factor was added. Chromosome numbers were finally stabilized by
selection after backcrossing at the same number as .V. tabacum 48, and
after specific selection only a few traits were transferred from N. ghi-
tinosa. All but the resistance to disease were eliminated. As an am-
phiploid then, the new A', tabacum with only the disease-resistance
characteristic added can hardly be considered as an independent t\pe.
Stability of a new amphiploid is proportional to the gene exchange
between the two parental genomes. Lack of interchange favors rela-
tive constancy; conversely, interchange promotes instability. Ex])cri-
mentally produced aniphiploids of all gradations from those with
much interchange to others with very little, offer excellent oppor-
tunity to explore certain basic propositions controlled and observed
after selection, ^f''^' ^ either in nature or under guidance.
Doubling of the chromosomes among sterile diploid hybrids may
be done either through gametic j^rocesscs, i.e., production of un-
reduced gametes, or by somatic doubling. The accidental doubling
in nature has occurred largely by the gametic processes. On the other
hand, colchicine is most effectively apj)lied to somatic tissues. The
differences between these methods of doubling the chromosomes are
imj)ortant and should be compared when such comparisons can be
made.
12.2: Amphiploidy in the Gramineae
Economically, the grasses comprise the most important family
among all plants. Polyploidy is common in many groujis including
agricidtural species. Generally, their origin has been through hybridi-
zation and doubling of the chromosomes. Autoploidy is limited as
a method of speciation-"'-^ in grasses compared with amphiploidy.^'*-^
Polyploidy among grasses presents problems-"'"' i*^- ^i- -">• ''^ that in-
volve both theoretical and practical aspects."- -•'• ■*'• ■'■*■ ^^'' '^*'' '^"' ^"^
The origin of hexaploid wheat^'"^ has many theoretical phases,'^*'' ^"'^
and no one can escape the practical importance attached to this one
species, Triticurn aestivutn L.^^^
The Amphiploids 295
12.2-1: Origin of hexaploid xvheat. Bread wheat, Triticiim aesti-
vum L. {T. vulgare Vil.) is mankind's most important single species
in culti\ation. Millions of people depend on the annual grain produc-
tion ol this plant. As an achie\emcnl in agriculture, the accession ol
this one species alone is man's important contribution as a plant
breeder.
Historically, in terms of the long period of agriculture, the 42-
chromosome wheats are relatively new. Certainly the tetraploid
Avheats antedate hexa))loids, while diploid species preceded the tetra-
ploids. No hexaploids are known out of cultivation, whereas diploids
and tetraploids are represented by wild and cultivated species. Full
knowledge of the origin of bread wheat probably will never be ob-
tained, but some phases can be closely inspected by observing the
experimentally produced poly]:)loids. Colchicine has been a useful
tool in tracking down certain stejjs in the origin of the hexaploid
species, notably Triticiim spelta and related species. 122
First, consideration should be given to Tritiniin monococciim L.,
a 14-chromosome sj^ecies, to gain some idea of the oldest species of
w^heat in agricidture today. Another diploid, Agropyrou triticeum
Gaertn., is suspect in the hybridization with Triticiim which created
the tetraploid, or 28-chromosome, species.'*'- ^*"* These two parental
types may be called the A and B genomes, representing Trilicum and
Agropyron , respectively.^^
A large group of cultivated tetraploids, having either free-threshing
or invested grains, remain in cultivation as valuable economic species.
The emmer and durum types play an important role in agriculture.-*"
One of the most interesting tetraploids is the free-threshing Triticiim
persicum.-'^
Let us return to our hypothesis that Triticiim monococcum is the
genome A, and that the diploid genome B came from Agropyron
triticeum. ^^^ The true contribution made by Agropyron may now be
so remote that one cannot hoj:)e to retrace these steps. Let us assume
these diploids combined to make the tetraploid wheats. The evolu-
tion fiom tetraploid to hcxaj:)loid may be repeated more easily than
that from diploid to tetraploid. Bv crossing tetraploid Triticum
dicoccoides, 28-chromosomes, with diploid Aegilops squarrosa, a sterile
triploid hybrid was obtained.""- ^'^ This plant had 21 chromosomes,
was sterile, and resembled hexajiloid Triticum spelta, or spelt wheat.
Upon doubling the chromosomes, a 42-chromosome wheat was de-
veloped. This synthesized hexaploid hybridized with the natural
hexa])loid T. spelta. The selfed ]3rogenies from this hybrid did not
thro^v segregates as one might expect from a wide cross. In fact, no
segregation occurred. Pairing at meiosis among the F, hybrid did not
indicate widely differentiated cluomosomes of synthetic T. spelta
296 Colchicine
against natural T. spelta.^""- '" On the contrary, a close homology was
suggested. There was more difference between synthetic T. spelta
and natural T. spelta when amphiploids were obtained after gametic
doubling''"* than those irom somatic doubling.""
Crossing with Aegilops squarrosa so improved the plant and the
grain that one might expect a naturally occurring fertile plant like
the resulting hybrid to be recognized as a new variant."" The geo-
graphic range of A. squarrosa should show in general where the
original hybridization took place.''"' This species grows today in the
northwestern Himalayas, the Caucasian region, and over an area
where hexaploid wheats could have originated as a result of the con-
tact of A. squarrosa with tetraploid species of Triticuui. Diploid
Aegilops, known as goat weed, is a very unpromising agricultural
plant;!"^ yet its contribution to connnercial wheat by a species like
A. squarrosa must be very specific and is apparently necessary. The
genome is called the D genome."'" 1 herefore, hexaploid wheats are
now identified by genomes A, B, and D, each representing a genus and
each sharing one-third of the 42-chromosomes.i""- ''^- '^^ An isolating
mechanism has been discovered in Triticuin associated with the D
genome. ""*
Between the dawn of agriculture and some time not too long ago,
the hexaploid wheat evolved. Exactly when and how many times the
hexaploid species appeared remain luisolved problems. Let us say
at some time between 2000 and 10,000 years ago. Or perhajjs the
cross between diploid Aegilops squarrosa and tetraploid wheat is
happening today. Ihe amjjhijjloid Triticum jyersicum X Aes,ilo}ys
squarrosa, which is very similar to hexaploid Triticuyn, is a species
obtained from Russia.^^ If more hexaploid cases could be found in
the areas where Aegilops squarrosa grows, sucli additions to our
knowledge would be of great interest. •''''
We know there are parts to the story that must be sketched with
certain reasonable assumj)tions. It was remarkable that two research
teams,-"'**' '^" working entirely inde))cndent of each other, came so close
to each other in an agreement that Aegilops squarrosa is suspected
as one of the diploid species.
Evidence that some other diploid species of Aegilops contributed
to wheat now becomes a burden of ])roof by using a cross involving
other species, or else by other methods to demonstrate how the hexa-
ploid wheats came into existence when they did. For the present at
least, the independent contributions of Japanese and American geneti-
cists that Aegilops squarrosa contributed genome 1) still stands.
An important character of Triticuin aestivum is the free-threshing
feature. Ihe synthetic T. spelta, like natural T. spelta, was an in-
vested type. How the free-threshing types such as T. aestii'uni L.
The Amphiploids 297
evolved remains lor lurther study. Answering the question whether
this type arose as a segiegate, or directly from a diploid-tetraploid
hybridization requires more data.""- ^"" A jjattern for research has
been established.'""^
Another method for converting the tetraploid species into hexa-
ploids has been reported. ^-^ Planting the 28-chromosomal species in
the autumn instead of spring, a regular procedure for these hard
wheat types, after two, three, or four seasons the durum spring wheats,
28-chromosome species, suddenly change into the vulgarc or 42-
chromosomal soft wheat sj^ecies. There was no evidence of hybridiza-
tion, and no intergrading forms. This method obviously differs from
the two explanations given by Japanese and American geneticists for
the origin of hexaploid species.
12.2-2: Other aiuphipJoids among Triticluae. The amphiploids
made from interspecific and intergeneric hybridization among Aegi-
lops, Triticiim, and Agropyron ha\e increased many iold.''' "•'• i^--^"' «"• "■*■
88, 100. 101, 118. 66. 6s. 86. w^. 98. 110 ^^^^^^ ^\^^. flj-^t fertile Triticinn-Agropyron
amphiploid was produced with colchicine in 1939.-'^ A wealth of
material is at hand to solve the basic problems that determine the
progress to be made in using amphiploids.'"' '-" Since all the cases
cannot be reviewed, a selection will be made to point out theoretical
and practical problems.
Among Aegilops, the species have evolved by interspecific hybrid-
ization and chromosomal doubling.'*' There are diploid, tetraploid,
and hexaploid species rejjrcsented by haploid numbers, yi ^7, n ^ 14.
?7 =: 21, respectively. Since Aegilops has contributed to hexaploid
wheat, a knowledge of these species is important even though the
group has little economic value of its own.
In 1913 Cook discovered a hybrid in Palestine involving the Emmer
Triticurn dicoccoides and some form of Aegilops. Later, Percival
jjointed to Aegilops rylindrica as a contributor of the spelt characters
in the tetraploid Triticurn. Evidence accumulated suggesting that T.
aesiivum L. arose as a segregate out of a cross between T. dicoccoides
and A. cyliudrica. The amphiploid [n ^ \A) , Aegilops cylindricd
(n ^ 14) , was synthesized by crossing Aegilops caudata (n := 7) X A.
sqiKirrosa (n = 7) and doubling the chromosomes with colchicine.'""
Now three sets of data come into focus. First, earlier taxonomic
wf)rk brought tetraploid Tritidim and the tetraj)loid Aegilops cylin-
(irira together. Second, the tetra]:)loid A. (\li}i(lric(i evolxed Irom two
diploid species, one being A. s(jiiarrosa. Ihird, the synthetic amphi-
ploid, Triticurn di( <)< ( oides var. spontaneoxnllosmn X Aegilops sr/uar-
rosa is similar to natmal Triticurn spelta.-'^'^^ In 1931 a sj)eltlike
sterile hybrid between tetraploid Triticuin diioccuni and Aegilops
sqiiarrosa was made by McFadden, l)ut for want of a ready method to
298 Colchicine
convert this sterile hybrid to a fertile one, the necessary evidence le-
mained hidden until fertile hexaploids could be made.^^*^
The D genome represented in hexaploid wheat and the genomes
of modern diploid Aegilops squarrosa are probably very close in their
homologies. Also, this genome is not found in any species of wheat
tested that had fewer than 21 chromosomes. Tetraploid wheat lacks
this genome. Finally, taxonomic characters in Aegilops squarrosa
correspond to those traits that distinguish the hexaploid wheat from
tetraploids.^"" These are: the square-shouldered inflorescence, hollow
stem, and articulation of rachis, differentiating Triticum spelta from
the tetraploid Emmcr wheats.'"
Taxonomic characters were used to trace the probable origin of
hexaploid wheat before cytogenetic evidences were at hand. The
fact that diploid Agropyron triticeum Gaertn. has features distinguish-
ing dijiloid T. monococciDU from tetraploid wheat arouses interest. ^"^"
Discovering more specifically how genome B was contributed and what
its relation to Agropyron is, becomes more involved. This genus also
has a polyploid series in its evolution. The base is ?; = 7 (Table 12.1) .
Some intergencric hybrids involving Agropyron have been made. 5-
11-9 Wey^A^iloid T. aestivum {ri=:2\) -And Agropyron gknicinn (n =^
21)^*^ were combined to make an amphiploid with 84 chromosomes.
Strong perennial tendencies arise with these high polyploids. In
another case, vigorous plants with 70 chromosomes were derived by
adding the hexaploid complements, 42 chromosomes, to the tetra-
ploid Agropyron intermedmm, 28 chromosomes. This particular 70-
chromosome fertile hybrid was the first amphiploid to be reported
from tests with colchicine.''^
The genus Triticum, represented by three chromosomal levels,
n r= 7, n =: 14, and ?/ = 21, provides much material following inter-
specific hybridization. A tetraploid, T. timopheevi, has the genome
G not common to other well-known species.-*' Another free-threshing
tetraploid species, T. persicutn, produces an interesting series when
crossed with Aegilops squarrosa/'^ Unquestionably, these amphi-
ploids have free-threshing hexaploid bread wheat features.
Within short intervals after colchicine was discovered, more than
80 different amphiploids, involving tetraploid and hexaploid, as well
as diploid species of Triticum were produced in Russia. ii'^ Some
higher numbers proved to be interesting in their hybridization charac-
teristics in subsequent generations. Generally the sterility increased
when hybrids above the hexaploid level were created. The ordinary
wheat, usually self-pollinated, changed into a cross-fertilizing type as
higher-level amphiploids were reached.
1 he complexity of sterility-fertility relationships appear in the
intergencric and interspecific hybrids among 'rriticinae.^^- i**' ^"" ■^"' ^^
The Amphiploids 299
Cliioinosonial pairing in the tli|>l()itl hybrid, or the lack oi pairing is
not necessarily an index of homology. The intergeneric aniphiploid
Aegilops iimbelhdata X Hayuoldia villosa has a reduced lertility.^""
The particular strain made a difference in pairing; environmental
and genetic factors, also, influence pairing of chromosomes. 1 wo dis-
tantly related species may introduce physiological upsets that cause
TABLE 12.1
Divergent and Convergent Evolution of Hexaploids
(Adapted from McFadden and Sears)
Primary Form
Diploid
Divergent Form
Diploid
Convergent Form
Polyploid
Agropyron genome B .
Unknown
Trilicum genome A
/
, AB
Trilicum
tetraploid
Trilicum
hexaploid
ABD
'Aegilops genome D — Aegilops
diploid
D
meiotic irregularities." The rule cannot be established that uni-
\alen(y in the F, is j)rcdictable evidence for obtaining good fertile
amphijjloids.
Evolution in wheat that finalh led to hexaploids may be charted
as a divergence in the early period following convergent evolution
giving rise to the tetraploid and hexaploid sj)ccies. Some tmknown
diploid form evolved into three basic genera: (1) Agropyron, (2)
Triticuni, and (3) Aegilops. The first two hybridized and gave rise
to a series of tetraploid species. A second step in evolution involved
the combinations between tetraploid Triticum and Aegilops. A chart
is used to help \isualize these evolutionary patterns (Table 12.1) .
Since such valuable species have arisen throtigh combinations of
genomes, this approach was suggested as a "radical" method of wheat
breeding. Desirable characters would be transferred to T. aeslnnim L.
by using specific series of synthesized amphiploids. Four were sug-
gested. The first series involves the D genome from Aegilops squar-
rosa added to various tetraploids because the hybrids are more fertile
than crosses between tetraploids and hcxa]:)loids within Triticum. A
second series involves combinations between tetrapltjid wheat and
300 Colchicine
Aegilops other than A. squarrosa. Third, the combined genomes A
and D united with various species oi Agropyron would lead to ways
for introducing genes from the latter genes to the present B genome
of hexaploid wheat. Fourth, the synthesized B and D genomes added
to diploid Triticum would allow transfer of einkorn characters to the
hexaploid wheat. Such a program is exceedingly involved; however,
it merits serious attention, (cf. Chapter 11. Ref. No. 49) .
72.2-5; Triticum aestwum L. X ^ecaJe cereale L — Triticale. In
1876 the first hybridization between wheat and rye was made. About
4 per cent of hybridizations between wheat and rye give some idea
of the success to be expected. Under unusual circumstances a fertile
56-chromosome Fo can be obtained. An unreduced gamete most
likely explains the mode of doubling. Since colchicine became avail-
able, new methodsii^ have been developed to increase the production
of Triticnles.^^- ^■'- "''
There are five well-known strains,2i (1) Rimpau 1891, (2) Meis-
ter 1928, (3) Lebedeff 1934, (4) Taylor 1935, and (5) Miintzing
1936. Since 1936 many more have been made. Actually no accurate
record can be given because of the number of unpublished cases.
Biologically the 56-chromosome plant is of interest because the
constant number has been maintained in the Rimpau strain after
more than fifty generations. Backcrosses to wheat give some index
of the stability that Tritirales can maintain. The 56-chromosome
plants survive better, are taller, and maintain a stable genetic
mechanism in spite of some meiotic irregularities.21 At meiosis in the
Fi very little pairing has been observed, 0-3 pairs; and upon dou-
bling, mostly bivalents are seen with as high as 6 unpaired chromo-
somes in some strains. There is practically no homology between the
wheat and rye chromosomes. -^
Among backcross progenies a pair of rye chromsomes have been
substituted for one pair of wheat chromosomes (cf. Chapter 14, Ref.
No. 37) , so there would appear to be slight possibility for gene ex-
change under selection. In nature the Triticale could evolve as a new
species because there is some degree of difference between the strains
regarding fertility and segregations in the subsequent generations.
However, the Triticale would remain at the octoploid level, and con-
sequently, a group ol new species could evolve with 56 chromo-
somes^i (cf. Chapter 14, Ref. No. 37, 27, 46, 51) .
Economically these species bring into one plant two of the world's
important bread-producing species, wheat and rye. Since doubling
the chromosomes can be done with colchicine, a serious attempt to
improve Triticale on a large scale should have possibilities.
An all-out attack on this ])roblem was begun in 1939 in Holland;
it involved the processing of hundreds and even thousands of combi-
nations.ii^ A new method of clonal division and vegetative propa-
The Amphiploids 301
gation of the Fj plant was devised so that several hundred plants
coidd be obtained in one season. These were treated by soaking the
roots in colchicine. ^^^ Fertile spikes indicated .56-chromosome plants.
The work was progressing satisfactorily until in 1944 the research
jjlot became the scene for \V'orld War II. Because of considerable loss
of material and change in personnel, the original plan had to be
modified radically.
It is encouraging from the viewpoint of polyploidy that Triticales
are now regarded as potential breeding material instead of a genetical
curiosity, as it was for a good many years.
12.2-^: Artificial and natural polyploids in Graniineae. Large-
scale synthesis of polyploids by colchicine can be of use theoretically
and practically.^**^ Newly created polyploids in grasses were placed for
testing on range, pasture, and luitended habitats. Following such an
introduction, continuing records will show up the potentialities for
adajjtation of the new species, for the competitive success or failiu'e
would become evident after several generations. To a degree, princi-
ples governing success apply to polyploidy among intensively culti-
vated situations, as well as in pastures or wild habitats. ^^'-^
Among Triticales we mentioned the maintenance of constant 56-
chromosomal plants after fifty generations of cidture. Backcrosses to
wheat always favored the more vigorous 56-chromosomal plants. Ap-
paiently a stabilizing mechanism operates in the Triticales complex.
Undoubtedly this is true for many polyploids among grasses where
70 per cent of the species are natural polyploids. Therefore, new
polyploids with high numbers and complex genomic additions shoidd
bring important facts to our attention. -^
Such projects involving artificial and natiual polyploids carried
out by Stebbins and his associates have already added important in-
formation, i*''^- ■'- Further research based on long-range objectives will
surely advance our knowledge of polyploidy.
In the valleys and foothill regions of California, agricultural prac-
tices have created three ecological situations into which natural and
artificial polyploids shoidd show differences in adaptation. First, the
once native grasslands that have been there are heavily grazed and
are now covered with annual species from Europe. Second, luigrazed
fields nearby are filled with introduced species. Third, there are
pastmes suitable for reseeding forage crops or grasses and for con-
trolled grazing. Obviously this is a tuiique situation representing
three unstable plant associations. Into these habitats artificial as well
as natural polyploids can be introduced by seed and/or vegetative
starts.i"^
Large ])opidations of artificial polyploids, both autoploid and
amjjhiploid, were made by colchicine methods. ^'^'•'' One successfid
autoploid, Ehrharta erecta, will be discussed in the next chapter. Here
302 Colchicine
general outline of the amphiploids ^vill be sketched. Polyploids from
24 interspecific crosses involved six genera: Bromus, Agropyron,
EJymus, Sitanion, Melica, and Stipa. Major emphasis was given to
Bromus because thirteen combinations were taken from this genus.
Considerable cytogenetical information has already accumulated for
three out of five recognized sections. Representative species are na-
tive to the American continents; perennials and annuals and natural
polyploidy series exist. i"''
A polyploid ^vith 112 somatic chromosomes involving Bromus
carinatus and B. marginetus exceeds the 84-chromosome level, highest
known for the genus under natural conditions. The artificial poly-
ploid into the C4 generation was vigorous, apparently more than the
Fi hybrid as shown by considerable vegetative growth that occurred
in the garden. A successful allopolyploid wdth 112 chromosomes was
a remarkable new case testifying to an effective use of colchicine when
combined with an appropriate hybridization. ^"^-^
Even more notable were the polyploids B. cannatus-trinii and B.
maritimus-irinii, which apparently combine the genomes from seven
different ancestral diploid species, thereby being 14-ploid, containing
98 somatic chromosomes. The immediate success demonstrated by
these polyploids is of exceptional interest when viewed together with
the implications about amphiploidy mentioned in the first section of
this chapter. The hyl)rids were very vigorous and mciotic processes
were irregular after doubling; plants in the C;:. and C4 generation
showed seed fertility in the range from 70 to 94 per cent. In all
probability this is a successful polyploid. i^*^
As shown by this work and an increasing number of other cases,
sterility-fertility relationships cannot be predicted in advance. Of all
the problems that confront polyploidy breeders, sterility-fertility
status among the newly created polyploids may well be the most
significant.^- The lowered fertility in autoploids has been confirmed
again and again. A conclusion that amphiploids necessarily have
higher fertility can be very misleading. A breeder using artificial
polyploidy must face the problems of sterility. Accordingly, two fac-
tors stand out as deserving primary consideration: vigor and fertility.
12.3: Gossypium
Special methods were devised for treating interspecific, sterile
hybrids of Gossypium with colchicine.^- 7. 27, 34, 54, eo. 106, iis) since
fertile amphiploids would be produced at once upon doubling the
number of chromosomes, a theory of the origin of tetraploid species
could be tested. Skovsted proposed that the American tetrajiloids in-
volved genomes from an Asiatic dijjloid and an American wild di-
ploid species. By hybridization between the Asiatic and American
The Amphiploids 303
diploids, and dou Idling of chromosomes, a tetraploid species like G.
Iinsiilinn arose in natnre. Now the test could be repeated experi-
mentally, and those investigators who had been studying species hy-
brids at the time promptly ajjplied colchicine. The synthesis was
announced independcnth from two laboratories."' ^^ G. arboreiitii
(n = 13, Asiatic diploid) X ^^- tlnnhrri (n = 13, American diploid)
was changed from a 26-chromosome h\bricl to a 52-chr()mos()mc amphi-
ploid. The plants were cytologically similar to G. hivsiiiiiin. The
synthetic amphiploid hybridized with natural tetraploids, and sur-
prisingly good pairing at metaphase was obtained. A concltisive ex-
periment had been performed. The hypothesis of Asiatic-American
origin of tetraploid cotton was confirmed.'- ^•'
A useful classification" was formulated to bring together data
about geographical distribution, morphology, chromosomal pairing,
numbers, and chromosomal structine differences. The genomes from
each region were gi\'en letters as follows: (1) Asiatic species, A-^ and
A./, (2) African diploids, B; (3) Australian species. C; (4) American
dijjloid species, D^ to D^r, and (5) Arabian-India diploids, E. The
Asiatic species represent a central position with affinities to American,
Australian, and Arabian-Indian sj^ecies. They are closer in relation-
ship to African species than the other grotips. Arabian-Indian species
are distant to all and jjarticularly farther front the American diploids.
One advantage of this system is the code that can be used for describ-
ing amphiploids." If the American tetraploids were derived from an
Asiatic and an American source, the amphij^loid should read 2 {AD)
with an appropriate subscript to indicate the species of tetraploid.
Accordingly the G. hirsutiun would be 2 (AD) ,. Table 12.2 illustrates
the use of genomes and some of the important species with their geo-
graphical distribution.
Experimentally produced amphiploids are potentially new species
because the duplications made by hybridization of diploids and dou-
bling the chromosomes do not exactly replicate the natmal one.^'' Some
kind of differentiation occurred after the first amphiploids arose. A
spontaneously occurring amphiploid, ^^ G. davidsonii X G. anornalum,
showed how a new species might have arisen in nature and become
isolated from other types. A counterpart of tliis spontaneously oc-
curring cotton was made by colchicine. The data for these cases were
similar.'""
Problems in polyploidy among species of Gossypium were well
known before colchicine was discovered."*^ Gene systems were con-
cei\ed to account for the way in which diploid and tetraploid species
became differentiated. By the use of experimentally produced amphi-
ploids, relations between genomes and the problem of speciation could
be studied more extensively. Specialists in Gossypium began to realize
more specifically that problems remained unsolved. i*^"
304 Colchicine
Interspecific hybrids between the two tetraploid species are vigor-
ous and fully fertile in the first generation. These species, G. hirsutum
and G. Ixirbadense, both carry desirable qualities.is Attempts to com-
bine the best features of each in a new variety have not been as success-
ful as one might wish.^"'' The second generation and subsequent ones
give rise to weak, sterile, and undesirable types. Backcrossing to
either parent has not led to new levels of improvement. One might
well ask if the combining of characters from other species, which are
TABLE 12.2
Genomes of Gnssjpium
(After Brown and Beasley, and Menzel)
Natural Species and Tetraploid Genome
Tri-species Hybrid Descriptions Formula
Gossypiim herbaceum L Asiatic 1 3-chromosome 2Ai
G. arboreiirn L Asiatic 1 3-chromosome 2A2
G. anomolum Wawra. and Peyr African 1 3-chromosome 2Bi
G. sturtii F. Muell AustraHan 1 3-chromosome 2Ci
G. thurbni Tod American 1 3-chromosome 2Di
G. aimouriamnn Kearney American 1 3-chromosome 2D2-1
G. harknessii T. S. Brandeg American 1 3-chromosome 2D2-2
G. davidsonii Kellogg .American 1 3-chromosome 2D3
G. klotzchianum Anderss American 1 3-chromosome 20.,
G. arulum (Rose and Standley) Skovsted American 1 3-chromosome 2D4
G. raimondii American 13-chromosomc 2D5
G. slocksii M. Masi Arabian-Indian 1 3-chromosome. . . .2Ei
G. hirsutum L American 26-chromosome 2(AD)i
G. barhadense L American 26-chromosome 2(AD)2
Hcxaploid G. hirsutum X herbaceum X G. harknessii 2(AD)iAi X 2D2_2
Hexaploid G. hirsutum X arboreum X G. harknessii 2(AD)iA2 X 2D2-2
Hexaploid G. hirsutum X anomalum X G. harknessii 2(AD)iBi X 2D2-2
Hexaploid G. hirsutum X stocksii X G. armourianum 2(AD)iEi X 2D2-1 X 2D2-2
G. harknessii
Hexaploid G. hirsutum X stocksii X G. raimondii 2(AD)iEi X 2D5
The Amphiploids 305
possible now that many fertile amphiploids can he produced, will
not face the same difficidties confronting a breeder who tries to com-
bine the characters of the already \vell-kn()\vn Upland and Sea Island
cottons.
If some chromosomal mechanism prevents the recombinations of
genes contributed by each parent, then merely growing large prog-
enies and exercising selection can hardly be expected to yield re-
sults.^'"'' The evolution of the tetraploid from dij)loids can be ex-
plained by the hybridization and doubling of chromosomes. This
does not explain the difterentiatirjn of the tetraploid species after
they once originated as an amphiploid. An argimient supported by
considerable data^*"' asserts that a structural differentiation of chromo-
somes was basic to speciation and this was of the cryptic type, that is,
in very small segments, so that a differentiation could not be ob-
served by pairing or irregularly arranged chromosomes at meiotic
metaj)hase. Therefore, a genetic hybridity and a hybridity caused by
the differentiation of small chromosomal segments could not be de-
tected by the ordinary genetic and cytological methods. The nature
and extent of chromosomal differentiation may be measmed by trac-
ing marked genes in subsecjuent generations and recording the rates
at which the genes are lost by successive backcrossing. Such chromo-
somal differentiation may be important in Gossypirim.'^^^ At least,
the suggestion has led to inflection on these problems in polyploidy.
Among the second generations of the interspecific hybrid between
G. hirsiitum and G. barbadense, asynaptic genes account for the ste-
rility observed, notably when certain parents are used." Genes for
asynapsis have been foimd in both genomes A and D. By the use of
trisomies, additional data about these asynaptic types have been col-
lected. The fully sterile plants eliminate the completely asynaptic
types, but partial asynaptic types are carried along.^^ Some of the
j)hen()mena attributed to a cryptic structinal hybridity might be ex-
])lained on the basis of asynaptic and partially asynaj)tic genes. ^'''
Sterility resulting from asynaptic genes is a kind of genic-*^ sterility
and may well be important in such sterility that causes failure in
chromosomal pairing. The extreme sterility at the diploid hybrid
level can be overcome by doubling the chromosomes. But a sterility
due to asynaptic genes is not cmed through doubling the nimiber of
chromosomes. Later generations introduce new problems in maintain-
ing the fertility level as well as the characters brought together in the
hybrid. If by selection some desirable characters contribtited into
the hybrid are eliminated and inidesirable ones retained, polyploid
breeding is faced with a difficult task. To incorjx)rate into commercial
varieties the desirable characters foimd in other sj^ccies can be ])ut
306 Colcbicina
down on paper more easily than producing die plants. One step is
hybridization and the doubling ot chromosomes; the next procedure
requires some new approaches.
Certain species are totally incompatible.^"' The tri-specics''' hy-
brids have overcome these difficulties, for some genomes can be
brought together in a tri-species hybrid not possible in a regular
hybridization. Gossypiuni arhoreum and G. harknessii have not been
brought together except when the hexaploid G. hisutum X <^- ^"^^o-
reum was crossed w'ith G. harknessii. In this manner a tetraploid
brought together genomes {AD) i A^ D. representing G. hirsiitiinu G.
arhoreum, and G. harknessii, respectively. Six new tetraploid tri-
species hybrids were developed by this method^'' (Table 12.2) .
From a plant-breeding standpoint, amjihiploids incorporating
genomes of G. anomahim, G. raimondii, and G. liarknessii with the
commercial strains of Iiirsutum are promising and represent a new
attack on the problem of cotton improvement.''-^ Increases in fiber
strength are possible; however, a problem arises when one tries to
gain hi fiber strength and also maintain the good qualities necessary
for commercial varieties of hirsiitum. Much cytological work is
needed; integrating the theoretical knowledge with practical testing
appears to be the outstanding problem at the moment. A final j>rac-
tical contribution resulting from the incorporation of characters from
other species is promising. Numerous amphiploids have been made
in a short time. Much has been done with colchicine as a preliminary
to the larger work of sorting out, by polyploid breeding, gains from
accumulated knowledge.
Among polygenomic hybrids, mosaics in flower and leaf appeared. ^^
Increasing the number of chromosomes shows some increasing tend-
ency toward mosaicism, but number alone does not determine the
degree. This is a side problem with no specific explanation except
that the polyploids exhibit such characters.'- ^^ Another side prob-
lem is the somatic reduction in numbers of chromosomes within a
hexaploid species hybrid. An original plant with 78 chromosomes
developed sectors that were triploid, having 39 chromosomes. Per-
haps the method offers a way to extract useful components from a
complex hybrid. i^'' "-
Aneuploids in Gossypixnn are readih de\eloped because the trip-
loids and jxntaploids are unbalanced types. Backcrossing and selec-
tion for trisomies and tetrasomics are possible among the synthetic
polyploids. Resultant ancuploid types have their effects upon leaf
texture, color, and structure. New lines with an extra pair of chromo-
somes, 54 instead of 52, may include Asiatic or American chromo-
somes placed into the opposite germ plasm.^'' 7?zh77specific and inter-
specific trisomies and tetrasomics were obtained. Such lines may be
partially stable, fertile, and morphologically distinguishable.^-^
The Amphiplo'ids 307
12.4: Nicotiana
A theory of evolution was experimentally verified when N. digliita
was made in 1925. 1 he parental species, N. tahacum, a natural tetra-
ploid with 48 chromosomes {n = 12), and the diploid N. glutinosa
were hybridized to make the sterile triploid with 36 chromosomes.
A fertile hexaploid was isolated that had 72 chromosomes. This num-
ber was a new and high one for the genus. Previous to the develop-
ment of A', digliita, 48 chromosomes was the highest number.i^. 4o, 4i
Using colchicine, A', digluta was resynthesized. Since then numbers
higher than hexaploid have been built into polyploids of Nicotiana.^^
These polyploids were made by bringing together the proper species
in hybridizations and doubling the chromosomes of the hybrids. A
combination of three natural tetraploids included 144 chromosomes
in one plant.s*' Another report of 176 chromosomes has been made.^o
The development of plants with high numbers is not the sole
objective. Of particular significance is the combining of widely diverse
genomes in order to establish higher polyploid-amphiploids that are
fertile, vigorous, and relatively stable in later generations of propaga-
tion.^'^ The changes that take place in subsequent generations of these
polyploids show what mechanisms might operate genetically when
new species at new levels of chromosomal numbers become estab-
lished. Furthermore, the effects of selection upon these types are of
basic importance. i'^-^' ^
An important development that resulted from the synthesis o£ N.
digluta was the eventual transfer of mosaic resistance to the com-
mercial varieties of tobacco." ^ The necrotic factor from N. glutinosa
was transferred to the N. tahacum genome.^o. 38 An example of poly-
ploid breeding is illustrated by this program. After full review of
the work necessary to make the transfer, one becomes convinced that
these methods are not short cuts.
Realizing all iliat \\as involved in the requirements for transfer
and the cyt'ological and genetic data at hand as late as 194.S, there
was no complete assurance that the factor for resistance in A\ glu-
tinosa could be incorporated in the genome of N. tahacum:-- Each
time the transfer was tried, disadvantageous traits were carried along
with the chromosome contributed by A', glutinosa. Therefore, the
problem was one of maintaining the good features of commercial
tobacco varieties and utilizing only the disease resistance of the
glutinosa type. Fortunately, some chromosomal change occurred
during generations of selection, and a true tobacco type with mosaic
resistance of the kind noted for A^ glutinosa ap):)eared in the cultures.
The plant had 48 chromosomes and possessed the resistance factor
incorporated in the tahacum genome. ^^ Perhaps one might call the
new varietv. N. tahacum var. 77)// after a type made by Kostoff.^''^ No
308 Colchicine
doubt only a small segment of the chromosome from A^. glutinosa was
transferred to a chromosome of A^ tahacxim. If more than a small
segment were involved, greater disturbance to the genotypical balance
of the tabacum genome might be expected/''^
Evidence that parts of chromosomes were involved was given by
the fact that homozygous, low-blooming, mosaic-resistant segregates^^^
that were different from the Burley tobacco appeared in backcrossing
A^. digliita to A^. tahacinu. These segregates in one case appeared in
the fifth backcrossing generation. Similar segregates were obtained
when Gerstel's 50-chromosomc "alien additional race," which had a
pair of A^ glutinosa chromosomes, was backcrossed to N. tabacum.
The nimiber of chromosomes during crossing was reduced to 48. In
the process these homozygous, low-blooming, mosaic-resistant plants,
that diffeied from Burley tobacco, appeared much the same as when
A^ digluta was the starting material. ^^^
The assumption may be made that an interchange had occmred
between the two genomes. In this case a segment was transferred
from one chromosome of a genome to another chromosome of the
opposite genome. The exchange was small, and transfer was limited
to the disease-resistance character. When whole chromosomes of A^
glutinosa were substituted for a whole chromosome of A^ tabacurji,
the differences were such that substitution races differed from regular
varieties of tobacco. ^^^
Morphologically and genetically distinct popidations were isolated
among specific amphiploids as well as diploid hybrids. If the selection
was directed to a j^articular character, the progress made toward a
certain goal was faster at the diploid level than the amphipUjid.^*'-"^
Generally, the amphiploid populations were less fertile. The tre-
mendous power of selection that is possible among amphiploids can
be demonstrated if the ])articular type has some intergenomal ex-
change.^
Among species of Nicotiana the genetic systems are close enough
to permit hybridization, yet removed from each other and sufficiently
differentiated to provide sterile hybrids between species. Upon
doubling the number of chromosomes, the amphiploids are fertile
and partially sterile.^' «• i-^ -^s. 32, .ss, 3.^, 4i. ss. 102. 118 There is enough
pairing at the diploid level to indicate that in some combinations of
species, exchange between genomes can occur. Such exchange leads
to interspecific segregation in the Fo and subsequent generations.
Pairing of chromosomes at the diploid level of interspecific hybrids
is not a true picture of pairing when the amphiploid is derived. Five
cases with some bivalents at the F, stage had no nudti\alcnts in the
polyploid. ^^
The Amphiploids 309
By interspecific hybridizations and doubling of chromosomes, syn-
thetic tetraploids liave been made that resemble N. tabacum, yet lack
the same genotypical balance that exists in the natural species. Even
though the diploid species, A^ sylvestris, and certain diploitls of the
tonu'ntosa group may be combined to make a polyploid that re-
sembles A^ tabacuNi. the exact genetic duplication has not been ac-
complished.''*^ Usually the sterile hybrids doubled somatically are
female-sterile. Sterility is caused by failure at the embryo-sac stage.
When a long procedure of backcrossing was involved, a fairly fertile
synthetic A^ tabacum was obtained.*"' AVhen the synthetic was crossed
with a natural species, the segregation ni the second generations was
like the variability found between varietal crosses.
A list of the amphiploids made with colchicine is necessarily
large. There are more objectives involved than have been out-
lined in this section. Nicotiana provides some good material for the
study of polyploidy both from a practical and a theoretical point
of view.'*'^' ■^i' -'^'' •^**' i*^^' ~^' -^' •'• ^' -' ^-' ^-' ^•^' *^- ^'■^- ^"'-
12.5: Dysploidy Combined With Amphiploidy
Within the Cruciferae a natural group called the Brassica com-
parium by Clausen, Keck, and Heisey, form a dys})loid series as fol-
lows: 71 = 8, n = 9, u =z\0. ?/ = 11, n = 12, n = 17, u = 18. If the
artificial amphiploids are added, the series rises to the hexa)3loid
level, i.e., dysploid, // = 27 and // = 28. At once some fundamental
problems can be predicted from what has been said before.
Some notable historical events in cytogenetics occurred with this
groujj. The first cross between radish and cabbage was produced by
Sageret in 1826. One century later, Karpechenko demonstrated fertile
Raphanobrassica plants. -^ After Sageret's time, the cross was re-
peated by others. With colchicine, autotetraploid Raphanus was
crossed with autotetraploid Brassica thereby repeating the intergeneric
hybrid by another method.-"- •'^"- "-^ Previously the sterile diploid hy-
brid was made, and fertile plants were selected after unreduced
gametes united.'*^
Fruit structure in the Raphanobrassica polypkjids is j^rojjortion-
ally radish or cabbage, depending on the genomes present. Accord-
ingly, diploid, triploid, tetraploid, and pentaploid series can be ob-
tained with different doses of whole genomes.-^
Judging from the total lack of ])airing in the Fj hybrid at diploid
levels along with the independence maintained in the amphi|)loid.
gene exchange at dij^loid level is exceedingly limited. Hyi)ridi/ation
and the synthetic amphiploids have raised the level above tetraploidy
370 Colchicine
as illustrated by amphiploids of the Brassica comparium.^^' '^' '^'^' ^^•
50, 19, 36. 37, 124, 125
Three basic genomes are represented by diploid species of Brassica;
B. campestris, n = 10, or a: B. tiigra, n = 8, or b; and B. nleracea, n =
9, or c. There is some evidence of homology between a and r, but no
bivalents are formed between b and either a or c. The tetraploid
species B. carinata would have genomes ac cc; B. juncea aa bb; and B.
carinata bb cc. Accordingly, the hexaploid B. cJunensis X B. carinata
would have aa bb cc as genomes, or 27 bivalents. "^o
Economically these genera of the Cruciferac comprise one of the
most important groups with world-wide distrilnition. The number of
amphiploids made at the tetraploid level has increased with the use
of colchicine. ^''- •^«' ■"• ^^^ ^""' ^3- ^'^- n''- 1'". i^i
Synthesized amphiploids, comparable to the natural tetraploid
species of Brassica, can be hybridized readily and show possibilities
for selection in the succeeding generations. A large ninnber of pro-
genies are under study by Gosta Olsson at Svalof, Sweden.
12.6: Other Interspecific Hybrids and Amphiploids
Four species of Galeopsis, two diploid and two tetraploid, became
sul)ject to colchicine methods as soon as the drug was announced for
its polyploidizing action. Since tlie first Linnean species Galeopsis
tetrahit L. was produced by hybridizations with the two diploid
species, following doubling by gametic non-reduction, one of the first
uses for colchicine was a repetition of Galeopsis tetrahit L. By first
inducing autotetraploid G. pubescens and G. speciosa, the amj:)hiploid
was produced with little difficulty. Within a short time nuich poly-
ploid material was at hand for this genus. "-^
Cross combinations between diploid and tetraploid Galeopsis
usually fail, but genomes of dijiloid species can be hybridized at the
tetraploid level, using induced autotetrajiloids with natural tetra-
ploids.""' These crosses succeeded. Quantitative conditions control
the hybridization. More crosses were made to confirm this point."'*
The octoploid number, 64, exceeds the optimum number for these
genotypes, for octoploid G. tetrahit and G. bifida are much inferior
to the natural tetraploids of these species.'^'' Basic cytogenetical data
have been increased many fold with the use of colchicine.
Cytogenetical data from certain interspecific hybrids among Sola-
num suggested that there may be small structural differentiations be-
tween chromosomes of diploid species.^*' Such changes may have
significance in the evolution of species within Sohvitim. At least,
considerable data for interspecific hybrids have been accunudated
already, and more can be expected.
The case presented for GTOSsypiuin proposing "cryptic structural
differentiation" as a speciation mechanism was recalled as an inter-
The Amphiploids 317
pretation for problems in Sola mini:*''' Certain species ol" potato carry
valuable economic traits, e.g., specific resistance to phytophora, and
these would be desirable to incor|)orate in the present jxilyploid
species, S. tuberosum.
A study oi meiosis in hybrids between S. demissum and S. rybinii
as well as in haploid S. demissum shows pairing and suggests similar-
ities coujjled with these observations; the backcrossing of Fj S. demis-
sum X ^- tuberosum to .S'. tuberosum showed increased seed set with
each backcross.^*' One is led to recall the well-known elimination of
donor jjarent genotypes in certain interspecific backcrosses involving
Gossypium hirsutum and G. barbadense.^^^ These species have been
studied extensively, and recombintions on a gene-for-gene basis that
would permit transfer from one species to another runs into serious
difficulty after backcrossing. If a similar situation holds in Solanum,
then the program of amphiploidy and species h) bridization requires
further analysis."*^
Enough similarity exists between genomes of .S'. rybinii, S. tubero-
sum, and .S'. demissum to produce bivalents. By multiple crosses other
species like 5. antipoviczii can be crossed to S. tuberosum through the
amphiploid .S'. antipoviczii X S. chacoense}'"-' Another case, S. acaule
and .S. ballsii, can be introduced through appropriate amphiploids
crossed to S. tuberosum when the species in question cannot be crossed
alone. For practical work such an approach appears promising,ioT of
course, dependent upon chromosomal differentiation, which may in-
crease the difficulties considerably.^"'^' ^'^^- ^•^' ^^
Three amphiploids can be made within the genus Cucurbita.^^
These are: C. maxima X C. pepo, C. maxima X C. mixta, and C.
maxima X C. moschata.^'^-^ The first is self-sterile; the second is
slightly self-fertile and segregates noticeably; the third is self-fertile
and cross-sterile with parental species. A relatively stable population
develops from the third ami:)hiploid with slight segregation. The
am])hiploid carried insect resistance to squash vine borer (Melittia
satyri)iiformis Hubner) , contributed by C. moschata, plus flavor and
fruit characteristics, contributed by C. tuaxiina. Diploid varieties,
Buttercup, Banana, Golden Hubbard, and Gregory, represent C
maxima; Butternut, Golden Cushaw, and Kentucky Field, C. mos-
chata. According to tests carried out at Cheyenne, Wyoming, Burling-
ton, Vermont, and Feeding Hills, Massachusetts, insect resistance was
stabilized. The fruits compared favorably with the comparable vari-
eties, in general, tliis particular combination may be regarded as a
"potential new species" with prospects of becoming \aluable eco-
nomically (cf. Chapter 13) .^-^
Theoretical problems must not be disregarded.^''' A \'ariaut like
C. pepo appeared sporadically in the first and later generations of the
Eastern material. Taxonomic similarity to C. pepo raises the ques-
372 Colchicine
tion of interspecific segregations. Some lack of uniformity showed up
in the fifth and hiter generations, where the early stages were uni-
form and did not segregate for fruit color, shape, and size. Some inter-
genomal pairing may have occurred. A homology between certain
chromosomes was demonstrated with some pairing in the diploid
hybrid. Such amphiploids shoidd make excellent material to test the
principles basic to amphiploidy and their practical possibilities. ^^^
The interspecific hybrid Trifoliinn repens X T. nigrescens was
made by crossing two colchicine-induted polyj^loids of the respective
species involved.!^ By special culturing methods the hybrid was saved
in the seedling stages. The explanation for incompatibility at the
tetraploid level can be adapted from the case in diploids. i'* Par-
ticularly interesting in the amphiploid TrijoUum is the fact that the
incompatibility apjilied to diploids and to autoploids holds for the
polyploid that brings the two species together. 1 he loci of genes which
determine incompatibility must be at the same place in both species;
furthermore, intergenomal pairing must occur in order to explain
the genetic mechanism of incompatibility through oppositional alleles.
A new species, Ribes nigrolaria, was created by the use of colchi-
cine and hybridization. Two Linnean species, Ribes nigrum, the
black currant, and R. grossiilaria, the gooseberry, were the diploid
parents. 1 hus genomes from two important horticultural species
were combined. These were developed and are under observation
at the Alnarp Horticultural Station, Sweden, under the direction of
Professor Fredrik Nilsson.
Among these and other cases there should come into prominent
use new plant breeding materials that combine the genie composi-
tion from two or more natural and artificial species. In some in-
stances only a specific trait such as disease resistance may be desired.
The key to a new jjlateau for plant breeders can be found among
artificial amphiploids.
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117. Vamada, Y. Some field observations on the tetraploid strains of limssica
jirkincnsis. Jap. Jour. Genet. 18:177-7«. 1942.
lis. /iniiRAK. A. rroduction of amphidiploids of Tr. (luiu))i x Tr. tinioplieevi.
C. R. Dokl. Acad. Sci. IRSS. 2r):3(i-.59. 1939. Production of a T. thiiopheevi x
T. durum v. hordciforine 010 amphidiploid by colchicine treatment. C. R.
Dokl. Acad. Sci. L RSS. 29:604-7. 1940. Experimental production of Triticuiu
pnlouicum x Tr. durum amphidiploids through colchicine treatment. C. R.
Dokl. Acad. Sci. URSS. 29:400-403. 1940. Production of T. persicum x T-
tiinnplu'cvi ani])hi(lipl()ids. C. R. Dokl. Acad. .Sci. I'RSS. 31:485-X7. 1941.
Colchicine-induced amphidiploids of Triticum turgidum x Triticum tiino-
pheevi. C. R. Dokl. Acad. Sci. URSS. 31:617-19. 1941. Comparative fertility
of amphihaploid and amphidiploid hybrids T. timopheevi x T^- durum v.
hordrifonnr OK;. C:. R. Dokl. Acad. Sci. URSS. 30:54-56. 1941. Synthesis of
new species of wheats. Nature. 153:549-51. 194 1. Production of am])hidi])l()ids
of Triticum oricntalc x Triticum timopheei'i \)\ colchicine treatment. C. R.
Dokl. Acad. Sci. URSS. 42:352-54. 1944.
119. , AND RzAF.v, M. Mass production of amphidiploids bv colchicine treat-
ment in cotton. C. R. Dokl. .\cad. Sci. URSS. 26:159-62. 1940.
120. Zhirbin. a. Com]xiratiye studv of cell sizes of auto and allopolyploids. C. R.
Dokl. Acad. Sci. URSS. 18:467-70. 1938.
ADDITIONAL REFERENCES
121. Frandsf.n, K. The experimental formation of B)assica napus L. var. Olcifera
DC. and Brassica carinata Braun. Dansk. Bot. Ark. 12:1-16. 1947.
122. K.U1ARA, H., ('/ al. Morphology and fertility of fi\e new synthesized ^\ heats.
Rpt. Kihara Inst, for Biol. Res.. Kyoto Seiken Ziho. Xo. 4:127-40. 19,50.
123. Lamm. R. In\ estimations on some tuber-bearing Solatium h\briiis. Hereditas.
3!):97-112. 1953.
124. NrsHnA-MA. I. PoUploid studies in the Brassiceae. Mem. Research Inst. Food
Sci., Kyoto Univ. 3:1-14. 1952.
125. Xisni\AMA, I., AND IxAMORi. 'i . P()i\ploid studics in the Brassiceae. III.
Mem. Research Inst. Food Sci., KM>to Uni\. 5:1-13. 1953.
CHAPTER 13
The Autoploids
13.1: Autotetraploids
Oejiothera lamarckinua. var. gigas, discovered by Hugo de Vries
at the beginning of the twentieth century, proved to have twice the
number of chromosomes found in a rehued species. After colchicine
became known, this classic polyploid was repeated. -o'^ Plants with
the doubled number of chromosomes are not considered mutants,
even though originally the concept of mutation advanced by de Vries
was in part taken from his experiences with Oenothera. Increasing
the number of chromosomes increases the number of genes, not the
kind. No one would consider as nnitations the production of diploids
from monoploids,3i or of triploids from hybrids between tetraploids
and diploids. Colchicine is not a mutagenic agent in any sense, either
for production of chromosomal changes or in its capacity as a poly-
ploidizing agent. ^^
Without exception, the autoploids produce fewer seed than the
diploid from which they originated by doubling. Great variations in
fertility are found from species to species, from almost total sterility
to values as high as 75 per cent.-"'^ In subsequent generations the
fertility level can be raised. Among tetraploid Melilotus alba two
groups of tetraploids have been isolated, high-fertility and low-
fertility lines.91
Many comparisons have been made between diploids and the re-
lated tetraploids, on a physiological, morphological, chemical, ana-
tomical, ecological, as well as cytogenetic basis. The differences are
well known, and the original gigas features have been demonstrated
over and over.
Certain problems relating to chromosomal mechanisms and fer-
tility have not yet been solved. Less and less agreement is found on
the causes for lowered fertility in the autotetraploids. Autotetraj^loids
from homozygous lines of maize are less fertile than the correspond-
[318]
The Autoploids 319
ing types from heterozygous diploids. ^"^ Comparative studies in Autir-
rhiniim showed that between intravarietal and intervarietal tetra-
ploids the problem of fertility involves something more comjilex than
a mere analysis of meiotic distiubances created in the tctrajiloitls.-"!
The ecological requirements of autoploids are not as distinctive
from the diploids as are these requirements in amphiploids and their
parental diploids.''^ Hybridization does not activate processes in auto-
ploidy, and evolution at the tetraploid level must occur through gene
and chromosomal changes -which arc imdoubtedly very slow.
From a practical standpoint, the lowered fertility at once placed
the tetraploid at a yield disadvantage. But these facts were well
known before colchicine was discovered. The problem in using tetra-
ploids becomes one of balancing the advantages against the disad-
\antages, and then measuring the net gain, in comj^arison with the
accepted competing diploid varieties. The use of polyploidy is not
a quick way to tlevelop new and improved varieties. Some projects
were undertaken with high hopes that revolutionary methods were
at hand. By now most of those concepts have been re\ised. For some,
polyploidy has been totally dropped as a method for improving vari-
eties. These are instances where the techniques should never have
been started; in others, the programs are stopping short of probable
success. Revised progiams using polyploidy are in progress in man)
laboratories throughout the world.
i^.i—i: The cereals and polyploidy. In the aiuumn of 1951, large
quantities of seed of autotetraploid steel rye were distributed to
farmers in Sweden. ^•^- The first tetraploid rye was made before colchi-
cine was discovered and it proved to be inferior. Therefore, one
might suspect other polyploids in rye to be poor. Several more poly-
ploid varieties induced by colchicine have also proved inferior to the
best diploid varieties. There were variations in the different tetra-
ploids as well as variation among plants. Finally a superior tetra-
ploid was derived from a diploid variety of steel rye, and this formed
the beginning of this valuable series. i-'- A report on the cytogenetics
and practical value of tetraploid rye is a good guide for steps neces-
sary to develop tetraploid varieties.
Testing the performance of tetraploid rye and diploid varieties
was difficult because plots coidd not be planted side by side. The
diploid pollen falling on tetraploid flowers greatly reduced the seed
yield of the tetraploid. Therefore, special tests had to be woikcd out
before a demonstration of practical value for the tetraploid rye was
possible.
Like all autotetraj^loids. the cell size was larger than that of the
dijjloid. Pollen measurements were a reliable index for tetraj^loidy,
l)ut even less complex for practical selection was the size of seed,
320 Colchicine
which was larger among tetraploids. When large populations were
studied, the diploid and tetraploid spikes could be separated by using
the size of seeds for comparison. This was quite as safe as making
pollen measurements, so the need for counting chromosomes in the
preliminary stages of sorting was not required.^- Such rules can be
adopted for other projects.
In regard to vegetative and floral characters, the tetraploids were
taller and of stiffer straws; the degree of tillering was lower; and the
number of flowers was reduced. But kernel size and weight ex-
ceeded ihat of the diploid. However, the hectoliter weight values
were lower. Tetraploid steel rye had good sprouting ability and was
able to stand the winter conditions as well as diploid rye. There
were no marked differences in maturity values between the two types.
The baking quality of the flour of the tetraploids was superior to the
diploid in the preparation of both the soft and the hard breads.^
Morjihologically, the tetraploid rye, like most autoploids, showed
the following differences from the diploid: (1) stems were thicker
and stouter; (2) tetraploids were taller; (3) leaves were larger; (4)
leaves were thicker; (5) leaves were somewhat shorter and broader;
(6) leaves were greener; (7) floral parts were larger; and (8) seeds
were larger.^^-
From a practical standpoint, the advantages gained by tetraploid
steel rye over the diploid arose from a favorable balance of two positive
properties as against the four more or less negative characteristics.
The lower seed setting (20-25 per cent) , reduced tillering, lower
number of flowers per spike, and tendency to shed basal spikelets,
were counterbalanced by the superior baking quality of the Hour and
the improved sprouting ability of the seed.^^-
Artificially produced tetraploids in rice have been made with a
number of important varieties.!"-^ The tetraploids were distinctly
larger-grained, heavier-awned, and more robust generally. While the
grains were heavier, a reduced fertility counterbalances the gain in
weight per grain. Here again tetraploids manifest the usual disad-
vantage. These raw tetraploids were without immediate practical use
for the reasons already well known. Moreover, there was nnuli doubt
that by further selection the fertility could be raised high enough to
overcome the yield disadvantage from a reduced fertility.
Another approach to polyploidy as a means for improving rice
was made. The F^ hybrids Oryza sativa var. indica X O. saiiva var.
japonica are very sterile in some combinations. This sterility has
blocked the j^ossible utilization of a hybrid between the subspecies.
There is no apparent meiotic irregularity in the hybrid, and the
causes of sterility remain unknown. Autotetraploids seldom exceeded
The Autoploids 321
fiO per cent fertility, while in the parental diploid fertility was over
90 per cent. Yet the hybrid between the subspecies japonica and
indira may even drop to 11 per cent when fertility is measured bv
seed formation. Sterile F/s, if doubled, immediately raised the seed
formation higher than autotetraploids.^'^ As the fertility decreased in
a oi\en Fj hybrid, the fertility increased in the corresponding tetra-
ploid. That is, the more sterile the diploid F, hybrid, the higher was
its restoration of seed fertilitv after doubling.-'"' Pollen sterility ap-
proximated the same rides. Thus the disadvantage met by strict auto-
tetraploidy seems to be overcome in this type of program. Some real
obstacles may yet be encountered in trying to stabilize the polyploid
that combines japonica and indica genomes. Further segregation must
be studied.
No quick results can be expected in spite of the apparent solution
to the fertility problem, for the tetraploids from hybrids are, like all
tetraploids, unselected. Judging from the high midtivalent formation,
segregating progenies in F^ and later generations can be expected.
This fact may offer exceptional plant breeding opportunities along
with serious obstacles. Obviously, these plants and such methods will
receive attention in the future as another approach toward plant
improvement in rice.
An c\tensi\'e literature is devoted to autotetraploid barley.-"'^
Some spontaneous An races have been isolated. Also, colchicine has
been used by several investigators. Morphological characters that
change with polyploidy are well catalogued along with several ex-
cellent physiological studies. The progress has been summarized in a
comprehensive review, and little more need be added. The practical
uses for barley have not come up to those of autotetraploid rye.
Autotetraploid maize has been followed over a long period, since
the earliest strains were made by heat treatment, before colchicine
methods were available. Fertility differences cannot be correlated
entirely with chromosomal processes at meiosis. The slower growth
and reduced fertility are disadvantages of the tetraploid. The dou-
bling of monoploids to autodiploids ^vill be developed in another sec-
tion.
Other cereals of economic importance, being natural jK)lyj)loids,
require other approaches. The autoploids are inferior to diploids and
provide genetic materials only.
1-^.1—2: Forage, range, and pasture spcdes. Raw polyploids in
some species of TrijoUum showed an immediate advantage over the
diploid in forage production. ^^-^ The data were obtained from limited
scale testing. \\'hen the tetraploids were distributed for larger scale
trials, the difficulties not encountered ^\•ith small tests then appeared.^
322 Colchicine
Atter revising the methods for making tetraj3loids and choosing much
larger samples, 50 commercial varieties of red clover, new tetraploids
superior to the first, were developed.
In Scandinavian coimtries notable progress has been made with
red clover, T. pratense. Twenty-eight chromosomes does not appear
to exceed the optimal nimiber. The yield of forage is also indepen-
dent of seed production. The seed setting becomes important for
propagation purposes but not yield of forage. At least five major
tetraploid varieties have been tested over several areas in Denmark,
Norway, and Sweden. The results are encouraging as a method for
improving red clover by jjolyploidy.^i'^- ^^' ■'-• --^ It is of interest that
the new tetraploids in rctl clover do not necessarily come from the
best diploid strains. Only by testing the tetraploids can their true
value be judged.
In addition to gigas features valued for forage production, the
earlier and more rajjid growth in the second year was better than in
diploids. Undoubtedly, the tendency toward a perennial habit in
polyploids would seem to be correlated with this trait. Susceptibility
to insects and diseases are a weakness in most strains, diploids as well
as tetraploids, but there were some red clover tetraploids with ex-
cellent insect and disease resistance. One red clover strain, Sv. 054,
from a diploid \aricty Merkur had good yielding capacity and re-
sistance to the nematode, clover eel.
Diploid alsike clover, T. hyhridinii, made tetraploid, showed
promise at once, giving consistent increases in forage from 15 to 25
per cent. For overwintering capacity the alsike clover was good from
the start. --'J Continued successful performance stimulated a change
to breeding on the tetraploid level. VV^ithout doubt, these two tetra-
jjloid clovers have made satisfactory performance.
A third species, T. repens (white clover) , was not successful, biu
as this is a natural tetraploid, 32 chromosomes, finther increases pre-
siunably took the niniiber to 64, a niunber above the optimum for the
species. We must conclude that one cannot draw a general rule for
all cloxer breeding (ct. Chapter 1 1, Ref. No. 4) .
The tetraploid Melilotus suffered from a reduced fertility and
was not as promising for practical purposes, althotigh there were
enough differences in fertility among eight plants of tetraploids to
make jjrogress in selecting toward higher fertility. '^i Crosses and
selections demonstrated that higher levels of self-fertility coidtl be
obtained. If interspecific hybridization could be effected, the com-
bined germplasm would open another avenue for analysis.
Polyploidy has been olitaincd in MecUcago satixia, M. media, M.
lupuUna, and M. denticulata.-''' Vigorous strains appeared among
these polyploids; however, the usual reductions in seed setting were
The Autoploids 323
met. Since there are diploids as well as naturaltetraploids within the
group, some .hybridization Avould appear possible. The crossing of
autotetraploids with natural tetraploids offers a method to be tried. i^*
Plihinu pratcii.sc was made uj) in chrdmosomal series, ranging Irom
di])loid to twelve-jjloid.^i'^ Analyses ior vigor, lorage production, and
quality were clone to check the optimum number, below or above
which poorer performance was noticed. Progenies with 5() to 64
chromosomes were more vigorous than the 42-chromosomal plants or
the polyploids with 84 chromosomes. This principle of optimum
numiicrs must be recognized in polyploidy breeding. Hexaploid
Phleuin nodosum was made by first doubling the chromosomes with
diploid P. nodosutn.^-'- The tetraploid was treated again and a hexa-
ploid was isolated. Of special interest is the close correspondence
between the natural species, P. pratense L., and the hexaploid, P.
nodosum.
Lolium perenne in the tctrapU^d state was compared to the dip-
loids.i'^"' Morphological and physiological studies brought to atten-
tion characteis such as winter injury, sugar content, dry matter, mois-
ture, leaf structure, tillering, and flowers. The autotetraploids of
seven species of grasses were compared in regard to both morjiho-
logical and cytological details. No specific advantages were demon-
strated for the tetraploids.
Autotetraploid Sudan grass, Soio^lnim vulgare var. sudunense. and
Johnson grass, .S'. halopense, were hybridized to make a j^asture
species.-'' Autotetraploid Sudan grass incorporated better forage
characters into the hybrid. One observation confirmed that the auto-
tetra])loid would hybridize while the diploid Sudan grass always
failed. Later generations followed for this hybrid segregated for the
dry and juicy stalk quality. The segregations were closer to 35:1 than
20.8:1, meaning that random chromosome segregation had occurred. ^'^
These polyploids showed a tremendous possibility for selection.
/^./• — 9; Polyploidy in fruit, xn'getable, jloivcr. and forest species.
Polyploidy and fruit improvement in the United States have been
summarized in this way. The problem is like that of a "i)uilcler sur-
veying the possibilities of his materials and the usefidness of his tools."
Materials are enormous and tools are now available. Ciolchicine is
one of those im])ortant tools, while the materials include an abun-
dance of i^lants in nature and under cultivatic^n. "4 he onl) limits are
his blueprint, his time, and his industry. "•^•'
The diploid, woodland strawbcrrx. Fragtiriu I'csca. 2}i = If, is
found in many parts of the northern hemisphere. Cultivated varieties
are octojiloids, 8^; =: 56. Autotetra])loicls from F. vesca, 4n ^= 28, ^vere
made and crossed with 56-chromosome cultivated strains. Such hy-
brids were 42-chromosome hexajiloids. These were crossed back to
324 Colchicine
cultivated types and ]Mo\ided material for selection. i^*^ Further search
for natural species useful in polyploidy is underway. Disease re-
sistance, flavor, quality, and size have been incorporated into hexa-
ploids. There were reportedly 24 breeding projects in the U.S.A. en-
gaged in various aspects of strawberry work. There are important
cytogenetical strains in polyploid series at hand in the Botany Depart-
ment at the University of Manchester, England.'^^
Including wild and cultivated varieties, chromosomal series from
2n = 14 to \2r} = 84 exist among the blackberries and raspberries.
Perhaps no other fruit can be correlated any more directly to poly-
ploidy than this one. The Nessberry, Logan, Boysen, along with
hundreds of forms of polyploid blackberries are in existence. Since
there are polyploids at hand, artificial doubling is not so necessary.
Where faster progress may be required, or the changing of sterile hy-
brids to fertile ones, colchicine serves as a useful tool.^^
Many cultivated cranberries are diploid, and in nature, tetraploid
as well as diploid species exist.^-' ^'^ Some sterile hexaploids have been
reported. By doubling the number of the cultivated diploid, a paren-
tal stock was made for crossing with the wild tetraploid. Selections
from all the important cultivated diploid varieties were doubled.
These types were selfed and hybridized. Such tyj^es have been grown
on large scale since their origin, and raw polyploids are being con-
verted into genotypically balanced types.
Perhaps polyploidy as a direct mode for improvement in grapes
has advanced as far as any fruit crop of the United States. Here
naturally occurring sports, often chimeras, proved to be tetraploid.
They occurred in sufficient abundance, so that artificial doubling by
colchicine has not been necessary. Giant fruited sports from the vi-
nifera and bunch grapes are tetraploid. ^''"^ These studies have pro-
gressed to a stage where newly named tetrajjloid varieties now com-
bine important characters and are distributed as improved types.
Named tetrajiloid varieties of summer radish were released in
Japan and tested widely enough to demonstrate a superiority for the
new polyploid. In vigor and growth the tetraploid exceeded the dip-
loid. Outstanding resistance to the common club root disease was
obtained with the tetraploid. The usual gigas features accompany
these autotetraploid radishes. ^^^
Polyploidy in water cress increased the succidence of leaves, which
feature made the tetraploid strains more desirable for salads. ^^ In-
creased content of vitamin C in the water cress, which is expected
in tetraploids, was an advantage over diploids. One disadvantage was
the slower-growing characters of tetraploids. Like the autotetraploid
rye, apparently a balance between the positive characters against the
negative ones is needed. When an immediate su])eriority in favor
The Autoploids 325
ot tctraploids, such as leaf size, succulence, ami vitamin content in-
crease can be demonstrated, the promise for future polyiiloidy breed-
ing offers some hope. Without some initial advantage or promise, the
use of polyploidv nnist be questioned for practical purjxjses.
Direct autotetraploidy in tomatoes has not brought imjirovements.
There seem to be hybridization possibilities.-^ Similarly, within the
large group of Sohniinn. an interspecific hybridization is probably the
most useful aj^proach.-"'' .S. tuberosum, the commonly cultivated
species, is already polyploid: doubling is therefore of no value. S.
antipoviczii X ^- chticoense amjjhiploid was fertile with S. tuberosum.
By this procedine the disease resistance to phytophora from one
species, S. antipoviczii, should be transferable into a polyploid hy-
brid.^i^^ The advantages gained from such work can be maintained
because vegetative propagation fixed the features once obtained.
The quality of tetraploid muskmelons, Cucumis meh> I... was
definitely superior to the comparaljle diploid variety. ^^ Enough seed
can be produced to propagate the tetraploid adequately. These poly-
ploids were made in several laboratories; each reported improve-
ments. In one instance, taste tests were conducted in such a way that
identity of ploidy was not revealed. Without exception, the choice
fell to the tetraploid. Since ten different varieties were made tetra-
jiioid, a larger number of them were used in comparison Avith the
polyploid and diploid.
A new potential economic species of Cucurbitn Avas developed by
doubling the chromosomes of a hybrid between C. maxima and C.
moscJiata. One species, C. moschata, carried insect resistance to the
hybrid while fruit characters were contributed by the other parent.
These characters were not entirely stable in the hybrid, but showed
more stability in the polyploid. Fruits matured earlier in the amphi-
ploid than in either parent. In the first generation of the amjjhijjloid
there was little or no segregation. Later, up to the fifth generation,
there appeared segregation for fruit color, shajje, and size. Evidently
some intergenomal pairing occinred, and occasional bivalents could
be observed during meiosis of the diploid interspecific hybrid. A
variant that resembled another species, C. pepo, appeared. This type
was completely sterile to either the 2?? or 4/? lines. Since the same
variant has reaj^peared, considerable theoretical interest becomes at-
tached to this segregate. Large-scale tests in several locations showed
that a new jjotential economic species of Cucurbit a has been made
(cf. Chapter 12).
The gigas characters accompanying induced polyploidy became
attached to colchicine as soon as the effectiveness of this method was
annoimced. Probably the first plantsmen to give serious attention to
colchicine were those interested in developing ornamentals. The rea-
326 Colchicine
sons for this appeal oi larger (lowers are easily understood. One
hundred and nine varieties chosen by iris fanciers from a total of 12
best selections were studied for chromosome numbers. Not one was
dijiloid. but 108 were tetraploid, and one was triploid. Practically
all these were developed and selected without studying chromosomes,
but in this case the potential of polyploids was forcefully demon-
strated."'-'
It is no surprise to find many persons attracted to the possibilities
to be gained from colchicine. Larger flowers were anticipated.
Among the first colchicine-induced tetraploids to be distributed
were snapdragon, phlox"'-, and marigold. VV^ork with carnation-'"',
poinsettia-"", day lilies-'-', and lilies''^ has yielded tetraploids. There
are numerous projects under way with many ornamentals, annuals,
perennials, and shrubs. Improved flower size, darker and more com-
pact plants, with greater drought resistance were obtained with tetra-
ploid J'nud rosea LJ""' Also the llo\\'ering period was extended longer
than in the diploid. While seed production was reduced, this disad-
vantage was balanced with other positive characters in the tetraploid.
1 3.1-4: Plants yielding special products of economic importance:
fibers, oils, latex, drugs, beverages. Autotetraploids increased the size
of seed, fruit, leaf, stem, and root, and larger plant organs should
yield more substances of economic importance.--^" Oil-bearing seeds
such as sesame, Brassica, and flax, all have lower seed production as
tetraploids. Flax is a notable case where the fertility drops extremely
low. Rubber increase in Koh saghyz and Hevea are objectives. Fiber
improvements in Hibiscus, cotton, flax, jute, and hemp have been
sought via polyi^loidy. Anabasine in polyploid Nicotiana increased
with polyjiloidy.
13.2: Triploidy
Hybrids from a tetraploid seed parent crossed with a diploid
pollinator are triploid. As such these are not stable, and both male
and female gametes are sterile from unbalanced chromosomal dis-
tributions. The vegetative vigor is not lowered, in fact many triploids
are extremely vigorous. Among the good varieties of apples, triploids
are common. In nature some triploid species are widely distributed.
Polygouatuin rnultiflorufn is an example of a triploid having a range
from the northwestern Himalayas throughout Eurojje.
The two kinds of triploids are the autotriploid and allotriploid.
The former arises from an autotetraploid crossed back to the parental
diploid, whereas the allotriploids involve two species. In these cases
bivalents and univalents are found at meiosis. Triploids offer the
opportunity for increasing the frequency of aneuploids since the trip-
loid female gametes are viable with one or two chromosomes above
The Autoploids 327
and Ixlow the hajjloid number. Another conuuon jMaetice is dou-
1)1 ing the triploid to make hexaploids. Such a bridge is regidarly fol-
lowed in Gassy piuni, where the hybrid between American tetraploid
and a species becomes a sterile triploid.
Certain advantages may be gained from triploids thai are not
possible otherwise, if the optimum chromosomal number is closer
to tri})loid than tetraploid, production may i)e increased over either
diploid or tetraploid. If rij^ened seeds can be eliminated or reduced,
as in the trij^loid watermelon, a new type fruit is obtained. These
features in triploids are limited but seem important.
Finally trijjloidy raises problems of seed production: an extra
propagation of parental stocks to preserve the two types, as well as
a specific hybridization to produce the seed for each generation. Suc-
cess may depend upon solving these problenrs. Triploid seeds do not
germinate as well as those of other polyploids. Finthermore, the
cross between tetraploids and dij)loids cannot be readily made for all
autoploids.
i^.2-i: Triploids i)i xixitcrniclons. Reasoning from the lact that
seedless fruits in nature are due to certain reproductive failures, the
idea was conceived that seedless watermelons woidd result if triploids
were made. The female sterility notable among trijjloids would lead
to this achievement. Such work was initiated in japan in 19.H9. Ten
years later the first triploid watermelon fruits appeared on the market
in lai^an.-'"- '""• '"^ This may be regarded by practical breeders as a
very short time for the production of a new variety. Triploid water-
melons were a new concejjt in\olving hybridization and polyploidy
breedi ng procedmes.
The tetraploid parents are produced by colchicine applied at the
seedling stage. These plants have 44 chromosomes and are easily dis-
tinguished from the diploid by seed size, ))ollen size increase, and
other characteristics. After the tetraj^loids are produced, these varieties
become the seed parent with the tliploids as jjollinators to make the
triploid.""- '""• ^•^•'''
Seeds obtained from a tetraploid fruit and pollinated b\ the dip-
loid are triploid. Upon planting such triploid seed, fruits without
seeds may be had. Early in the season, and late, the ovides develop
hard coats that resemble seeds. These are emjjty. but the term seed-
less becomes meaningless when Iruits show these cores or empty seeds.
Therefore, the term trij^loid is far more desirable. To avoid these
difficidties, the fnst pistillate llowers are removed to elimiiiaic ihe
fruits with seed shells."'
When triploid plants are growing, pollinations must be made by
diploids because the pollen of triploids (fio\vers) is not sufficient to
induce fruit development. 1 hus, interplanting diploids with trip-
328
Colchicine
loids causes iruit development among triploids. However, the ste-
rility of the female precludes seed setting even though viable diploid
pollen is present. This is the general scheme in producing triploid
watermelons that under specific circumstances set seedless fruits.
The general procedure of formation of triploid fruits is set forth
diagrammatically in Figure 13.1. Only crosses involving the female
2x X 4x 4x X 2x
i
2x
empty
seeds
Fig. 13.1 — Triploid watermelon. Propagation of triploid seed by crossing diploid and
tetraploid lines. Use of colchicine to make tetraploid stocks. Fruits from diploid,
triploid, and tetraploid stocks. (Adapted from Kihara)
as tetraploid and the male as diploid pollinator are successful. Re-
ciprocal procedures do not succeed.
As in autotetraploids, the size of flowers increases in proportion to
the increase in chromosome number. This relation holds for tetra-
ploid pollen and stomata. Triploid pollen is variable in size and can-
not be made to fit the proportional increase as chromosome numbers
increase. Many grains are empty while others are full and may be
huge.
The 3X seed is a tetraploid seed with triploid embryos obtained
from a diploid pollination. The SX seeds are slightly thinner, averag-
The Autoploids 329
ing 1.7 inni. in thickness as compared with about 2.7 mm. for the 4X
seeds. This feature is of practical vahie in sorting 3X and 4X seeds
if the tetraploids are left to open pollination from tetraploid and
diploid pollen in the same field. In Figure 1.S.2 the sizes of diploid
and tetraploid seeds are contrasted.
If longitudinal sections are made of mature seed, the diploid, or
2X, seeds show a completely filled cavity, while the 3X and 4X seeds
fill the space up to 82.5 and 90.1 per cent, respectively. Accordingly,
a weaker germination is a characteristic of the ?>X seeds. This becomes
a point of considerable practical importance and must be overcome
^\ith j)roj)er cidturing conditions. Such seed cannot be j)lanted in
the field with dijiloid and be expected to produce the same field
stand for both varieties.
Genetic markers are helpful to distinguish triploid fruits from
tetra])loid and diploid. Dark -green, parallel striping is dominant over
smooth color, therefore fruits pollinated by diploid with the stripe
character show in the triploid if tetraploid fruits are non-striped.
Tetraploid fruits may have this mark (Fig. 13.2) .
Yielding capacity of triploid plants exceeds the diploid by almost
twice. Variations a])pear de])ending upon the particidar varietal
combinations. Ihe increase in number of fruits per unit area is
particularly significant both as to number and weight.
Triploid fruits are seedless because chromosome distribution to
gametes is irregular. Trivalent associations form among the 33
chromosomes. At reduction division, less than 1 per cent of the
gametes obtain a complete set of 1 1 chromosomes necessary for a bal-
anced gamete. Ninety-nine plus per cent have numbers ranging from
1 1 to 22 chromosomes. Sterility is induced, and pollination with
viable pollen does not produce seed because of female sterilitx. \\4ien
pollinations are prevented on triploids, fruits do not set.
Special cultivation procedures are necessary for triploid A\ater-
melons: soil shoidd be sterilized, seed planted in beds kept at 30°C.,
and transplantation procedines carried oiu to insme a field stand of
vigorous plants. Once the triploid is established, its growth exceeds
that of the diploid and continues longer during the season. A ratio
of 4 or 5 triploid plants to 1 dijjloid provides adequate pollen to set
fruit on triploids: the latter become parthenocarpic.
A sLuiimari/ing j:)aper by Professor H. Kihara of the Kyoto Uni-
versity, Kyoto, japan, on triploid watermelons. ]niblished in the Pro-
ceedings of the American Society lor Horticultural Science,-'" was
recognized as an outstanding contribution to horticidiinal science.
Accordingly, this jniblication was chosen to receive the Leonard H.
\^aughn Award in \'egetable croj)s. The published works from \'ol-
iinies 57 and 58 of the Proceedings were considered in the competition
for this honor.
2x1 (
(mL^ ^^ J0 ^1
'^^
<l& ^ # it;
— .^
1
A
^ (1 % B
i-Yamai
to" tetraploid
Fig. 13.2 — Photographs of diploid, triploid, and tetraploid fruit and seed. (Photographs
furnished by Professor H. Kihara, Kyoto, Japan)
The Autoptoids 331
111 japan, production of tiiploids as a method for improving
watermelon production has been successfully explored. The opinions
of American horticulturists on this subject vary with the experiences
gained from testing the Japanese varieties. Success is reported in per-
sonal conmiunications from Professor E. C. Stevenson, Purdue Uni-
versity, Lafayette, Indiana, and Professor W. S. Barham, North Caro-
lina State College, Raleigh, N. C. Undoubtedly other unpublished
reports in America and elsewhere concur in many of the general
observations published by Kihara and his associates relative to yield
adxantages, disease resistance, and improved quality.
Seed production and wide-scale commercial growing will increase
as l)etter adapted varieties are made available. Some problems pecul-
iar to cultivating triploids and to seed production need attention in
the American system. If watermelons of better quality can be ol)-
tained. fruits produced without seeds, or almost so, and if triploid
varieties are placed in the hands of commercial groovers who can pro-
duce melons more profitably than by present methods, the problems of
seed production and triploid cultivation will eventually be solved. The
time required for this transition in America is difficult to calculate;
however, the records of acceptance of h)bridi/ation in mai/e set a
standard that might well obtain in watermelon seed production and
commercial growing of this fruit.
The application of colchicine to the problems of watermelons
represents a most specific and outstanding i)ractical advantage gained
from the use of this drug.
1^.2-2: Triploid sugar beets. Early in the colchicine era poly-
ploidy breeding was directed at the improvement of sugar beets. Raw
tetraploids did not prove to be as good as the parental diploids. This
was to be expected for reasons outlined in the section on jirinciples
of polyploidy breeding.^- e^- ^^^' n-*- '--• "-• ^'""^
A significant rejjort was made that triploid plants yielded more
sugar than diploids because the larger roots maintained the same
percentage while the diploid tended to reduce the percentage of sugar
per hundred grams as the larger-sized beets developed. An additional
set of chromosomes raising the number from 18 to 27 did not \noxe
detrimental to volume of sucrose per acre of plants. 1 his represented
an imiKjriant advancement in sugar beet breeding'- (Fig. 13.8).
11 triploids were superior — and this has been shown in several
cases — then special procedures were required to produce triploid
seed. Tetraploid seed parents are made, and then pollinations are
carried out with the dij^loid. Studies by Jajjanese workers show prac-
tical plans for making triploids.-"-'
The increase in sucrose per unit area of cultivated triploids justi-
fied the additional work to make triploids which produce more su-
332
Colchicine
crose than either diploid or tetraploid, in this case, the 2X or 4X sugar
beets. Intervarietal 3X hybrids between high-yielding tetraploids and
disease-resistant diploids will prove better than any of the present
tri|)loids.
Large-scale production of SX seed remains a serious problem.
However, the self-incompatibility of the species can be used to ad-
0 0_
'- X:!pjo,
lO
15
20 23
Individual beet weight ^100GM. units
Fig. 13.3 — Weight of root and percentage of sucrose production does not decrease at
same rate as in diploid when large roots are produced. The addition of another set of
chromosomes does not pass the optimum for sugar production per acre. (After Peto
and Boyes)
vantage. This alternate planting of 4X and 2X varieties can be used.
Seventy j^er cent of the seeds from the 4X plants are triploid on an
open pollination basis. About 30 per cent from diploid are triploid
seed. Other factors arc involved, such as maturity dates, jiollen tid^e
growth, and environment that inlluences seed production. The
optimum number of chromosomes has not been exceeded in the trip-
loid.i^ii-^
Through the cooperative activities of the National Institute of
Genetics Laboratory of Plant Breeding, Hokkaido University, the
Hokkaido Agricultural Experiment Station, and the jajKUi Beet Sugar
Manufa(tiuing Company, improvement of sugar beet by means of
induced polyploidy has progressed very satisfactorily.--"
The Autoploids 333
The SX beets aie more vigorous; ihey grow better and always yield
more than other beets. Large-scale tests in 1919 and 1950 proved the
superiority of the 3X beets.
/9.2-3.- TripJoid fruits. Some ol the best varieties ol ajjplies, Stay-
man, ^\'inesap, and Baldwin, are widely known. Since giant sports
can l)c- produced by colchicine, in similar fashion to the natmally
o((iuring types, the drug has ready application in apple breeding.
Trijjloids can be made from hybrids between tetraploid and regular
diploid varieties. These have possibilities for winter hardiness ac-
cording to tests by special laboratory equipment."^ Among 31 tetra-
ploids, two \'arieties were exceptionally hardy. Mains barrafa, a dip-
loid species, has been polyploidized and might \\ell l)e the start for
breeding stock.
Triploid guavas have been reported occurring in natme. Such
tvpes are seedless. Tetraploids induced by colchicine were promising
soiuces for making crosses between diploid and tetraploid.'"' Assum-
ing that other qualities cotdd be controlled, polyploidy for this eco-
nomic crop and particularly seedless fruit jModuction should repre-
sent an important improvement.'^''
13.3: Monoploids and Autodiploids
The fust monoploid plant discovered in 1922 proved that plants
existed with one set ol cliromosomes. More than 30 genera have been
added to the list \vith monoploids reported for one or more species."''
The impro\ement of methods for detecting monoploids is an impor-
tant part of the program. At once geneticists recognized that doubled
monoploids became homozygotis diploids, lire theoretical and prac-
tical use for breeding jnnposes should not be underestimated. Since
the first monoploids were reported, the practical value for homozygous
breeding stock to produce hybrid maize has been developed ex-
tensively."^
1 he frequencies of the appearance of monoploids are low. Their
propagation after isolation from diploid cultures depends u|>on the
doubling of chromosomes in tissues that develoj) the pollen and egg
(clls. Colchicine serves adequately for increasing the sectors that
double to give rise to fertile tissues. The problem that remains is
to find -ways to increase the frequency of producing monoploids,
apjjlicable to a large number of plants.
A prediction was made that the discovery of methods to increase
the frequency of monoploids woidd mark another period in the his-
tory of polyploidy breeding (cf. Chapter 11, Ref. No. 43) . According
to this scheme the Drosera research by Rosenberg marked the be-
ginning: a distinction between allopoly|}loid\ and autopolyploidy was
the second phase: and colchicine in 1937 was the beginning of the
334 Colchicine
third period. Large-scale production of nionoploids is a discovery
for the future.
The frequency of increasing nionoploids has been improved by
special methods adapted for a few species. Twin seedlings proved to
have a high incidence of monojiloids in Hax, cotton, and peppers.
The nionoploids derived from the twin embryo method were isolated
and doubled to make the homozygous diploids. i'^'^*- -'" As a basis for
improving commercial varieties some application has been made in
this direction.-'' Since many seeds can be rini over the germinators,
more nionoploids are discovered than was jjossible by field selection.
Gossypiuin was treated by these methods.'''
Significant differences in the frequencies of nionoploids ha\e been
found with certain stocks of maize. Previously selected strains were
better than imselected ones. Oi:)en-pollinated varieties, generally,
were comparatively low for production of parthenogenesis.-''^ By ap-
propriate genetic markers, introduced from the pollen parent, the
detection of nionoploids at seedling stages is improxed. Color genes
from the pollinator are expressed in the diploid, but not those from
the maternal nionoploids. Cytological confirmation of the niono-
ploids among the colorless seedlings proved that the marking system
was reliable.
Monoploid sugar beet obtained from seed taken from a colchicine-
treated shooting plant has been found. Their occurrence is quite
rare. In another instance, the nionoploids were derived from colclii-
cine-treated populations. An interspecific hybrid of Nicotiana pro-
duced two niono))loid ])lants. One of the plants was like one parent,
N. gJutinosa, and the other like A^ rcpauda. In the original cross the
former parent was the female type and latter was used as the polli-
nator.
An important use for colchicine arises for making autodiploids
from monojiloids, thereby increasing the niunber of plants that can
be proj^agated. By spontaneous doubling some sectors regularly pro-
duce viable pollen and eggs. Injecting 0.5 ml. colchicine into the
scutellar node of the monoploid seedling jjroved to increase the
amount of good pollen ]jroduced. an index of doubling."'' A luiique
feature and application of the autodiploids of maize arises from the
fact that genetic systems are fixed as gametes and testable as such.
1 hereafter the autodiploid reproduces the fixed system of genes.
13.4: Conclusion
The nmnber of autoploids is larger than that of the amphi|)loids.
Rel'erence niunbers in this chajjter and other chajiters will be uselid
to check the many kinds of plants already studied. The \()liunc of lit-
erature has de\ eloped so extensively that every example coidd not be
The Autoploids 335
cited in the sj^ace alloted. Only selected examples that pointed out
])iin(iples and basic features about polyploidy were chosen tor the
text discussion.
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197. SiMONET, M. De rol)tention de varietes polvploides a grandes fieurs apres
a|)|)lication de colchicine. Tribune Hort. 23:61;5-46. De lobtention d'un
l.iinun iisitatissiimini tetraploide apres application de colchicine. Rev. Hort.
110:159-61. 1938.
198. SiNoro, v., AND Sato, D. Colchicine polyploids in Faoopyi iiin. Bol. aiul Zool.
7:1398-1402. 1939. Polyploidi de colchicine in Fa i>;opy ru m . Sci. Genet. 1:354.
1940. Polyploids and aneuploids in Trirxrtis foinnisaua. jap. jour. Genet.
lS:H8-90. 1942.
199. Smith, H. Induction of polyploidy in Nicotiaiia species and species h\l)rids l)\
treatment with colchicine. |our. Hered. 30:290-306. 1939.
200. Smith, L. Cytology and genetics of barley. Bot. Rev. 17:1-355. 1951.
201. Sparrow, .\., et ah C:omparali\e cytology of sterile intra- and fertile inter-
\arietal tetraploids of .//;//r;7//ni///; iiiajtis L. Amer. join. Bot. 29:711-15. 1942.
The Autoploids 343
202. SiAiR. E.. AM) Showai-TIR. R. Tetraploidv in tomatoes induced l)\ liic use of
colchicine. Pioc. Amer. Soc. Hoit. Sci. 40:383-.S(). 1942.
2().'5. Sinu5i\s. G. The sis^nificance of polyploidy in plant evolution. .\mer. Nat.
71:r)l-()(i. C;oni])aiati\e "growth rates of diploid and aiilotetraploid Stijxi lepkla.
Amer. [om. Bot. 2S:()s. 1941. The cytological anahsis of species hvbiids. II.
Hoi. Re\. 11:463-86. 1945. Types of polyploids. /;; Advances in genetics. \'ol.
I. Acad. Press, Inc., New York. 1947. The cxoliitioTiaiy significance of naliiral
and artificial polyploids in the family Gramineae. Hereditas .Snppl. \'ol. Pp.
Kil-H,"). 1919. \'ariati(>n and exoliition in i)lants. Cdlumhia Iiia. Press. New
^ork. 19.")U.
204. .Sri iNF.r.GKR, E. Pohploich researches on medicinal plants. Mitt. .Natiir. Forsch.
Cies. Rem. 6. 1949. Der Alkaloidgehalt tetraploider Datura Spezies. Pharm.
Acta Helv. 26:188-94. 19r)l.
205. .Stf.phkns, S. (sec Rcf. No. 106, C4iap. 12).
206. Stkwart, R. Colchicine-induced tetraploids in carnations and ponisettias. Proc.
Amer. Soc. Hort. Sci. 57:408-10. 1951.
207. SioMi'S. T. l''l)er die kiinstliche Herstellung von Ociiolhrxi Itnniixkiinia gigas
de \ ries. Ber. Deutsch. Bot. Ges. 60:125.^1942.
208. SiT^AiB, J. (.hromosomenimtcrsiichinigen an polvploiden Bliitenpfian/en. I.
Ber. Deutsch. Bot. Ges. 57(10) :531-44. 1939. Die Beseitigung der Selhsislerili-
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209. SvvAMiNATHAN, M. (scc Ref. No. 107, Chap. 12) .
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21 1. lATt'so. S.. AM) ToMiNAC.A, V. fbcr die kihistlicben Polvploiden hei Ruftlianus
sativus L. Jap. Jour. Genet. 22:31-32. 1947.
215. Tho.mi'son, R., ami Kosar, W. Poh|)loidy in leltiue induced bv colchicine.
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344 Colchicine
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ADDITIONAL REFERENCES
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88. 1951. . .
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,Sha, 5 Higashi-kalaniaclii, Rinikyo-kii, Tokyo, Japan. 131 pp. 1953.
CHAPTER 14
The Aneuploids
14.1: Aneuploids Among the Treated Generation
Ihe variations in numbers oi chromosomes through loss or gain
of extras were first appreciated for their possible value in fundamental
cytogenetics by Belling and Newton.-*' Since then the aneuploids have
been acctnnulating in large numbers for many genera. A new group of
ancujiloids ^vas developed when colchicine was used with large j^opu-
lations of treated jjlants. Certain plants were deficient for a chromo-
some, and among the diploid species these losses were very rare but
significant.^' All diploid deficient types, including the 2n — 1 Datura
stranioiiinin plants, failed to set seed. The origin of such types is an
interesting j^roblem, for the action of colchicine must be interpreted
somewhat differently from the usual doubling of chromosomes.^ Ap-
parently a mitotic disturbance, the loss of a chromosome at the time
of treatment, is transmitted through mitotic processes tmtil meiosis,
when these types are discovered.
1 hat di])loid deficient plants are rare is emphasized w'hen we learn
that only 55 spontaneously occurring 2.S-chromosomal tyj^es (2)i — • 1)
have been recordetl from among more than 2 million Datura plants
recorded over a period of years." From a standard line / of Datura, the
frequency of a 2)i — 1 plant is 1 out of 20,879 offspring, compared
with 7 such types foimd among 2135 plants growing from treated
cultures." The frecpiencics are increased fjy colchicine more than 70
times over the naturally occurring rate. Since the records were made
from pollen mother cells, only the diploid deficiencies from the
sube}jidermal layer that lell in the germ line were calculated. There-
fore, the incidence of 2/? — 1 tissues created by colchicine was higher
than these figures show.
Out of 88 plants in the deficient class, 81 were tetraj^fiMcl de-
ficient kinds, i.e., 1// — 1 or 4/? — 1 — 1. Similar to the dij^loid de-
ficient plants, the tetraploid deficient cases arose from the effects of
colchicine.^
[ 345 ]
346 Colchicine
One other fact is striking. There were, in all. 17-^ chromosomes
lost; and the largest type, known as the L chromosome, was missing
more often than other types. Previous data tor spontaneotisly occur-
ring Datura showed that the 1 + 2, or L chromosome was missing
more often than any other type. Special morphological traits are
fairly reliable for recording Datura progenies.^
Before these data were reported, missing chromosomes were known
in Drosophila. Nicotiaita^'^ heteroploids were obtained by other treat-
ments, and a genetic demonstration proved the loss of chromosomes
in a culture of Hyocyauius niger. Since the Datura work was pub-
lished, deficient types have been recognized in Nicotiana,^'' LiliumP
and Eruca.^^ There must be many that have escaped notice and also
records that are not specifically listed here.
If one looks at the recovery stages from colchicine, the explanation
for the tetraploid deficient types can be seen easily. One or two
chromosomes are left outside the restitution tetrai)loid nucleus. The
causes of a diploid deficient case require additional examination be-
cause a c-mitosis leading to a tetraploid restitution nucleus would not
have taken place unless a distributed c-mitosis of unequal distribution,
23 and 25 respectively, occurred. The 23-chromosome cell would lead
to a deficient cell and the 25 to extra-chromosome types. There is
yet another explanation. When grasshopper neuroblasts were treated
at certain concentrations that did not completely destroy the spindle,
certain chromosomes were lagging. Presumably an incomplete in-
hibition could cause one chromosome to lag. The fact that the larg-
est chromosome of Datura was the one most often missing is of in-
terest.^ To assume that tetraploid deficient tyj^es and the diploid
deficiencies arose from a similar action on the spindle appears to be
oversimplification of the problem.
Among the progenies of these treated plants there appeared also
extra-chromosomal types.^ The fifteen-year breeding record for Da-
tura showed that 0.16 -f .019 jier cent of the 2// jilants recorded were
extra-chromosomal types.'' Among the 2135 plants, 0.52 + .105 per
cent hatl one or more chromosomes. This value is 3.36 times the
probable error, and combining data for two years leads to a value 4.42
times the probable error.^ An increase caused by colchicine seems a
reasonable explanation. Of the extra-chromosomal types induced by
colchicine, ten plants had 2" + 1 chromosomes, one had 2n -U 1 + 1,
and three were 4» -(- 1. If colchicine increased the frequency, the
action had to occur at mitosis during treatment. A specific action on
the spindle directed to one chromosome is suggested.
Aneuploids from treatments in Lilium lon<riflurufn were analyzed
from root tips and not the jjollcn mother cells.-" Out of 500 plants
treated and analyzed, 303 cases from roots were counted. Eight aneu-
The Aneuploids 347
ploick were louml: these were either 4ii deficient or 1/; phis one
chromosome.^** Among hctcroi)loids in Nicotiana, deficient types
(2;/ — 1) Hke those in Datura were found. Simihirfy, in Enira sativn
the ]jhint was facking two chromosomes, 2// — 2. No explanation
difierent from that ad\anced for Datura has been made. The devia-
tion originated when colchicine acted on somatic mitosis.
In \iew of these cases we are prompted to suggest that the sub-
type of exploded c-metaphase, the distributed c-metaphase, shoidd be
studied further with respect to unecpial distributions of chromosomes
following treatment with colchicine. Activity of this type was often
observed in pollen tubes of Polygonatu)ii . but the relation to such
phenomena has been for the most part overlooked. As a basis for an
action of colchicine on mitosis that leads to numbers other than the
true i)olvploids, illustrations are abundant in cultures of pollen tuf:)es
Avhich account for a variety of deviating numbers that might occur
^\■hen colchicine acts on mitosis.
14.2: Mixoploidy From Colchicine
The action of colchicine upon individual cells was emphasized in
the first studies with Allium roots. .\ single root tip treated for 72
hoius may yield cells with many chromosomes while other cells re-
main dipioid. It has been confirmed many times that within one
meristematic group there may remain diploid cells alongside tetra-
ploids. Such tissues are described as mixoploid. These cases should
not be confused with sectorial chimeras since the word means mixed
together.
A cyto-histological study of maize after treatment with colchicine
showed that different areas may become tetraploid more readily than
others.^i Treatment of maize plants with colchicine rarely gives rise
to a completely tetraploid plant.*^ Certain branches of the tassel show
tetrai:)loid. and others, diploid pollen. ^Vhether these are true sec-
torial chimeras or the result of mixoploid conditions has not been
decided.
Another case of mixoploid tissues from treated plants was fol-
lowed through enough generations to prove that mixoploids were
involved rather than sectorial chimeras. =^ Lolium pereuue L., 2/? =
14. was originally treated by subjecting seed to colchicine. -^ Plants
with tetraploid cells, determined by measurements of pollen grains
and chromosomal counts in root tips, were isolated. Sujjposedly tetra-
ploid tillers were being separated and transplanted. Also some clones
were separated as progenitors for control diploid clones. Selections
-^vere again made for diploid and tetraploid clones.-^ As before,
chromosomes were coiuited. For two generations such propagation
was continued, yet mixoploid tissues persisted into the seventh gen-
348 Colchicine
eration ot vegetative propagation in spite of well planned and care-
fully followed methods of determining numbers of chromosomes.
These seven generations were preceded by four vegetative generations
in which two were selected after chromosomes were determined to
guide the selection.
In some cases individual anthers yielded diploid and tetraploid
microspore mother cells.-" Clearly a mixoploid tissue gave rise to
these anthers. Remembering that tested plants were remo\ed from
the tetraploid progenitors by several generations of propagation, the
persistence of diploid and tetraploid cells with neither one crowding
out the other is of particular interest. Liliinn is considered to be
tetraploid on the basis of chromosome counts; yet diploid and tetra-
ploid pollen mother cells have been found in the same anther of
lilies.-" In one test a generation was grown by scale propagation and
ten plants were selected. One plant from scale propagation and three
plants obtained by dividing the original bulb yielded flowers with
anthers that had both dij^loid and tetraploid cells. The parent plant
was supposedly a tetraploid.
Both cases mentioned here, Li Hum and LoUuui. represent vegeta-
tive propagations, and in each instance colchicine created a mixo-
ploid tissue. Projects that involve vegetative increase present complex
problems, the true nature of which remains unsolved.
14.3: Chimeras Induced by Colchicine
In longitudinal section, the apical meristem of Vinca rosea L.
shows a distinct la\ering of cells. '^ These are clearly illustrated with
the photomicrograph in Figure 14.1, A and B. Using terminology
promoted by plant anatomists, the first layer is called T^ and the next
To. These, then, refer to the first and second layers of a tunica. The
third layer and cells deeper in the apex are called the corpus, initialed
C'l and C.J. Lower than C., no specific layers can be observed. ^^
From species to species the limits of the tunica and corpus may
vary. For example, J'ijica minor L., obviously related to V. rosea, was
described with three layers of tunica and a fourth as the corpus. If
the older terminology of Hanstein is related to the tunica-corpus
concept using Vinai )nin()r as an example, then T, is ecjuivalent to
Hanstein's dermatogen, r._, and T->, are the same as periblem, and Cj
is the plerome. Another and different labeling has been used in re-
cent cyto-chimeral studies following j^olyploidy induced by colchicine.
The layers are called L-I, L-II, and L-III, etc. without reference to a
tunica and corpus. ^^
The point to be strongly emphasized here is not the terminology
but the fact that the various layers make a definite and precise con-
tribution to the shoot axis and to such parts of shoot as the flower
■>
V
•• •«
^
V
E
c^./^J."' " • r
Fig. 14.1— A comparative study of Vinco rosea L. diploid and tetraploid strains. A.
Shoot apex of tetraploid plants showing layers of cells, outermost is the first tunica or
T , second layer L, third layer C, and deeper strata become C, etc. B. Shoot apex of
diploid plant and foliar primordia. C. Brush method for treating ycung plants with
colchicine. D. Size differences between the tetraploid and diploid flowers. Larger Hower
is tetraploid. E. Tetraploid pollen mother cell, n— 16. F. Diploid pollen mother cell, n—S.
(Contributions from the Botany Department, University of Oklahoma, Norman, Ok.a-
homa. Adapted from Schnell)
350 Colchicine
parts and leai. Since the cells ot the first layer at the aj^ex always
divide anticlinally and not periclinally, all epidermal cells trace their
origin back to the first layer as seen in the shoot apex. Accordingly,
the second layer divides anticlinally, and tissues originating from
the second layer will be independent in genetic make-ui) from the
first, and in many cases from the third. If colchicine changes the cells
of the first layer to tetraploidy while the second layer remains diploid,
then the epidermal cells will be tetraploid and the pollen grains dip-
loid, because the sporogenous tissues originate from the second
layer. This condition is called a periclinal diimcra. Various com-
binations can be had.
When geneticists realized that the treated plants might look like
tetraploids yet reproduce as diploids, the significance of periclinal
chimeras began to be tridy appreciated.'^' '^ Moreover, developmental
problems can be traced with closer attention to the origin of tissues,
because specific periclinal chimeras shoidd yield certain results in
the matme organs.^-- ^•*- ^^ If the pollen develops from the second
layer, T^, just beneath the epidermis, which is T,, then diploidy and
tetraploidy will be loiuid in pollen and epidermis according to the
changes in T^ and T-,. I hat is to say, a tetraploid second layer, Tj.
should produce tetrajjloid pollen mother cells while diploid guard
cells originate from tliploid Tj. The situation has been j)ro\ed to be
ijust that way. These are periclinal chimeras.
; An important series in Datura was clearly described showing that
the development of petals, sepals, pistil, ovules, and stamen coidd be
traced back to specific layers of the apical meristem. Similar periclinal
chimeras were found in the cranberry. i" Cyto-histological changes
were described in detail. One important conclusion "was reached.
Stem and lateral bud apices were seldom converted into total poly-
ploidy. Therefore, semiwoody and woody plants propagated follow-
ing treatment with colchicine, required special attention ^vith care
given to the nature of polyploidy induced."' Periclinal chimeras fol-
lowing treatment with colchicine have been reported many times
since the first cases were reported for Datura.^-- ^•''
By induced polyploidy, specific and discrete layers were demon-
strated for Datura sirainoiiiuni L.^- The leaf and flower were traced
back to the shoot apex. One important type useful in detecting
origins was a diploid outer layer, an octoj)loid second layer, and a
diploid third layer. ^- Any tissue that originated with an octoploid
layer was unquestionably marked by the size of cells. Development
of the carpel was traced in Datura^- The periclinal chimeras Avere
used to discover specifically how the style, stigma, calyx, and corolla
differentiated. In questions regarding axial or foliar origin for such
parts as the stamen it can be stated more precisely how development
takes place.
The Aneuploids 351
When numerous periclinal chimeras were demonstrated among
well-known varieties of apples, interest was again intensified because
the breeding behavior dejiended upon the specific chromosomal nature
of a ])articular chimera. i'^- i" If the layer that produced pollen was
dijjloid, triploid, or tetraploid, then entirely dilferent results in hy-
bridization could be expected. Periclinal tetraploid giant sports of
Mcintosh should be of great interest since tetraploids in subepidermal
layers breed on the tetraploid level. i*"' Some important varieties are
trijjloid, many are dij^loid, while some sports are chimeras. Two
naturally occurring chimeras in apples are: (1) the 2-4-2 type and
(2) the 2-2-4.
The pomological curiosit) known as "sweet and sour" from the
Rhode Island Greening is meaningfully interpreted as a periclinal
chimera. The sour portion originates from the outer layer and the
third layer, whereas the sAvect ])ortion takes its origin from the second
layer.1'5
Seven years after colchicine treatment, a Mcintosh tree bore fruit
that was giant-like, and similar to the diploid-tetraploid periclinal
sport which occurs in nature. The induced type proved to be a peri-
clinal chimera. By adventitious buds that originate from deeper
layers, a com])lete tetraploid stock can be obtained. When crossed
^vith diploids, this becomes breeding material for new triploid vari-
eties. AVith better knowledge of periclinal chimeras, breeding in
many fruit trees can be expected to advance.
Another kind of chimera is the sectorial chimera. As the name
imjilies. sectors are either diploid or tetraploid. The changes occur
in a mass of cells not limited to layers. This type was studied in
Datura.'' One branch becomes tetraploid and another diploid, de-
pending on the origin of a specific branch.'^ '
The A\ ide distribution of periclinal chimeras in polyploids derived
from colchicine shows that the change is not unusual. \Vhile our
discussion is limited to only a few species, important work has been
done with Lilium, Solanurn, and many other plants. The principles
as outlined with fruits and Datura are basic to all chimeras.
14.4: Sex Determination and Polyploidy
As was stated in the introduction to this chajner, jiolyploidy and
special j^roblems in botany did not arise suddenly \\hen colchicine
became known for its use in research. At this time, however, there
was an inmiediate increase in papers dealing with such problems. A
notable case was the relation between sex and polyploidy in plants.-"*-^^
One mav erroneously conclude that new ideas were conceived as soon
as colchicine was discovered. A proper persj^ective is needed here
to e\aluate projicrly the role played by an imj^roved methcxl such
as colchicine proved to be. Whether the colchicine technique had
352 Colchicine
been developed then or not, a proof that dioecious races in phmts
could be established as ]3olyploids would certainly have been re-
ported when it was, in 1938.'^''
As early as 1925 the similarity in ploidy between animals and
dioecious plants was obscr\ed.''-^ Both cases were generally dijiloid.
Among many plants jjolyploidy was a mode of sjiecies formation.
These were not dioecious. Therefore, an explanation for the lack of
polyploidy in animals and in dioecious plants seemed to be related
to the diploid state. When a polyploid species of Empetru^n hennapli-
r<Hlituin was found to be hermaphroditic, the fact was particularly
interesting because there was a related diploid species, dioecious
Empetniin )iignim.'''' Conflicting evidence accumulated when a dioeci-
ous tetraploid strain of J'aUisneria was reported. Briefly this was the
state of affairs when Westergaard decided to test the hypothesis by
making tetraploids from diploid dioecious species of MeUnidriiun. He
began the project in spite of the fact that no well developed methods
for making polyploids were available at that time. Colchicine had not
been announced. i*^'' ^''' '*-^
In America, polyploidy and sex determination in plants were
started because colchicine should quickly lead to the evidence needed
to test the question raised by Muller about sex determination as
limited to diploidy in animals and dioecious plants.'' The projects
in Denmark and America were started about the same time and first
results from each came close together.-^^ Yet there was no a\vareness
that either was studying the same problem.
Soon other work began in japan,-^*^- '^^ and there were additional
studies in America. •^■■' A large volimie could be compiled from this
problem after only a few years of investigation. Some excellent work
was done and colchicine provided enough breeding material to demon-
strate conclusively that sex determination was not limited to a dip-
loid state when plants were under consideration. Howe\er. male
and female plants are not strictly comparable to maleness and female-
ness among animals. In plants there are three kinds, with respect to
production of flowers: (1) plants producing staminate, or pollen-
bearing, flowers, (2) some giving ]jisiillate, or seed-producing, flowers,
and (3) plants that have staminate and pistillate structures in the
same flower. These are called male, female, and hermaphroditic, re-
spectively.''''
Adopting the sex-determining code used for animals, notably
DrosophiJa, diploids are XX as females and X}' for males; in addition
there are other chromosomes called autosomes. A tetraploid female
carries the chromosomes XXXX and male XX)T with a tetraploid
set of autosomes designated AA. At once, it can be seen that another
combination XXX)' may exist at the tetraploid level. If further
The Aneuploids 353
crossing between tetraploicls and diploids and between tiiploids and
dijiloicis were carried out, combinations could be extended to XYY,
XXX}', XXXry, XXXX)'. obviously, a great range may be pro-
duced. Everyone agrees that the Y chromosome is a determiner for
maleness because the j^resence ol this chromosome once or twice clearly
imjircsses its influence on the j^lant. Only when four X chromosomes
are opposing the one }' does the flower change to a hermaphrodite.
This tendency begins to show slightly among the XXXF type. The
XY and ATT are male without exception. ''•'
The I^anish"*-^ and American''"- polyploids differed with regard to
the possible iniluence of autosomes and the role of the X chromosoine
as a female determiner. Some of the differences may be due to sources
of diploid plants and some difference to method as well as interpreta-
tion. Two critical j^apers must be studied if one wishes to weigh the
evidence: one by \Varmke.'^-' and another by ^\^estergaard.■■'•"'
Cytologically the Y chromosome can be distinguished from the
smaller X. In turn, the X is larger than any autosomes. This feature
is highh desirable because certain problems woidd be difficult to
interjjret otherwise. The hybrid generation between tetraploid XXXX
and tetraploid XX}'}' throws 1 female to 12 males. The diploid sex
ratios are 1:1. Looking at the chromosomes, it can be seen that most
males are XXX}" (89 per cent) and only a few XX}'}' (4 per cent).
The association between XT and Y-Y is more frequent than between
X-}' and X-}'. A high proportion of gametes were X}' and the XX
and }'}' classes were low. If a male with chromosomes XXX}' was
crossed \vith a female XXXX. the offsjjring showed 50-50 male: female
ratios. Similar results were obtained with Acuida lamariscina (Nutt.)
\\ood,-'-^ and for Mehmdrnim dioecum var. alhuiii described above. •''^
In nature, the excess 4;/ males that are XXX}' instead of XXYY
would iertilize a large majority of the 4/7 females XXXX; hence, equal
populations of males and females at the tetraploid level could be
expected. From an evolutionary standjioint tetraploids differing on
the basis of X and }' determining maleness and femaleness could be
established much the same as a diploid species. A tetraploid race of
Ritinex acetosa has not been demonstrated as a stabilized dioecious
type.''^
Autotctraploid hemj) gave an excess of females in the second
generation follo^ving jjolyploidy.-'^ This was a reversal over the dip-
loid male-female proportions. Less cytological attention has been
given to this species.
Polvjjloidy provides a method for deciding whether the male or
female is heterogametic. that is. carrying the X'}'. A test was made
for Silene otites since cvtological methods did not give a solution in
this case."'"' Polyploid plants would become XXXX and XX}'}^ but
354 Colchicine
the designation ot male or female remains unknown. Crossing these
tetraploids gives three types ol offspring, XXXX, XXXY, and XXYY.
About 5 males to 1 female are ol^tained. The female is tested by
making triploids, mating tetraploids with diploids. A female XXXX,
the 3» pojndation crossed to male XY, should be 1:1, male, female.
If the -hi population is 5 males to 1 female the constitution would be
XXYY. The tests showed 1:1 ratios; thus females were homogametic
as in Mehnidriinn.
14.5: Aneuploids and Colchicine
Aneuploids can be created by colchicine in two ways. One pro-
cedure involves direct action on dividing cells in meristems.^ The
other method is indirect, following specific breeding procedures after
polyploids have been made. Until ccjlchicine was discovered, the first
types were very rarely seen, particularly the diploid deficient plants,
2?? — 1. These were discussed on page 347. In this section ihe Ijetter-
known, indirect method for developing aneuploids is discussed.
The scope has been expanded to more species because colchicine
has stinudated the production of tetraploids. It is well known that
tetraploids crossed with diploids create triploids. These in turn,
when crossed back to diploids, become a rich source for off-type
plants, those with extra chromosomes. Among the higher levels,
pentaploids are excellent sources for aneuploids. Propagating auto-
tetrajiloids regularly throws plants with somatic numbers deviating
from the euploid value.
Distribution being unequal at meiosis, the chromosomes in the
megaspore mother cell and the pollen mother cell cause the numeric-
ally different types. Sometimes transmission of extra types can be
done through the seed parent only. In other cases the transmission
of certain aneuploids is known only at high levels of polvploidv. If
a particular morjjhology of the plant can be identified -with aneu-
ploidy, spontaneously 'occurring cases are usually high enough to
create a large reservoir of extrachromosomal types.
Aneuploids among Datura. Zea, Nicotiana. Tyitiruin, and other
genera have been studied extensively and have i)een used for specific
genetical tests before colchicine methods came into prominence. In
other instances, such as Gassy pi iim.^- ^^ their isolation in large num-
bers began when this ready method for producing polyploids was
discovered.
14.=^-!: Trisomies and tetrasomics. In 1915, A. F. Blakeslee found
a mutant in the cultures of Datura stramonium. This was called the
"Glofje nuitant" because this plant had a globose capsule distinct
from the usual patterns. Five years later, in 1920, John Belling
The Aneuploids 355
demonstrated cytologital evidence that this plant and others lound
between 1915 and 1920 each contained a single extra chromosome.
In 1938, a summary covering 60,000 field-grown ofFsj^ring from types
with extra chromosomes was published.'' The term trisornic, as the
extra chromosomal j^lant was called, is used in cytogenetics.
With the use of colchicine in polyploidy and in Beta there arose
an opportunity to study the effect of chromosomal variation in sugar
beets. •'"^* It is one of the most intensively studied species as well as
one of great practical importance in many coinitries. The large-
scale ])roduction of tetraploids in 1938 with subsequent triploids
opened opportimity to study variation in regard to chromosomal
numbers. Since trijjloidy was discussed in the chapter on autoploidy,
that will not be repeated. Here the influence of separate chromo-
somes, the trisomies, are of special consideration.''"
Progenies from triploids intercrossed, and backcrossed to diploids,
included plants with chromosomal numbers from diploid to tetra-
ploid and beyond. One or more plants ranged from 18 to 36 chromo-
somes.^o Between 37 and 45 several classes were missing. This
material arose from colchicine-treatcd seed of the Hilleshog strain at
Svalof, Sweden. When the seed j^arent was a triploid and the pollen
parent diploid, all numbers from 2x to 3x were recovered. A recipro-
cal cross yielded an excess of diploids (77 per cent) with classes from
21 to 25 missing. The transmission difference between seed and
parent confirms what had been learned long ago. Extensive pollen
tube studies by J. T. Ruchhol? demonstrated the effect of extra
chromosomes in Datura upon the male gametophyte.
Effects of different chromosomal classes upon a whole series of
morjihological and physiological characters in sugar beet were com-
pared. Since this study permitted analysis of the entire population,
certain advantages Avere presented that had never been jjossible be-
fore this time. Every chromosomal class from 18 to 36, inclusive, was
analyzed as follows: (1) field estimation, (2) weight of tips and roots,
(3) refractometer determinations, and (4) leaf development. The
trisomies were distinct in plant characteristics, and the particular
chromosome stamped its influence on growth habit. An interesting
problem that requires more attention is the possible correlation be-
tween vigor increase and decrease in the size of the extra chromosome.
This point becomes important when transfer of characteristics by
single chromosomes is attempted. In addition to single trisomies, two
plants with 20 chromosomes were studied. Plants beyond the 36
chromosomes, including a 42-chromosomc plant, had good \igor.
Finally the optimal niunbers as would be predicted have three modes;
these are diploid, triploid, and tetrajiloid. Maximum viability occurs
at the euploid number.'"*
356
Colchicine
Five different chromosomes from Nicotiana langsdorffii, a small
flowered species, was studied as trisomic in relation to corolla size.
The background into which the extra chromosome was introduced
was the hybrid between N. lano;sd()rffii and N. sanderaea, a long-
flowered species. ^'^ Since each trisomic could be detected by plant
appearance the influence upon particular structures could be ana-
Control 2n.2n,2n
8n , 2n. 2n
2n,8n, 2n
2n, 2n,8n
Fig. 14.2 — Diagrams of longitudinal sections through the shoot apex of diploid Datura
stramonium t. and three layers of periclinal chimeras. Upper left, diploid layers of
tunica and corpus. Upper Right, octoploid tunica and diploid layers beneath. Lower
left, first tunica diploid, second tunica octoploid, corpus diploid. Lower right, tunica
diploid and corpus octoploid.' (After Blakeslee and Satina)
lyzed. Three of the five chromosomes, when in trisomies, reduced
the corolla in all regions, but two chromosomes decreased one region
and increased another. This method was apjjlied to find the relation
between whole chromosomal additions and size effects. The con-
clusion was reached that size is determined by genes according to a
geometric proportion. Eventually, size in Nicotiana flowers can be
resolved as a "cumulative geometric effect." ^^
Hexaploids combining two species of Gossypium crossed back to
G. hirsuium lead to aneuploids with one or two chromosomes from
the diploid species introduced in the hexaploid. The characters in-
fluenced are: leaf, floral parts, size and shape of bolls, as well as fiber
The Aneuploids 357
and seed coat. Cytological study of these trisomies is valuable for
determining the nature of chromosomal differentiation among specific
chromosomes.^^
Some fertile, partially stable i)lants can be derived by selling
inter-species trisomies instead of the tetraploid number or the extra
chromosome; morphologically distinguishable 54-chromosome lines
were produced. The interest in these types lies in their constitution
because the extra pair may be Irom an Asiatic-American wild or an
African species. This pair is added to the naturally occurring G.
hirsutum, a tetraploid 52-chromosome plant.^^
Another type, the intra-specics trisomies, arises from polyploids of
G. Jiiysutinn. By selling and appropriate crossing between various
trisomies in this class, both double trisomies and tetrasomics were
developed.
There are then tAvo types of tetrasomics identifiable by the extra
pair, the intra-species tetrasomic and inter-species tetrasomic. As
the word suggests, the latter pair is derived from strains from another
species, whereas the intraspecific tetrasomics are limited to one
species.ii Morphologically both types may be distinguishable from
the species. A remarkable fertility is retained when a pair comes
from another species, but the intraspecific tetrasomics are almost com-
pletely sterile. A great many cytological problems can be solved with
these types. Trisomies and tetrasomics have been obtained in A^
sylvestrus. Among the off-type plants from a progeny of monoploid
pollinated by diploid, trisomies were derived in wheat. Further self-
ing yielded tetrasomics. These added chromosomal types are not
easily detected in hexaploid wheat. Some homozygous speltoid wheat
proved to be 44-chromosomal plants. Tetrasomics and trisomies may
have been involved in the dwarf and subcompactoid types.-^^
7^.5— 2; Nullisomics and monosojnics. Chromosomes lost in dip-
loid plants do not survive. This was reviewed in an earlier section.
Tetraploids in Datura also lacking a chromosome or two failed to set
seed. Additions in diploids have been propagated extensively, but
these are often transmitted only through seed parents.
At the polyploid level, missing chromosomes are tolerated.-" For
that reason some imj)ortant work can be done with two general types:
(1) monosomies, those plants lacking one chromosome, and (2)
nullisomics in which a pair is missing.^^i The latter are well known
among hexaploid wheat.^^ In Gossypium and Nicotiaua a success
similar to that for hexaploid wheat has not been achieved with nulli-
somics.^-
Monosomic plants have been found in Gossypium spontaneously,
through nondisjunction in trisomies, and after intergeneric |)ollina-
tion.ii Since the transmission of haplo-deficient gametes fails in Gossyp-
ium. ihe further utilization of monosomies is stopped. In contrast
358 Colchicine
to this situation, monosomic analysis developed for Nicotiana has
proved most useful in many genetic tests, notably in establishing link-
age groups; surveying amphidiploids for specific genetic characters. i-
The technique applied to Nicotiana suggests that other groups might
profit from these methods.--^ There are limitations to this method
among such a group as Gossypium, where polyploids are common;
yet the use of monosomies is limited. No nullisomics are reported for
GossypiiiN}.'^'^
Quite another situation exists in hexaploid Triticuni aestiinim L.,
where nullisomics and monosomies can be applied to genetic prob-
lems.''^ As we mentioned for trisomies, the number of different types
with one whole chromosome extra should equal the haploid number.
For Datura, 12 primary trisomic types exist. In Nicotiana the total
monosomies possible is 24. Accordingly, 21 nullisomics would be ex-
pected or ecjual to the 21 pairs representing hexaploid wheat. ^^
For each pair missing, the 20-chromosome plant has specific
characteristics. Nullisomics may be numbered from I to XXI. ^^ None
is completely sterile, and certain are fertile in both male and female.
Some are female-fertile only, others male-fertile only. Some nulli-
somics pollinated by normal plants give more monosomes of a par-
ticular type, as well as irisomes. The incidence is more than a random
occurrence. For example, nullisomic III produced more monosomic
IV and XV than other types of monosomes.
Particular tetrasomics may cancel the effects of certain nullisomics.
Such compensating cases are known for wheat and oats. For example,
tetrasomic II compensates for nullisomic XX so that the plant is very
nearly normal even as the male gametophyte.^'' There does not seem
to be a competitive advantage between pollen-deficient for chromo-
some XX and duplicated for II. Common properties in the segments
of these chromosomes would appear to be a cause for the compensa-
tion. There seems to be no pairing between tetrasome II and nidli-
some XX. These are. in very brief sketch, problems related to poly-
ploidy.
Seven chromosomal pairs corresponding to the D genome in hexa-
ploid wheat are dwarf nidlisomics and differ from each other accord-
ing to the specific pair missing. These nullisomics were derived from
among offs]:)ring of Trittcum pojomiciim, genomes AABB, X T.
spelta, AABBDD. These 7 nullis(jmics are lettered a, b, c, d, e, f, g,
respectively. Twenty-one nidlisomics from a Chinese wheat (T. aesti-
vinn L.) should throw light on the D genome by hybridizing the
dwarf nidlisomics and those from T. aestiviuit. which had a different
origin. -^1
Success has been achieved in transferring mosaic disease resistance
from one species to another in Nicotiana. 1 he commerical tobacco re-
The Aneuploids 339
ceived a (hromosomal pair from .V. ghttijiosa, \\hi(h contributed the
necrotic factor tor resistance. Alien additional races included a pair
from one species and 24 pairs from N. tabacum. By another series of
crosses, alien substitution races were formed, whereby a pair of
chromosomes were substituted in the N. tabacum genome.^^ Other
species carry factors that can be traced by successive crosses into the
interspecific hybrid, then by a backcrossing procedure through a
number of generations. The monosomic method of analvsis lias been
\\orked out with good success in Nicotiana}-
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3. Beaslev, J., AND Brown, M. (see Ref. No. 8, Chap. 12) .
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360 Colchicine
22. Franzke^ C, AND Ross^ J. Colchicinc-iiuiuced valiants in sorghum. Jour. Hered.
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23. Gerstel, D. (see Ref. No. 38, Chap. 12) .
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28. Kerns. K., and Collins, J. Chimeras in the pineapple; colchicine-induced
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31. Matsl'MUra, S. Genetics of some cereals. Ann. Rpt. Nat. Inst. Genet. Japan.
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32. MuNTZiNc;, A. Ne;v material and cross ccml)inatit)ns in Galeopsis after col-
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Polvploidv and sex determination in Melandrium. II. The effect of polyploidy
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.Studies on artificial polvploidv in liops. Bot. and Zool. 10:63-68. 1942.
39. Rajan, S., et al. (see Ref. No. 171, Chap. 13) .
40. Ramanujam, S., and DrsuMtKii. M. (see Ref. No. 168, Cliap. 13) .
41. Sass. J., and Green, J. Cvtohistology of the reaction of maize seedlings to col-
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42. Satina, S. Periclinal chimeras in Datura in relation to development and struc-
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493-502. 1944. Periclinal chimeras in Datura in relation to the development
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Bot. 27:895-905. 1940.
44. , AND Blakeslee, a. Periclinal ciiimeras in Datura stramonium in rela-
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species and 4n for another. Amer. Jour. Bot. 36:802. 1949.
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The Aneuploids 361
48. Smith, H. Effects of genome I)alaiuc. pohploidy, and single extra chromosomes
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49. Smith, L. (see Ref. No. 200, Chap. 13).
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CHAPTER 15
Criteria for Judging Polyploidy
15.1: Sterile Hybrids Made Fertile
In the final analysis, pohploidy is determined bv connting the
niunber of chromosomes, and comparing this number with the dip-
loid or untreated plant. Some rapid and accurate methods are
available for judging polyploids indirectly.
If a sterile species hybrid begins seed production after treatment
with colchicine, the evidence is good that polyploidy has been in-
duced.^i Geneticists knew that doubling the number of chromosomes
in a sterile species hybrid was a critical test for demonstrating the
effectiveness of the drug.^. 3i, 41, 14, 47 Species hybrids of Gossypium
were treated immediately.^ Plants that flowered, yet failed to set bolls
and seed, began seed production in those sections of the plant treated
with a proper concentration of colchicine. Therefore, without count-
ing the number of chromosomes, the preliminary efficiency of a treat-
ment could be estimated. The chance doubling that might have oc-
curred through unreduced gametes is of such low frequency that the
effects of colchicine were not obscured by natural or spontaneous
doubling.
Amphiploids among Nicotlaua were made in large numbers.^- The
list of artificially induced polyploids increased within a few years.^^
Combining the first data from Gossypium and Nicotiana proved the
value of colchicine beyond doubt.
Many combinations of interspecific and intergeneric hybrids were
converted into amphiploids within the Triticinae.i- ^ From one pro-
ject, 18 amphiploids involving 10 species were created within two
years.^i The production of good pollen and eventually seed in the
sectors of treated plants that showed the effects of doubling was re-
liable criterion for amphiploidy. Estimates of how effective colchi-
cine was upon these plants could be checked on a percentage basis.
Some modifications were necessary because the monocotylcdonous
species had to be treated differently from the dicotyledonous types.
[362]
Criteria for Judging Polyploidy 363
After the amphiploids in Triticinae were produced in such large num-
bers, it was demonstrated that both monocotyledons and dicotyledons
were being doubled by the use of colchicine.
A barrier in plant breeding had been removed or considerably
reduced by the discovery of a ready technique for making sterile hy-
brids fertile and estimating the effectiveness by seed production. In-
compatibilities such as failure to make hybridizations must now be
overcome. Some work on embryo culture has been used to excellent
ad^■antage.
15.2: Appearance of Polyploids
New leaves and stems that grow from treated sectors are usually
wrinkled, thicker, and darker green, and have coarser texture, as
compared with the untreated plants.^' '• ^^ An increase in thickness of
the tetraploid leaf can be judged by holding the leaves between
thumb and forefinger. By such methods a rough sorting of tetraploids
can be made among large populations of treated cultures. Those
that have not responded can be quite accurately eliminated.
Specific marks on the leaves such as veins, hairs, and glands are
valuable references for the first sorting of possible changed types.
The outline of the leaves changes; they are usually shorter and more
rounded than the diploid leaves.
Flowers of the tetraploid plants are larger (Fig. 15. IB) and more
compact than the diploid (Fig. 15.1/4). These changes were corre-
lated with chromosomal determinations (Fig. 15.1C,D). Tetraploid,
triploid, and diploid flowers form a decreasing series in size of flower.
These proportionate changes are illustrated for watermelon strains.
At the tetraploid level, optimum size is reached, and beyond that
point the increase in sets of chromosomes actually reduces the size
of the flower. Among the best varieties of Iris, polyploids are favored
over diploids.^" The increase in size of flower has been a goal for the
improvement of ornamental species.
A tetraploid plant has a more rugged appearance, looks sturdier,
and has certain giant-like features. Usually the rates of growth are
slower, but even the final growth does not produce a plant as tall as
the diploid. Among polyploid watermelons, the vine remains green
over more days than among diploids, disregarding disease factors.
Another difference between the stems of diploids and those of tetra-
ploids is the shape of the apex as viewed in longitudinal section (cf.
Chapter 14) .
15.3: Fruit and Seed
The development of larger seeds from tetraploid lines is a con-
sistent macroscopic characteristic that has been confirmed for hun-
t
•
• f
^
•
«
Fig. 15.1 — Flower, pollen, stomata, pollen mother cells of diploid and tetraploid strains
of Phlox drummondii. A, B. Diploid and tetraploid flowers, respectively. C. Pollen mother
cell with 7 bivalents. D. Tetraploid pollen mother cell, n — 14. Note quadrivalency. E,
F. Stomata of diploid and tetraploid respectively. G, H. Pollen grains of diploid and
tetraploid, respectively. (After Eigsti and Taylor)
Criteria for Judging Polyploidy 365
clreds of cases. -^ The sizes can be judged by volumetric measure-
ment, weights, or length and ^vidth measurements. As a sorting
method for choosing the tetrai:)loid rye plants in the treated genera-
tion, size of seed is a reliable feature.-" The grain weights of letra-
ploid rye were distinctly separated from dii)loids. Table 15.1 shows
the increase based on thousand-grain weights for diploid and tetra-
ploids. A mean weight of 30. 'M ^vas obtained for diploid and 46.50
for tetraploid.''-
Increasing the size of seed has been used as an argument to im-
prove the crop yield of diploids through polyploidy. The fallacy lies
in the fact that the seeds of tetraploids may be larger and heavier,
but the reduced number of seeds per plant prevents complete use of
the increase. Reduced fertility in autoploids is the most common
cause of decreased yield in number of seeds. Decreased seed produc-
tion in watermelon brought out this relation. A comparison of ten
fruits, diploid and tetraploid, showed avarages of 290.0 and 92.7 per
fruit, respectively.-i Since cultivation was similar and the varieties
were strictly comparable, the reduction was directly correlated with
tetraploidy. For reasons discussed in the previous chapter, triploids
are without seeds.
Amphiploids do not show the same consistent increase in seed
weight or size compared with the parental species. A comparison be-
tween amphiploids and parental types was made among species of
Broinits of the Gramineae. On the basis of weight for 200 seeds, the
amphijiloid increased as much as 75 per cent, while other increases
w^ere not more than 17 per cent^^ (Table 15.2). Genetic factors and
the contributions by each parent have a greater influence than merely
doubling the number of chromosomes.
A given kind of plant may regularly show specific marks among
the tetraploid seeds. Close inspection of the tetraploid seed of water-
melon showed that fissures developed in the seed coat upon drying.-^
A rupture of the outer layers of ovules creates this condition. These
marks as well as size of seed are good criteria for making preliminary
sorting of the tetraploid. Another distinction was the thickness of
"triploid" seeds and tetraploid. Seed from tetraploid fruit pollinated
by di])loids are called "triploid" seed and are thinner than the seed
from tetraploid fruits pollinated by tetraploids. -^ Other marks such
as coarseness, special spines, ridges, and color differences, once noted
can be reliably used as guides in selection auiong treated plants and
the tetraploid generations.^' i^- ^^- ^^'' "' ^^
Fruits of tetraploids are not necessarily larger than those of dip-
loids. Nevertheless, distinguishing marks can be found among teira-
jDloid fruits. The external marking, shape, and attachment to plant
are some of the features that have been used. Parthenocarpic fruits,
such as ihe triploid, may be somewhat triangular. -^ llie Iksin por-
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Criteria for Judging Polyploidy 367
lion of tetraploid tomatoes may be coarser, and for that reason the
polyploids are less desirable than the diploid. Many fruited plants
of horticultural importance show direct correlation between fruit size
and polyploidy within certain limits. A'aluable tetraploid varieties
of grapes are larger and superior to diploids. Tetraploid muskmelon
TABLE 15.2
Seed Weights of Species and Allopolyploid Deriv.\tives of the Hexaploid
Species of Bromus ( Ccratochloa )
Species of Polyploid
B. catharticus
B. catharticus
B. catharticus
B. haenkeanus
B. haenkeanus
B. stamineiis
B. catharticus-haenkeanus . . .
B. catharticus-haenkeanus . . .
B. catharticus-haenkeanus . . .
B. haenkeanus-stamineus ...
D. haenkeanus-stamineus ...
B. haenkeanus-stamineus ....
Strain
Waite
Berkeley
San Antonio
Carmel
Sparks
Berkeley
Waite-Carmel
Berkeley-Sparks
San Antonio-Carmel
Carmel-Berkeley
Sparks-Berkeley
San Antonio-Berkelev
Weight of
200 Seeds
[grams)
3.1
^
Increase
Over Arith-
metic Mean
Between Parts
(per cent)
52
17
25
41
75
64
fruits were more promising than the diploids according to sampling
methods made in one sttidy.
Pistillate flowers of tetraploids pollinated with pollen from diploid
strains may reduce the size of grain in such a plant as rye. Normally
these species are cross-fertilized, so planting side by side gives the
diploid pollinator an advantage over the slower-growing pollen from
tetraploid flowers." Yield is at once reduced. The effect of diploid
pollen upon fruit development in watermelon is quite the opposite.
The triploid plants must be interplanted with diploids to insure
pollination, for the diploid pollen stimulates parthenocarpic or seed-
less fruit formation. The number of fruits produced by triploids may
be double the number for a representative diploid. Yield trials
showed that this feature favors the polyploid.
15.4: Physiological Differences
Excellent reviews have been made to differentiate the diploid and
tetraploid plants.^-* An ever-increasing nuniber of autotetraploids
adds more material for such study, including physiology, incom-
patibility,-'•*» morphology, and anatomy. Final superiority of the
368 Colchicine
tetraploid depends upon the physiology of the ixuticuhu' strains. •'• ^•■'•
-f"' Advantages such as protein content,^' vitamins,^^ yield of su-
crose,3'^ and other valuable characters-'*- -*• ^*' are products of the func-
tioning plant.
A superior baking flour was produced by the tetraploid rye varie-
ties. Bread with better texture and color, as well as a larger volume
of bread per sample of flour was made from the tetraploid flour. The
value for tetraploid was 279 in contrast to the value 260 for a diploid,
or an increase of 10 per cent in favor of the tetraploid. Higher pro-
tein content was correlated with the improved baking properties and
these were in turn correlated with the tetraploid varieties.
Increased sugar content in triploid watermelon and tetraploid
muskmelon improved the eating quality. Increases from 8 to 9 per
cent for dijiloids were shown to be raised to 12 per cent in the trip-
loid. The (juality and final test of any variety depends upon the
genetic nature of the diploid or the hybrid, so that variation exists
between tetraploids quite as much as between diploids. The induc-
tion of polyploidy does not automatically guarantee improved fruit
quality.
In a j^revious chapter, reference was made to the significant in-
crease in amount of sugar produced in the larger sizes of triploid
roots compared with the diploid. Tetraploid sugar beets are gen-
erally lower in yield of sucrose per unit weight of root. Other plant
products, such as latex prodrijced by Taraxacum holisaghyz and trans-
lated into rubber production, gave the tetraploids an increase of three
times the diploid. Drug production in Datura stramonium showed
increased atropin in the tetraploid. Another species, Cannabis sativa.
showed increased potency of the marihuana content when additional
sets of chromosomes are built into a variety. Environment influences
potency of drug production as noted in Chapter 5, but the addition
of chromosomes also causes changes in production of special plant
products.
The superiority of tetraploid red clover and alsike clover may be
correlated ^vith an increase in forage production. The amount im-
proves in the second year over the first. Enough tests have been made
with these forage crops, and on a sufficiently large scale, that the
conclusion of increased leafage is reliable.^
15.5: Microscopic Characteristics
Pollen size may be used for preliminary sorting of polyploids be-
fore the final chromosomal counts arc made for a particular plant.
This microscopic classification permits one to handle large numbers
of individuals. After the macroscopic identifications are completed,
a logical step is to measure the pollen grains.
Criteria for Judging Polyploidy 369
True autotetraploids have larger grains than the diploid (Fig.
15.1//, G). Microscopes are equipped with measuring oculars that
make this procedure routine. The correlation between the size ot
the pollen grain and the number of sets of chromosomes has been so
well established that no further discussion need be made on this
point. Triploid jiollen grains are notable for their irregular dimen-
sions and are useful in separating triploid and tetraploid plants on
a field scale basis.
The mean diameters for the diploid and tetraploid watermelon
\aricties ^vere 57.3 and 67.5, respectively. The smaller grains in trip-
loids averaged 62.1 and the larger sizes, 67.5. Similar size com-
parisons have been made for the guard cells of epidermal cells. A
photomicrograph (Fig. \5.\E,F) gives a visual picture of the dif-
ferences between the larger tetraploid and smaller diploid. Also the
distribution of guard cells varies; the diploid cells are closer together
than the tetraploid.
The relation between the size of pollen grains and guard cells of
a given plant are important for the reasons discussed in the previous
chapter under the subject of periclinal chimeras. If the pollen is tetra-
ploid and the guard cells are diploid, treatment with colchicine has
produced a chimera in which the deeper layer that produced the
pollen was made tetraploid and the outer layer remained diploid. A
reverse situation may occur. In these instances the guard cell would
show tetraploid characteristics and the pollen, diploid. The breeding
behavior of such a plant would be that of a diploid. Seed from this
plant would not lead to the expected tetraploid types, according to
information based on the guard cell sizes. Sometimes, a mixture of
diploid and tetraploid pollen exists in the same anther, or mixtures
of diploid and tetraploid guard cells appear on the same leaf. These
cases are a result of mixoploidy, a direct action of colchicine.
In cross section the leaf of the diploid is not as thick as that of
the tetraploid. Usually extra layers of cells of the mesophyll are
present.
Pollen mother cells undergoing meiosis are universally used for
counting chromosomes and determining the associations between
chromosomes during pairing. Acetocarmine stains have speeded up
such cytological work. Photomicrographs in Figure 15.1 show the
differences in numbers of chromosomes and some difference in the
association. Section D shows the multivalents in contrast to the one
in C (Fig. 15.1) .1"
Other cells, such as the generative cells in pollen tube cultures,
root tips, and leaf cells, may be used for counting the number of
chromosomes. At the second meiotic division and the division of the
microspore, chromosomal counting may be easier than at the first
meiotic metaphase.
370 Colchicine
Comparisons at meiotic metaphase of diploid sterile hybrids and
the amphiploid are important for an understanding of the possible
associations that form between chromosomes of opposite genomes.
While this evidence is not infallible, correlations may be obtained
between pollen fertility, possible intergenomal exchange between
chromosomes, and reasons for the failure in seed setting of the poly-
ploid.
15.6: Ecological Considerations
The success of a polyploid in nature or in agriculture depends
upon how closely the new variety meets the requirements for each
situation. Productivity or adaptation are measured in terms of the
responses such as yield, disease resistance, drought resistance, and
cold tolerance. The elimination in nature occurs through competi-
tion and in agriculture at the hands of the agronomist. Wide dif-
ferences exist between diploid varieties, and considerable improve-
ment can be done at the diploid level without stepping up to the
tetraploid. Adaptation problems increase, rather than decrease, with
the use of tetraploids. Autotetraploid rye clearly showed that the
kind of plant used to make the diploid may be as important as any
other feature.
Trying to measure the rates at which artificial polyploids become
established under natural conditions strikes at some basic problems
in polyploidy. Already differences have been recorded for the success
of the tetraploid over the diploid, or vice versa. An unusually high
seed production, about 75 per cent, in autoploid EJiroluita erecta
played some part in the establishment of the new type under natural
conditions. This situation held for ungrazed conditions, but where
grazing occurred, the low-growing habit of the diploid assured sur-
vival better since the flowers, being closer to the soil level, were not
destroyed as readily. This is one example of the critical differences
that determine success or failure of the tetraploid. ^^
Wilt diseases are devastating to watermelons in Japan. Appreci-
able resistance to Fusarium niveum was exhibited by the triploid and
tetraploid varieties. By selection, notable progress can be made for
insect and disease resistance if an initial advantage is provided
through the jjroduction of tetraploids. Autotetraploid radishes were
more resistant to the common club root disease, yielded more, and
had greater vigor than diploids.
The succulence of water cress leaves was improved by increasing
the number of chromosomes, but the growth rates being slower among
the tetraploid reduced the yield. Fewer cuttings can be made per sea-
son with tetraploids. The slower growth and prolonged flowering
period for ornamental species is advantageous. No single trait can be
Criteria for Judging Polyploidy 371
established as a rule that ^\ill hold for all polyploids. In the above
cases a lew instances are cited Avhich indicate that each problem must
be dealt with independently according to the requirements.
15.7: Fertility
Two general methods are used to judge the fertility level of a
specific polyploid: (1) percentage of good pollen as demonstrated by
microscopic test, and (2) the amount of seed set. Fertility differ-
ences and chromosomal phenomena at meiosis have been correlated,
but no general rule that explains the total possibility has been estab-
lished.^^ Unequal distributions of chromosomes in the meiotic stages
from first metaphase do cause unbalance in chromosomes in the pol-
len, and ultimately in the gamete. Triploids are notoriously bad with
respect to chromosomal balance. -^ When the percentage of pollen that
appears to be good is used to express the fertility ultimately judged
by seed production, some reservations must be made.*
Female sterility in the ovule arises at meiosis and may or may not
be the same as for pollen. Some polyploids are female-sterile and
pollen-fertile, and vice versa. The embryo-sac stages are difficult to
study because an involved cytological technique is required.^
Among progenies of amphiploids the first generation may be quite
fertile, while later generations may segregate due to weak and low-
fertility. By successive selection the fertility level may be raised, or
there may be mechanisms for improving fertility by elimination of
those genotypes that are deficient or have no survival value.
Perhaps no other aspect of polyploidy is more controversial than
this subject of fertility in the immediate product of doubling and in
the subsequent generations. Practically and theoretically the prob-
lems are unsolved at this point.
REFERENCES
1. Akerman^ a. (see Ref. No. 1, Chap. 11).
2. Atwood, S. (see Ref. No. 9, Chap. 13) .
3. Blakeslee, a. (see Ref. No. 11, Chap. 11).
4. Brown, M. Pohploids and aneuploids deii\ed from species hybrids in Gossyp-
iu»i. Hereditas Suppl. \'ol. Pp. 15-16. 1949.
.5. Chin, T. (see Ref. No. 18, Chap. 12) .
(). Clausen, J., et al. (see Ref. No. 18, Chap. 11) .
7. CuA, L. (see Ref. No. 20, Chap. 11) .
8. Das, B. Cytoloy,ical and enil)rvolos^ical basis for stcrilit\ in antotctra]iloid
sweet clover Melilutus alba Desr. Iowa State College Jonr. Sci. 27:537-61. 1953.
9. Dermen, H. Detection of polyploidy by pollen-grain size. (I) Investigation
with peaches and apricots. Proc. Anier. Soc. Hort. Sci. 39:96-103. 1938.
10. Ek.sti. O. The effects of colchicine upon the di\ision of the generative cell in
Pol\o;())uitu»i. Tradescantia. and Lilium. Amer. Jonr. Bot. 27:512-24. 1940.
11. _, AND Taylor, H. (see Ref. No. 52, Chap. 13) .
12. EiNSET, J. (see Ref. No. 19, Chap. 14) .
372 Colchicine
13. Ekdahl, T. Gigas properties and acreage yield in antotetraploid Galeopsis
pubescens. Hereditas. 35:397-421. 1949.
14. Emsvveller, S. {see Ref. \o. 30, Chap. 11) .
15. Ernould, L. [see Ref. No. 59, Chap. 13) .
16. Frandsen, K. {see Ref. No. 63, Chap. 13) .
17. Hakansson, a., and Ellerstrom^ S. Seed development after reciprocal crosses
between diploid and tetraploid rve. Hereditas. 36:256-96. 1950.
18. HoFMEVER, J. [sec Rcf. No. 79, Chap. 13) .
19. JuLEN, G. {sec Ref. No. 92, Chap. 13) .
20. Kehr, a., and Smith, H. {see Ref. No. 56, Chap. 12) .
21. KiHARA, H. {see Ref. No. 97, Chap. 13) .
22. , AND NiSHiYAMA, I. {see Ref. No. 100, Chap. 13) .
23. , AND Yamashita, K. {see Ref. No. 101, Chap. 13) .
24. KosTOFF, D. Cytogenetics of the genus Xicotiaua. States Printing House.
Sofia, Bulgaria. 1073 pp. 1943.
25. Krythe, J., AND Wellensiek, S. {see Ref. No. 44, Chap. 11).
26. KucKUCK, H., AND Levan, a. {see Ref. No. 45, Chap. 11) .
27. Lang, A. Beitrage zur Genetik des Photoperiodismus. II. Photoperiodisnuis
und Autopolvploidies. Z. Naturforsch. 2b:36-44. 1951.
28. Levan, A. {see Ref. No. 113, Chap. 13).
29. Mann, L. Fruit shape of watermelon as aftected l)\ placement of pollen on
stigma. Bot. Gaz. 105:257-62. 1943.
30. Mrkos, H. Uber Erfahrungen bei der Herstellung von 4 etraploiden mit Hilfe
von Colchicin imd Schnellmethoden zur Untersuchung der Chromosomenan
zahl. Bodenkultur, Vienna. 4:138-41. 1950.
31. Muendler, M., AND ScHWANiTZ, F. (see Ref. No. 131, Chap. 13) .
32. MiJNTZiNG, A. {see Ref. No. 51. Chap. 11) .
33. Myer, W. Meiosis in autotetraploid Loliuiii perenne in relation to chromo-
somal behaviour in autopolyploids. Bot. Gaz. 106:304-16. 1945.
34. Ncx;gle, G. The physiology of polyploidy in plants. I. Review of the litera-
ture. Lloydia. 9:155-73. 1946.
35. NoRDENSKjoLD, H. Gcuetical studv in the mode of segregation in hexaploid
Phleiim praterise. 9th Internat. Cong. Genet. No. 54. Bellagio, Italy. 1953.
36. Olsson, G., and Rufelt, B. {see Ref. No. 157, Chap. 13) .
37. O'Mara, J. {see Ref. No. 37, Chap. 14) .
38. Peto, F.. and Boyes, J. {see Ref. No. 58, Chap. 11) .
39. Rajan, S., et al. {see Ref. No. 171, Chap. 13) .
40. Randolph, L. Personal commiuiication. 1951.
41. Sears, E. {see Ref. No. 64, Chap. 11).
42. Smith, H. {see Ref. No. 199, Chap. 13) .
43. Smith, L. {see Ref. No. 200, Chap. 13) .
44. Stebbins, G. (see Ref. No. 66. Chap. 11) .
45. Steineggar, E. {see Ref. No. 204, Chap. 13).
46. Stewart, R. {see Ref. No. 206, Chap. 13) .
47. Stephens, S. {see Ref. No. 106, Chap. 12) .
48. Stout, A., and Chandler, C. Hereditary transmission of induced tetraploidy
and compatibility in fertilization. Science. 96:257-58. 1942.
49. Unrau, J. {see Ref. No. 51, Chap. 14) .
50. Wexelsen, H. {see Ref. No. 73, Chap. 11) .
CHAPTER 16
Techniques of ColchlcLne
Treatment
A. In Animals
16A.1: Solutions
It has been explained in Chapter 5 that the substance which has
been repeatedly called colchicine in this book may have differed from
author to author. One reason tor this discrepancy is the factor of
crystallization. Whereas pure, amorphous colchicine is very soluble
in water, crystallization from aqueous or chloroformic solutions yields
complex crytals which are less soluble and may have other biological
properties. ^'"^ Colchicine may crystallize Avith i/C molecide of water,
A\ith i/o niolecule or 1 molecide of chloroform. This last form of
crystalline colchicine is only soluble in water in the proportion of 4
per cent.^'^ It has often been used in experimental research. In
botanical work, results may be modified by the presence of chloro-
form, which is itself a mitotic poison. ^-^ In experiments on animals,
where the amounts of colchicine used are far smaller and the solu-
tions much more dilute, the presence of chloroform does not appear
to have any importance. But, for any quantitative estimation of the
activity of the drug, it must not be forgotten that crystalline colchi-
cine with 1 molecule of chloroform contains 25 per cent by weight of
the solvent.55 On the other hand, chemical work has demonstrated
that the plant Colchicum contains many alkaloids closely related to
colchicine, but with different pharmacological properties.^i- °~ One
of these, desmethylcolchicine, is found in the colchicine preparations
of the U.S. Pharmacopeia. •^'5 In the most recent work on colchicine,
care has been taken to purify the alkaloid before testing it.-^- ^ This
applies only to a very small number of the papers, and some results
may differ because the injected drug differed in its mode of prepara-
tion froiii the plant.! ^Vhile the above-mentioned differences are only
[373]
374 Colchicine
of importance for quantitative work, the changes that colchicine may
undergo in solution are far more important, especially for work with
warm-blooded animals or tissue cultures. Colchicine solutions should
always be freshly prepared, or kept protected from the action of
oxygen and light. For work on plants, where rather concentrated
solutions are used and where no problems of general toxicity arise,
this is not so important. In animal work, and especially for all work
on birds or mammals, it is most important to use freshly prepared
solutions.43 Standing in the presence of air, colchicine appears to
undergo a slow oxidation about which little is known (cf. Chapter 7) .
This decreases the spindle-inhibiting action, but may not affect simi-
larly the general toxicity, which is increased in cold-blooded animals
such as frogs.-^^ These remarks apply to solutions, whether in water
or fatty solvents. The latter have been mainly used for local applica-
tions in cancer chemotherapeutic tests. i*^- '^
The important point is that each paper should mention clearlv
the origin of the colchicine, whether crystalline or not, whether puri-
fied and how, the method of preparing the solutions before the ex-
periments, and the temperature at which these are conducted. It is
only in this way that a valid comparison of results is possible.
16A.2: Temperature
In Chapter 7, several instances have been given of the effect of
temperature on the action of colchicine. This has long been known,
but has often been overlooked.^^^ Most workers mention that the
alkaloid docs not influence cell division in unicellular organisms
(cf. Chapter 4) . However, while Paramecium is unaffected by colchi-
cine solutions at a one per cent concentration at 15°C., the same
solutions kill the paramecia in less than 4 hours at 33°C. Exposure
to this temperature is in itself not harmful to the organisms.^-^
These temperature effects are not yet understood properly. They
explain the considerable differences between colchicine pharmacology
in cold-blooded animals and in birds and mammals (cf. Chapter 7) .
For instance, colchicine-arrested metaphases remain intact for hours
and even days (Fig. 2.2) in amphibia; in mammals, on the contrary,
the nucleus of a cell arrested at metaphase by a spindle poison under-
goes rapid destruction. In all in vitro ^\ork, the temperature should
be constant and checked carefully.
16A.3: The Study of Mitosis
Colchicine may be utilized for many different purposes when
analyzing mitotic growth, and techniques may considerably differ.
For instance, in studies on the morphology of chromosomes or pseudo-
spindle in arrested metaphases, quantitative data, except those about
Techniques of Colchicine Treatment 375
effective colchicine concentration, may not be of paramount impor-
tance. Tlie same may apply to some work where colchicine is mainly
a tool for increasing the "visibility" of cellular division. ^VHien the
topography of mitotic gro^^■th is the main purpose, several instances
of which have been given in Chapter 9, precise data about the mitotic
rate may not be important. On the contrary, when using colchicine
to assess the importance of cellular proliferation, either in complex
tissues or in tissue cultures, it is indispensable to understand the
complex action on the mitotic count. This point will be considered
further.
Special techniques for the production by colchicine of abnormal
gi'owth in embryos have been mentioned in Chapter 8. The experi-
mental creation of polyploid animals has been one aim of colchicine
research. The methods used and the results obtained merit some dis-
cussion, which Avill be found in the last paragraph of this chapter.
i6A.^-i: In vivo studies. Many methods have been utilized in the
study of c-mitosis in animal cells; they are all variants of two: viz.,
placing cells in contact ^vith colchicine solutions, or injecting these by
various routes into the cell or into the animal.
The intracellular injection is of great interest, for it was possible
to demonstrate by this procedure that some cells were resistant to
colchicine since the alkaloid did not penetrate into the cytoplasm.
Such experiments have been performed only on one unicellular.
Amoeba sphaeroiiiiclens. Mitotic division of this species is not affected
when it is grown in colchicine solutions. \'ery minute quantities of
a one per cent solution of the akaloid were introduced in the cyto-
plasm with a micropipette. Typical mitotic arrest, together with for-
mation of polyploid nuclei, restilted when the timing of the injection
was properly related to the mitotic cycle.--
Many cold-blooded animals, invertebrates, fish, amphibians, have
been studied after immersion in colchicine solutions. One important
pathway of absorption is through the branchiae. In such experiments,
care should be taken to avoid svmlight and to replace the colchicine
solution which may lose its activity through chemical changes.
Injection is often the easiest way to administer colchicine to pluri-
cellular animals. In the study of hematopoiesis in the chick, colchi-
cine was simply injected into the egg yolk through the shell. ^ In
adtdt animals, subcutaneous or intraperitoneal injections are theniost
frequently used. One most important point, if a quantitative study
of the number of mitoses is needed, is to inject all animals at the
same hour of the day, so as not to be disturbed by the diurnal varia-
tions of mitotic rate.^-'' This is also influenced by feeding the animals,
more precisely by the blood glucose level, and experimental animals
should be kept under standard and specified dietetic conditions. ^^
376 Colchicine
In mammals, and especially the small rodents, which have been
widely used for colchicine work, some tissues are most favorable for
the study of mitosis and the influence of colchicine and similar poi-
sons. The skin lends itself to repeated biopsies, for instance the ear
of the mouse, from which small fragments may be punched out at
hourly intervals.i-^- " However, the mitotic activity of the skin is low,
and counting is long and tedious, even after colchicine. The num-
ber of mitoses is increased little by mitotic arrest, probably because
under normal conditions they are of long duration, up to three hours.
The influence of the sexual cycle is considerable (Chapter 9, Fig. 9.6)
and must not be overlooked. i" The cornea may be studied by stain-
ing whole mounts and counting the number of mitoses per thousand
cells; this method has only been utilized in mammals by one group
of workers,is though it appears to offer many advantages over the
skin. Bone marrow and intestinal crypts are zones of maximal mitotic
growth in mammals. They both provide excellent material for study-
ing the action of colchicine. In bone marrow, comparative studies
may be made between the white-cell- and the red-cell-forming tissues.
In the intestine, quantitative estimation of mitotic growth is possible,^^
though the counting of mitoses may be difficult because of their rapid
destruction of pycnosis. The intestinal mitoses have been one of the
best tools for the study of mitotic poisons at Brussels. Contrary to
the mitoses of lymphoid tissue, which are strongly affected by hor-
monal influences such as those of the "alarm-reaction" or pituitary-
adrenal stimulation,^! the intestine provides a tissue with uniform
growth,"*'^ not affected by the adrenal cortical hormones.-^ Intestinal
fragments should always be taken from the same location, for the
mitotic activity is greater in the duodenum, and decreases gradually
towards the large intestine, where few mitoses are seen. The gastric
mucosa of the mouse has also been proposed;**' ^^ it offers an interest-
icing comparison between squamous-celled and glandular epithehum
in a single organ. The regenerating liver is a favorable material in
rats, and quantitative estimations of mitotic growth are possible. ^^
However, it has been shown that the repartition of mitoses was not
uniform throughout the remaining liver.^^
Local applications of colchicine have been most useful in the study
of c-mitosis and regeneration in amphibians." The study of recovery
after a prolonged colchicine impregnation (five days) has been dis-
cussed in Chapter 2 (cf. Fig. 2.7) .^^ The inhibition of regeneration
of the tail of Xenopus larvae has been illustrated in Chapter 9; the
technique involved a local application of an aqueous solution of
colchicine to the amputated tail.44 Local apjjlication has also been
found useful in studies on the mitotic activity of genital tissues in
rodents-^s and of the human vagina before removal of a fragment by
Techniques of Colchicine Treatment 377
biopsy;^^' ^^ this is one ot the methods for treating human tumors
with the alkaloid, prepared in a vaseline-lanoline paste (Chapter
10) .If'- ^ Local applications of colchicine-im])res;nated agar cut into
small fragments have also proved useful in studying the origin of col-
chicine malformations in eggs;^" this technique does not seem to have
received the attention it deserves.
Another method by which colchicine is brought into direct con-
tact with the cells is the use of the so-called "ascites-tumors" in mice.
These are neoplasms freely growing in fluid gathered in the ab-
dominal cavity. Colchicine is injected intraperitoneally, and re-
peated observations of the cells are possible by removing a small
amount of the ascites fluid. *-
j6A.^-2: In vitro techniques. For many studies, it is preferable
to keep precise amounts of colchicine in contact with the cells which
are studied. This enables the results not to be disturbed by general
toxicity reactions and other pharmacological side-effects of colchicine
(Chapter 7) . More concentrated solutions may be tested, which, in-
jected to ^vhole animals, would have brought death through nervous
and respiratory paralysis. These techniques apply especially to warm-
blooded animals.
In invertebrates, however, some remarkable results, discussed in
Chapters 2 and 3, have been obtained by the study at 38°C. of the
isolated nervous system of the grasshopper, Chortophaga viridifas-
ciata De Geer. Embryos, at an age equivalent to 14 days' development
at 26°C., are removed from the egg in artificial culture medium. The
maxillary and thoracic appendages, the head, and the posterior half
of the abdomen are discarded, and the embryo is mounted ^vith the
ventral nervous system close to a cover slip, which is sealed. These
hanging-drop preparations may be observed for several hours under
oil-immersion objectives'^' ^i (cf. Chapter 3, and Fig. 3.1). This has
proved to be one of the most interesting techniques for the study of
the spindle destruction by colchicine and of the mitotic cycle. '^ Iso-
lated eggs of invertebrates, for instance Arbacia,^ should also be men-
tioned here, although the techniques do not differ from those used in
experimental embryology (cf. Fig. 3.3 and Chapter 8) .
In mammals, two tissues have provided excellent material for the
study of mitosis in x'itro. Fragments of the ear of mice may be in-
cubated in AVarburg flasks, and the action of various chemicals on
mitotic growth studied on the epithelium, the mitoses of ^vliidi ])er-
sist for several hovns, provided that glucose is added to the medium. '^
Bone marrow is readily available in many mammals, including man,
and its mitoses may most simply be observed in cover-slip prepara-
tions at 37°C. Glucose does not appear to be as necessary as for
epidermal cells.- This technique has provided most useful data on
378 Colchicine
the physiology of cellular division in bone marrow and on the actions
of various substances on rate of cell multiplication (Chapter 9) . The
cells, which are suspended in homologous serum, are able to divide
regularly for more than 24 hours after explantation.-
A method for iii vitro cultivation of immature rat ovaries has been
described" and should be of great interest for endocrinological re-
search.
Colchicine has been used with the main techniques of tissue cul-
ture, especially with hanging-drop preparations, wdiich enable a con-
tinuous observation of growth. i- Some estimation of the quantitative
amount of newly formed cells may be made by planimetric measure-
ment of the whole culture, but the influence of cell migration must
not be neglected. 1- Tissue cultures are especially favorable for cine-
micrographic methods. i- A very thorough study of the action of col-
chicine on the rate of mitotic growth and on the repartition of the
various types of abnormal or arrested mitoses has been made possible
by this technique!-' ■>- (Chapter 9, Fig. 9.1). Tissue cultures are also
most useful for comparing normal and neoplastic cells,^! for the
study of synergists or antagonists of colchicine, and for testing other
mitotic poisons42 ^^f. Chapter 17) . It should, however, be mentioned
that cultures of chick fibroblasts will not always behave like fibro-
blasts from mammals.^^ For the study of colchicine derivatives or
other spindle poisons, cultures of various types of cells from different
animals should be compared.
i6A.^-^: Mitotic counts. When colchicine is used as a tool for
studying growth (Chapters 9 and 10) , when the problem of mitotic
stimulation by colchicine is considered (Chapter 9) , or when sub-
stances acting synergically or as antagonists to the alkaloid are studied
(Chapter 17), a precise estimation of the number of mitoses in con-
trols and at various intervals after mitotic arrest is indispensable.
Some of the methods outlined in the preceding subsection provide
excellent material for counting cell divisions, but even with tissue
cultures, the problem may be complicated because only the periphery
of the explanted fragment grows rapidly. Precise counts of the total
number of cells in mitosis are possible both with the ear-clip tech-
nique^^' !■* and the methods of bone-marrow explantation.^ In more
complex tissues a reliable standard may be difficult to find. For in-
stance, many authors define the "mitotic index" as the number of
mitoses found in a given area, i.e., so many microscopic fields, of
tissue. This is a good method when dealing with uniform and fairly
simple tissues, for example, the regenerating liver,ii but not when
complex tissues are considered. In the small intestine of mammals,
for instance, it is preferable to count the number of mitoses per
Techniques of Colchicine Treatment 379
hundred glandular crypts. This method has been widely used by the
junior author in studies of mitotic poisoning.-^
Many data obscuring the problem of possible mitotic stimulation
by colchicine result from the difficulty of comparing tissues before
and after the action of the alkaloid. To cite one instance, the great
increase in mitotic activity in the crop-sac of pigeons injected with
prolactine and colchicine has l)een mentioned (Chapter 9) . Is it
possible to compare quantitati\ely the mitotic counts in this tissue?
From the figures which ha\'e been published one may conclude that
it is not, for after prolactine and colchicine, there is not the same
number of cells in a given area of tissue as in the same area of normal
epithelium or of prolactine-thickened crop-sac.^*' A quantitative re-
sult could only be correct if it were possible to count a very large
number of cells, and not only the mitoses in a given area. Such
counts are not often reported in this type of work (Chapter 9) .
Another error is that of injecting a hormone at a too short interval
before colchicine. Theoretically, the mitotic index should remain
constant; that is to sav, the niunbers of cells entering prophase should
not vary during the period of action of colchicine. It has been
pointed out that this is not often so with hormone-stimulated
growth. 1^' 23 Considerable errors may result from hasty interpretations
of the significance of mitotic increases.
Any quantitative work supposes also that the exact number of
cells arrested at metaphase by colchicine is known. In warm-blooded
animals, and apparently also in amphibia,^' this is never so, even
with large doses. Increasing the dosage of alkaloid is never a good
solution either, for it increases secondary, nonspecific toxic reactions
and the percentage of destroyed arrested mitoses, and may also depress
the number of prophases. It is often very difficult, especially in mam-
mals, to know exactly how many metaphases with clumped chromo-
somes undergo degeneration, for this is rapid, and the nucleus breaks
down to many small fragments. The data about the duration of
c-mitosis in animals are scarce and widely divergent, as pointed out
in Chapter 2.^^ It is also necessary, when planning an experiment
with colchicine acting as a tool, to know how long after an injection
of the alkaloid the animal should be killed. Many factors complicate
this estimation: There may be a period of latency like that observed
in tissue cultures (Fig. 9.1) ;^- some anaphases may persist even with
large doses. Recovery starts after an interval which is not always
known. In some tissues this may be rather short, and in the study of
epidermal mitosis it is recommended to kill the animals six hours
after colchicine. This duration appears favorable for many experi-
ments on mammals, but it is obviously too short in cold-blooded
380 Colchicine
animals. Here again, temperature may play a great part, but no
quantitative work relating temperature to the duration of action of
colchicine exists. In tissue cultures, colchicine may be left to act
much longer, and 24 hours is often mentioned in work with bone
marrow. -
This brings in another problem which we have not yet dealt
with: the duration of interphase. It is evident that, if colchicine were
acting longer than a normal interphase, no more new prophases
would be available and the mitotic index would cease to rise. While
most data on grasshoppers, i'' tissue cultures,^- and complex tissues
indicate that interphase is far longer than mitosis, precise information
is often lacking. It has been suggested that colchicine itself may pro-
vide a means for measuring the duration of interphase. ^'^ If new pro-
phases were indefinitely provided by the tissues, i.e., if interphase
diuation did not interfere with mitotic counts, the number of
arrested mitoses would increase until all the cells would be in a con-
dition of c-mitosis. This is never observed, and even in the fastest
growing tissues never many more than 50 per cent of the cells show
c-mitoses. This is because after a certain time no more interphasic
cells are ready for prophase. On the curve of the numbers of mitoses
in function of time, the time which elapses between the beginning
of mitotic arrest and the leveling of the number of mitoses is related to
the duration of interphase. Theoretically, under ideal conditions, it
is equal to interphase. ^'^ This is of interest for workers handling
colchicine and certainly deserves further study. In the preceding
chapters, enough has been said about the comi:)lexities of c-mitosis
to prevent conclusions to be drawn hastily. One fact remains true:
In colchicine experiments, the duration of the action of the alkaloid
should be much shorter than the interphasic duration of the cells
which are studied.
Considering the great variations in mitotic duration which are
mentioned in the literature (from about 30 minutes to three hours
in the mouse) , our ignorance about the duration of interphase, the
difficulties of accurately counting mitoses, and the complexities of
colchicine's pharmacology, it is evident that quantitative conclusions
are only possible in a few instances. 1 he advantages of tissue cultures
are obvious.
16A.4: Polyploidy
Polyploid animals have been produced experimentally, -•■^- -^- ^ but
colchicine has not yet proved very effective in doubling the chromo-
some number. This is prol^ably only a question of technique, though
cellular destruction, nondivision of the centromeres, and restitution
during early development (Chapter 8) may be factors which prevent
Techniques of Colchicine Treatment 38 J
colchicine from acting on animal cells as in j)lants. I7nder the head-
ing of polyploidy should be considered only doubling or multiplying
by 2, 3, 4, . . . the numbers of chromosomes (cf. Chapter 11). Most
results obtained with colchicine are related to trijjloidy.
Any experimental change in the numbers of chromosomes should
be checked by chromosome counts. This point may seem quite obvi-
ous, but in early reports of "polyploidy" in mammals, changes in
cell volume alone were mentioned. It is known from previous experi-
mental data, mainly on amphibians,-' that the size of the polyploid
animals remains the same, or is even smaller, than the diploid size,
though individual cells become larger and larger with increasing
numbers of chromosomes. However, to deduce from measurement of
cell size alone the degree of -ploidy cannot be accepted as a valid
scientific method.'"' Considerable error may be involved; for instance,
making smears of red blood cells and comparing the diameters is
incorrect and cannot bring evidence of triploidy, as has been
claimed. •'^2' ^^ The red blood cell volumes would be a better choice,
but these were not measured, either by indirect calculation from the
diameter, or by measuring the packed red blood cell volume in a
hematocrit tube. Some "polyploid" mammals have been claimed to
be larger and to grow faster than the euploid ones.^^-' •''•"' This is in
contradiction with all data on amphibia, and as the numbers of
colchicine-polyploid animals which have been studied is very small,
and as they were not of pure breed, the data lack the necessary
statistical significance.*''
In the work on the unicelhdar Amoeba sphneronucle'iis, poly-
ploidy was assessed without counting the chromosomes, which are
very numerous and small. Here, the action of the alkaloid injected
intracellularly at metaphase could be followed under the microscope.
A single nucleus resulted from the arrested metaphase, and its volume
was roughly double that of normal amoebae. Checks were made
possible by grafting these abnormal nuclei into normal amoebae, and
vice versa. ^^ The cellular voliune became proportional to the size
of the nucleus. However, even in these experiments, mitotic abnormal-
ities were observed in the "polyploid" species, and it is not possible
to assert with certainty that a true doubling of the chromosome num-
ber and not aneuploidy had resulted from the injections of colchi-
cine. Claims of colchicine-induced polyploidy in frogs, rabbits, and
pigs have been repeatedly published. ""^2' ^^' ^^ The females were artifi-
cially fertilized by sperm mixed with colchicine. The alkaloid is sup-
posed to reach the e^g at the time of the second maturation division,
which ^voidd be arrested. The egg woidd thus remain dij:)loid, and
after fertilization with haploid sperm, triploid animals would be
expected. Monstrous development in frogs treated similarly had pre-
382 Colchicine
viously been reported in a short note.-" A frog sperm suspension with
2.6 X 10~* M colchicine was most toxic to eggs, and only 8 per cent
of these developed normally. It has been claimed that this did not
result from a direct action of the alkaloid on the eggs at fertilization.^^
The production of triploidy deserves close attention."*-- •^•'' ^' A sur-
prising fact is that the rabbits and pigs were considered to have an
abnormal growth with increased Aveight and size. In the first papers,
triploidy was deduced from the increased size of red blood cells and
spermatocyte heads. The accuracy and significance of these measures
have been severely criticized.** However, chromosome counts were
later published. In frogs, tetraploid, but also diploid, triploid, and
pentaploid cells Avere found.^e In rabbits, a considerable variation of
chromosome number was found. While the diploid one was the most
frequent, it is clear from the results published that the animals were
heteroploid.46 The same applies to the single triploid pig. While in
a preliminary note about this animal it was claimed that the mitotic
count in the testicle was "certainly over 45 and not more than 48,"
and that the animal resulted from the fusion of a spermatozoon with
15 chromosomes ("Old Swedish" race) and an egg with a doubled
chromosome complement of 32 (mixed race) , the results of a later
publication are by no means so clear.'***- ^^
It is already evident that in producing artificial "polyploids" one
should deal with animals with a well-known number of chromosomes
and should not cross two varieties with different and imperfectly
knoA\n numbers.3 The detailed stud\ of the testicular mitoses of the
abnormal pig shows chromosome numbers varying between 19 and 51,
Avith an "average" of 49. It was assumed that the probable number of
49 was correct.'*^ This should result from the fecundation of a diploid
egg w:ith 2 X 15 chromosomes by a spermatozoon with 19 chromo-
somes. EA-idence for this is given from the chromosome count of a
normal l^rother of this pig. Avhich had 34 (19+15) chromosomes.
HoAveAcr, one of the authors mentions as an interesting point that
ancuploid cells could be ol>scrved in the so-called triploid.^'*
From these descriptions it is apparent. (1) that colchicine may
have altered the second meiotic division of the egg, but that only in-
direct evidence is produced, and that the concentration present Avhen
the sperm reached the eggs is unknoAvn: (2) that no polyploid ani-
mals have been produced by colchicine, Avhile other methods have
proved quite efficient in amphibia; (3) that triploidy is not proven,
and that aneuploidy is possible.
It remains possible that colchicine may prove as useful in poly-
ploidy breeding in animals as in plants, but the premature claims of
the Swedish authors do not rest on firm ground. The technique of
insemination Avith colchicine is open to criticism, and even more, the
Techniques of Colchicine Treatment 383
absence of repeated chromosome counts in various organs. It ap-
pears surprising that the bone marrow, the skin, or the cornea was
not chosen for chromosome counts and that so many pubHcations
and claims rest on such meager technical data.
B. Techniques in Plants
16B.1: Solutions Used
Compared with warm-blooded animals, cells of plants tolerate
relatively strong concentrations of colchicine. The substance diffuses
rapidly through plant tissues and may be translocated in the plant
through the vascular system. Active concentrations remain in con-
tact with the cells for a longer time than is recorded by the total
exposure to the drug. Apparently tlie effects of colchicine are re-
tained for a long time. Penetrability, its low toxicity, and retention
in the cell, along with the complete recovery through reversibility by
the cell, are unique qualities of colchicine for doubling the number of
chromosomes in plants.
Successful procedures have favored stronger solutions applied for
shorter periods over the dilute ones applied during long exposure.^- ^•
9. 11. 13, 1.5, IS, 21, 22, 24, 2.5, 26, 27, .30, 17, 3.3 Schedulcs with specific concen-
trations advocated and exposure recommendations are given in the
papers. If a universal c(jnccntration were selected for treating plants,
the strength would be 0.2 jjer cent acjueous solution. This con-
centration, or one close to it, has been used more frequently than
any other. Wide ranges are effective, but there is an optimum which
produces the highest percentages of changed cells. Generally, one
gram of colchicine is dissolved in 500 ml. water. The length of time
for keej)ing cells in contact with the drug varies from 24 to 96 hours.
In addition to concentration and exposure, the growing conditions
of a particular tissue are important. Cells must be in a high state of
cell division for maximum effective use of colchicine. i-
A study of the action of colchicine iqoon mitosis requires the use
of wide ranges in concentration in order to obtain mininuun, opti-
mum, and maximum effects. The objectives are somewhat different
from using the drug as a tool for making polyploids.
The carrier used for colchicine in treating seed plants may be
water, emulsions, agar, or lanolin. Whetting agents have been used
effectively. Sometimes the addition of glycerine has been recom-
mended.'^ The enudsions are sprayed on to the plants or lanolin
pastes applied, as suitable. Aqueous solutions are applied by drop-
384 Colchicine
ping, brushing, or total immersion oi the phmt in the sokition. The
latter method has been used efEectively for root systems and seedlings.
16B.2: Seed and Seedlings
One of the most convenient ways to treat plants uses the ger-
minating seed placed in solution. The seed may be presoaked or
placed directly into the colchicine. Different lots may be removed
after given intervals. Then some exposures will not cause doubling;
others will prove lethal; and other lots will be at the optimum ex-
posure. In this way the most effective concentration and time of ex-
posure can be determined by the survival of treated seeds trans-
planted alter treatment. Overexposures kill the seedlings, and under-
exposure does not lead to new polyploids.
Plants, when young, are well adapted to treatment. If only the
plumule is treated, the roots remain unharmed, and plant growth is
not so totally harmed. The growing point may be immersed in col-
chicine, or the solution applied to the plant by brush treatment. By
sowing seeds in rows, and treating each row with different exposures,
the differences between too much treatment and too little will show at
the time seedlings are ready for transplanting. Selections for probable
polyploids can be made at this time.
Seedlings of monocotyledonous plants are difficult to treat with
colchicine. Special methods'- ^s- 1^. s had to be devised for these cases.
Admitting the drug to the growing tissues that lie beneath a coleop-
tile sheath has been the chief problem.
16B.3: Root Systems and Special Structures
Soaking entire root systems has been effective for many species of
the Gramineae.i''- ^^' -^ An alternate period of soaking in colchicine
12 hours and in water 12 hours has ^\•orked out with good success.
The number of exposures depends upon the particular experiment,
material, and concentration. Reference to specific schedules in the
literature shows what directions have been most successlul. The
technique was developed for sterile species hybrids of grasses and
specifically for wheat-rye sterile hybrids to make fertile amphiploids.^"*
Scales of liliaceous plants,!^ bulbs, corms, and rhizomes represent
structures that call for modifications in method. Usually a large mass
of meristematic tissues arc present, and unless the whole group of
cells responds, the production of mixoploids and chimeras becomes
an inevitable result.
Expanding buds of woody stems require proper timing in order
to introduce colchicine when the cells are in their peak of division.
In this way mature woody plants can be treated when dormancy is
Techniques of Colchicine Treatment 385
being broken. By grafting the changed sectors, the new polyploids
can be propagated.^ Periclinal and sectorial chimeras are frequently
pioduced in treating Avoody species. These chimeras may be propa-
gated for generations through grafting. Their role in horticulture
is being more fully appreciated from a breeding point of view.
16B.4: Special Techniques for Studying the Action of Colchicine
Pollen grains that can be used for artificial culturing work serve
well for testing the action of colchicine upon mitosis and growth
processes. The specific morphology of somatic chromosomes were
studied in Polygonatum, and discovery of natural polyploidy was
made directly from these observations. Another valuable feature is
the small amoimt of chemical that can be tested. Other mitotic
poisons soluble in water can be adapted for testing ^vith the pollen
tube methods.
Several modifications have been made in pollen tube studies since
the original paper was published in 1931 by Trankowsky. The par-
ticular conditions for an experiment must be worked out and fol-
lowed thereafter. In pollen tube studies the detail is not as im-
portant as a routine which, once successful for an operation, is always
done in that way.*'
Mitosis in the cells of staminal hairs of Tradescantia can be studied
in vivo. Single cells may be followed through the stages of mitosis.
When such cells are growing in agar containing colchicine, the total
time required for a c-mitosis can be measured. Special chambers for
keeping the cells alive for long periods were designed for these studies.
While the general technique for observing mitosis in the living cell
of Tradescantia has been known for many years, the adaptations for
experimental cytology are new.'^-^
Colchicine was used so effectively with root tips of Allium ccpa
that the test has become known as a method for experimental work,
the Allium cepa test. Threshold concentrations in relation to solu-
bility are some of the contributions from this method. Standardiza-
tion of procedures have been devised so that a variety of chemicals
can be measured for properties of mitotic inhibition or chromosomal
breakage. The time for exposure, for recovery, and for fixation after
treatment are important parts of the routine method.
Allowing roots to germinate when suspended over a test solution
is a modification of the Allium cepa method, and more specifically
known as the onion root germination test.
Tissue cidtures for excised roots, virus tumor tissue, proliferating
cells, and regenerative tissues generally may be adapted for the use of
colchicine. In vitro and in vivo studies are made by these methods.
386 Colchicine
16B.5: Chromosome Studies
The pollen mother cells stained by acetocarmine are universally
a most important sovnxe for studying chromosomes in plants. The
procedure for determining the number of chromosomes is rapid.
More important than deciding what the number might be, are the
pairing characteristics at meiotic metaphase, chiasmatal frequencies,
lagging of chromosomes at meiotic anaphase, configurations due to
translocations, and the irregularities of meiotic jMocesses generally.
These are the problems associated with polyploidy that must be
studied at the pollen mother cell stage.
Root tips are used for a check of the somatic numbers of chromo-
somes. Pretreatment of roots before fixation with chemicals that
arrest mitosis at metaphase facilitates the study.- Distributions of
chromosomes in an arrested metaphase are easier to count and com-
pare for size and morphology. i^- '*■ ^^- -
Leaf cells in division combined with acetocarmine and Feulgen
technics are another source for counting chromosomes in polyploids
and related diploids. The longer period of time during which leaf
cells provide material and the abundance and availability of ma-
terial are favored in this test.
Pollen tube cells that undergo mitosis in the tube rather than
inside the pollen grain can be treated with colchicine in sucrose-agar
media. Scattered chromosomes are easily counted, and the morphology
of somatic chromosomes in haploid sets can be measured. ^^
Causes of sterility in pollen and pollen mother cells may not be
the same when viewed in the embryo-sac stages, or among megaspore
mother cells. Frequently the polyploid may be pollen-sterile and
female-fertile, or vice versa. Transmission of certain extra chromo-
somes occurs only through the female and not through the male
gametophyte. Cytological methods to measure chromosomal varia-
tions in the female gametophyte are long and difficult procedures,
but they are important to a full knowledge of why some strains are
lower in fertility than others.
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Techniques of Colchicine Treatment 387
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10. Broderson, H. Mitosegifte und ionisicrende Strahlung. Strahlenther. 75:196-
254. 1943.
11. Brues, a. M. The effect of colchicine on regenerating liver. Jour. Physiol.
86:63-64. 1936.
12. Bucher, O. Zur Kenntnis der Mitose. VI. Der Einfluss von Colchicin und
Trypaflavin auf den Wachstumrvthmus tuid auf die Zellteilung in Fibrocyten-
kulturen. Z. Zellforsch. 29:283-322. 1939. Le role de la culture des tissus
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10:245-70. 1951.
13. BuLLouGH, W. S. Mitotic activity in the adult female mouse, Mus niiisciilus L.
A study of its relation to the oestrus c\cle in normal and abnormal conditions.
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to waking and sleeping. Ihid. 135:212-33. 191S. 1 he effects of experi-
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adult male mouse, Mus musculus L. Ibid. 135:233-42. 1948.
14. , and Eisa, E. a. The effect of a graded series of restricted diets on epi-
dermal mitotic activity in the mouse. Brit. Jour. Clancer. 4:321-28. 1950.
15. , and Johnson, M. A. simple technicjue for maintaining mannnalian
epidermal mitoses in vitro. Exp. Cell Res. 2:415-53. 1951.
16. BiiRCKHART, E. Z. A study of the effects of androgenic substances in the rat
with the aid of colchicine. Doctoral Dissertation. The University of Chicago
Library. Chicago, 111. 1940. .\ study of the early effect of androgenous sub-
stances in the rat by the aid of colcliicine. Jour. Exp. Zool. 89:135-66. 1942.
17. Bureau, V., and Vilter, \. Action de la colchicine <;tudiee sin les cellules
epith<;liales de lAxolotl. C. R. Soc. Biol. Paris. 132-553-58. 1939.
18. Buschke, W., Friedenwald, J. S., and Fleischmann, W. Studies on the mitotic
activity of the corneal epithelium. Methods. The effects of colchicine, ether,
cocaine, and ephedrine. Bull. Johns Ho]ikins Hosp. 73:143-68. 1913.
19. Orlson, J. G. Effects of X-radiation on giasshopper chromosomes. Cold
Spring Harbor Symp. Quant. Biol. 9:104-12.' 1941.
20. Chanc;, M. C. Artificial product ion of monstrosities in the rabbit. Nature.
151:150. 1944.
21. Ceearkin, p. a. The effect of colchicine on noiiual and neoplastic tissues in
mice. Jour. Path. Bact. 44:469. 1937.
22. Comandon, J., AND DE FoNBRUNE, P. Action de la colchicine sur Atnoehu
sphaeronncleus. 01)tenlion de varic-tcs gcantes. C. R. Soc. Biol. Paris. 136:
410-11. 1942. £tude \c)lvmctriciue comparative d'Anioeba sphacronucleus et de
deux varietes obteinies par Taction de la colchicine. Ibid. 136:123. 1942.
Anomalies de la division observces a partir de noyaux atypicjues, che/. Amoeba
sphacronucleus et ses varietes colchicinicpies. Ibid. 136:460-61. 1942. Greffes
nucleaires croisees entre Amoeba sphaeronucleus et I'une de ses Narit'tes col-
chicinicpies. Ibid. 136:746-47. 1942. Modifications hc-reditaires de volume
provoquees par lechange du noyau entre Amorl)n sphaeronurleits et ses
varietes colchicinicjues. Ibid. 136:747-48. 1942.
388 Colchicine
23. Di'STiN, A. P. Recheiches sin le mode d'actioii des poisons stathmocinetiques.
Action de hi colchicine siir riiteiiis de lapine impnbere sensibilisee par injection
preahi hie dill ine de femnie enceinte. Arch. liioL 54:111-87. 1943.
24. Dlistin, p., Jr. The action of mitotic poisons on normal and pathological
blood cell formation. Le Sang. 21:297-330. 1950.
25. Fankhauser, G. Indnction of polyploidy in animals by extremes of tempera-
ture. Biol. Svmp. 6:21-35. 1942. The effect of changes in chromosome number
on amphibian development. Quart. Rev. liiol. 20:20-78. 1945.
26. Ferguson, F. C. Colchicine. I. General Pharmacology. Jour. Pharmacol. Exp.
Ther. 106:2(51-70. 1952.
27. FiSHBERG, M., AND Beattv, R. A. Heteioploidy in Mammals. II. Induction of
triploidy in pre-implantation mouse eggs. Jour. Genet. 50:455-70. 1952.
28. Freud, j., and Uyldert, I. E. The influence of colchicine upon mitoses in the
intestine in normal and adienalcclomized rats. Acta Brev. Neerl. Physiol.
8:16-18. 1938.
29. FiJHNER. H. Die Colchicingruppe. In Heffters Handbuch Exp. Pharmakologie.
2:493-507. 1920.
30. Gabrike, M. E. The effect of local applications of colchicine on Leghorn and
pohdactylous chick embryos. Jour. Exp. Zool. 101:339-50. 1946. Produc-
tion of strophosomy in the chick embrvo 1)V local ajiplications of colchicine.
Jour. Exp. Zool. 101:351-54. 1946.
31. Gaulden, M., and Carlson, J. Cytological effects of colchicine on the grass-
hopper neuroljlast m t'itro, with special reference to the origin of the spindle.
Exp. Cell Res. 2:416-33. 1951.
32. Haggqvist, G. Pol\ploidy in frogs, induced by colchicine. Proc. Kon. Nederl.
Akad. Wetensch. 51:3-12. 1948. Induktion triploider Schweine durch Kolchi-
zin. Verb. Anat. Ges. 49:62-65. 1951. Uber polyploide Saugetiere. Verb. Anat.
Ges. 48:39-42. 1951.
33. , AND Bane, A. Polyploidy in rabbits, induced by colchicine. Nature.
165:841-43. 19.50. Chemical induction of polyploid breeds of mammals. Kungl.
Syenska Vetenskapakad. Handl. IV Ser. 1:1-11. 1950. Kolchizininduzierte
Heterploidie beim Schuein. Kungl. Svenska Vetenskapakad. Handl. IV Ser.
3. Pp. 14. 1951.
34. Hall, T. S. Abnormalities of amphibian deyclopment following exposure of
sperm to colchicine. Proc. Soc. Exp. Biol, and Med. 62:193-95. 1946.
35. Hausemann, W., and Kolmer, W. Uber die Einwirkung kolloidaler Gifte auf
Paiamacien. Biochem. Z. 3:50.3-7. 1907.
36. HoRowrrz, R. M., and Ullvot, G. E. Desmethylcolchicine, a constituent of
U.S.P. colchicine. Science. 115:216. 1952.
37. Jacobj. C. Pharmakologische Untersuching fiber das Colchicumgift. Arch.
Exp. Path. 27:119-57. 1890.
38. Jaun, I', liuluktion \erschiedenei I'ohploidiegrade bei Rana temporaria
mit Hilfe von Rokhi/in und Sulfanilamiil. /. Mikr.-anat. Foisch. 58:36-99.
1952.
39. JouRNOUD, R. Recherches sur iin element pen coniiii de Ihcmatopoiese: la
duree des mitoses des cellules nncloi'des. Le Sang. 24:355-63. 1953.
40. Leblond, C. p., and Allen, E. Emphasis of the growth elfect of prolactin on
the crop gland of the pigeon by arrest of mitoses with colchicine. Endocri-
nology. 21:455-60. 1937.
41. '-. AND Si-XAL, G. Action de la colchicine sur la surrcnale et les organes
hmphatiques. C. R. Soc. Biol. Paris. 128:99.5-97. 1938.
42. Lettre, H. iJber Mitosegifte. Eigebn. Plnsiol. 46:379-452. 1950.
43. Lns, F. Recherches sur les reactions ct lesions cellulaires provocjuces par la
colchicine. Arch. Int. Med. Exp. 11:811-901. 1936.
44. Lusc;her, M. Die Hemmimg tier Regeneration durch Colchicin beim Schwanz
der A'e/io/ji/v-larve und ihie entwicklungsphvsiologische Wirkungsanalyse.
Hel\. Physiol, et Pharm. Acta. 4:465-94. 1946.
45. Malinskv, J.. AND Lang, B. Hvpcrplasie du foie de rat apres hepatectomie
partielle et influence des corps colchicines sur ceUe-ci. C. R. .Soc. Biol. Paris.
145:609-12. 1951.
Techniques of Colchicine Treatment 389
-\(\. Mii.AMiFR. ^■. (liiomosome l)clia\i<)Ui of a tiiploid adiill ral)l)ii, a^ ])roclu(ed
h\ Ha^i^ip ist and Banc after colchicine nealniciil. Hcreditas. ,'}(): 3;5r)-4 1 . 1950.
Polyploidy after (okhitine treatment of pigs. Hereditas. 37:288-89. 1951.
47. Osf.oOD, E. E. The culture of human marrow as an aid in the exaluation of
therapeutic agents. Join. Lab. and Clin. Med. 24:954-62. 1939.
IS. I'ARMKNrnR, R. Personal communication.
49. Peikrs. I. T. A cytological stud\ of mitosis in the cornea of T)iturus vnidcscens
during recovery after colchicine iieatment. Jour. Kxp. /.ool. 103:3.3-()0. 1946.
50. PuNDEL, M. P. Etude des reactions \aginales hormonales che/ la femme par la
mcthode colchicinique. Ann. Endocrin. 2:659-64. 1950.
51. ,S.A.\T.\vv, F., AND Reichstein. \ . Isolieruug neuer Stofte aus den Samen der
Herbstzeitlose, Colchiciun nutuimiale L. Helv. Chim. Acta. 33:1606-27. 1950.
52. , Lang, B., and Maejnsk^. ]. L'action mitoticjue et la toxicitc- des nou-
velles substances isolees du co]chi(|uc. Arch. Int. I'harmacodvn. 84:257-68.
1950.
53. Sentein, p. La degeneresence nuclc!'aire apres stathmocinese. (;. R. Soc. Biol.
Paris. 139:585-87. 1945.
54. Shorr, E., and Cohen, E. J. Lse of colchicine in detecting hoinional ellects on
\aginal epitheliiuii of menstruating and castrate women. Proc. Soc. Enj}. Biol,
and Med. 46:330-35. 1941. '
55. Steinegger, E., and Levan, A. The cytological effect of chloroform and colchi-
cine on Alliiun. Hereditas. 33:515-25. 1947.
56. Tier, H., Schauman. A., and Sindfee, B. Mitotic ratio and colchicine sensiiixily
of the stomach epithelium of the white rat. .\cta .Anat. 16:233-44. 1952.
57. ViLTER, V. Inibition of colchicinique de la mitose chez les mammiferes C. R.
Soc. Biol. Paris. 138:605-6. 1944.
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the female mouse to the local application of colchicine. Yale Jour. Biol, and
Med. 13:841-46.
REFERENCES — SECTION B
1. Belling, J. The iron-aceto carmine method of hxing and staining ciiromosomes.
Biol. Bull. 50:160-62. 1926.
2. Bhadiri, p. Improved smear methods for rapid doidjle staining. Jour. Roy.
Micr. Soc. 60:3-7. 1940.
3. Brewbaker, L. Personal commimications. 1951.
4. Brown, M. Personal commimications. 1951.
5. Chase, S. Production of homo/\gous diploids of niai/e from nionoploids.
Agron. Jour. 44:263-67. 1952.
6. Conger, A. Personal communications. 1953.
7. CuA, L. A newlv devised colchicine method for inducing pol\pl()id% in rice.
Bot. Gaz. 112:327-29. 1951.
8. 1)ari,in(;ton, C, and LaCour, L. The handling of chromosomes. Ihe Mac-
millan Co. New York. 165 pp. 1942.
9. Dermen, H. Personal communications. 1948.
10. E1G.STI, O. Methods for growing pollen tubes for plnsiological and c\ tological
studies. Proc. Okla. Acad. Sci.' 20:45-47. 1940.
11. Emsweller, S. Personal commiuiications. 1950.
12. Fvee, J. The action and use of colchicine in the production ')f pohploid
plants. Imp. Bur. Plant Breeding and Genet. Pid)l. No. 57(). 1939.
13. KiHARA, H. Personal communications. 1948.
11. 1.aC;oi!R, L .Improvements in ])lant cvtological icchniciue. II. Koi. Re\ . 13:
216-40. 1947.
15. .Manlev, r. Colchicine techniciues. Hemerocallis .Soc. M)k. 195(»:l(i-I9. 1950.
16. Meyer, J. Modification of mitosis by chemicals. .Science. 108:2799. 1948.
17. Morgan, D., and Rai'PLETE, R. I win and triplet ])epper seedlings. .V study of
polyembryony in Capsicum frulfsrciis. Jour. Hered. 41:91-95. 1950.
18. MiiNTZiNG, A. Personal commimications. 1949.
19. Navashin, M., AND CiERASsiMov A, H. Production of polyploids i)\ admiiiistei ing
colchicine solution via roots. C. R. Dokl. Acad. Sci. URSS. 2():681-83. 1940.
390 Colchicine
20. Nebel. B., and Ruttle, M. The cvtological and genetical significance of col-
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Hort. Sci. 57:310-22. 1951.
22. Randolph, L. Personal communication. 1948.
23. Rasmlsson, J., AND Levan. a. Tetraploid sugar beets from colchicine treat-
ments. Hereditas. 25:97-102. 1939.
24. Rosen, G. Problems and methods in the production of tetraploids withm the
genus Beta. Socker. Handl. 5. Hafte 10:197-217. Landskrona, Sweden. 1949.
25. ScHNELL, L. The induction of polyploidy in J'iiua rosea. Amer. jour. Bot.
28:5s. 1941.
26. Sears, E. Personal communication. 1948.
27. Smith, H. Personal communication. 1951.
28. Smith, L. The aceto-carmine smear technique. Stain Tech. 22:17-31. 1947.
29. Stebbins, G. Personal communication. 1951.
30. Stewart, R. Personal communication. 1949.
31. Tjio, J., .A.ND Levan, A. The use of oxiquinoline in chromosome analysis. Anales
Estacion Exp. Aula Dei. Zaragoza, Spain. 2:21-64. 1950.
32. Wellensiek, S. Method for producing Triticales. Jour. Hered. 38:167-73. 1947.
33. Wada, B. Eine neue Methode zur Lebendbeobachtung der Mitose bei den
Tradescautia-Haa.ize\\cn. Cytologia. 13:139-145. 1943.
CHAPTER 17
Mechanism, of Colclucine' Mitosis
17.1: Introduction
While many activities of colchicine have been discussed in the
previous chapters, it is evident that this alkaloid would be known
merely as an effective treatment for gouty patients (Chapter 7) had
it not been for its remarkable property of destroying the spindles of
mitotic cells. The consequences of this, both in animal and botanical
work, have been described. As a polyploidizing agent alone, colchi-
cine has become of world-wide importance and has opened new vistas
in experimental agiiculture. The scope of the work which has been
published since 1934 is so great that all its aspects cannot be covered
in this book. More detailed information on some aspects of the
colchicine problems may be found in several review papers to which
the attention of the reader is directed. i-*- ^^^ -''• •''-• ^•■' '•"• ^'~- ^'^- ^^■
77, 81, 97, 102, 18, 111
Many still unsolved problems have been mentioned in the text,
and it would be useless to discuss again their various aspects. How-
ever, the main action of colchicine, as evidenced by microscopy and
by the production of polyploids, is in changing the properties of the
spindle. Other chemical or physical agents are also capable of de-
stroying the spindle and preventing mitosis from proceeding. The
uniqueness of colchicine appears with greater clarity when it is com-
pared with the other "spindle poisons." AV^hile no attempt will be
made to cover spindle poisoning, this great field of cellular pharma-
cology, it appears evident that the mechanisms of c-mitosis may be
better imderstood from the study of other agents altering mitosis like
colchicine. Many chemicals closely related to colchicine ha\e been
studied, and relations between their chemical structure and their
spindle, activity throw light on the possible action of colchicine.
ly.i-i: Historical. Spindle poisons were kno\vn long before col-
chicine, and the fact that none of them was so successful is in itself
[391]
392 Colchicine
a demonstration of the singularity of colchicine. The action of nar-
cotics on divisions of sea-urchin eggs was studied by Hertwig in
1887,^* two years before the discovery of c-mitosis by Pernice;^^ in-
activation of the spindle was conspicuous. Phenylurethane in "nar-
cotic" doses was later used in experimental work to study the in-
fluence of mitosis on the respiration;'--^ the latter was not modified
when the spindle was inactivated. In plants, Nemec''*' studied another
narcotic, chloral hydrate. Figure 17.1, which is from a later paper,9s
demonstrates how similar the arrested mitoses after chloral hydrate
are to c-mitosis. The induction of polyploid plants was, however,
never recorded, probably because of the too great toxicity of this
narcotic. This points to one of the principal qualities of colchicine
and explains most of its success in practical botanical work: its low
toxicity and high efficiency.^^
A classical monograph dealing with animal ceils was written by
Politzer,'"' who had done important work in the years 1920-1930.
Several basic dyes appear to influence the spindle, but Politzer's work
is mainly concerned with chromosome poisons, which act somewhat
similarly to the ionizing radiations (so-called "radiomimetic" drugs) ,
and he mentions only occasionally metaphase poisoning and spindle
destruction.
In 1929, in A. P. Dustin's laboratory, Piton"« demonstrated the
action of various arsenical derivatives on mitoses in mice. These ex-
periments were later extended to grafted tumors.-^ However, the
concept of t-mitosis did not yet exist, and observing the gradual in-
crease in the numbers of mitoses, it was thought that a mitotic
stimulation was taking place. Actually, it was only after the study
of colchicine that it was clearly realized that arsenicals were also
spindle poisons, and much later, that they also influenced jilant
mitosis. Another curious observation is that of Rosenfeld,"'* who
noted arrested metaphases in cells treated with ammonia.
On the other hand, it was demonstrated by Lewis'^^ that heat alone
could inactivate the spindle. Sax observed a similar behavior of plant
mitoses in Tradescatilia^*'^ This research opened a way for the suc-
cessful production of polyploid plants (cf. Chapter 11) and poly-
ploid vertebrates (cf. Chapter 16A) , but it was not linked to the
other observations of what came to be called c-mitosis.^" After the
discovery of colchicine, and mainly after the observation of its action
on plant cells, a host of new spindle poisons was described, and other
chemical and physical means of arresting metaphases were found.
None Avas more efficient than colchicine, with the exception of some
derivatives closely related to colchicine.
77.7-2 .- Colchicine and tlie spindle. Before discussing further
other mitotic poisons, it is imj^ortant to stress the peculiar properties
Mechanism of Colch'icine-Mitosis
393
of colchicine. These have been analyzed at length in Chapters 2, 3,
and 4, and only a short summary is necessary at this point. Colchi-
cine is a mitotic poison; that is to say, it belongs to the vast and
rajiidly increasing group of substances which act spccificallv on divid-
ing cells. In Chapter 7 many other actions of the alkaloid on "rest-
ing" (intermitotic) cells were mentioned, but these are limited to
Fig. 17.1 — Root tips of Vicia faba treated for three hours by a 1 per cent solution of
chloral hydrate and replaced for 24 hours in water. Pseudo-metaphases and pseudo-
anaphases. (After van Regemoorter,""' Fig. 1)
some specialized tissues and to some groups of animals. Effects on
cell-shape, apart from mitosis, have also been recorded in Chapter 4.
These arc most interesting for a proper understanding of the c-mitotic
effect, but are mainly side-effects, usually brought about with strong
concentrations of the alkaloid.
On the contrary, the sjjindle action is remarkably sjiecific, and
solutions of colchicine diluted to one part in one billion, may still
exhibit spindle poisoning: colchicine has Jiiij^h ncthnly. This is ex-
pressed as the inverse of the activity threshold. Colchicine is also of
great efjiciency; that is to say, it acts over a wide range of concentra-
394 Colchicine
tion. This is especially visible in plant cells, where the general toxic
reactions of strong closes described in animals (Chapter 7) are
avoided. No other spindle poison is at the same time so active and
so efficient, though some of the colchicine derivatives may poison
animal spindles at concentrations lower than colchicineJi- "• ^•^' »-• ^'^
The changes of the mitotic spindles under the action of colchicine
have been described at length in Chapter 3. Suffice it to recall here
that the fibrous and polarized spindle is very rapidly changed into
an amorphous "pseudo-spindle" or "hyaline globule," which is in-
capable of moving the chromosomes.-*"- ^^ Much evidence is at hand
to demonstrate that the action of the alkaloid is proportional to its
concentration and is totally reversible, two facts of great importance
in the interpretation at a molecular level of spindle inactivation.
Chromosome changes are usually only a consequence of the arrest of
mitosis, especially in warm-blooded animals. In plants, the continua-
tion of the normal chromosome-cycle in cells devoid of spindles is
the basis of colchicine polyploidy. Cytoplasmic changes have been
recorded in plants and animals, especially a decrease in the general
viscosity, or rigidity, as evidenced by centrifugation.^s This may be
a consequence, and not the cause, of spindle inactivation.
Most of the other cellular changes are indirect consequences of
the spindle inhibition. Short and thick chromosomes are frequently
met in arrested metaphases. In plant cells, the cycle of chromosome
reduplication is not disturbed by the alkaloid, while in animals, only
a few instances of polyploid cells resulting from the multiplication
of chromosomes in colchicine-treated cells have been recorded. Here,
the prolongation of metaphase leads often to degenerative nuclear
changes. Alodifications in the shape of cells and in the growth of cell
membranes have been recorded (cf. Chapter 4) . These involve fi-
brous proteins, and may be of a similar nature to the spindle changes.
Considering the many data that have been gathered, it can be
stated here that colchicine appears to be one of the most specific and
least toxic of all the spindle poisons. Hence, any work which helps
to solve the problem of spindle inactivation by this complex mole-
cule may throw more light on the mechanism of cell division and
on the physiology of the peculiar fibrous protein which constitutes
the sjMndle. The importance of this cannot be underestimated, for
all cellular growth in nucleated cells involves the separation of the
two groups of chromosomes by the fibrous strands of the spindle.*
* Whether similar mechanisms exist in bacteria is still open to discussion,
though nuclei ha\e been recognized bv many authors, and at least one group has
tentatively identified a mitotic spindle.-* It may be that tiie plurinucleated bac-
terial forms which arise under the influence of some antibiotics, e.g. penicilhn, are
true polyploid cells. Some antibiotics have been shown to be spindle poisons ni
warm-blooded animals,^ and future work may lead to the extension of the concept
of mitotic poisoning to microorganisms.
Mechanism of Colchicine-Mitosis 395
ij.i-y. Materials and inelhods. W^hile ihe problems ot colchicine
technique have been reviewed in C:hapter IC), it is necessary to say
something more about this subject in introducing a chapter on
s])iiidk- poisons. The fundamental processes of mitosis are very similar
in all nucleated cells, but it would be an error to think about cell divi-
\ ision as an identical phenomenon in all nature from the unicellulars
to higher plants and animals. Though the changes brought about
by exposure to colchicine are nearly identical, it has been pointed out
in previous chapters that Amoeba reacts only when the alkaloid is
injected with a micropipette into the cytoplasm, that in plant cells,
chromosome division proceeds for a long time in the absence of any
spindle, and that in animals the hormones and other influences
regulating cellular nudtiplication interfere with the action of colchi-
cine (cf. Chapters 7, 8, and 9) .
Spindle poisons have been studied by a small group of research
workers, and each laboratory has used the celhdar material which
appeared the most convenient. It would l)e unwise to compare un-
critically results obtained on Allium root tips or on sea-urchin eggs
with those observed in fibroblast cultures or in mammals injected
with colchicine, or to comjiarc colchicine and spindle-poison effects
in normal and neoplastic cells, in embryos or in adults, in slow-grow-
ing cells or in tissues stimulated to cellular multiplication by the
action ot hormones — both in plants and animals. These facts may
seem e\ident from previous chapters. The great mass of data that
has accunuilated for twenty years about spindle poisons can only be
discussed with catition. It is clear that the time is not yet ripe for a
single theory covering all types of cells. This important point should
be kept in mind when, in the next pages, different and apparently
conflicting theories are considered. The only firm ground is that of
the experimental facts, and this alone provides a varied and interest-
ing insight into the action of spindle poisons.
17.1-4: The problem. The jnnpose of this chapter can now be
defined more clearly. The fundamental problem is that of spindle
inactivation by colchicine, a highly specific property of a complex
molecide. Other spindle poisons will be considered as far as they
help to understand colchicine, and also the modifications of the
fibrous properties of the spindle, as evidenced by its structine and by
sLibmicroscopic evidence (polari/ed light) ^••'^- ''i (Chapter 8) .
The following jjoints will be considered:
(1) Like most biological activities, spindle formation and modifi-
cations during mitosis may be under the control of enzymes. Most
work on the effects of colchicine on enzyme systems does not bring
much tiseful evidence, fjut should be pmsued. Some of the latest
theories, discussed in Subsections 17.5-2 and -4, point to enzymes as
the targets inhibited l)y colchicine.
396 Colchicine
(2) A great amount of work on plant cells with a large series of
chemicals has indicated that the destruction of the spindle was most
closely related to physical properties such as solubility. Jn short, c-
mitosis appeared as a "narcotized" mitosis, and the theories of nar-
cosis explain many findings. It will be seen further whether colchi-
cine fits into such a theory (Subsection 17.3-5).
(3) Work with a molecule as complex as colchicine benefits from
experiments with related chemicals having simpler structures. These
have clearly indicated which, in the molecule of colchicine, are the
groups necessary for the production of c-mitosis. Other substances
"that inactivate spindles and have definite chemical properties which
may explain their action, are of varied structure and range from the
simple inorganic arsenic salts to complex molecules, alkaloids, or anti-
biotics. Though no chemical explanation of spindle destruction by
all these substances can be given, the comparison of their structures
and activities with that of colchicine throws some light on the singular
properties of this alkaloid.
(4) Another approach to the problem of colchicine and the
spindle is through the study of antagonists and synergists. Some of
the work done in this field has given rise to controversies, but it can-
not be ignored. It is evident that the discovery of a substance capable
of preventing colchicine from destroying mitotic spindles might at
least throw some more light on the biochemistry of the alkaloid and
the spindle and on the complex reaction which apparently takes place
between them.
From all these studies, however scattered and incomplete they may
yet be, emerges an outline of a new cellular j)harmacology which
should ultimately not only explain why colchicine is a mitotic poison
but help, by what can properly be named a "biochemical dissection
of mitosis," to explain the mechanics of cell multiplication and of
growth.
17.2: Metabolic Actions of Colchicine
We will consider under this heading only the facts which help to
explain c-mitosis. Other properties of the alkaloid have been de-
scribed in Chapters 4 and 7. The resistance of some plants and ani-
mals to colchicine will be mentioned. While the mechanism of re-
sistance is very imperfectly understood, it may be related to the in-
Huence of the drug on cellular physiology.
77.2-7.- Enzymes. The work done in this field has been conducted
with quite different purposes, some authors being interested in mi-
tosis, others in possible mechanisms of colchicine treatment of gout,
the origin of hemorrhages observed in malignant growths (Chapter
10) , or the formation of c-tumors in plants.
Mechanism of Colchicine-Mitosis 397
An over-all decrease in tumor respiration was one of the first bio-
chemical observations on colchicine. Its relation with the inhibition
of mitosis is not evident.^"' ^'"''
It has been demonstrated that a 1.2 X K* " '^^ soluiion of colchi-
cine inhibits dephosphorylation and the deamination of desoxyribonu-
clcotidev Desoxyribonuclease is also inhibited; however, the relation
of these facts to mitosis is by no means clear, and the concentrations
of colchicine are far greater than those effective in spindle poisoning.«o
In rats injected 0.2 mg. of the drug, a decrease of the alkaline phos-
j)hatase activity was recorded in liver tissue; there was no increased
disintegration of ribonucleic acid (RNA) .'^i The RNA content of
fibroblasts erowine ni vitro was decreased by colchicine.--^ Pyrophos-
phatase, an enzyme which was found in great quantities in a benzo-
pyrene-induced sarcoma in a rat, was inhibited after a colchicine in-
jection, though no action on the enzyme could be detected i)2 vitro. ^
Other work on changes in pinine metabolism, possibly linked
with the curative effect of colchicine in gout, demonstrates that, while
the nucleotidase of the intestine of calves was not affected, that of
human serum was inhibited. Xanthine-dehydrase was also inhibited
in guinea pigs, but the concentrations of colchicine (50 per cent and
more) A\ere far larger than those effective both in spindle poisoning
aiul in therapeutics.''^
Inhibition of dehydrogenase acti\ity by colchicine and sodium
cacodylate. another spindle poison, was reported in 1938,''^ but no
further data on this subject have been published since. A strong de-
crease of liver dioxvi^henylalanine-decarboxylase in rats, and of the
pressor amines of the adrenals,^' may be related to the general toxicity
reactions of the alkaloid (Chapter 7) . In vitro studies of rat liver
slices demonstrated an inhibition of creatine synthesis, and blocking
of the formation of p-aminohippuric acid from p-aminobenzoic acid.
The methylation of nicotinamide was also inhibited. There appeared
to be a relation between amount of drug and degree of inhibition.
The formation of creatine from guanidoacetic acid and L-methionine
was inhibited by 65 per cent by a lO'^M solution of colchicine.^-^
In plant material, enzymatic reactions, /// vitro, of malt diastase
were accelerated by the addition of colchicine; however, the rates of
conversion of sucrose by invertase were not influenced. ""^ In the ger-
minating grains of Triticinn acstivinn L., the acti\ity of amylase was
increased by 10" Af colchicine. No significant changes of photosyn-
thesis ha\e been detected. *-
Some further results will be considered in the paragraphs on the
action of meso-'mo'^hoX (17.5-2) and adenosinetriphosphoric acid
(17.5-4) . It is evident at this point that no significant relation be-
tween enz\nie inactixation and spindle poisoning has been detected.
398 Colchicine
jy.2-2: Resistance in plants and animals. Cells of Colcliinnn
aatumnale L. yield as much as four parts per thousand of alkaloid.
Thus, some of the mitoses of the plant may be in close relation to
large doses of colchicine, and the questions arose by what mechanism
these mitoses are protected, and whether c-mitosis is possible in Col-
chicum. The first experimenters used as a test the bulbous enlarge-
ments of the root tips of Colchicum and concluded that large doses
of colchicine were active. However, as mentioned in Chapter 4, this
is only presumptive evidence, and c-tumors may arise -without any
mitoses taking place (C:hapter 4) . Cytological work was carried fur-
ther on several species of Colchicum and with various concentrations
of the alkaloid.-o xhe results were compared to those of the spindle
poison, acenaphthene (cf. Subsection 17.3-2). No true resistance in
excised root tips grown on agar with strong concentrations of colchi-
cine-o was observed, though the concentration of alkaloid necessary to
induce full c-mitosis was considerable (5 per cent in water) . The
possible influence of the chloroform present in crystalline colchicine
has been ruled out; chloroform is only a weak spindle poison.^ i' It
is clear that mitoses in Colchicum are considerably more resistant
than any other plant mitoses towards the alkaloid. This type of re-
sistance appears somewhat similar to that of venomous animals to-
wards their own venom, but in the case of the plant, the basic mechan-
ism is not understood and further research would be useful. Evi-
dently, this is linked with the other unsolved problems of the role
and metabolism of colchicine in Colchicum sp. The glucoside found
in the plant, colchicoside,^- may be of some significance (cf. Subsec-
tion 17.4-1) .
During routine laboratory tests the discovery was made that golden
hamsters resist very large doses of colchicine,"" considerably greater
than the lethal doses for rabbits, guinea pigs, mice, and rats. The
tests yielded no c-mitotic values, but only toxicity values which j^roved
beyond doubt that natural resistance exists with the hamsters. Another
similar case is the resistance of rabl)its to aconite.
Hamsters are native to the region where species of Colchicum are
abundant (cf. Chapter 1). Through a long period of evolution the
hamsters may, by the processes of survival of those animals that lived
after eating the Colchicum, have passed this resistance on to succeed-
ing generations. Any part of the Colchicum, leaf, flower, seed, fruit,
corm, would contribute generous portions of colchicine that would
be lethal to an animal without resistance.
Such resistance displayed by the hamsters is of interest in con-
nection with the evolutionary problems involved. Further \\'o\\ should
be done with the mitotic processes to make comparison of the action
of colchicine upon these features.
Mechanism of Colchicine-Mitosis 399
17.3: Physical Action
All inhibition ot spindle function and the destruction of its fibril-
lar structure can be the consequence of physical agents acting on the
cells during division. On the other hand, it appears most probable
that many of the spindle poisons which have been described do not
act by combining in the chemical sense of the word with the spindle
proteins, but by altering some of the physical conditions necessary
for the proper development of mitosis.
iy.3-1: luliibition of the spindle by physieal ao;eiits. That modifi-
cations of the physical environment of the cell, without any mitotic
poison being present, may induce c-mitosis is evidenced from the action
of heat, cold, and high hydrostatic pressiires.
The reversible changes of the mitotic spindle under the influence
of an increased temperature were described in 1933.'- Before colchi-
cine, heat-shock was perhaps the most reliable method for producing
polyploid plants (cf. Chapter 11) .lo^ It is also one of the most efficient
methods of inducing polyploidy in mammals, as mentioned in Chap-
ter 16A. In Triton inilgaris, on the contrary, larvae kept in water at
3°C. show a typical metaphase arrest, with chromosomes grouped in
a single star. The only difference with colchicine is that the alkaloid
does not depress prophases, and that ball metaphases (ct. C;hapter 2)
are more frequent.^ The hypothesis that cold should mainly affect
the ccntrosomcs and centromeres and prevent the orientation of
spindle fibers at their contact^ is interesting and deserving of further
study. Cold may have played a significant part in the evolution of
polyploid species, especially during the periods of glaciation.
The action of high hydrostatic pressures, about 200 atmospheres,
is similar to that of temperature changes in that it brings reversible
changes of the sj^ndle, ^vhich loses its fibrous appearance.-'^ 1 his has
been demonstrated both in animal cells (Urechis) and in j^lants (pol-
len mother cells of Tradescantia) . The exact significance of these re-
sults is far from being understood and need not be discussed here.
Evidently, the proper functioning of the spindle is only possible
within a limited range of physico-chemical conditions. It is thus not
surprising that changes induced by chemicals of various and unre-
lated structures may also arrest mitosis by inhibiting the spindle. Re-
search in this field will now be discussed, and the "narcosis theory" of
c-mitosis explained. Most of this work, for obvious experimental rea-
sons, has been conducted on plant cells, mainly the Allium root tip,
and on eggs of invertebrates or vertebrates. A k-w observations have
been made on tissue cultures.
I J. ^-2: Simple aromatic and aliphatic mitotic poisons. A very ex-
tensive Mudy on plant cells has been condudetl by several groups of
400 Colchicine
workers, that happened to be widely separated by the e\ents of the
second World War. lire similar conclusions which were reached have
thus an added significance. The names of Gavaudan (Marseille,
France), ^i- *•■ Schmuck (U.S.S.R.) ,i^"- 1""' i""' and Levan and Oster-
Ostergren (Lund, Sweden) ""• "^- "^- •'- should be mentioned at this
point, lliis work began with the search for some i^olyploidizing agent
more eflfecti\e than colchicine and led to an intensive study of chemi-
cals and of the relation between their structure and their activity. One
of the first sulxstances demonstrated to be eflfcctive for the induction of
polyploidy in plants was acenaptJiene (I) . 1 his was discovered in
1938,i"'' '^•^- "•'■ -^^ and the simplicity of its chemical structure, ap-
parently without any relation to that of colchicine, quite naturally
led other authors to investigate various aromatic derivatives.
In the following years, haloid derivatives of acenaphthene were
also found to be effective c-mitotic poisons, as well as later haloid
derivatives of other aromatic compounds, "i- ^'- i'-^- ^^^ and various de-
rivatives of benzene and naphthalene. All of these were soluble in
lipids and, contrary to colchicine, had low water solubility. In
France, mairy mono-substituted derivatives of benzene and naphtha-
lene were tested by the Gavaudans on Triticutn. This extensive work
can only be briefly reviewed here. It appeared that, Avhile benzene
was only weakly active, it was necessary only to add some side-chains
to obtain effective c-mitotic poisons. One exception was hexamethyl-
henzene, the inactivity of which was linked with its high degree of
symmetry. Nitro- and halo-derivatives of benzene and naphthalene
were studied, and many found to be mitotic ])oisons. Ho\\e\er, total
inactivation of the spindle was not always observed, and partial c-
mitosis (mero-stathmokinesis) or abnormalities of spindle (jricniation
(tropokinesis) were often the only cellular changes. C-mitosis was
also observed under the influence of anesthetic drugs, such as phenyl-
urethane, acetophenone, or anesthesine.^^- ^-^
It soon became evident that no definite chemical structure was
necessary, but that nearly all aromatic derivatives were c-mitotic
poisons under proper experimental conditions, except those Avith a
carboxyl, for instance, benzoic acid, or an amino-group. It ^^•as e\ i-
dent that an increased solubility in water was unfavorable for spindle
poisoning. More recently, however, amino-acenaplitJieue was demon-
strated to be a spindle poison for fibroblasts in tissue culture.''*'^- ^'^
In 1944, the French authors linked their observations with Fer-
guson's notion of tliennodynamic activity, which expressed the tend-
ency of a given substance to escape from the ])hase in which it is dis-
solved. It can be measured by the relation between the lowest active
concentrations of a sufjstance and its highest solubility in water. The
conclusion was reached that with only a few exceptions, all the chemi-
cals which had proved to arrest spindle activity acted like chemically
Mechanism of Colchicine-Mitosis
401
indifferent poisons, and that their influence on mitosis was quite
similar to the changes brought aljout in the nervous system l)y the
so-called indiftcrent narcotics. Physical changes appeared prominent,
and c-miiosis was called a "narcoti/ed" mitosis. The suljstances listed
as not following the rule included aniline, phenol, hexanilrodipheji-
ylduinir. and coUhiciue. The activity of phenol and aniline, two
CH2-
-CHo
H
H
H H
(I) Acenaphthene
simple derivatives of ben/ene, demonstrated that in the series of ben-
zene derivatives, the hypothesis that the substances with high tliermo-
dynamic potential and high solubility in lipids were the most active
spindle poisons, could not be accepted without some corrections.^^- ^^
The Swedish authors,'"- '^^^ '•!■ ''- studying the Allium root tips,
came to nearly identical conclusions, linking lipoid solubility with
the mechanism of c-mitosis. They studied a large number of com-
poiuids, listed in the papers of Ostergren, (cf. also ^''■>) who proposed
a theoretical explanation of "narcotized mitosis" which will be dis-
cussed in Subsection 17.3-4. It should be pointed out here that all
these exjjeriments could easily be carried out on root tips, but that
the (onclusions cannot be too rapidly extended to animal cells, which
would not resist treatments with strong concentrations of lipoid-
soluble substances, often of high toxicity. It is however evident that
some drugs known as narcotics in animals, do possess c-mitotic proper-
ties.
i-j.^-^: Narcotics and indifjerent inorganical substances. Among
the chemicals capable of inducing narcosis in animals, we have already
mentioned chloral hydrate,^-*'- •'•'*• •^•* which is a spindle poison, as
shown in Figtne 17.1. Ethylcarbamate (ethylurethane) is a narcotic
in animals antl a s]Mndle poison in the egg of Paracentrntiis lividus
LK.,'-*-^ in amphibians and in plant cells. -^ In other animal cells, e.g.,
the intestinal mucosa and the l)one marrow of mannnals, ethylcarba-
mate acts like a chromosome poison.^" Chloroform'" and ether are
known to arrest cell division in ]jlants and in some eggs of ani-
mals.^'^- "' In the corneal cells of Sahniunidra. ethyl alcohol, ether,
and chlorethone also prevent the proper activity of the spindle.-'^
402 Colchicine
None of these substances, however, has an activity comparable to that
of colchicine, and their mitotic effects are only visible in relatively
concentrated solutions.
These facts, demonstrating that no evident relation exists between
the chemical constitution and the c-mitotic action, and that lipoid
solubility is always present, confirm the theory of c-mitosis as a nar-
cotized mitosis. Lipoid solubility is one of the foundations of Over-
ton's well-known theory of narcosis in animals. The wide use of gase-
ous narcosis in medical practice prompted some workers to study this
group of narcotics on the root tips of Allium cepa. These were kept
humid in a mixture of atmospheric air and the gases, which were
under pressure. Propane, nitrogen, nitrous oxide, methane, argon
(under a pressure of 75 atmospheres) , and hydrogen (200 atmos-
pheres) induced c-mitosis and typical c-tumors. However, only pro-
pane, nitrogen, and nitrous oxide induced polyploid cells, for the
other gases depressed too much the number of new mitoses.-^'' This
observation of c-mitosis under the influence of an inert gas like argon
definitely demonstrates that the chemical structure may be cjuite in-
different to the production of inactive spindles, and that physical
changes j)lay a great part. C-mitosis appears at this jjoint to be a
general reaction of the spindle under the most varied conditions.
Work discussed further will show how far these results may explain
the action of colchicine.
17.3-4: Narcosis and colchicine. The facts gathered so far point
towards a close relation between metaphasic (spindle) poisoning and
lipoid solubility or thermodynamic activity. The precise relation be-
tween lipoids and the function of the spindle is by no means clear,
and narcotics appears to modify mitosis somewhat like cokH or high
hydrostatic pressure.^"* It is not surprising that the problem appears
complex, for very little is known about the main target of all these
poisons, namely, the spindle. That it is fibrous and anisotropic is
evident and is no longer discussed. ii"' ^o How it functions is the sub-
ject of much controversy, for it is not yet demonstrated whether the
fibers "ptUl" the chromosomes towards the poles (after gathering
them at the equator of the cell) , or if the chromosomes are "pushed"
polewards by a "Stemmkorper" lying at anaphase in the center of the
cell. The results of colchicine research indicate (Chapter 2) that trac-
tion must play an important role in the movements of the anaphase
plates, but how this traction takes place and on what sujjport the fibers
are anchored are still unsolved problems. The shortening of the
fibers involves most probably changes from fibrous to globular pro-
teins, as evidenced by the polari/ed light data.'^i These changes
probably take place first between the two anajihasic plates, where all
Mechanism of Colchicine-Mitosis 403
fibrous structvires disappear and later between the poles and the cen-
tromeres, where ihcy bring about a shortening of the fibers. The
biochemical basis ot this complex mechanism is unknown. The
chemical constitution ot the fibers themselves has not been deter-
mined, with the exception of some histochemical Avork indicating
that their proteins are rich in sulfhydryl groups (cf. Subsection
17.4-2).
Any theory linking "narcosis" to spindle changes requires ad-
ditional investigations with a wider use of specimens from both ani-
mals and plants. The Swedish author Ostergren"' -♦- has presented
evidence for the "narcosis theory" using AUiuni root tip cells as a
major testing material. The relationship demonstrated to exist be-
tween lipo-solubility and the c-mitotic activity for many substances
fits the hvpothesis quite well, but there are unanswered questions that
do not give us as much supporting evidence as everyone woidd desire.
Therefore, the hypothesis put forward by Ostergren at this time re-
cpiires additional testing. Rejjcating from the preceding paragraph,
it is to be stressed that the lack of specific biochemical evidence
drastically limits our understanding, particularly when trying to
formulate basic mechanisms for reactions such as the c-mitosis.
Colchicine is a spindle poison with a low thermodynamic activity
and extremely high solubility in water. Therefore, this chemical is an
exception to the general rule that applies to simpler aromatic deriva-
tives.^i These relationships are clearly illustrated in Figure 17.2, as
drawn from experiments with cells of Allium and/or Triticnm. The
proposed theory of a narcosis, while interesting from the standpoint
of the biochemistry of the spindle, cannot at the same time apply to
colchicine, "which aj^pears to act on a chemical basis rather than
physically. This conclusion was reached independently by the French
authors.^^ Certain results will now be considered to show that ideas
of a chemical relation between alkaloid and spindle appear promising
for the ultimate explanation as to how a c-mitosis is accomplished.
17.4: Chemical Action
Two lines of research indicate that s])indle poisoning may be re-
lated to definite chemical structures, and probably to chemical inter-
ference between pcjisons and spindle fibers. The first is the study of
derivatives of colchicine and related molecules. This indicates that
minor changes in this complex atomic structure may considerably
affect the cytological activity. The second is the study of other mitotic
poisons: while those Avhich have been considered so far acted more
physically than chemically, there is a small but imjiortant group of
substances which inactivate the spindle and which possess specific
404
Colchicine
chemical reactivity. After studying these simple spindle poisons, some
other substances acting like colchicine, or those with complex mole-
cular structure will be examined briefly. The properties of colchicine
will then be compared to those of other poisons.
1J.4-1: Colchicine derivatives. These have been studied from
three main points of view: their toxicity, their antimitotic activity,
C
O
u
o
o
£
_o
c ""
.0
'**
u —
o
O -
o
ji —
«o
Colchicine
-.7 -6 .5 _4 .3 .2 .1 _0
Solubility(log. mol. fraction)
Fig. 17.2 — Relation between c-mitotic activity in the Allium test and solubility in water.
Each dot or triangle corresponds to a different substance. The singular behavior of
colchicine is evident. (After Ostergren, 1951 '")
and their inhibition of tumor growth.-^ The spindle poisoning will
mainly interest us here, and it should be made clear that this is not
necessarily paralleled by other properties of these molecules. For in-
stance, it has long been known that colchiceine (II) is less toxic, and
also a weaker mitotic poison than colchicine. But desacetylcolchiceine,
trimethylcolcliicinic acid (III) ,'"'-^ does not interfere ai all with cell
division in animals, while it may, like colchicine, kill frogs by central
nervous paralysis. The opposite is also true; and results to be discussed
further point to the possibility of synthesizing derivatives with lower
toxicity and greater mitotic-poisoning effects than colchicine.
Mechanism of Colchicine-Mitosis
405
In [he AJIimii test, I) I mctli\l((>!(lii( ii)ic ociil (III) has been sh()\vn
to induce c-niitosis, but it is thought that the incchanisiii is (|uite dif-
Icrent from that of colchicine, and rchitcd to the amino grotij) of ring
B.i^' 1 his derivati\c has a marked toxicity, while even 20 j^er cent
solutions of colchicine are only slightly toxic for these plant cells.
Before considering in some detail artificial colchicine derivatives, it
is important to remember that other closely related alkaloids exist in
CHsO
CH30
COCH;j
CH30
CH30
NHo
CH3O
O
CH3O
= 0
OH
OH
(II) Colchiceine
(III) Trimethylco!chicinic Acid
Coh liicuNi, and also that colchicine is probably present in chemical
combination with a glucoside. DesmethyJcohhicine has been found
in preparations of colchicine;^^ it differs from colchicine only by one
methyl group missing in ring A. It has been proved that it jjoisons
mitosis like colchicine, and demonstrates that two methyl groups are
sufficient for this. It is probable that at least one is indispensable.
Work by Lettre is interesting in this connection.'"'' This author,
searching for mitotic poisons with a simpler chemical structure, and
basing his researches at the time on the old formula of \V^indaus in
^vhich rings B and C are 6-membcred, showed that on fibroblasts in
tissue culture, niescaJine (IV) was without action, while a-phcnyl-^-
{^,4, ytrimethoxypliefiyl) -etJiylatnine (V) is active. Further simplifi-
cation demonstrated that spindle poisoning was retained in a-i)henyl-
(5 (p-metholxyphenyl) -ethylaminc (VI) , which was the simplest ))os-
sible poison of this group.
Ihe exact chemical structure of several other substances from
Colchuum and closely related to colchicine is not known yet; they
probably differ from the parent molecule by relatively minor changes,
'""■ ""•''•■'' and are all more or less active against mitosis.
In Colchicutn, a substance named colchicoside, resulting from a
glucosidic linkage of colchicine, the exact chemical nature of which
has not yet been established, has been isolated.''- It is of interest to
note ihat this poisons spindles, but is 40 times less active than colchi-
cine towards plant mitoses. With diluted solutions, it is observed
U"
\
^
\
o
n
X
a
/^
>
/^
o
o
X
o
CO
/Mechanism of Colchicine-Mitosis
407
that c-tumors (root-tip swellings) occtir with solutions which arc de-
void of any mitotic action. The hypothesis has been put forward that
colchicoside may be some kind of detoxication product of colchicine,
a fact which may help to explain die resistance of Colchiciun towards
colchicine (cf. Subsection 17.2-2) .
The principal changes affecting the action of colchicine are those
affecting the .V-substituted radicals in ring B and the esters of ring
NH.COCH3
CH30
= 0
0CH3
NH.COCH3
OCH3
(VII) Colchicine
Isocolchicine
C. Before considering some of these derivatives, it is important to
stud\ the residts obtained with an isomer of colchicine, isocolchicine,
(VII) in which the positions of the O and O-CHo radicals of ring C
are reversed. i^*'- ^'^'- *'•"'
The activity of /.socolchicine has been studied on Allium root
tips^^' and on fibroblast cultures.*"'^ Solubility and thermodynamic
acti\ity differ considerably from those of colchicine, ^\'hilc the latter
is soluble in approximately all proportions in water, /5ocolchicine
has a solubility of oO.OOOx iO'M// 1. The activity thresholds stand at
150 for colchicine and 14,000 X 10''iU/l for the /io-compoiuid, the
thermodynamic activity of which is 0.28, that is to say, about a thous-
and times higher than that of colchicine. As a conclusion of this
work, it appears "that colchicine, with its low thermodynamic activity
is a typical representative of the chemically acting substances, while
/.^ocolchicine with its 900 times higher thermodynamic activity be-
longs to the type of unspecifically acting substances." ^i' /iocolchicine
interferes thus w'nh mitosis like the many substances mentioned in
the previous paragraph of this chajitcr. In fibroblast cid tines, the
difference is not quite so great, for ?5ocolchicine is only 50 times less
active than colchicine. Two other similar molecides, ethyl-colchi-
ceine and isoethylcolcliiceine, were compared on the same material:
the second was about 200 times less active than the first. These sub-
stances have been isolated from C.olrliicinn. Other iso- derivatives of
408
Colchicine
66
colchicine have also proved to be without action against neoplasms.
It is premature to discuss the reasons for the weak activity of the
iso- compounds. One reason which has been jnit forward is the forma-
tion of hydrogen bonds between the side-chains of ring C and ring B,
because of the closeness of the methyl groups of these chains in the
iso- forms. (VII) It has been suggested that the weak antimitotic
activity of colchiceine may be the consequence of the iso- form of this
molecule.*'-^ Other data prove that the activity of colchicine on mitosis
is related to both these side-chains.
The substances to be studied now can all be considered as de-
rivatives of trimeUiylcokhicinic acid (III) . This compound was dem-
onstrated in some of the first work on colchicine derivatives and
mitotic cells in mammals, to be inactive. In cultures of fibroblasts
and of neoplastic cells also, no activity could be detected (Table
17.1).^^
Substitution on ring B alone does not yield effective mitotic poi-
sons. On tissue cultuies, Is-acetyl-colchicol and its methyl ether (VIII)
have only slight activity. Tables 17.1 and 17.2 give further evidence
TABLE 17.1
LD 50's OF Colchicine Derivatives in mg kg
(After Goldberg et al.**)
Substance
Mice
Rats
Cats
.V-Benzoyl-TMCA*
TMCA .
>700
200
84
56
46
32
3.5
200
30
200
>10
Colchiceine .
>12.5
,V-Acetvl-colchicol
10
TMCA-methvl-ether
5
;V-BenzoyI-TMCA-methyl-ether . . .
..V-Acetyl-TMCA-methyl-cthcr . .
<25
5.0
0.5
* TMCA = trimethvlcolchicinic acid.
of this. I he activity of this derivative is comparable to that of col-
chiceine.
However, when ring C remains as in colchicine, it is evident that
A^-substitution in ring B is not of great importance for activity. In
tissue cultures, desacetylcolchicine, trimetliyh olchicinic acid methyl
ether (IX) , is an effective spindle poison, while the parent substance,
desacetylcolchiceine (=TMCA) , is almost inactive. N-betizoyl-tri-
Mechanism of Colchicine-Mitosis
409
NH.C0CH3
CH30
CH30
CH30
0CH3
(VIII)
(IX)
= 0
0CH3
methylcolchicinic iiictliyl cllier has been demonstrated to be one of
the most effective derivatives in arresting mitoses in the stomach
epithelium of mice."- ^-- -^^
Substitutions in ring C are the most important, for they yield
substances with a greater antimitotic activity than colchicine."^- '^^
These are derivatives of colchicamide (X) . (This abbreviated spell-
ing is to be preferred to colchicine amide or colchiceinamide, which
are to be found in the literature.) Thirtv-fi\c derivatives of this type
have been studied by Lettre,''*' who found A -methyl-, N-ethyl-. and
A^-dimethyl-colchicamide to be most effective in tissue-culture work,
the activity decreasing when longer side-chains were added to the
amino-group (Table 17.3).
Other derivatives with more extensive changes in ring C, for in-
stance with a six-carbon aromatic ring C, coJchinol series (XI) , or
TABLE 17.2
Minimal Effective Antimitotic Dose of TMCA Derivatives on Corneal Mitoses
OF Mice, Six Hours After Injection, Expressed as the Fraction of the LD 50 In-
creasing the Mitotic Index Above That of Controls and Minimal Effective
Antimitotic Doses in Various Tissues of Mice
(After Goldberg et al .^^)
Minimal
Antimitotic
Dose/
LD 50
Minimal Effective Dose
Substance
Cornea
^mg/f^g)
Regenerating
Liver
(mg/kg)
Tissue
Cultures
ifJ-g't^g)
Colchicine
1/10
1/2
1
>1
0.01
1 .0
4.0
inactive
0.21
9.01
8.01
inactive
0.35
A'^acetylcolchicol . .
28.0
Colchiceine
84.0
TMCA (trimethyl-
colchirinic acid)
inactive
470
Colchicine
K-be}2zoyI-coIchicinic anJiydride (XII) , have been tested on tumors.'^^
None has shown an activity comparable to colchicine, and the reader
should refer to the papers of the National C;anccr Institute group for
detailed data on this subject.^- i- ^^' «^' ^'^
Although colchicine derivatives have been tested on few materials,
the main pinpose of the work having been a search for substances of
NH.C0CH5
CH'jO
= 0
NHo
(X)
CH3O
CH3O
NH.COCH3
CH3O
CH3O
CH3O
NH-CO
c = o
o = c-
-0
%/ OH
(XI) N-Acetylcolchinol
(XII)
interest in cancer chemotheraj))', the following conclusions can be
drawn for the papers published:
1. The /^ocolchicine derivatives, and /^colchicine itself, are con-
siderably less active. It appears important that the esterified side-
chains of rings B and C are at a proper distance one from another.
2. At least one methoxy group appears indispensable in ring A.
.H. The amino group of ring B does not need to be esterided, though
this increases the activity.
4. Ring C must be seven-membered, and the hydroxyl grouj) esteri-
fied, or better, replaced by an amino group itself esterified (colchi-
camide derivatives) .
These facts help to reveal which are the active groups of the
colchicine molecule. However, they are yet of no help in explain-
ing how these react with the spindle. Results obtained with spindle
Mechanism of Colchicine-Mitosis 411
poisons ot very different chemical structure, and indicating relations
between this structure and their action, throw further light on the
subject of spindle inactivation.
ij.-f-2: Sulfhydryl poisons. \Vith a few exceptions, most of the
work in this field has been done on tissue cultures^^ or in intact
warm-blooded animals.^^^^ This method has an advantage in that, be-
TABLE 17.3
Smallest Antimitotic Doses (fig/ml) Effective in Arresting
Mitoses in Cultures of Chick Fibroblasts
(After Lettre «")
Derivative Dose
Colchiceine 5.0
Colchicine 0.01
Colchicamide 0.01
jV-methylcolchicamide 0 . 0025
A'-ethylcolchicamide 0.003
JV-propylcolchicamide 0 . 08
.\ -butylcolchicamide 0.9
.Y-methyl-propyl-colchicamide 0.5
cause of the necessity for avoiding toxic side-effects, only small doses
may be used. Hence, substances acting as narcotics or producing a
"physical" change ot the spindle will not be found to have mitotic-
poisoning properties.
The most extensively studied in mammals,''^'- -»• ~^' ^" in inverte-
brates,-*'' on tissue cultures,'^' i-^' ''^ and in plant cells'**- -- are simple
derivatives of arsenic. Arsenious oxide and sodium nrsenite arrest
metaphase by destroying the spindle, and these star metaphases are
very similar to those described in Chapter 2. The most effective of the
organic arsenicals appears to be sodium cacodylate, or dimeihylarsin-
ate (XIII).
In mice, it has been demonstrated that this action was reversible,
that is to say, that arrested metaphases could be detoxicated and pro-
ceed to a normal telophase."'*' The inactivation of the spindle is thus
the consequence of a labile combination of its proteins with arsenic.
The detoxicating agent was dimercaptopropanol (BAL, British Anti-
Lewisite) (XIV) , a substance which combines rajMdly and strongly
with arsenic and other metals. This action of a chemical with two
-SH functions suggested that arsenic may have combined \vith similar
412 Colchicine
SH groups in the spindle.""' This hypothesis was in agreement with a
theory of spindle activity in which reversible changes of SH to S-S
functions were supposed to play a prominent part in the "contractile"
properties of the spindle. The further discovery that -SH substances
themselves were also sjiindle poisons, for instance, dimercaptopro-
panol and sodium dielliyldilJiiucarbainale, was in agreement with this
CH — SH
yCHg I
y/ CH SH
0=AS CH3 I
\ CHOH
^O — N a
(XIII) (XIV)
hypothesis, if it was considered that a proper equilibrium l^etwcen
reduced and oxydized sulfhydryl functions was indispensable for
spindle activity.-^"
This theory of chemical action on the spindle received further sup-
port from the discovery that many metals, known to combine with
-SH groups, are mitotic poisons.'^" Ethylmercurychloride is an ex-
ample of an organic poison of this type, active on plant cells, ■■^•'' ''"
while cadiniuiu salts are most effective in arresting mitosis in mam-
mals.122, 30, 2 -fhe inhibition of metaphase by beyyUium salts, which
has been considered to be the result of nuclear phosphatase inhibi-
tion,i" may possibly be explained by the combination of this metal
with sulfhydryl groups.
It has been further demonstrated by work on tissue cultures and
in injected mice, that the typical -SH poisons, chloracetopheyione,
iodoacetic add, and iodoacetajnide, arrested mitoses at metaphase.^^"-
50 However, these substances are very toxic, and have strong inhibi-
tory actions on glycolysis, which may be important in explaining
their action on cell division. Some of the complex molecules con-
sidered in the next Subsection may also act as -SH poisons.
This does not close the list of mitotic poisons which appear to act
chemically on the cells. The most remarkable is etJiylcaibylaminc
(C.H-.CN) , which has been demonstrated to modify the course of
mitosis in tissue cultures exactly like colchicine. 1-" Total inactivation
of the spindle with exploded metaphase and, later, formation of
numerous micronuclei were conspicuous. Ethylcarbylamine reacts
chemically with metals; this chelating property is shared by diethyldi-
thiocarbaiuaie, another spindle poison.'"' These results point to some
further complexities of the problem; the action of other organic
spindle poisons will show how far we are from understanding the
basic changes involved.
Mechanism of Colchicine-Mitosis 413
i-i-^: Complex organic inolccitlrs. The mechanism ot action of
most of the substances mentioned in this subsection is unknown;
molecular structures are widely different. However, these drug;s are
all very active, and it is felt that they modify the spindle more by a
chemical than by a physical change. The resin of Podophyllum sp.
(mandrake) contains several toxic substances, the principal ones being
podophyllotoxin, a- and {^-peltatius, and quercetin. The crude resin
was a popular remedy against warts in the United States, and this
observation led to a scientific study of the active substances-^^- -' (XV) .
These jjroved to be efficient spindle poisons, and to act most similarly
to cokhicine, both in skin tumors of man, and in various animal
materials."'^ From a chemical point of vie^v, they are complex lac-
tones.'•'• Another instance of a lactone acting as a mitotic poison is
the antibiotic patuUu (Bacitracin, clavacin) (XVI) . 1 his inhibits
remarkably the spindles of erythroblasts in the chick and in many
tissues of mice.^
It is interesting to compare the formula of patulin with that of
roumarin (XVII) , which has been described as a weak metaphase poi-
son in Allium and Lilium. Its action may be of the "physical" type,
though combination with -SH groups is also possible. ^-^
Other substances of plant origin have been foimd to inhibit mitosis,
mainly in tissue cultures of fibroblasts. Chelidonine^^ is of interest
because of its use in cancer chemotherapy (Chapter 10) . In an ex-
tensive study of alkaloids, it has been shown that the only active ones
were found in the group which is chemically related to stilbylamine,
and thus to a-phenyl-i^ {p-metho\yphenyl) -ethylamine (cf. 17.4-1).
These are narcotin, gnoscopin, chelidonine, liomocJielidonnie, meth-
oxychelidonine, and protopin."'^ Many other substances may yet be
discovered ^vhcn further systematic studies are conduced. This is al-
ready underway, and has demonstrated c-mitotic activity in extracts
of Clumapliila maculata and Sassafras albidum.'
Other complex substances extracted from plants are anctliol*''- and
apiol,^^ which may induce polyploidy. This has also been observed
in Allium root tips treated -with veratrine.^-^ Sanguinarine and cryp-
topleuriue are also spindle poisons, and the second, extracted from
Crypt oca ria pleurospora, has been considered as effective as colchi-
cine.'' Positive effects on mitosis have also been found with extracts
of the following plants: Ervatamia augustifolia, Aristolocliia clegans,
Euphorbia peplus, Bulbina bulbosa, and Strychnos arborea. Proto-
a)icmonii)i is an interesting poison,-^^' ^-^ for its action on the spindle
may be prevented by dimercaptopropanol (BAL) ; this is evidence of
a chemical reaction.
The list of c-mitotic active substances is much longer, and among
chemicals of animal origin or related to the growth of animal cells,
adrenalin^''''- "^ has been found to arrest metaphases in fibroblast ( ul
>
X
>
X
X
o
>
X
Mechanism of Colchicine-Mitosis 415
tures at a concentration of 0.1 mg/ml, and the antifolic drug-, niiiino-
pterin (^-amijiopteroylglutamic acid) arrests mitoses in tissue cul-
ture.-^2 Tfhis is a remarkable fact, for this antimetabolite when in-
jected into mice, behaves as a strong and typical poison of the "radio-
mimetic" tvpe, inducing chromosome breakages.'''^
77.^-7; Colchicine compared witJi other spindle poisons. The
spindle structure, which can be destroyed by purely physical means,
is evidently adversely influenced by a series of substances which appear
to act through their chemical reactivity. Arsenic, the heavy metals
(mercur\ and cadmium) , and the sulfhydryl poisons of the iodoacet-
amide type indicate that -SH groups may play an important role in
metaphase dynamics. Some more complex substances, such as the
antibiotic patulin, and protoanemonin, may owe their antimitotic
properties to the lactone structure, and perhaps also to interference
with sulfhydryl. Podophyllotoxin may possibly belong to the same
group, but the difficulties of understanding clearly the action of such
complex molecules are formidable. There is no indication that colchi-
cine may fit in this type of chemical theory, though the facts gathered
by the protagonists of the "narcosis" hypothesis, as well as the study
of colchicine derivatives, point towards a chemical combination of
the alkaloid ^vith some intracellular receptor.
The comparison of colchicine with other spindle poisons makes
clear t^\'o facts: the great amount of work which is still necessary to
understand the action of this drug, and the notable specificity of
colchicine. For, if several chemicals have been quoted as acting
similarly, fe^v have been capable of inducing polyploidy, and still
none has pro\ed comparable in the practical work on polyploidy in
plants. The extraordinary fact is the great efficiency and activity of
colchicine, which will lemain active when highly diluted, but con-
centrated solutions of which will not kill the cells. This points to
some singular relation between the alkaloid and the spindle.
Further research about the biological activity of the tropolone
compounds should help to understand better the chemical action of
colchicine in the cell. Thus far, it has not been possible to "simplify"
the molecule and obtain spindle poisoning. The few^ reports on trop-
olone derivatives indicate some action on mitosis, in Tradescantia
staminal hair cells, far weaker than colchicine. ^-^ The necessity for
such a complex molecule to achieve with the utmost efficiency what
can be done by such simple agents as cold, arsenic, and ethylcarbyl-
amine, is most puzzling. The solution of this problem should bring
some important new insight on the submicroscopic and (hemical
mechanics of mitosis.
Often the mechanism of thug adivity has been solved when a
proper antagonist could be found, for instance p-aminobenzoic acid
476 Colchicine
and the sulfonamides. Some work in this direction has been carried
along and ^\'\\\ he sunmiarized now.
17.5: Synergists and Antagonists
A possible synergism between animal gro^\■th hormones and col-
chicine has been considered in Chapter 9. In plants, some changes
visible alter colchicine have been interpreted as evidence'^- -'^^ ^*^'- '^'
79. 87 of hormonal action of the alkaloid. This has not been jnoved
(cf. Chapter 4) . In animal and i)lant cells, the antagonism of
7neso-'mos\to\ and colchicine is still a subject under discussion which
merits to be reviewed here. Mention will also be made of a long series
of experiments on fibroblasts in tissue cultures. These have led to a
novel theory about c-mitosis which ^vill l)e pioperly considered in
the light of all the facts already gathered in this chapter.
77.5-/.- Meso-inositol. y-HexacJilorocyclohexant' ("Gammexane") ,
a widely used insecticide, has been reported by several authors to
induce c-mitosis in Allium and other plant cells.--' ■^•'^•■''-'' Both the y
and the b isomers ha\'e been found to be active, i"' while the first only
is of use as an insecticide. Polyploidy and chromosome fragmentation
have also been recorded. Gammexane is probably an antagonist of a
naturally occurring substance. meso-inositoL having the same stereo-
isomeric structure as this sugar, the biological significance of which
appears from its presence in many types of cells.
It was thus not surjjrising that in 1948 it was announced that
?/K'50-inositol, (but neither d-inositol nor D-sorbitol) prevented, in
proper concentrations, the c-mitotic activity of Gannnexane in Allium
cepaJ*' It was, howe\er. more surprising and most interesting that
7neso-inosito\ was claimed to jjrevent also the sjjindle effect of col-
chicine. The results were gi\en as j^ercentages ol the different stages
of mitosis, and it is to be regretted that no counts of the total num-
ber of cell di\isions were recorded. Inositol alone did not interfere
with mitosis. The formation of c-tumors, both by Gammexane and
colchicine, was also prevented. ''' 1 hese results were checked over a
wider range of concentrations and times by another author, who lonnd
that meso-inosho\ merely delayed the c-mitotic eftect of colchicine,
which was visible, as in the controls, after 24 hours.-- Similar delays
were observed with other sugars, a solution of saccharose (0.95 mg/ml)
suppressing all colchicine mitoses in root tijjs observed after tour
hours of treatment, while after 24 hours the c-mitotic effect was
normal.-- Modified cell permeability was thought to explain the re-
sults obtained with //(07>-inositol. A confirmation of these findings
was found in the observation that colchicine and jjodophyllotoxine
effects were antagonized in the egg of the sea urchin Lyt echinus varie-
gatus by glucose.-" The antagonism was never total; it was suggested
Mechanism of Colcbicine-Mitosis 417
thai inositol may become changed into glucose in the cells. How-
ever, in Allium, it was demonstnitecl that the isomer ol hexacyclo-
chlorohexane, which could not act as an antagonist to ineso-inos\io\,
was also a spindle poison, and that no true protection was offered by
meso-'\no^\io\ against the effects of Gammexane.i'' y^g different tem-
Cl Cl OH OH
CH CH Cl CH CH OH
/ \l / \l
CH Cl CH CH OH CH
i \l / I \i /
Cl CH CH OH CH CH
Cl OH
(XVIII) -'-Hexachlorocyclohexane (XIX) Meso-lnositol
("Gammexane")
peratines at which the experiments were conducted may explain the
conflicting results.
Two papers published in 1951 renewed interest in this problem.
In the first, the authors who discovered the action of //u'5o-inositol
first in plants, brought forward evidence that a similar antagonism
existed in rat fibroblast cultures. '^^ Here, for the first 12 hours, no
difference Avas observed between colchicine alone and colchicine -j-
inositol, but in the following hours, while the colchicine mitoses re-
mained arrested, the cultures treated with inositol recovered almost
completely. 1 his period of 12 hours during which, quite contrary to
the plant experiments, inositol does not prove to have any effect, ex-
cept that of lowering the total numbers of mitoses, is considered to
correspond to the duration of interkinesis. The authors suggest that
?nejo-inositol may "allow the cell to prepare for a new mitosis," which
is surprising, for this would lead one to think that there is no true
detoxication of c-mitoses, similar to that of arsenite by BAL, and that
these degenerate, and are no longer counted, while other cells enter
mitosis. However difficult the interpretation of these results may
seem to be, it is significant that neither sucrose, glucose, ribose, sor-
bitol, nor even rf-inositol, ineso-'xno^ose or e/?/-inosose are capable of
altering the action of colchicine.'^^
This result is also in contradiction with the facts observed in plant
cells, and no conclusion can be drawn at this time. One interesting
report, given only in a short note, is that some enzymes of bacterial
origin capable of oxidizing inositol are inhibited by colchicine and
the parent substances, tropolone and 4, 5-tetramethylene-tropolone.^^'-
^-^ Further results on this aspect of the colchicine problem are eagei ly
418 Colchicine
awaited; they may help to understand better the biochemistry of the
spindle and the physiological functions of //?f'^o-inositol.-'^ As for the
action of y-hexachlorocyclopropanc, it may of course be of a "physi-
cal" type, similar to that of the numerous other c-mitotic and poly-
ploidizing substances studied in plants. ^^^
77.5-2; Other anlagonists and sy}iergists. In tissue cultures of
rabbit heart fibroblasts, 1-ascorbic acid was found to prevent, to a
certain extent, the action of colchicine.^^ The numbers of arrested
mitoses were smaller, and a careful study of the different types of
mitotic abnormalities indicated that the vitamin decreased the amount
of sjiindle inactivation. This was not the result of an action as a vita-
min, lor d-(iraboascorbic acid, whose properties as a vitamin are 20
times weaker, had the same effect. The two substances are equally
reducing, and the interpretation of these residts is difficult, lor ])-
qiiinone, an oxydant, also depressed colchicine inhibition of mi-
toses.^^ An antagonism between colchicine and "soluble prontosil"
(sulfanilamide) has been reported in plants,'" but the effecti\e concen-
trations of the sulfa drug were about a hundred times those of col-
chicine, and solubility effects were unavoidable. In animals, sulfanil-
amide has been claimed to influence colchicine-leukocvtosis, but this
was only remotely related to mitosis^-' (cf. Chapter 7) .
An extract from hearts of embryonic warm-blooded animals has
been reported to delay the cytotoxicity of colchicine in fibroblast and
myoblast cultures. A colchicine concentration of 2 X 10'^ ^^ ^^'^s with-
out effect after 10 hours in cultures previously treated Avith the ex-
tract. If this was added after the alkaloid, no antagonism was vis-
ible.i--^ Another more recent obser\ ation is that glycosidic substances
endowed with cardiotonic activity decrease the action of colchicine in
tissue cultures of chick heart fibroblasts. s'*
It appears evident from these data that no true antagonism has
yet been found between any substance and colchicine, on a molar
basis, and that the only effects observed depend on the presence of
substances either of unknown chemical nature or in concentrated
solutions.
On the contrary, the search for synergists of c-mitotic activity has
yielded important results.*^'"' -'^ Some synergists act mainly by increas-
ing cellular i:)ermeability to the alkaloid, and the reader is referred
to tlie paper of Deysson-'^ for a detailed study of this type of false
synergism. It has been observed only in plant cells. In fibroblast
cultures, Lettre has conducted a very large series of experiments, and
has discovered that many substances increased the action of colchi-
cine, though having no c-mitotic activity of their own. These syner-
gists belong to the most dissimilar groups of chemicals: alkaloids,
steroid hoimones, and carcinogenic agents (benzopyrene) . The
Mechanism of Colchlclne-Mitosis
419
amount of the synergist is always far greater, on a molar Ixisis, than
that of colchicine. For instance, while 5.5 mitoses per hundred were
found after 0.01 mg/ml of colchicine, the addition of 5 mg/ml of
Inilbocapnin increased this figure to 23.8. Forty times this dose of
bulbocajinin had no action on control cultures. With phlorizin the
results are very striking also.
More than 8 times more mitoses are arrested when a solution of
phlorizin, which has no antimitotic action, is added to a concentration
of colchicine, which is only weakly antimitotic. Ihis is truly a syner-
gistic eflect. "' Its study may most probably increase oiu- knowledge of
the physiological action of colchicine, and further work along similar
lines with different types of cells is to be expected.
Another interesting colchicine synergist has been reported by P.
Rondini and A. Necco (Tumori, 39:161-63, 1953). Italcliitie, an
acridine derivative, is itself a mitotic poison, affecting spindle and
chromosomes. Small doses, which do not affect mitosis, increase
markedly the action of colchicine on chick fibroblasts cultivated ///
xntro. Ihe principal results are apparent from Table 17.4.
ly.^-^: The role of adenosme-lriphosphoric acid (ATP). That
the spindle functions, partly at least, as a fibrous contractile structure
has l>een aliirmed repeatedly. The contraction which takes place has
TABLE 17.4
Synergic Action of Italchine and Colchicine on Tissue Cuitures of
Chick Fibroblasts
(Mitoses counted after 48 hours' incubation with the drugs)
(After Rondoni and Necco)
Substances and
Concentrations
Pro-
phases
Meta-
phases
Ana-
phases
Telo-
phases
Total
Italchine (1/300,000)
2.5
18.8
5.03
7.7
34.03
Colchicine (0.0033 ;ug /ml) . . .
4.9
42.3
8.86
5.06
61.12
Italchine + colchicine
(same concentrations)
0.5
79.4
2.15
1.07
83.12
Controls . .
4.9
13.8
8.00
10.5
37.2
also been compared to that of muscle. While biochemical data about
the nature of the spindle proteins are lacking entirely, it could be
imagined that colchicine acted on the contraction mechanism. Most
cytological data (cf. Chapter 2) point to an action on the fibers them-
selves, which can be observed to "dissolve" into a "pseudospindle" or
"hyaline globule" luuler the inlhience of the alkaloid. In muscular
420 Colchicine
contraction, the role of ATP is well known. Observations of colchi-
cine synergists and theoretical considerations led Lettre to suppose
that ATP may also be indispensable for spindle contraction and
mitosis, and that colchicine acted on the cell by modifying this
mechanism. •^'•*
Experiments in vitro demonstrated that strong concentrations of
colchicine inhibited the viscosity fall of complexes of actomyosin and
ATP.'" It was further observed that ATP-ase w^as inhibited by col-
chicine at concentrations of lO-^ and 10^ M. However, more dilute
solutions (lO-'^Af), which arrested mitosis, did not affect the en-
zyme."^^
A direct antagonist action of ATP and colchicine was difficult to
j^rove, because of the rapid destruction of ATP in fibroblast cultures.
Only with very small doses of colchicine was such an antagonism
visible. Cultures were grown for 24 hours, and then colchicine, at a
concentration of 0.04 mg/ml was added. *58 This arrested, after 24
hours, 55 per cent of the cells in mitosis. When 1 mg/ml of ATP was
added at the same time, mitotic inhibition did not start until four
horns later. The results are given in Table 17.5. It is concluded that
the higher the amount of ATP in a cell, the smaller the action of
colchicine, and vice versa.*''-'
ATP may play an important part in the conservation of cell form
in cultured fibroblasts. The "resting" cells have been considered to
be in a condition of permanent contraction, while cells intoxicated
with various drugs, such as Victoria blue, have a lower content in
ATP, and display a rounded form with rapidly moving surface blebs.
If ATP is added to a fibroblast culture, the cells assume a spindle
shape, even when dividing. In this condition, ATP would provide
the energy necessary for this contraction, and would also protect the
sj)indle against mitotic poisons. ^'^
This hypothesis is only a tentative one, and it is not yet proven
that colchicine acts by depressing ATP in the cells. Further experi-
ments will be needed to explain the relation between cellular respira-
tion and the formation of the spindle fibers, and also between ATP
and the physiology of the spindle. It is apparent that more funda-
mental knowledge about the dynamics of mitosis is needed before the
effect of colchicine and its various synergists may become clear. While
these effects are still difficult to understand, there is no doubt that
the discovery of the colchicine-mitosis has provided a considerable
impetus to such fundamental studies.
17.6: Conclusion: the Singularity of Colchicine
From this chapter it has been made evident that destruction of
the fibrillar properties of the spindle, and mitosis arrest at metaphase
Mechanism of Colchicine-Mitosis
421
or pro-metaphase, is by no means limited to colchicine or even to
chemical agents. From some angles, it appears as an entirely non-
specific reaction of metaphase to agents as different as cold, nitrogen,
hydrostatic pressure, lipid-solublc hydrocarbons, or heavy metals.
However, that it is in most cases more than a "narcotized" mitosis
is evident from the data about sulfhydryl groups, colchicine deriva-
TABLE 17.5
Percentage of Mitoses After Colchicine and
Adenosine-triphosphoric Acid (ATP) in
Cultures of Fibroblasts
(After Lettre and Albrechti^*)
Hours
Colchicine
id. + ATP
1
2.0
2.0
2
7.7
3.0
3
11.2
3.3
4
13.0
5.0
5
16.4
8.3
9
27.4
9.4
14
38.4
23.2
tives, and synergic activities. It is also evident at this point that fur-
ther progress will only be possible when the biochemical and physio-
logical properties of the spindle are better known. Mitotic poisons
are useful tools for this purpose, and it may well be that the solution
of this problem will lead rapidly to an understanding of the {proper-
ties of colchicine. The difficulties of this task are great, and resemble
in many aspects those of the study of muscle contraction. The spindle
structure is however relatively simple, as far as can be known at this
time, and its contractility and reversion to a nonfibrous "hyaline
globule" are problems of which a solution appears possible in the
not-too-distant future.
Colchicine, from all that has been said in this chapter, must be
considered a singular substance. Not only does it possess remarkable
side-effects, such as its action on gour, the colchicine-leukocytosis, its
action on the nervous system and on muscular contraction, its induc-
tion of specific malformations in embryos; it is also the most efficient
and active of all mitotic poisons known — with the exception of de-
rivatives of the colchicamide series. It is also the mitotic poison to
which the largest amount of work has been devoted. While some
substances like podophyllotoxin have received great attention, others,
such as the arsenical derivatives, have hardly been studied from the
angle of mitosis. It is not because colchicine was one of the first-
discovered spindle poisons that it received such attention. Chloral
422 Colchicine
hydrate, acenaphthene, and arsenic may have deserved more detailed
studies. Colchicine was investigated from such diverse standpoints
because it was not only a mitotic poison like others, but also an ideal
tool for the study of growth, and, last but not least, the best poly-
ploidogenic agent in plants. As the creation of new polyploid species
was taken up with enthusiasm, chemists and morphologists studied
more and more the structure and the properties of the alkaloid. It is
probably more than mere chance that the vmique structme of this
tropolone derivative is associated with so many physiological activities.
It is reasonable to prophesy that colchicine will long retain its
prominent place in the vast chapter of mitotic poisons. Many ob-
servations point towards a high degree of specificity in the reactions
between the alkaloid and the spindle; if these reactions covdd be
properly imderstood, that fimdamental process of all growth and
evolution, mitosis, would appear in a new light.
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Mctaplasma. Protoplasma-Monographien. 11. Gein'. Borntrager, Berlin. 1937.
106. SdiNHTZ, H. Zur Ikcinliussung des Zellstoffwechsels durch Alkaloid. Z. Krebs-
lorsch. 57:405-22. 1951. '
1U7. Sc;hmuc:k, \. The chemical nature of substances inducing polyploidy in
plants. C. R. Dokl. Acad. Sci. URSS. 19:189-92. 1938.
108. , AND Gi'SSEVA, A. .\ctive concentrations of acenaphthene inducing
alterations in the processes of cell-division in plants. C. R. Dokl. .\cad. Sci.
I'RSS. 22:441-43. 1939. Chemical structure of substances inducing jxilvploidy
in plants. Ibid. 24:441-46. 1939. The biological activity of isomeric com-
pounds. I. The action of isomeric naphthalene derivatives upon plants. Bio-
428 Colchicine
chimija. 5:129-32. 1940. Haloid derivatives of aromatic hydrocarbons and
their polvploidogenic activitv. C. R. Dokl. Acad. Sci. URSS. 26:674-77. 1940.
Metliowl deri\atives of benzene and naphthalene studied ^vith regard to their
polvploidogenic action on plants. Ibid. 30:639-41. Activity of polvploidogenic
compounds as influenced by hydrogenation. Ibid. 642-43. 1941.
109. ScHMUCK, A. AND KosTOFF, D. Bronie-acenaphthcne and brome-naphthaline as
agents inducing chromosome doubling in rye and wheat. C. R. Dokl. Acad.
Sci. URSS. 23:263-66. 1939.
110. ScHRADER, F. Data contrilniting to an analysis of metaphase mechanics.
Chromosoma. 3:22-47. 1947. Mitosis. Columbia University Press, New York.
1944.
111. ScHULER, H. M. Le probleme de la colchicine, substance stathmocinctique, en
relation avec ses proprietes physico-chimiques et spectrales. These. Uni-
versite de Strasbourg. Imprimerie Mont-Louis, Clermont-Ferrand. 1942.
112. Sentein, p. Arret de la segmentation, blocage de la mitose et polyploidie par
Taction de lethvlurethane sur loeuf de Batracien. C. R. Acad. Sci. Paris.
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113. SiMONET, M. AND GuiNOCHET, M. Obteutiou, par les a-monochloronaphtalene
et a-monobromonaphtalene d'effets comparables a ceux exerccs sur les caryoci-
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Sur I'apparition dans les tissus vcgetaux de cellules polvploides sous I'influence
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paradichlorobenzenicjues par un derive nitre des carbures cycliques: le m-nitro-
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118. Sullivan, B. J., and Wechsler, H. I. The cvtological effect of podophvlhn.
Science. 105:433. 1947.
119. SwANSON, C. P. The use of acenaphthene in pollen tube technic. Stain, lech.
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120. Tennant, R., and Liebow, A. A. The actions of colchicine and ethylcarbyl-
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Author Index
Abe. S., 338
Ahe<4g, F., 335
Al)raham, A., 335
Akerljerg, E.. 335, 342
Akciman. A.. 288. 335. 359, 371
All)o. G.. 154, 159, 172
Albrecht, M., 426
Alcaraz, M.. 312
Allen, B., 272
Allen, E., 19, 21 1. 221 , 249. 252. 253, 254,
388, 389
Allodiatoris. I., 213
Alweli, L.. 252
Amerio. G., 132
Amiard, G., 174
Amin, K., 312
Amoroso, E.. 17. 270
Ancel. P., 211
Anderson, A.. 154, 174
Anderson, E., 288, 312
Anderson, K., 313, 336
Andersson. E.. 315. 340
Ander\onl. H.. 270
Andres, G.. 62
Andres, J., 335
Arenkova, D., 335
Arloing. F., 198
Armstrong. }., 312, 335
Arnstein, H., 172, 173
Artschuager, E., 335
Arvy, L., 198
Aschkenasv, .\., 199
Ashlev. J.. 159. 172,386
Astakii.G., 211, 223, 225, 219, 270, 386,
422
Atwood, S., 288, 335, 344, 371
Avanzi, M., 133, 422
Avery, A., 21, 289, 335, 359
B
Bachniann, H.. 198
Back, A., 135. 198,422
Badenhnizen. X.. 335
Baer. F., 219
Baillif. R., 249
Bain, H., 336, 359
Baker, D., 249
Baker, R., 335, 359
BaJdensperger, A., 135
Baldnini. M.. 198
Bamford, R.. 313
Bane. A.. 61. 212, 388
Bannan, M., 335
Bannon, L., 137
Baranv, E., 422
Barber, H., 98, 115, 132,422
Barham, \V.. 331
Barigozzi, C., 98
Barnard, C., 422
Barnes, R., 198
Barros, R., 132
Bariek, J.. 158
Bartolncci. A.. 312
Barton, N., 162, 173
Bartosova, 1., 158
Bass. A., 270
Bassett, S., 199
Bastenie, P., 229, 233, 250
Bates, G., 288, 335
Batra, S., 335
Banch, R., 132, 422
Beal, G., 156
Beal, J., 288
Beams. H.. 75, 95, 98, 100, 132. 135. 211.
386
Beaslev, J., 288. 312, 335, 359
Beatty, R., 387, 388
Beer, A., 155, 157
Beikin. M.. 422
Bell, G., 288, 313
Bell, M., 273
Belleau, B., 155
Bellet, 157, 172
Belling. J., 57, 60, 288, 335, 345, 354, 389
Beneke, E., 132
Berger, C., 60, 85, 98, 133, 422, 428
Bergner, A., 21,60,98, 359
Bernardelli, E., 211. 249, 270, 386
Bernelli-Zazzera. A.. 270
Bernhard, \V., 250
Bernstrom, P., 335
Berrian, J., 250, 251, 387
Bertolotti, G., 198
Besson, S., 1,35
Bhaduri. P., 60, 98, 133, 335, 389
Bianchini, E., 253
Bichlbauer, U., 199
Bimes, C., 250
Biraben. J.. 1.31
Bishop, C.. 198
Black, A., 270
[429j
430
Author Index
Blakeslee. A., 21. 23, 133, 289, 316, 335,
354,356. 359. 360, 361, 371
Bla/ek. /., 155
Bloch-Fnuikcnthal, L., 270, 122
Block, W., 200
Bloom, W., 273
Boas, F., 98, 133
Boatnev, R., 133
Bock, H., 143
Boekelheide, \'., 174
Bogvo, T., 335
Bo'hn, G., 317, 336
Bohren, B., 212
Bond, L., 133
Bonner, W., 428
Bonnetli, E., 133
Bordonaro, P., 250
Boighetti, U., 247, 250
Bonrg, R., 270, 387
Bovd, J.. 133
Boves, J., 290, 313, 315. 332, 341, 372
Bovland, E., 133, 198, 270.422
Bovland, M., 133, 198, 270, 422
Bovle, D., 200
Bradlev, P., 133
Bragdo, M., 289
Brain, A., 98
Branch, C, 270, 387, 422
Braungart, D., 60
Brebion, G., 134, 424
Breckler, I., 249, 252
Bremer, G., 336
Bretscher, A., 211
Bretschneider, L., 198, 250
Brette, R., 271
Brewbaker, f., 288, 313, 389
Brierlev, P. ,336
Brock, X., 98, 211
Brodersen, H., 98, 270, 387
Broun, G.. 21
Brown, M., 289, 313, 315, 359. 371, 389
Brown. N., 270
Brown. W., 198, 270, 272
Briicke. E.. 270
Briies, A., 19, 21, 44, 57, 60, 98. 198, 216.
250, 253, 270, 387
Brumlield, R.. 60, 133, 270
Brunner, T., 134
Bryan, C., 21
Bryan, J., 155
Buchanan, G., 164, 173
Bucher. O., 30, 60, 99, 215, 216, 250, 387,
422
Buchholz, J., 355
Buchnicek, J., 152, 155, 157
Bulfinch, T., 21
Bullough, W., 220, 231, 250, 387, 422
Bureau, v., 60, 251, 387
Burkhart,Z., 219, 251, 387
Burrell, P., 60
Burrill, M., 251
Bursian, K., 161, 172
Buschke, W., 99, 387
Bushnell, R., 211
Busquet, H., 198
Cafiero, M., 133
Callan, H., 98, 422
Calvino, E., 336
Camara, A., 336
Cantarow, A., 200. 253
Capelletti. C.. 289
Caridroit, F., 198
Carlson, ]., 61, 66, 99, 289, 387, 388, 424
Carnot, P., 251
Carpentier, ,8., 423, 426
Carr, ]., 270
Carvalho, A., 339
Casady, A., 313, 336
Castelnuovo, G., 251
Castro, D., 336, 342
Cattelain, E., 155
Cavallero, C, 60, 244, 249, 251
Caventou, J., 158
Cech, J., 167, 173
Cernoch, M., 174
Chaigneau, M., 155
Chakravarty, A., 335
Chandler, C., 372
Chang, M., 60, 211, 387
Chapman, J., 273
ChargalT, E., 133, 134, 423, 124, 426
Chase, S., 336, 389
Chemnitius, F., 159, 172
Chen, S., 336
Cheng, K., 64
Chevallier, P., 270
Chevremont, M., 423
Chin.T., 313. 371
Chodat. F., 133
Chodkowski, K., 251,423
Chopinet, R., 313, 336
Chopra, R., 21, 155
Cisnev, M., 22, 163, 173
Clark, W., 198
Claus, P., 99, 271
Clausen, J., 276, 281. 289, 313. 336. 359,
371
Clausen. R.. 289. 313, 359
Clavton, E., 313
Clearkin, P., 270, 387
Clewer, H., 155, 159, 172
Cohen, A., 15, 21, 60, 98. 173, 198, 250,
253, 389
Cohn, C, 201
Colin, M., 270
Collins, J., 360
Colombo, G., 211
Comandon, J., 60, 133, 387
Conant, C, 20
Conger, A., 389
Conn, J., 200
Constantin, T.. 133
Constantinesco. D., 273
Author Index
431
Constantinesco, M., 273
Cook, J., 15, 21, 60, 99, 155. 1()2. 163. 161.
170, 172-73, 423
Corfield, C, 157
Cormack, R., 133
Cornil, L.. 133
Cornman. I.. 99, 133. 123
Cornman, M., 99, 423
Coufalik, E., 158
Courtois-Suffit. 198
Cowdry, E., 272
Crane,M., 2.S9. 336
Crisan, C, 60. 99. 134
Cro.ss, G., 359
Csik, L., 61
Cua, L., 289, 313, 336, 371. 389
Cuanv. R.. 313
Culting. W., 252
Czekalouski. J.. 133
DallHo, R.. 336
Dalla. X., 132
D'Amato, F., 60. 99, 133. 423
DAncona. V., 251
Dangeaid, P.. 133
Dan'ielsson. B.. 336. 338, 360
Daoust. R.. 253
Darlington, C... 289, 389
Dai row, G., 289, 313, 336, 359
Darwin. C 17
Das. B., 371
Das Gupta. C, 198
Da\ id, M.. 99
Davidson, J., 423
Da\ies. E., 155
Dawson, A., 100, 212
Dawson. R.. 336
De Castro, D., 99, 336
Decoux, L., 336
DeFonbrune. P., 60. 133. 387
De Lam, H., 426
De Lamatcr. E., 423
Delay, C. 291
Dekourt. R., 29, 60, 99, 198, 246. 251
Delioux, 198
De Mol. W.. 340
Denissenko, 270
Deodikar, G., 313
Dermen, H., 21. 60. 114, 133. 270, 289,
336,359, 371,389
Desclin, L., 251
Deshmuk. M.. 290. 316. 341, 360
De Vries, H., 318
De Vries. J., 169, 174
Dewar, M., 15, 168, 173
De\vey, V.. 273
Deysson, G.. 60. 62, 99. 100. 133. 136.
156, 423. 426
Devsson, M., 134,423
Dicker, S., 198
Dickinson, L.. 270
Dickson. G., 173
Dighv. 279
DiGuglielmo. 1... 249
Di Ouattro. C:.. 200
Dirschel. W.. 234. 235. 251
Dixon, W., 17, 184, 186, 187, 198
Dobzhanskv.r.. 289, 313, 3.36
Dode, M.. 99
Doering, W.. 21. 174
Doig, J., 336
Dol'hv, D., 133
Doljanski.L.. 100.200
Doolev. T., 99
Dornfeld. E., 250. 251, 387
Dorsev, E., 336
Dorst,' J., 289
Dott. D., 155
Doutre, L., 134
Douwes, H., 313, 3.36
Downing, \'., 270, 423, 425
Doxev, b., 424
Dragoiu, J., 61,99, 134
Drochmans. P., 61
Druckrev. H.. 98. 211
Du Bilier, B,. 271
Duff, J.. 201
Duhamet, L., 134,423
Dusseau, A., 289, 313. 336
Dustin, A., Sr., 17. 21. 24, 25, 26, 27, 29,
61. 99, 216, 217, 218, 240, 251, 255,
256. 271. 388.423
Dustin. P., Jr„ 22, 61, 99, 134, 177, 184,
185, 19'8, 270, 271, 272, 289, 313, 336,
387, 388. 423. 424
Duvvene de Wit, J., 198, 250
Ebner, H., 423
Ehrenberg, L.. 99
Eigsti, O.'. 18, 19, 20, 22, 25, 37, 61, 99,
108, 134, 289, 313, 336, 364, 371, 389,
424
Einset, J., 359, 371
Eisa, E., 250, 387
Ekdahl, I., 372
Eklundh, C. 338
Ellerstrom, S., 372
Emsweller, S., 289, 313, 336, 359, 372, 389
Erickson. R.. 424
Ernould, L., 337, 372
Estelmann, W., 425
Estes, S., 213
Euler, H., 134
Evans, A., 337
Evans, T., 60, 75, 98, 211, 386
Eyster, W., 337
Fankhauser, G., 61, 388
lanta. P., 164, 173
lantoni, L., 98
Fardv, A., 289, 313, 316
432
Author Index
Fanar, G., 156
Farren, A., 158, 171
Fatalizade. F., 313, 424
Felfoldv. L., 135
Felix, M., 422
Ferguson, F., 183, 199, 388
Ferguson, J., 424
Fer'nholz. H., 167, 169, 173
Fierz. H.. 254
Finn, E., 156
Firket, H., 423
Fishberg, M., 387, 388
Fitzgerald, D.. 422
Fleischmann, W., 249, 251, 387, 424
Fogg, L., 270, 387, 422
Foreman, D., 253
Forlani, R., 314
Foster. C, 99
Foinment, P., 155
Frahm-Lelivcld. J.. 337
Frandsen, K., 289, 314, 317, 337, 372
Frank, H., 164, 173
Franzke, C, 360
Franzl, R., 134,424
Fred, L., 199
Frei, W.. 134
Freud, J., 199,251, 388
Friedenwald, J., 387
Friedlander, R., 199
Friedrich, A., 174
Fromageot, C, 423
Fiihner, H.. 14, 175, 195, 199, 388
Fukushima, E., 314, 337
Funke, G., 134
Furusato. K.. 337, 344
Fiuukaiti, S., 337
Fwa-TiMig, 1,., 155
Fyfe, J., 389
Gaal, G., 155
Gabaev, G., 337
Gabriel, M., 211,388
Gal, E., 424
Gardner, D., 17, 22, 214, 249
Garner, W., 198
Garofalo, F., 134
Garrigues, R., 134, 271, 424
Garrod, A., 14, 22
Gatz, A., 252
Gaulden. M., 61, 66, 99, 289, 388, 424
Gavaudan, \., 61, 99, 134
Gavaudan, P., 20, 22, 27, 61, 99, 134, 424
Gay, H., 100
Gay-Winn, N., 22, 99
Geiger, P., 157
Geiiing, E., 201
Geissler, G., 134
Gelber, S., 62
Gelei, G., 61
Gellhorn, A.. 273
Gerassinuna, H., 340, 389
Gerstel, D., 314, 360
Gineste. D., 252
Ginsberg, D., 174
Giordano, A., 99
Gistl, R., 98, 133
Glotov, v., 314, 337
Goehausen, M., 21
Gohar, M., 134
Goldberg, R., 409, 424
Golubinskij, J., 337
Gompel. G., 424
Goodspeed, T., 289, 313, 314, 336
Gordon, W., 135
Gorini, P., 270
Gorter, C, 128, 129, 130, 135
Gottlieb, D.. 137
Grace, X., 135
Graham, W., 162, 173
Grampa, G., 99, 271
Granel, F., 252
Graner. E., 337
Granhall, I., 337
Greadick, R., 249
Grebel, M., 21
Greef, H., 174
Green, J., 137, 360
Green, "S., 159, 172
Green, W., 271
Greene, E., 22
Greene, R., 251
Gregoire, C, 17, 22, 60, 271, 423, 424
Gregorie, C, 252
Gremling, G., 106, 135
Grewe, R., 170, 173
Grier, J., 155
Grimme, C, 155
Gross, R., 143, 148
Grun, P., 344
Guichard, A., 271
Guinochet, M., 135, 428
Gunther, R.. 22
Gurtl, 206
Gusseva, A., 427
Gustaffson, A., 60, 61
Giithert, H., 252
Gutman, A., 199
Gutsche, C, 174
Guver, M., 99, 271
Guver, R., 155
Gyorffy, B., 314. 337
H
Haas, H., 99, 199, 212
Haase, E., 136
Hadorn, H., 62, 100. 212
Hager, V., 21
Haggquist, G., 61, 212,388
Hakansson, A., 372
Halberstaedter, L., 135
Hall,T., 171,212,388
Author Index
433
Harland, S., 314, 337
Harris. J,. 159. 172. 38(5
Hartmair. \.. 337
Hartwell, J.. 174, 270. 423, 425
Hartuig, E., 169, 174
Hausemann. ^V., 135, 199. 388
Hauswalci, R.. 200
Havas. L., 20, 22, (il, 79. 99, 135, 199 '>r^
271,289,424
Haukes, J.. 62, 99, 135
Hawkins, J., 424
Hawkins, S., 424
Hecht, A., 337
Hcidusclika, A.. 155
Heinzlcr, J.. 157
Heise, F.. 135
Heitz, E., 135
Hejtmanek, M., 155
Hellinga, G.. 337
Hcllman. L., 21, 199
Herken, H., 98. 211
Herlant, M.. 239, 241, 242, 252
Hertwig, O., 424
Hertwig, R., 424
Herzog, H.. 156
Hession, D.. 249
Hiesev, \\'.. 276
Higbee, E., 62
Higgins. G., 253
Hill, H., 289, 337, 360
Hill. K., 341
Hiravoshi, I., 337
Hirobe, T., 62
Hirschfeld, J., 271, 272
Hirschler. H., 174
Hitier, H., 313
Hoffman, F.. 253
Hofnieyer, J., 337, 372
Hollaender, A., 100
Hooper, E., 155
Horning, E., 166, 173
Horning, M., 173
Horowitz, R., 155. 172, 173, 174. 388 424
Hoscalkova, Z., 158
Hosoda, T., 314, 337
Hottcs, C, 19
Houde, A., 14, 157, 175, 176, 425
Howard, H., 314, 338
Huang. H., 172, 173
Huant, E., 271
Hiiber, E. v., 270
Hudson, P., 289
Hughes, A.. 99, 425
Hunt, H., 201
Hunt, T., 252
Hunter, A., 338, 360
Hunziker, J., 314
Huskins, C. 289, 360
Hutchinson, C., 212
Hutchinson, J., 314
Hwang. T., 136
Hyde, B., 338
I
Inamori, Y., 317
Illcnvi, A., 133
Inoba, F., 62
I none, S.. 314,425
Inoue. v.. 338
Isch-Wall. P.. 271
Iwasa. S., 314
Iyengar, \., 314
Jack. J.. 170, 173
Jackson, E., 57, 60. 99, 250. 270
Jacobj, C., 14, 175, 199, 388
Jacobson, T., 199
Jacobson, ^V., 425
Jadassohn, \V.. 254
Jahn, v., 62. 100, 212, 388
Jailer. J.. 252
Jakol), H., 135
Jakob, K., 314
Janaki-Ammal, E.. 338
Janot. M., 155
Jarctskv, R., 338
Jenkins. \V., 212
Jennison, N., 135
Jensen, H., 338
Jennstad, A., 155
Johannv, S., 172
Johansson, E., 315
Johnson, I.. 338
Johnson, M., 250, 387
Johnson, T., 359
Johnsson. H., 338
Johnston, T., 159, 173
Johnstone, F., 315
Jones, D., 20
Jorgensen, C, 57, 289
Josefsson. A., 344
Joshi, A., 341
Journoud, R.. 388
Julen, G.. 338. 372
K
Kadlec. K.. 200
Kahan. J.. 199
Kahn. .S.. 251
Kallio, P., 135
Kanter, M., 100
Karapetyan, S., 155
Karivone, T., 155
Karpecbenko, G.. 62, 279, 290, 309, 314
Karsniark, K., 155
Kartashova, N., 135
Kasparyan, A., 314, 338
Kassner, H., 155
Katterman. G., 360
Katz, J., 200
Kaufman, B.. 100
Kawakami. K.. 340
434
Author Index
Kavser, F., 135
Keck, D., 276
Kedharnath. S., 315, 338
Keeser, E.. 425
Kehr,A„ 290, 314,338, 372
Keibl, E., 199
Keim, W., 313
Kelsev, F., 201
Kemp, A., 173
Keppel, D., 100, 212
Kerns, K., 360
Kerr, T., 212, 252
Khoshoo, T., 22, 156
Kidder, G., 273
Kicllander, C, 338
Kihara, H., 290, 314, 317, 33U, 331, 338,
360, 372, 389
King, C. 135
King, H., 425
King, J. Jr., 155
King, K., 155
King, L.. 271,425
King, M.. 169, 174
King, R., 60, 98, 100, 135
Kirkpatrick, H., 155
Kirsclibauni, A., 272
Kishimoto, E., 338
Kisselew, W., 157
Kjellgren, K., 212
Klein, E., 271
Klein, G., 155. 159, 172, 271
Klein, H., 199
Kline, I.. 425
Kneedler, W., 271
Knox, L., 21, 174
Knutsson, R., 63, 427
Kobavashi, T., 342
Kobozieif, N., 134, 272
Kolda, J., 1.56
Kolmer, W., 135, 388
Kolthoff, I., 1.56
Kondo, N., 314, 315, 338
Kondo. v., 338
Koo. J., 173. 174
Kosar, W., 343
Kostoff, D., 135, 307, 314, 338, 372, 425,
128
Kramer, \V'., 271
Kremers, E., 22
Krishnaswamy, N., 339
Kropp, K., 251
Krng, C., 339
Krythe, J., 62, 131, 135, 290, 315, 372, 425
Kuckuck, H., 290, 372
Knhn, A., 156
Kumar, L., 339
Kurivama, H., 343
Kuzell, AV., 252
Laborde, }., 425
Lacour, l.., 389
Lafay, B., 263, 272
Lahr, E., 19, 252
Lallemand, S., 207, 208, 211. 212
Lambers, K., 199
Lamm, R., 317, 339
Landolt, R., 199
Landschiitz, C, 425
Lang, A., 372
Lang, B., 158, 174, 252, 388, 426. 427
Lang, K.. 135, 425
Langeron, L., 198
Langham, D., 339
Lapin, V.. 315, 339
Lapslev. R.. 173
Larsen. P.. 3.39
Lattin. G.. 339
Lannoy. L., 156
Laur, C„ 136
Lauter, W., 155
Lawrence, C., 173
Layani. F.. 199
Lazure\skii. G.. 156
Leblond, C., 199, 252, 253, 388
Le Camus, H., 272
Lecomte, J., 199
Lee, T., 136
Lefevre. J.. 136.425
Lehmann. F.. 62, 100, 212, 245
Leibbolz, 199
Lein, J., 136
Leiter, J. ,270, 423,425
Lenegre, }., 271
Lesi:)re, 206
Leslie, L, 423
Lettre, H., 22. 100, 135, 136, 169, 171, 171,
252.271. 388,425
Levan, A.. 21. 22. 28. 49. 62. 100. 101. 113,
136, 138, 290, 338, 339, 341, 360, 372,
389, 390, 426, 427, 428
Levin, M., 199
Levine, H., 199
Levine, M., 62. 136, 271
Levine, R., 201
Levring, T., 136
Levy, M., 157
Lewis, D., 289, 339
Lewis. M., 426
Licbtman, 148
Liebow, A., 63. 101, 253, 273. 428
Lilienfeld. F., 314
Limarzi, L.. 200. 272. 426
Lindstrom, 57
Lipova. J., 158
Liptak. P., L56
Lison, L., 424
Lits, F., 17, 22, 61. 62. 79. 100. 199. 212.
272, 388
Little, T.. 339
Livermore, ).. 315
Loeper, M., 272
Lofgren, N., 99
Loicq. R.. 200
Author Index
435
Loo, T., 136. 343
Lorthioii. 1'.. 200
Lorz, A., 315
Lotfv, T., 62, 100. 136
Loudon, J., 15. 21. 60, 99, 155, 156, 159,
162, 164. 165, 168, 170, 172, 173, 423
Louis, L., 200
Ludford, R.. 17, 22, 62, 100, 216, 252,
272, 425. 426
Lumsden, D., 313, 336
Lusclier, .\L, 62, 100, 243, 245, 252, 388
Lushi)auo;h. C. 271
Lutko\ . A.. 339
Lyons, A., 156
Lysenko, T., 62. 290. 315
M
Macak. \.. 158
McFadden. E.. 290. 297. 299, 315
MacFailane, E.. 426
Mack. H., 156
McKechnie. ].. 135
McKinnev. G.. 426
McKinnev, H.. 313
McKracken, J.. 200
McLeman, H.. 312
MacMillan. J., 164. 173
McPhail. M.. 200. 253
Magasanik, B.. 423
Mainx, F., 426
Mail-old, F., 136, 426
Majumdar, G., 22
Makkawi. M.. 134
Maiden. W., 184, 186, 187, 198
Malhotra, S.. 339
Maliani, €., 315
Malinsky, J., 158, 174. 252, 388, 426
Mallet, L.. 272
Mangelsdoif. P., 290
Mangenot. G., 34. 41. 62. 96. 100. 136,
426
Manlev, T., 389
Mann. H.. 200
Mann, L.. 372
Man ton, I., 314
Manus, M., 253
Marble, B.. 250, 270
Marchal. 117
Martens, P., 339
Martin, G.. 62. 100. 1.36
Martinez, L., 136
Martini, A., 156
Mascre, M., 62. 100. 136. 15(i. 12(i
Masima, I., 339
^L^sin()\a, W, 157
MasleiHiikova, V., 156
Matsubayashi, G., 290, 341
Matsumoto, K., 315
Matsumura. .8., 315, 339, 344, .360
Mauer, F., 315
Mauri, C., 249. 270, 386
Mav. R.. 251
Meguro, I.. 341
Me'hlquist. G.. 339
Mehra, P., 22, 100. 136, 1,56, 3.39
Meier, R., 143, 148
Meisner, N., 155
Melander, Y.. 62. 100, 389
Melchers, G.. 314
Mendes, A., 290. 315, 339
Mendes, L., 339
Mcnetrier, P., 272
Menschikow, G., 157
Menzel. M.. 304, 313, 315
Mever, H.. 148
Mever, J., 62, 389
Mever, k.. 160. 172
Mills, K.. 100. 212
Mis/urski, B.. 100, 200
Mi/ushinia, U., 315, 340
Modlibokska, 1.. 339
Moeschlin, 148, 272
Mokrantza, ^L. 156
Mol. W. de. 63, 136
Mollendorff, W. v., 63. 100
Monrov, A.. 100, 212
Montalenti, G., 100, 212
Morato-Manaro, J., 253
Moreau. F.. 22
Morgan, D., 340. 389
Morrison, J.. 156
Motizuki. A.. 315
Mouzon, M., 199
Mrkos, H., 372
Muendler, M., 340, 372
Mugler, A., 200
Mulilemann, H., 156
Mailer. H., 352
Miintzing, A.. 136, 279. 290, 315, 340. 360.
366, 372, 389
Murray, NL, 315. 340. 360, 426
Miisotto, G.. 200
Myers, \V., 289, 337, 340, 360, 372
N
Nadkarni, M.. 425
Xakajima. G.. 340
Xakatomi, S., 340
Xakayama, K., 156
Xathanson. I., 253
Xaundorf, G.. 136
Xayalikhina, X., 315
Xayashin, M., 340, 389. 427
Xebel, B., 20, 22. 63, 87. 100, 137 21"
290. 316. 342. .390
Xegodi. G., 340
Xcipi:). 1 18
Xeniec, B., 427
Xewcomer, E., 137, 340
Xcwton. 345
Xitholls. G., 169, 173
Xichols, C., 63
Xickell, L., 137.427
436
Author Index
Nicod. J., 272
Niemann, E., 156
Nihlsson-Ehle, H., 286, 340
Nihous, M., 137
Nilsson, F., 312, 315, 340
Nishivama, I., 290, 317, 338, 340, 360, 372
Nodule, G., 372
Nogiiti. v., 315,341
Noidcnskiold, H., 290, 315, 341, 372
North, E., 156
Northen, H., 137, 427
Nybom.N., 61,63, 427
Nygren, A., 341
Ohaton. P., 137
01)crlin, L., 157
Oka, H.. 315, 341
Okuma, K., 341
Olden. E., 337
Ollivier, H., 1,37
Olnio, H., 341. .360
Oksson. G.. 310. 341, 360, 372
Olsson, P., 339
O'Maia, J., 63, 137, 360, 372
Ono, T.. 341. 360
Oomen. H., 341
Orsini, M., 137. 127
Osgood, E., 389
Osol, A., 156
Ostergren, G., 62. 63. 100, 136. 137. 426.
427
Oswald. H., 425
Ott, G.. 60
Ott-Gandella, A.. 138
Oughterson. A., 271. 272
Owen, P., 200
Pair, G., 212
Pal. B., 290, 341
Paletta, F., 272
Palis, A., 422
Pansini. R.. 427
Panskv. B.. 137, 427
Pappo, R., 174
Parini, F., 247, 250
Parker, J., 173
Parks, F., 273
Parnientier, R., 272, 389
Parr, L., 137
Parihasarathy, N., 156, 290, 315, 338, 311
Paschkis, H., 156
Paschkis, K., 200, 253
Patton, R.. 137
Paul. J., 272
Pearson, O., 315, 390
Pease, D.. 427
Pellegrini, G., 251
Pellegrino, J., 100,205,213
Pelletier, P., 158
Pennington, F., 174
Pepinskv, R.. 169. 174
Perak, J.. 315, 341
Perje, \I., 134
Pernice, B.. 17, 39, 63, 427
Perrot, E., 156
Persai, D., 342
Pesez, M., 174
Pesola, v., 315
Peters, J., 43, 63, 100, 389
Petit, A., 174
Peto, F., 290. 315, 332. 341, 372
Pevron, A., 263, 272
Philippe, L., 271
Pienaar, R.. 341
Piettre, L., 63, 100, 137
Pincus, G., 63, 100, 212
Pirschle, K.. 341
Piton. R., 29, 272, 427
Podi\ inskv. R., 158
Poewer, F., 157
Politzer, G., 427
Pollauf, G., 155, 159, 172
Pomerat, G., 253
Pomriaskinsky-Kobozieff, N., 22
Poppe, \\'., 315
Postma, W., 137
Potesilova, H., 158
Pottz, G., 137
Poiilsson. K.. 272
PounieauDelille, G., 263, 272
Poussel, H., 99, 212
Praaken, R., 137
Pratt, J., 200
Probert, C, 270
Pundel, M.. 253, 389
Q
Quincy, J., 13
Raffauf, R., 158, 171, 174
Rajan.S., 341,360, 372
Rakoff, A., 253
Ramanujam, S., 290, 316. 341, 360
Randolph, L.. 58, 63, 290. 341, 372, 390
Rapoport, H., 22, 163, 171, 173
Rappleye. R.. 340, 389
Rasmusson, J., 341, 390
Raw, A., 316
Rawson, R., 253
Ra\niond-Haniet, 200
Rebaudo, G., 249
Reese, G., 63, 100, 137
Regemorter, D. van, 393, 127
P-egnier, P., 174
Regnier, V., 198
Re'hi)ein, M., 199, 200
Reichstein, T., 148, 157, 158, KiO. 172,
174, 389, 427
Reynolds. S., 249
Author Index
437
Rhazes, 12
RiclKiids, O., 137
Richharia. R., 342
Richmond, T., 290, 316
Riddle. O., 252
Ries, E..45, 63, 100,212
Rol)cro;, M.. ir)6
Rol)inson, \V.. 200
Rochette, 158
Rodenhister. H., 360
Roe, E., 15.21, 173
Rogers, P., 253
Rojahn, C, 156
Rondanelli, £., 211, 270, 386, 422
Roosen-Runge, E., 212
Roques, H., 155
Rosen, G., 390, 424
Rosenberg, 333
Rosendaiil, G., 137
Rosenfeld, M., 427
Rosenthaler, L., 156
Ross, H., 342
Ross, J., 360
Rossanda, M., 201
Rossbacii, M., 200
Rossi. S.. 200
Rotlienberg. M.. 253
Rudorf, AV., 316, 342
Rufelt. B., 341, 360, 372
Ruflilli, D., 272
Ruhe, D., 137
Runquist, E., 136, 340
Rutile, M., 20, 63, 87, 100, 137. 212. 289,
290, 316, 337, 342, 390
Rybin, V., 342
Rvland, A., 100
Rzaev, M., 317
Saccheti, C., 253
Sacharov, \., 290, 342
Sachs, L., 313, 316
Sageret, 309
Saito. K., 342
Salfeld, J., 174
Salgues, R., 156
Salomon, E., 342
Sampavo, T., 342
Sando,'W., 342
Sandwall, C., 136
Sanno, V., 200
Sansome. E., 137
Santavy, F., 15, 22, 100, 137, 143, 144, 146,
147, 148, 154, 155, 156, 157, 158, 160,
167, 170, 172, 173, 174, 200, 389, 427
-Sargent, L., 427
Sass, J., 137, 338, 360,427
Satina, S., 356, 360
Sato, D., 137, 342
Savignac, 198
Sawak. K., 157
Sawver, M., 360
Sax, K., 63, 427
Schafer, G., 156
Schairer, E., 272
Sthar, 143, 148
Schauman, A., 389
Scheiblev, C., 253
Schenk, Ci., 338, 342
Schildt, R., 342
Schjeide. O., 272
Schlosser, L., 342
Schmidt, F., 200
Schmidt, I., 253
Schmidt, W., 427
Schmit/. H., 427
Schmock, N., 426
Schmuck, A., 427
Schnack, B., 342
Schnell, L., 342, 349, 390
Schrader, F., 428
Schreiber, G.. 100, 205, 213
Schrock. O., 316, 342
Schroft, K., 200
Schuldt, E., 137
Schuler. H., 157, 428
Schuanitz, F.. 340. 342
Scott, D., 336. 342
Scott, G., 22, 172. 173. 174
Sears, E., 290, 299, 315, 316, 360, 372. 390
Sedar, A.. 101
Seed. L., 200. 272
Segal, G., 199, 388
Seifert, R., 157
Seldam, B., 273
Self, P., 157
Seligman, K., 174
Selve, M., 200
Sengbusch, R.. 342
Sentein, P., 22, 101. 213. 253. 273, 389,
428
Seris, L., 22
Setala. K.. 101. 273
Shalvgin. 1.. 342
Shapiro, D., 273
Sharp, G., 22
Shear, M., 270, 273, 423, 425
Shifriss, O., 342
Shimamura, T., 63, 101, 137, 342
Shimotomai, N., 342
Shorr, E., 253, 389
Show-alter, R., 343
Siebenthal, R., 138
Siebert, G., 425
Simonet, M.. 291, 316, 342, 428
Sinoto, v., 138, 342
Skipper, H., 273
Skoog, F., 22
Slaughter, D., 200, 272
Slou'f, A., 155
Small, L., 427
Smith, G., 19, 249
Smith. H., 290. 311, 316, 342, 361, 372,
390
438
Author Index
Sinith.L., 316, 342, 361,372, 390
Smith, P., 138,214,428
Soetarso, B., 273
Sokolow, L, 63, 101
Solacolii, T., 273
Sorkin, M., 160, 172, 428
Soulier, J., 271
Soyano, V., 101, 138
Sparrow, A., 138, 342
Speakman, J.. 156, 159, 172
Sreenivasan, A., 13,S
Srinivasachar, D., 31G
Stair, E., 343
Stalfelt, M., 138
Stebbins, C, 276, 291, 301, 316, 313 37'^
390 ■ ■ "■
Steenken, \V., 135
Stefaiiott, B., 8, 22, 157
Steiger, A.. 158
Stein, K., 253, 254, 389
Steinberg, R., 138
Steinegger, E., 63, 101, 136, 138, 343 379
389.426,428 ' "'
Stephens, S.. 291, 316, 343, 372
Sterzl, J., 138
Stetten, d. W., 200
Stevens, C, 252, 253
Stevenson, E., 331
Stewart, R., 337, 343, 359, 372, 390 4'>3
Stockert, K.. 169, 174
Stomps, T., 343
Storck, 13
Stout, A., 372
Straub. J., 138,343
Streckcr, H.. 423
Strong, L., 272
Strosselli, E., 422
Stiirte\ant, F., 138
Subbaratnam, A.. 158
Sugawara, l., 341
Suita, N., 63, 101, 138
Sullivan, B., 428
Sullivan, M., 271, 425
Sundeil, B., 389
Su/uka, O., 157
Svardson, 213
Swaminathan, N., 314, 316, 343
Swanson, C, 63, 428
Sweeny, \V.. 21
Sydenham, I., 14
Tabata, H., 343
lahmisian, I., 101
Takcnaka, V., 138, 343,361
Takewaki, K., 253
Talas, M., 158
Talbott, J., 198
ramaYo,'A., 312
Tandon, S., 343
Tang, v., 136, 343
Taran, E.. 157
Tarbell, D.. 22, 161, 164, 172. 173, 174
Tatsinni, J.. 201
latuno. S'., 343
Tavlor, H.. 336. 364, 371
Teir. H.. 253, 389
Tennant, R., 63, 101, 253, 271 ''7'^ '^73
428 ' ~ '
Tennev. B., 61. 289. 336
Thales-Martins. 254
Theophrastus. 1 1
Thimann, K., 428
Thorn. C, 138
1 homas. P., 273
1 homas. T., 249
I hompson, iM., 270
Thompson, R., 343
Thompson, W., 316
Thornton, C. 254
Tischler. G.. 291
Tislowitz, R., 254
Tjio, J., 390
Tobias. J.. 428
Tobler, R.. 156
Tominaga. "W. 343
I'onzig. S.. 138
4 oole. M., 343
loro, E., 254, 428
Toxopeus, H.. 316, 343
Trankowskv, 385
1 rastoin-. 198
Traub. H.. 157. 291, 343
Tschermak. E., 138
Tuchmann-Diiplessis. H., 253
Turesson. G.. 291. 343
Tutin, F., 159, 172
u
Ubatuba. F.. 138
I'chikawa, L, 343
I'eda. R., 139
I'elinger. E., 254
I'flelie, O., 157
I fter. A., 173
Ullyot, G.. 1,55, 158, 166. 171 17'^ 173
174,388,422,424,425
Umney, J., 157
Tmrath, K., 138
Vm-du, J.. 316. 361, 372
Ihbani, E.. 213
L'rdang, G., 22
Uveki, E., 198
I'yldert, I., 199, 388
Vaarama. A., 63, 10], 138, 316 313
Vaccari, F.. 201
\adasz, J., 254. 428
\ alleau, W., 316
Vandendries, R.. 138
\'anderwa]lc, R., 138
Author Index
439
Van Heeiswviif^hels, J., 201
Vail Ooidt.j., 250
Van Ros, (;.. 213
\avilov, N.. 2S(;, 2*)1
Ventnri, V., 157
Verne, J., 101,251
Vibert. C, 201
Vietez, E., 137. 138
Villars. R., 273
Vilter, v., 60, 63. 101. 254. 387, 389
Vies. F., 157
Vollnier, H.. 201
w
Waila, B.. 23. 63. 101. 1.3S. 171, 171. 291.
390, 428
Waddi nekton, C. 63, 100,212
Walas/ck. E.. 198.201
Walker. A., 1.39
Walker, (,.,313
Walker, G. N.. 173
Walker, J., 424
Walker, R.. 101, 111. 118. 138
Walklins, A.. 200
Wandrekar, S.. 138
Wang. P.. 139
Warburg. O., 428
Warmke, H., 316, 343, 361
Warren, L., 23
Warren, S.. 271
Waseilewski, W. \.. 428
Waterman. A.. 213
Wavmoulb, C, 423
Webb, M., 425
Weber, F., 136. 138, 139
Wcchsler, H.. 428
Weddle. C. 343
Weichsel, G., 139. 344
Weinland. R.. 157
Weiss, R., 273
Weisse, G. \.. 157
Weissenbock. K., 139, 344
Weizmann, A., 158
Welds, C, 213
Wcllensiek, S., 23, 62, 290, 291, 315, 317,
372, 390, 425
Werle, E.. 157
Werner. G.. 139
Wertb. E.. 157
Westendorft. W.. 340
Westergaard. 352. 361
Wettstein. 57
Wexelsen, H., 278, 291, 343, 372
Wcyland, H., 139
Wbitaker, T., 317
Widmann, H.. 201. 428
Wilbur. K.. 101,200. 213.253
Williams. A.. 22. 163. 171. 173
Williams, I., 23
Williams, W., 254, 389
Williamson, G., 273
Wilson. 87
Wilson, D.. 101
Wilson, G., 64, 132
W^ilson, J., 249
Wimsatt, W., 213
Windaus, A., 15, 161. 172. 173
Winge, 278
Winkler, H., 57
Winkler, R., 148
Wirtzung, 13
Witkus. E., 60, 85, 133. 1.39, 422, 428
Wokcr. H.. 64, 101,213
Wolcott, G.. 139
Wolf. O., 251
Wolfson, W., 201
Wolsky, A., 213
Woodside, G., 273
Woodward, M., 23
Woodward. \ .. 213
Wortbington, R.. 254
Wulf. w'.. 170. 171
^akuwa, K., 317
N ainada. Y.. 317
^amaguti. V.. 344
^amaiia. G.. 139
Namasaki, M., 344
Vamashita. K.. 338, ,344. 372
\'oumans. G.. 139
\oung. G., 315,341
\oung, M.. 133
Vii, T., 199
Yuasa, A., 138
Zajickova, A., 174
Zambruno, D., 133. 139
Zebrak, A., 287, 291.317, 344
Zeebuisen, H.. 157
Zeisel, S., 14, 158, 159, KiO, Kiy, 172, 171
/bnrbin. A., 317, 344
Zilliken, F., 251
/irkle. R., 273
/vllicrsac, S., 61, 99, 233. 250. 251
Subject Index
Abiine, 206
Acenaphthene, 398. 400
c-mitotic potential, 104
c- tumor, 104
compared to colchicine, 82
Acetocarmine methods, 19, 27. .^7, Aii. 3fi9
Acetophenone, 400
Acetyl chloride, 171
N-Acetvlderivati\e, 16.3
Acetvlamido-i;roiip. 160
N-Acetvl-colchicol, 408. 409
N-Acetvlcolchinol, 161. 167, 169
N-Acetvlcolchinol methyl ether, 161
N-Acetvliodocolchinol, 161. 164, 167
N-Acetyl-TMCA-methvl-ether, 408
P-Acetamidotropolone, 171, 27.")
P-Aminobenzoic acid, 397
P-Aminohippuric acid, 397
Achromatic sphere, 25, 27, 79, 80, 84
Acnida tamariscina, 353
Acriflavine, 193
ACTH; see adrenocorticotropic hormone
Action of temperature, 374
in birds, 374
in mammals, 374
Actomyosin, 420
/Vddison-Biermer anemia, 210-1 1, 224
.\denosine-triphosphoric acid (ATP) ,
419; see also \IY
.\drenal cortex, 177, 193, 226, 230, 232,
regeneration of, 241
Adrenal medulla, 230
Adrenal mitosis, 193
.\drenals, 226
Adrenocorticotropic hormone (ACTH) ,
197, 226, 229
Adrenalin, 181, 413
.Achentitious buds, 351
Aegilops, 295-99
species of: caudata, 291; cylindrica, 297;
squarrosa, 295-98; umbellulata x
Ha\tuildia villosa, 299
sterile triploid hvbrid, 295
Agranulocytosis, 2(J4
Agrobacterium , 121, 122
Agrofjyron, 295, 299
species of; glaucum, 298; i)ilennrdiinii.
298; triticeum, 295, 298
Alarm-reaction, 177, 178, 190, 197. 376
Alexander of Tralles, 12
.■\lkaline hydrogen peroxide, 167
j)hosphatase, 397
Alkaloid, 7, 160, 167, 179,418
classification of, 1()0
reagents, 160
Allergy, 197
Allium, 19, 69, 80, 403, 413, 116; sec also
C-mitosis
achromatic sphere, 91
c-mitotic threshold, 101
c-tumors, 102, 103, 110
cepa photomicrographs. 25-27, 79
rem num. 1 12
colchicine and X-ray, 267, 268
mitochondria in root tip. 26-27. 91
root tips. 51, 78. 81. 83, 84. 90, 94, 1U2,
104, 395, 399, 407
J//ocolchiceine, 167
,4//ocolchicine, 167
structure of, 169
Allomyces jai'aniciis. 123
Allopolyploid species, 367
Allo-syndesis, 276
Alloxan. 241,244
Alnarp Horticultural Station, 312
Ambhstoma
deyelopment, 203
opacum, regeneration in, 242
pinictatiun, regeneration in, 242
American Society for Horticuliuial
Science, 329'
Leonard H. \'aughn award, 329
Ami no-acenapht bene, 400
Aminocolchicine, 169
Aminopterin, 265, 415
Ammonia, 392
Amoeba, 395
species of; proteus, 126; sphaeroiiu-
cleus, 35.58, 126, 381
Amphiasiers, disintegration of, 74
Amphibia, 54, 68, 375
temperature and colchicine poisoning
in. 194
Amphiploidy
Aegilops, 294-301
Aegilops scjiiarrosa, role of. 29(5
Agropwon. 297-99
lirassica, 309-10
Bromus, 302
cataclysmic e\olution among. 277
classification of, 288
conyergent evolution, 299
Cruci ferae, 309-10
defined, 276
[441]
442 Subject Index
Amphiploich {(oiitiiiucd)
divergent evolution of hexaploids, 299
dysploidy superimposed on, 277, 309
ecological range of, 292
Elymus, 302
evolution by, 277-80
ferlilitv, 371
Galeopsis tetrahit synthetic species.
279
gametic doubling of sterile hybrids,
294
genomes of species comi)ined by, 310
Gossypiuin, 302-6
Gramineae, 294-302
hexaploid wheat, 295
hvbridization by-passing sterility
barrier, 284, 362
implications of, 292-94
intergeneric hybrids converted to, 362
intergenomal exchange prevented by,
292-93
interspecific hybrids. 310-12
interspecific segregation, 286, 308
limits between autoploids and, 292
list of, 309
Melica. 302
multivalents, 308-9
natural and experimental, compared,
303, 307
new species, 285, 292, 303
Nicotiana, 288, 294, 307-8
origin of new species, 278
origin of wheat, 295
pairing between genomes, 293
pairing of chromosomes, 281
pairing of hybrid and polyploid. 30S
Priinuhi kcivensis, 278
Rapluntobmssica, 279, 293
Ribes )iiii)olayia, 312
segmental allopolyploids, 281
significance of, to breeders, 293, 312
Sitanion, 302
Solatium, hybrids, 31 1
somatic doubling, 294
Spartiiia tuxvnsendii. 279
stability index of, 300
sterility changes, 281, 362
Stipa. 302
success of. 292-93
Tritictiles. 300-301
Triticinae, 297-99
Triticuin aestivum, 279
Triticuin spelta, 295
weight of seed, 367
Winges' hypothesis, 277-79
Anachromasis, 32, 51
Anaerobic glycolysis, 183
Analecla ])harmacognoslica, I 10
Anaphase, 80
Anaphase hi idges, 97
Anaphylactic shock, 197
in guinea pigs, 197
influence of colchicine, 197
Anatomical malformations, 275
Androgens, 232, 234
Androstendione, 235
Androsterone, 235
Anemia; see Addison-Biermer anemia
Anemia, hemolytic, 184. 185
Anesthesine. 400
Anesthetic properties. 180
Anethol,413
Aneuploids, 354
Aniline. 400
Aniostropical nature of fiber, 75
Anodonta, experiment with egg, 202
Antagonism, 378, 396, 415, 418
Anterior pituitary extract, 226
Antibiotics. 394
Anticlinal division. 350
Antifolic drug: see Aminopterin
Antirrhimun, 319
afterellects of colchicine in, 115
intervarietal tetraploid, 319
intravarietal tetraploid, 319
meiosis, 1 15
susceptibility of, 1 16
Apigenin. 117
Apiol.413
Apolar mitosis, 86
Apple, .351
Arabian medicine, 2, 3
d-Araboascorbic acid. 418
Arbacia. ()9. 74, 79
colchicine applied to eggs, 87, 377
egg, 202, 203
puru liilata. 74-75, 87
Arbacia, lakelike body in. 74. 79. 88
Archiroh liicuin. 8. 9
Aretacus. 1 1
Aristohx Ilia eleiroiis. 113
Aromatic compounds. 400
Arsenic, 415
derivatives of, 257, 392,411
Arsenious oxide, 41 1
Arterial constriction, 179
Artificial insemination, 59
Ascites-tumor, 259, 377
1-Ascorbic acid, 418
Ascorbic acid, in tumors, 260, 261
Aspergillus, 123
Asterias forhesii, 88
Astral ray, 74, 77
Asymmetrical development. 203
Asynaptic genes. 305
genie sterilitN, 305
in Gossypiuin, 305
partially asynapti( . 305
sterility, 305
ATP, 18l', 420. 421
Subject Index 442
Atiactoplasni. 73
Atropin, 181
Atiopin sulfate, 186
Anlacomnium audr()g\nuiii . 119
Autodiploid, 31^1.333
homozygous, 333
from monoploid, 333
Autoploidy, 318-35
abbreviated auto])oU|)l()i(h . 27()
appearance of, 363
autotetraploids as, 318
in barley, 321
borderline types between amphiploid,
281
in carnation, 326
categories of, 292
classification of, 280-82
comparison with diploid, 319
defined. 276
disadvantage in seed nunil)er per
plant, 326
doubling of diploid, 278
drug content increase, 368
ecological characteristics, 370
Ehrharta erecta, 301-2, 370
fertility reduced, 281, 371
fiber improvement, 326
forage production compared, 368
Fragaria vesca, 323
fruit and seed, 363-67
genomes interchanged. 292
gigas characters, 325
grapes, 324
guard cell sizes, 364, 369
improvement by, 324
increase proportional to jjloidv, 328
index for, 319
larger flowers, 326
less fertile than parental diploid, 28^
lilies, 325
Lolium, 323
maize, 321
making of, 278
marigold, 326
mean diameter of pollen, 369
Medicago, 322
microscopic characteristics, 368
morphological differences, 320
muskmelons superior, 325
pairing of chromosomes in, 281
Phlox, 364
physiological differences, 367-68
poin.settia, 326
pollen sizes, 319, 368-69
raw polyploids, 321
reversion to diploid, 285
rubber increase in Hei'ca and
Kohsagliyz, 326
seed weights of, 365-67
sex determination, 352-54
snapdragons, 326
Solanuin, 325
Sorghum, 323
steel rye, 319-20, .366
sterility of, 281
sugar beet, 331
technicjues for making, 383-84
Trifolium, 322
use of colchicine to make, 328
vegetative character differences. 320
I'iiica rosea, 326, 349
watermelon parental-types for trip-
loids, 327-31
Auto-svndesis. 276
Autotetraploids. 278. 318-26, 328
barley, 321
empty grains. 32(S
fertility correlation. 321
flowers larger, 328
maize, 321
meiosis, 321
morpholog) , 320
pollen larger, 328
practical value, 319-20
3 A' seed, 328
seeds larger, 328
segregation of, 321
steel'rve, 319
stomata, 328
testing performance, 319
thickness of seed, 328-29
triploid pollen, 328
A\icenna. 12
Azaguanine, 265
Azide, 181
B
Bacillus mesentericus, 121, 122
effect on growth, 121
Bacillus tumefaciens, 265, 266
effect of colchicine, 265-66
gall formation. 265
tiMiior formation, 266
Bacillus typhosus, 260
action on timiors, 260
extracts, 260
Bacterium megatherium, 122
Barbiturate, and colchicine toxicitw 179
Basal cell carcinoma, 264
Basic dves, 392
Bats, 194
Benzene, 400
X-Benzenesulphonvl trimethylcoldii-
cinic acid, 160
N-Iienzoylcolchide, 166
N-Benzoyl-colchicinic anhydride, 410
X-Benzovlcolchinic anhydride. 166
X-Benzoyl-TMCA-methvl -ether, 408
\ Benzoyltrimethvkolchicinic acid,
165,408
444 Subject Index
V
N-Benzoyl-t)iinethvlcoklii(iiiic methyl
ether, 409
N-Butykolchicamide, 1 1 1
Benzenoid ring, 161
ring A, 161
Benzoic acid, 400
Benzopyrene, 261, 418
Beryllium salts, 412
Beta T'a/i^rtm
characteristics, 355
diploid, 355
increased vigor, 333
production of sucrose, 332
root lieet weight, 332
seed production of, 332
triploids, 331-33
trisomies, 355
Bile ducts, 248
Bile elimination, 196
Bile secretion, 191
Bimctaphase. 42, 43. 45, 83, 84, 97
bipolar metaphase, 40
compared vvitli distributed c-mitosis,
42, 45, 46, 97
confused with c-anaphase, 45—46
recovery stage, 83
Binomial system of nomenclature, 7
relationship of Colchicum, 8
in Species Plantar\im. 7
Binucleate blastomeres, 203
Biologies, 140
Biopsies of human neoplasms, 259
Bipolar mitoses, 97
bipolar metaphases, 40
bipolar spindle, 93, 96
Birds. 190
Birefringence patterns, 75, 89
tracing of s])indle disappearance, 76
Blackberries. 324
polyploid series, 324
Black Sea, 3, 4
"Bleb" formation in nerol)lasts, 91
Blocked cleavage, 74, 87, 89
critical point, 87
inhibiting cleavage, 88
role of concentration, 88
Blood
chemical ciianges after colchicine, 183,
193, 191
defibrinized hog's blood, 195
Blood cells, white; see Leucocytes
Blood clotting, 194
Blood glucose le\el, 375
Blood level, 196
Body temperature, 194
Bone
endosteum, 247
periosteum, 247
repair, 247
Bone marrow, 176, 178, 183, 184, 189, 223,
376, 377, 380, 383
aplasia, 184
Botanical Review. 20
Botiytis cinerea, 123
Boveri, 87
Boysenberrv, 324
Bmssica, 309
comparium of. 309-10
species of: canipestris, 310; carinata,
310; cbinensis x ^- carinata, 310;
juncca. 310; nigra, 310; oleracea,
310
British Empire Cotton Research Station,
288
British West Indies, 288
Bromi nation
of colchiceine. 169
of colchicine. 169
of tropolones. I(i9
B ramus, 302, 365, 367
seed weight, 365
species of: carinatus, 302: carinatus-
trinii, 302; catharticns, 367; cathar-
ticiis-haenkeanus. 367; haenkea-
nus. 'Mil: liaenkeanus-staniineus,
367; inarilinius-trinii, 302; mar-
jinetus, 302; stamineiis, 367
Bronchioli, 181
Brownian mo\ement, 72, 89
Brush treatment, 384
Biifo vulgaris, 203
abnormal development of, 204
Bulhina buJbosa, 413
Buli)()capnin, 419
Bull)ocodiae, 9
Bulbocodiuni L., 8
ruthenicum, 143
source of colchicine, 143
species name, 143
Bulbs, 384
treatment of, 384
C-mitoses, 380
C-metaphase I and II, 114
unoriented. 97
C-mitosis, 18, 26, 28, 74, 83, 84, 86, 92, 188
in Allium, 36, 41
c-pairs, 4, 9; see also C-pairs
chromosome contraction independent
of, 46
concept of, 21-26
consequences in plants, 57
in contrast to mitosis, 31
cycles, 95
described first by Pernir.:, 18, 39
description of, 28
distributed, 42, 45, 84, 97
distrilnited melapliase, 84-85
chiration of, in animals, 48-50
exploded c-mitosis, 84-85
exploded metaphase, 42, 84-85
full, 51
Subject Index 445
limited in animals, 56
liver of rat, regenerating, 44
octoploidy, 95
onion root-tip, 34
other methods, 57
percentages, 30, 34
jjollen tube, 37
progression sequence, 55
recovery in animals, 56
recovery in plants, 56
reiteration, 55-56
restitution nuclei following, 57
setiuences, 31
tissue cultures, 56
in Triton, 40
in Triturus. 40, 43, 97
C-pairs, 25, 48, 85, 86, 95, 114
/i//n/m, 25, 41,49, 79, 85, 96
c-anaphase stage, 49
chromosomes dechromatizing, 85
cruciform, 47, 49, 85
evolution of, 49
neuroblasts, 66, 70
pairs of "skis," 49
photomicrograph of, 37, 79, 85
in pollen tubes, 37, 86
around pseudospindle, 84, 85, 91, 96
relational coils, 49
in Triturus, 43
C-telophase II, 115
conclusion of c-meiosis, 115
C-tinnor, 398, 416
proportional to concentration, 105
Cadmium, 415
salts, 412
Caffeine, synergisms \\'ith colchicine, 181
Callus tissue, 274
formation of, 274
Camphor, 122
Cancer, 17, 19, 255; see also Carcinoma,
Sarcoma
Ehrlich, in mouse, 258, 262, 267
Flexner-Joijling, rats, 261, 266
gastric, in man, 267
mammary, in mouse, 258
Yale carcinoma, 267
Cancer chemotherapeutic tests, 374
Cancer chemotherapy, 17, 260-65, 110
in man, 263, 265
Cannabis sativa, 368
marihuana content, 368
Carbohydrate metaljolism, 222
Carbon tetrachloride, 247
Carcinogenesis, 257, 269, 270
Carcinogenic agents, 418
Carcinoma
Brown-Pearce, 265
Ehrlich, in mouse, 258, 262, 267
Flexner-Jobling, in rats, 261, 266
gastric, in man, 267
mammary, in mouse, 258
Yale carcinoma, 267
Carcinoma, mammarv gland
in man, 2() 1
in mouse, 258
Carnation, 326
polyploids, 326
Carpel development, 350
effect of colchicine, 110, 350
Carrier of colcliicine, 383
Cartliainus tiiutorius. 110, 127
flowers, 110
Gynoecia, 110
ovules, 117
petals, 110
pollen grain, 115
pollen mother cells, 131
reduction, 115
Castration, 232
pituitary mitoses, 232
Cat, intracerebral injections of colchi-
cine in, 179
Catachromasis, 44, 45
Cataclysmic evolution, 277-80
examples, 279
origin of new species, 277, 278, 292
Cell '
permealiility, 418
surface changes, 202
type of, 81
Cell organites
centrosome, 90
mitochondria, 90
Cell plate formation in plants, 89
continuous fibers, 89
destruction by colchicine, 89-90
phragmoplast, 89
prevention, 89
rudimentary forms, 89
septa, 89
special technique, 89
wheat root -tip cells, 90
Celosomy, 206, 208
Central nervous system
effect on, 179
paralysis of, 178, 179, 404
Centrifugal wall, 90
Centrifuge tests
Allium root-tip, 90
cyclosis in Elodea, 90
cytoplasmic, 90
effect on viscosity, 90
Centriole, 83
Centromere, 47, 49, 83
non-di\ision of, 380
Centrosomes, 90, 204
Cereals, 319
Cerebral edema, 178
I7-Cestosteroids, 197
Chaetopterus pergaiuenlaceus, lb
egg, 82
meta])hasic spindle of egg, 76
Chelidonine, 255, 264, 41.3
Chclido)iiuiit niajus, 264
446 Subject Index
Chemotherapy of cancer; see Cancer
chemotherapy
Cheyenne, Wyoming, 311
Chiasmata reduced
crossing o\er, 113
frecjiiency decrease, 113
terminalized, 113
Chiasmatal frequencies, 386
Chick egg, malformations of, 206, 209
Chilomoiias, 216
Chimaphila nianilata
extracts of, 113
influence on mitosis, 413
Chimera, 350, 356, 384
induced bv colchicine, 348
periclinal,'350, 356, 385
reproduction of tetraploids, 350
sectorial, 351, 385
Chinese \vheat, 358
Chlainydoinouas, 126
Chloracetophenone, 412
Chloral hydrate, 392
Chloralose anesthesia, 183
Chlorethone, 402
Chloroform, 373, 398
Chorionic gonadotropic hormone, 217.
218, 226
Chortoplioga I'iridifasiata. 90. 377
embryos, 377
neuroblast cells, 66, 71-73
Chromatin bridges, 203
Chromatographic analysis, 153, 159
Chromatophores, 191
Chroniodoris sp., 88
Chromosomal breakages, 414
Chromosomal class, 355
Chromosomal orientation, ecjuatorial, 77
in Arbacia. 11
m Allium, 25, 79, 85, 96
destroyed by colchicine, 37, 43, 44
mechanism blocked, 65
neuroblasts, 66, 70
pollen tulje, 37, 108
in Triticuin, 95
Chromosomal pairing
diploid level, 281
measure of fertility, 28!
measure of homolog\. 281
polyploid leyel, 281
value of, 281
Clnomosome; see also Spiral coiling
of chromosomes
aberrations, 54
acetocarmine methods for, 19, 386
agglutination, 52
ball metaphase individuaUtv, 52
birefringence of, 75
breakages, 268
iM-idges, 203
c-pairs, 45, 85
carbon tetrachloride poisoning, 52
chimeras, 356
clumped, 240, 379
colchicine-treated, 47
Cold Spring Harbor studies, 19
contraction autonomous, 46-50
cruciform type, 47-50
destruction. 53
desynchronization of, 51
distribution in triploid meiosis, 329
distributions, 86
doubling of, 278
duplications, 37
e\olution of, 46-50
extra, 345-47
fibers of, 77
fragmentation, 416
fusion of. 52. 256
independent of c-mitosis, 46
intactness period, 46
lagging, 83, 346
lost, 40, 83
mammalian cells, 47
meiosis, 1 1 1-15
meiotic contraction. 113
metaphase, 37
micronuclei in mice liver cells, 237
nucleus and, 24
niunber of. in Colchicuin species, 9-10
ninnbers increase, 25
optimal luunbers of, 283
pairs of "skis," 49-51
phlox. 364
plants doubled number, 25-27
poison, 392
pollen tube. 108
polyploidy induced, 20, 274-75
precocious reversion, 47, 49, 73
prophase, 31-5
prophase arrangement, 35-42
pseudospindle, 79
pycnosis, 68
rearrangements, 268
reiteration of c-mitosis and numbers
of, 55-56
relational coiling;, 47-50
repulse each other, 41
restitution nuclei, 17-59
reversions, 108
scattered, 40—11
seedless fruits and, 329
sex determination. 352-51
star formations, 39, 40, 41, 43
stickiness, 47
structure altered, 52-55
studies of, 386
tetraploid numbers, 52
tetrasomics, 354
transfer of, 286
transformation of, 30-31
transmission of, 386
transverse division, 53
trisomic pairing to measine homology,
281
Subject Index 447
trisomies, 345-47
in Tiibifex, 53
I'inra pollen mother cells, 349
watermelon, 329-31
X and y, 352-54
Chronica Botanica, 287
Ciba, of Basel, Switzerland, 143
Cinematography, 67, 91, 378
Cladnphora, 119
Cleavage, 87
in eggs, 202, 203, 204
Cloacal epithelium, 226, 227
Clonal division method, 300
Closterium, 119
Cliii) root disease
in radish, 370
Cokhicamide, 409
Cokhiceinamide, 409
Cokhiceine, CiH.sOeN, 14, 16, 160-61,
167-69, 171, 404, 408,411
singularity, 420
structures, 168
tautomerides, 168
yields isomeric methyl ethers, 168
Colchicine
action of
algae, 124
Arbacia, 74-75
blood level, 196
circulation, 193-94
cleavage, 87-89, 202-3
differentiation processes, 125-27
feathers. 190-93
heart, 138
kidney, 191-93
liver, 191
liverworts, 1 18
meiosis, 110-17
mosses, 117
nervous system, 179
spindle. 65-98
striated muscle, 180-81
advantages o\er other agents, 275
agar impregnation, 377
Allium treated, 34, 41
anaphase, 45
anatomical changes in plants, 129-31
anesthetic properties, ISO
and anticarcinogens, 269
archesporial tissue, 110
bacteria treated with, 121
biological properties, 373
bivalents of c-meiosis in jilants. 1 10
cancer chemotherapy, 265
carbon-labeled derivatives of. 171. 196
cell plates inhibited, 89, 90
cell size, 103
chemical action concept of, 403-16
cliemistry of, 15. 159-74
chromosomal pairs, 28, 37, 41, 43, 49
chronic intoxication after re|)eated in-
jections, 193
in mice, 189
in rabbits, 187-88
classified disturbances on spindle, 86
clea\age processes, 75, 87-89
compountls of, 144—48, 153-54
concentration. 383
conversion of spindle form, 65
critical time-dose relation of, 34
crystallization of. 159
derivatives of, 15-17, 111-47, 159-74,
394
destruction of spindle fibers, 69-78
effect of temperature. 374
toxicity, 194
effect on bacterial enzymes, 417
effect on enzyme reactions. 131
diastase, 131-32
elimination of
bile, 196
in excretions, 194
intestine. 196
embryonic growth, 209-1 1
e\olution of cpairs. 49
experimental gro\\ th, 214-19
fad in research, 16. 21, 274
fate in animal body, 195-96
fixation in tissues. 245-55
gonads. 202-3
gout treated by, 196-98
hormotie-mimetic actions, 190-91
induced mutant seedlings, 55
and induction of:
amphiploidv. 292-311
aneuploid. 345-47
autoploidv, 318-33
chimeras, 348-51, 384
polyploidy, 274-75
inhibition of spindle, 68
inhibition of fall of complexes of acto-
myosin, 420
interaction with, at molecular level, 65
intramedullary injections, 264
leucocvtosis after, 183, 185, 189
light effects on solutions of, 154, 374
local applications of
on embryo, 209
on tumor, 263
male gametes changed by, 204-5
malformations in chickens bv. 206. 207,
208
mechanism of reactions upon mitosis,
391-421
megaspore mother cells, 1 10
meiosis following, 103. 110
metabolism effects induced b\ , 191 96,
395-96
methylation of, 171
mutagenesis by, 275. 318
named, 14
neoplastic growth changed bv
iir animals, 214-47
in plants, 269
448 Subject Index
Colchicine (continued)
other than Colchicitm producing, 150
parts of plants yielding, 144—17
pharmacology of, 175-80
pharmacological properties, 373
pharmacy of, 148-53
physical action to explain mechanism
of, 399-403
plant tumors and, 265-66
plants containing, 141-42
poisoning in man by, 175, 176, 178
pollen mother cells, 110
pollen tubes in, 107-9
polyploidy in plants; see Polyploidy
polyploidy in animals, 58-59! 380-83
purity, 159
radioactiye compounds of, 196
recovery from effects of
in animals, 56, 96-98
in plants, 56, 94-96
regenerating liver after injection of,
44,57
reversibility after, 91-98
singularity of, 420-22
solubility of, 159, 275
solutions of
for animals, 373-74
for plants, 383
sources of, 141-45, 150
specificity of reactions of, 67
spindle form changes, 78-81
technique for use in animals, in vitro
bone marrow, 377
duration of mitosis in, 379
ear of mouse, 377
eggs of Arbacia, 377
grasshopper embryo, 377
immature rat ovaries, 377
mitotic counts after using, 378-80
mitotic index, 380
tissue cidtures, 377
technique for use in animals, in vivo
adrenal cortex, 376
Amoeba. 375
amjjhibians, 375
ascites tumor in mice, 376
bone marrow, 376
chick, 375
cold-blooded animals, 375
cornea of manmials, 376
duodenum, 376
ear of mouse, 376
eggs, 376
fish, 375
gastric mucosa, 376
genital tissues in rodents, 376
human vagina 376
intestinal crypts, 376
intestine, 376
invertebrates, 375
lymphoid tissue, 376
mammals, 375
pluricellidar animals, 375
red-cell-forming tissues, 376
regenerating liver of rats, 376
regenerating tissues in amphibians,
376
skin, 376
small rodents, 376
Xenopus larval tail, 376
technicjue for use in plants
buds, 384
chromosomes, 386
root systems, 384-85
seed, 384
seedling, 384
solutions required, 383-84
X-ray and, 266-67
Colchicine-mitotic dose
in animals,
cold-blooded, 96
cold-blooded vertebrates, 97
critical time-dose relations, 34-35
duration of interphase, 380
in fibroblast cidtiues, 30
injection methods, 35
lethality of, 96
mitotic index a measure, 378
period of latency, 379
regenerating liver of rats, 44
Siiedon. 97
stage of mitosis and concentration of,
70-73
tissue culture, 96
Triton. 40
Tri turns. 40
in vitro study, 377-78
fn vivo study, 375-77
warm-blooded, 96
Xenojnis. 97
in plants
concentration of 0.2 '/r for poly-
ploidy, 383
for repetition of c-mitosis, 55-56
length of treatment, 56
onion root-tip, 34, 41, 55
pollen tubes, 37
related to concentration, length of
exposures, stage, kind of cell, 38
in Tradescantia, 33, 69
Colchicinetinnor, 25, 102
autonomy of, 104
cell size, 103
growth -promoting substances, 104
hair cell of root, 102
hvpocotyl, 102
independent of c-mitosis, 102
isodiamctric enlargements, 103
pollen tube, 102
region of elongation, 103
root, 102
somatic cells, 102
Subject Index 449
slylar cell of pistil. 102
test of autonomy. 105
time of treatment, 105
\()liimcs, 104
Cokliifineamide, 409
Colchicinic acid. KiO
Colchicoside. 117, 172,398.405
Colchicuni
geographic distribiilion of. 111. 1 12
history of. 1-14
species of. 9-10
variously named, (i, 7
Colchicum, isolating compounds from
bv boiling water. 153
by chromatography, 153, 154. 159
corm, 153
by degradation. 153
detection in pollen germination. 153
flowers, 4, 153
fruit, 5, 153
methods, 153
by polarography, 153
properties, 153
seed, 153
Colchinol, 167
Colchinol derivative. 167
Colchinol mcthvl ether, 163, 170
Colchis, land of. 3. 4. 7
Cold-blooded animals. 379
vertebrates, 96, 374, 375, 399
Coleoptile, 384
"Coliform bacteria," 122
Complex crystals. 373
Concentration effects, 70-73. 7(i
Concepts of metaphasic arrest, 16-21,
2-1-26, 26-29
Conidial hypertrophy, 124
Connecti\'e tissue
mitotic stimulation. 230
Contractile force. 181
Contracture, 181
Convoluted tubules, see Kidney
Cooley's anemia, 224
Coprimis radifnis, 123
Corcgouus. pol\|)l()idy in, 210
Cork, in plant tumors, 266
Corm powdered, 2
Corms of Coh lii( iiiii . 3. 5
Cornea, 383
healing of, 248
mitosis, 409
tissue mitoses, 40, 43, 82-83
Corpuscular body of spindle, 25. 66. 79.
80, 85, 96
Cortical la vers, 74, 89
Cortin, 180
Cortisone, 197, 227
Cotton, 26, 279: see also Gossxpiinn
evolution under domestication. 279
Cossypiiini, anijjhiploids of. ,30 1
natural tetraploids, 283
in nature. 279
Sea Island cotton. 305
speeded up. 2^0
Uplaiul cotton, 305
Coumarin, 413
Cranberries. 324
cultivated diploid, 324
natural tctraploid, 324
sterde liexaploid, 324
Creatine, 397
Criteria for judging jiolvploidv in plants,
362-77
Crop-sac, in pigeon. 22(). 228
Cryptic structural hybrichty, 277, 305
compared to genetic hybridity, 305
Ciyptoearia pleuiospoia. 413
Crvptopleurine, 413
Crvstallization of colchicine, 159, 37,3
Cucuniis III el o, 325
Cucurbita, 325
amphiploid, 285
Banana, 31 1
Buttercup. 311
Butlenuit. 31 1
Ciolden Cushaw. 31 1
Golden Hubbard, 311
Gregory, 311
Ken'tuckv Field, 311
new species, 285, 325
species of: inaxiiiia, 325, 285; ,nos-
chala, 325, 285; mixta, 285, 311;
pcpo, 2S5, 311
Cidturing chambers, 385
Cumulative geometric effect, in \'ico-
tiaiui polyploids. 356
Cupaniae, 9
Cyclosis, in El odea, 90
Cvtogenetic changes, 93
Cvtokinesis, 73, 86. 88
spindle of. 89, 92
Cytological artefacts, 77
Cytoplasm, 74
Cvtoplasmatization. 71. 89
C^ toplasmic \iscosity. 90
D-genome, 296-98, 358
in Aegilops squarrosa, 296
amphiploiils. 29(i
isolating mechanism. 296
Datura, 19, 20, 345, 350, 354, 357
apical meristem, 350, 356
chimeras. 356
cytohistology, 350
2n — 1 delicient types, 345'
drug production, 368
fifteen-year breeding recoixl, 31()
frecpiency of diploid deficiencies, 34.5i
increase bv colchicine, 34.>
L chromosome, 34(>
450 Subject Index
Datura (continued)
seedlings, 20
standard line 1, 345
stramonium. 356
tetiaploid deficient types, 345
Day lilies, 326
Deamination, 397
Deaminocolchinic anhydride, 166
Deaminocolchiiiol methyl ether, 162,
163, 170
iodo form, 164
Deformed spindle, 27, 79, 80, 84; see also
Achromatic sphere
hyaline globule, 79, 80, 81
pseudosjjindle, 79, 84. 193, 394
Dehydroandrosterone, 235
Demecolcin, 143, 146, 268
Demethoxylate deaminocolchinol methyl
ether, 164
Demjano^v -type, 170
Denmark, 322, 352
Dephosphorylation, 397
Dermatogen, 348
Desacetylcolchiceine, 404
Desacetylcolchicine, 408
acetylation of, 171
Desacetyl-N-methyl-colchicine, 143; see
also Substance F
Desh\clrogcnase, 397
Desmethylcokhicine, 373, 405
in U. S. P. colchicine, 141, 373
Desoxyribonuclease, 397
Desoxyribonucleotides, 397
Desoxyribose nucleic acid, 121
Di-aldehyde, 1()2
Diaporthe perniciasa. 123
Diarrhea from colchicine, 178, 179
Diazomethane, 171
Di-benzoylation of trimetlnlcolchicinic
acid, 16()
Dicotyledons, 363
Diestrus, 231
Diethyldithiocarbamate, 412
Differentiation processes, 103, 125-31
microbiological material, 103
modified by colchicine, 103
in plants, 103, 127-30
in unicellulars, 103
Dihydride, 166
Dihydro-2-met by 1 naphthalene, 166
DiliUe mineral acids, 159
Dimercaplopropanol, 411, 412, 413
Dimethylarsinate, 411
Dinitrodiphenic acid, 163
Dioecious races, 352
diploid, 352
Dioscorides, 4, 5, 7
lK)tanical studies, 4, 7
Diox) phenylalanine-decarboxylase, 397
Dipcadi, 68, 84
prophase arrangement of chromo-
somes, 35
Diploid. 382
Diploid hybrid, 278
daughter nuclei, 93
diploid interspecific, 293
roots, 332
Diploidized type of polyploid, 282
Disappearance of spindle bire-
frigence, 75
rate of disappearance correlated with
concentration, 76
Discoglossus pictus orth., 210
Displacement of chromosomes, 90
Distorted star metaphase, 38, 39, 83
Distributed c-metaphase, 347; see also
Distriljuted c-mitoses
Distributed c-mitoses, 42-46
Diurnal variations, 220
mitotic rate, 375
Dog
first c-mitoses observed by Pernice, 18
leucocytes in, 187
Dominici, 19
Dried flowers. 2, 3. 7
Drosera, 333
Drosophila, 346, 352
Drug evaluation
assay methotis, 141
biological. 141
of crude drug, 151, 153
microchemical, 141
microscopic, 141
organoleptic, 141
physicochemical, 141
Drug traffic, 3, 6
Drugs, 326
anabasine of Nicotiana, 326
Dryopteris, species of: felix-mas, 119;
subpubescens, 1 19
Duodenum, 376
Duration of c-mitosis, 379
in animals, 48-50, 379
compared with mitosis, 91-94
delay in neuroblasts, 46
intactness period, 46
in pollen tubes, 47-48
Duration of interphase, 380
Dwarf wheat, 357
Dysploidy, 277
combined with ampiiiploidy, 309-10
in Cruciferae, 309
Ear of mouse, 376, 377
Ear-clip technique, 378
Ebers Papyrus, 1, 3, 196
Ecological characters and polyploids, 370
disease resistance, 370
range, 292
requirements, 319
seed production, 370
Ehrliarta erecta, 301-2
successful autoploid, 301
Subject Index 451
Electronic niicrosc()i)\, si
El odea. 132
Ely mils, 302
Embryo culture, 363
Enil)r)o of grasshopper, 55
Eini)ryo sac develo])nicnt. 103
in Carthamiis. 117-19
effect of colchicine, 118
embryo sac sterility, 38fi
enlargement, 1 17
stages, 118
in Tradescantia. 117
Embryonic extracts, 224
action on bone marrow. 224
in animals, 202. 211
gonads, 202
malformations in chick, 20()-9
niegaloblasts, 210
tool for growth, 209-11
Embryonic growth, moditicd h\
colchicine, 202-9
Euipetrum, species of: hennaphiodituni.
352; nigrum, 352
Emulsions. 383
Endocrine glands, mitotic simulalion of,
232-36
Endocrine kidney operation, 238
Endocrine tissues of pancreatic gland,
177
assay methods of, 19; see also Groi\ th
research on. 214-16
Endosteinii: see Bone
Endothelial cells, mitoses. 246
England, 324
Enolone system. 161
enolone-methyl-ether system. 161
enolone properties. 167
tautomeric enol s\stem, 160
Enzyme reaction, 103
Enzyme system, 395
Enzymes, 396. 417
Ephedra, 74, 92
Epiieineron, 6, 11, 264
of Dioscorides, 1 1
for tumors, 11, 264
Epidermal cell origin from apex. 350
Epi-inosose. 417
Epidermal mitosis. 379
Epidermis for tests, 377
Epididymis, 232
Epinephrine. 183
Epoophoron, 227, 232
Equatorial orientation of chromosomes;
see Chromosomal orientation
Errors in experimental procedure, 379
Eruca, 346
Ervatamia aii'^iislilolia. 413
Erythroblasia. 186. 188, 224
erythroblastic cells, 189
erythroblastosis, ayian, 262
Escherichia coli, 121. 122
filtrates of, 261
Estradiol, 226, 235
Estrogens; see Hormones
Estrus cycle, 220
Ethanol, 122
Ether. 401
Ethyl alcohol, 104, 402
Ethylairbamate (ethylurethane) , 101
Ethylcarbylamine, 412, 415
N-Ethylcolchicamide, 41 1
Ethyl-colchiceine, 407
Ethylmercurychloride, 412
Eucolchicum, 8, 10
Euglena, 126
Euphorbia peplus, 413
Eyulution in wheat, 295. 299
Aegilops, role of, 296-97
Agropyron, role of, 295-99
divergent and convergent, 299
origin of hexaploid, 295-300
Triticuin, role of. 295
Exocrine tissues, 177
Experimental cytology, 385
Explantation, 219
Exploded c-metaphase, 38-39, 44, 347
in Allium. 25, 79.85,97
in Arbacia. 7-1-76
described, 40-41
diagram of Triturus, 43
in neuroblast of grasshopper. 66
in pollen tidie, 37
in regenerating li\er, 44, 57, 70
Exponential decay curve, 77
measured by birefringence, 77
Extra chromosomal types. 346; see also
Aneuploids
Extra chromosome transmission. 386
Extraction methods for colchicine,
154-59
alcoholic, 154-59
chromatographic differentiation, 151
petrol ether, 154
Fall-blooming meado\v saffron, 4
Feather gro\\th. action of colchicine on,
190
Feces, after (ohhidne injection. 194
Feeding Hills, Massachusetts, 311
Female gametophyte, 386
Female sterility, 386
Fern studies
germination of prothalli. 119
pro t hall ia, 119
sperms, 119
sporogenous tissue, 119
Fertility, 371
of am|)hipl()ids. 293
of autoploids, 319-25
female sterility in watermelon. 371
meiosis, 371
percentage pollen, 371
452 Subject Index
Fertility (cnnlinued)
seed set, 371
triploid sterility, 371
Fertilizing agents, chemical, 275
Feulgen technique, 386
Feulgen-positi\e masses, in cytoplasm, 53
Fiber destruction, 77
Fibers, 140, 403
chromosomal, 67, 77, 81
continuous, 67, 77, 81, 83, 89
suppressed continuous, 77
Fibroblasts. 91
Fibroblast cultures, 30, 407, 419
of chick, 378
of mammals, 378
in tissue culture, 215, 378, 417
Filifoliae. 10
Fish, 375
Flexner-Jol)liiig carcinoma of rat, 90
Flour, tetraploid rye, 368
Fluoroacetate, 181
Folic acid antagonists of. 247
Follicular cells; see 0\ary
Forage production in clo\'er. 358
Forage species, 288, 321
Forensic medicine, 176; see also Medicine
Formidaries, modern. 140
pharmacy handbook, 141
Fragaria I'escti. 323
autotetraploids, 323
diploid, 323
hexaploids. 323-24
octoploids, 323
polyploids of, 323-24
Fraoments, 97
Fritillaria, 115
chiasmata and colchicine, 115
Frog
colchicine dosage, 382
deyelopment, disturbances of, 204
heart of, 183
oyary of, 192
pattern of gro\\'th in, 210
polyploidy in, 381-82
sperm sirspcnsion, 382
striated muscle of, 180-81
tadpoles, 245
temperature effects and colchicine,
194-95
toxicity study, 194-95
Fruit
of Colchiruiu, production in spring.
5, 6, 7
of Cucurhita, 31 1
grain weight in rye, 366
improyement of, 331
larger size of tetraploid, 363
parthenocarpy in watermelon, 329
pericarp extracts, 144, 146, 147. 154
pollination by diploid. 367
polyploidy, 323
of Rihes, a new species, 312
and seed, criteria of polyploidy, 363
size correlated to polyploidy, 367
triangidar. 365
of watermelon, 327, 328, 330
Furrowing, in animals, 88
Fiisarium niveum, 370
resistance to, in watermelon. 370
Fusions
binucleate stage. 97
trinucleate stage, 97
Galen, 1 1
Galeopsis, species of: bifida. 310; puhes-
ceus, 310; specinsa. 310; tetrahit, 310
Galeopsis tetrahit synthetic. 279
amphiploids. 310
interspecific hybrids. 310
octoploid with more than optimal
niunber, 310
synthetic Linnaean species, 310
tetraploids, 310
Gametes
adult spermatozoa. 206
binucleated, 205
c-meiosis, 112-15
diameters of, 205
diploid, 278
female gametophytes, 118
frog sperm suspension, 382
male, 204-6
in pollen tube, 37, 117, 118
to produce triploids, 381-83
sperm material, 59
Gametic doubling
in nature, 294
in Nicotiaua, 307
Gametophytes
ferns, 119
liyerwort, 117-20
mosses, 1 1 7-20
polyploids, 117
Gametophytic development, 103. 110-18
Ganglionic nerve cells, 210
Gastric mucosa. 376
Gastrulation. 203
Gelation-solation phases, 89
Genetic changes, 275
Genetic markers, 329
Genital tissues
biopsy of, 376
or hinuan \agina, 376
of rodents, 376
Genome
amphijiloid stability depends on gene
exchange iDCtween. 294
classification by, 303
D genome, 296
Gossypium, 304
hexaploids, 299
incompatil)ilitv of, 293
intergenomal pairing, 277, 293
Subject Index 453
ill nature, 283
Or\z(i satiTd. var.. 321
parental genomes, 292
Germination of ]iollcn
effect of colchicine, 37, 107-9
special technique, 385-86
Germinative zones, 219
Glaiuhilar crypts, 379
Glandular cpithelinm, 376
Glomeruli; see Kidnev
Glucose, 223, 377, 416. 417
Gliicoside
colchicine in combination with, 405
siihstanccs of, 144-48
Glutamatc, 223
Glycerine, 383
Glycolysis, 181, 412
anaerobic, in muscle, 183
in tumors, 261
Gnoscopin, 413
iiiliil)it mitosis, 413
substance of plant origin, 413
Golgi bodies, in mice, 91
Gonadotropins; see Hormones
Gonads. 202
Cnnioptcris proUfera. 119
(lOiiiiim. 1 19
Gossypium, 277, 285, 288, 303-4
African species, 302-6
American species, 303
Arabian-Indian species, 303
Asiatic diploids. 302-6
Asiatic species, 300-302, 303
Australian species, 303
complex amphiploids, 304
doubling with colchicine, 302
extra chromosomes, 357
fertile after sterile, 302-6
fertility, 357
haplo deficient gametes, 357
hexaploids. 327
interspecies types, 357
interspecific hybrids, 302
intraspecies trisomic, 357
origin of ncAv species, 303
spontaneous amphi])loid, 303
tetraploid, 302-6
tetrasomics, 357
triploid a bridge to species, 327
Gossypium, 354. species of: anoiiuihuu.
'303. 304; arboreum. 303, 304; arbor-
eimi X lliiirberi. 303; ariduni. 304
armourianum. 304; barhadense
304; dnvidsonii, 303, 304; davidsonii
X anomalum, 303; harknessii, 304
herbaceum. 304; hirsutnm. 303. 304
356; hirsutnm x arboreum, 306
klotzcliiauum, 304; raimondii, 304
slocksii. 304; sturtii, 304; ihurberi,
303, 304
Gout
Alexander of Tralles and, 12
in ancient civilizations, 1-3
and cancer, 255
curative property of Colehicum. 196
Doctrine of Signatures and, 11-12
forgotten tlisease, 197
Garrod's study of, 14
heimodactyls, 12
Husson's preparation for, 13
Krauterbuch, 6
podagra recognized, 11, 12
reconnncndcd in pharmacopoeia, 13
Storck's work, 13
theories of, 14
treatment of, 11-13, 175-76, 196-98,
391
Grafted nuclei, 381
in amoeba, 381
cellular volume, 381
Grafting methods, 385
for chimeras, 385
to propagate, 385
Grain, 2
effect of pollination on, 319-20
weight of in rye, 366
Gramineae, 365
amphiploidv in, 294-302
artificial and natural polyploids in,
301-2
autoploids, 319-21
hexaploid wheat, 295-97
root systems treated, 384
Triticales, 300
Tritieum group in Russia, 287
valuable crop, 279
Granulation tissue, 246
Granuloblastic, 189
Granulocytes, 186-88
Grapes
improvement by polyploidy, 324
in nature, 279
vinifera tetraploid, 324
Grasshoppers
Bleb formation, 91
Brownian movement, 89
cytoplasmic constituents, 90
duration of interphase in, 380
hyaline globule in cells of, 80-81
lost chromosome, 83
mitotic stages, 70
neuroblast'ic cells of, 32, 46, 52, 71-73
photomicrographs from cmbrvo of. 66
spindle iuhil)ition, 68
viscosity changes, 89
in vitro technique, 377
Growth
algal cellular changes, 128-29
androgenic hormones. 232
aneuploids iufiuciue. 345
appearance of pt)l\pU)ids in. 363
bacterial changes caused bv, 122
binucleate spermatid, 205
bloating of pollen tube, 107, 108
454 Subjeci Index
Growth (coiili)ui('d)
Ijoiie repair and, 247
cellular intrusions, 131
cellular multiplication, 319
cclosomv in chick emhrvo, 206
colchicine tiuiior phenomenon of,
103-7
criteria for judging polyploids by,
362-71
effect of chimeras on, 348-51
embryo sac. 1 18
eml)r^onic, of animals, 202-1 1
endocrinological research, 214
experimental, in animals, 214-49
fruit markers, 363-65
gametophytes enlarged by. 118
gonadotrojiic hormones and, 229
hormone stimulated, 224-26
hormones in, 416
inhibition of, 236
liml) regeneration, 242
malformations. 206
malignant, 259
neoplastic, 255-73
nuclear diameters of spermatid, 205
overgrowths in plants, 26()
|)at terns, 21 1
pituitar\ activity and, 2-5-28
])lants, point of, 384
pollen mother cell abnormality, 127
pollen tube enlargement, 107-9
regeneration and hypertrophy. 236-46
regidation of, 236
root hair, 127
root-tip tumors, 19-25
sex determination and, 351
sex hormones, 230-32
shoot apex, 319-56
spearlike roots, 102
strophosomy in chickens, 206-8
study of colchicine and, 214-15
tetraploids show featiues in, 320-21
tri|)loids vigorous in, 32()
vascidarizalion in root cells, 128
virus tumor tissue, 132
in vitro studies of, 223-25
/n x'ivo studies of, 219--2
wound healing, 246-47
wrinkled leaves, 125-26
Ciuanidoacetic acid, 397
(.inns. 1 10
H
Hair
cells of root, 107-10
cells of stem and loot, 102
follicles, 269
growth, 189
loss, in man. 261
mitosis in follicles, 189
Halo-derivatives, 400
Hamster, golden, 398
accjuired resistance by, 107
under laboratory conditions, 107
resistance to colchicine, 107, 398
Hanging drop preparations, 378
Hanstein, terminology of, 348
Haploid sperm. 381
Heart. 183
action on libers of, 183
and circulation, 183
Heat-shock, 398
Helianthus tuberosus, 105
cellular inhibition. 105
hairlike cells, 109
heteroauxin, 105
test to measure phv tohormone
potency. 107
tissue ctdtures of, 105
Hematocrit tube, 381
Hematopoiesis in chick, 375
Hemoglobin, 188
Hemolvtic anemia, 184
Hemorrhage, 181, 194
in tmnors, 260-61
Hemp, 353
autotetraploid, 353
excess of females, 353
Henle's loops: see Kidnev
Hens, 190
Hepatectomv, partial, in lat. 97
Herbalists, 7
Herbs, 3
Hermodactyl, 7
Hcterogametic, 353
sex determination, 351-54
Heteroploid, 8
i:i animals, 97
in pig. 382
polvploids as, 58-59
as polvploids in animals. 380-83
7-Hexachlorocyclohexane, 416, 417
Hexacyclochlorohexane, 417
Hexahydrocolchicine, 161
Hexamethyl-benzene, 400
Hexanitrodiphenylamine. 401
Hcxaploid wheat, 279, 284
optimal number, 284
origin of, 295
T.\ndgare\i\., 295
Hilleshog strain of Bcla ■i'uhgdris, 355
Hippocrates. 2, 3, 6. 1 1
Histamine, 183. 198
Histiocytes. 189, 269
Histology, 153
Hodgkin's disease, 264
use of colchicine in, 14
Hofmann degradation. 162
Hokkaido Agricultural Experiment
Station, 332
Hokkaido I'niversitv. 332
Holland. 300
Tritir/iles project in. 300
Homochelidonine, 113
Subject Index 455
Homogametic, 354
in sex determination, ^r>\
Homologous chromosomes, i \'J.
Homologous serum, 378
Honey poisoned l)y Colchiciuii. 109
Hormicliitm, 119
leukophytic variant of, 124
Hormones
androgenic, 232, 235
estrogenic, 214, 226, 232
experiments on growth and, 226-27
gonadotropic. 191, 226. 227
mimetic actions, 190
ovarian hormones, 226
ph\ lt)liorm()nc: sec Plants
jMtuitary, 228; see also Hypophysis
sex, 230-31
stimulated growth, 224-27, 379
test of estrogenic, 19, 214
Husson, 13
Hyaline globule, 66, 68, 79, 80, Rl. 193,
394; see also Aciiromatic sphere.
Deformed spindle, Pseudospindle
change of spindle form. 78-81
diagram of, 70
in neuroblasts, 66, 70. 78-81
non-fibrous, 68
photomicrograph of, 6()
similar to pseudospindle, 80
Hydrodictyon, 119
Hydrogen bonds, 408
Hvdrogeuation, 161
Hvdrolvsis to colchiceine. 160
Hydrostatic pressures. 399
Hyocyamus niger, 346
Hyperglvcaemia, 193
Hyperthvroidv, 229
Hypertrophy, 124. 236
iniilateral, of kidney, 241
Hyphae, 124
Hypocotyl, 102
cortex, 105
swelling, 105
Hvpophysectomy. 190
Hypophysis, 177, 220, 226
in carbon-tetrachloridc poisoning, 248
mitoses in, 241
in pregnancy, 230
Hypothesis, Ax B. 278
'Winge's, 278, 307
I
Index for induced tetraploidy. 319
counting cinomosomes. 320
criteria forjudging. 362-71
floral parts, 320
pollen size, 319
seed size, 319-20
sprouting ability, 320
vegetative characters. 320
Indian Pharmacopoeia, 8
Indole acetic acid, 109
Indolebutyric acid, 104
Inhibition, 33-34
iTijcctions, 188
J-Inositol, IK). 117
wcvo-Inositol, 101, 416,417
role of temperature, 104
me50-Inosose. 417
Insect resistance, 311
Insulin, 244
and Langerhans' islets mitoses, 241
"Intactncss period" of chromosomes, 92
Intergeneric amphiploid, 299
Intergeneric hybrid, 279, 309
Intergenomal exchange, 292
no pairing between genomes, 293
Interkinesis. 417
Interphase, 50, 78; see also Mitosis
c-pairs enter, 108
duration of, 380
loss of chromaticity, 45, 50. 70. 85, 95
micronuclei of, 44
processes, 31, 50-52, 75, 95
prophase to, 32, 70
transformation to, 30, 75, 79
unraveling, 50, 85
yesicidating stages, 45, 51, 85, 95
Interspecific hybrids, 278, 293
Brassica, 309-10
classification problems, 280-81
Cucurbit a, 311
diploid, 293
Galeopsis. 310
in Gossvpiuin, 302-6
in Gramineac. 294-302
in Nicotintia, 307-9
Ribes, 312
Solanuni, 31 1
Tri folium. 312
Interspecific segregation, 285, 293, 308,
311, 312
Interspecific trisomies, 306
tetrasomics, 306
Intervarietal 3X hybrids, 332
Intestinal crypts, 376
Intestine, 181, 196
elimination of colchicine bv, 196
hemorrhage, 181
mitosis, 265
nnicosa.
iO
Intoxication, chronic, of mice, 193
Intracerebral injection, 179
Intramedullary injections of colchicine.
in man, 264
Intranuclear orientation, 33
Intraperitoneal injections of colchicine,
377
Intraspecific trisomies, 306
tetrasomics, 306
Invertasc. 397
Invertebrates, 54, 375, 377
lodoacetamide, 412, 415
456 Subject Index
lodoacetic acid, 412
4-Iodo-5-methoxvphthalic acid, Ifi?
Iris, 363
Irradiation, 154, 170
Irradiation of Allium root-tip pri-
mordial, 105
c-timior not inhibited, 105
Isocolchicine, 16, 160, 169, 407, 410
Iso-componnds. 1()2
Isodeaminocolchinol metiiyl ether, 162
Iso-derivatives, 407
Isodiametric expansion, 103
Isoethylcolchiccine, 407
Isomeric nieth>l ethers, 168
Isomeric nnsatnrated ketone, 163
Italchine, 419
Japan, 287, 330, 352, 370
Japan Beet Sugar Manufacturing
Company, 332
K
Karyokinesis (nuclear mitosis) , 86, 88
to excite, 17
Karyometry, of liver nuclei, 237
Kashmir hermodactyls, 2, 3, 6
Katachromasis, 51
Kernel weight, 366
Kew Gardens, 279
Kidnev, 177, 227, 229
colchicine and, 193, 195
connective cells, mitoses, 239
convoluted tubules, 237, 239, 240
emljryonic, in tissue cidtiue, 258
■"endocrine." 238
glomeruli, 237, 239
Henles loops, 237, 239
hypertrophy, 237-41
mitosis in. 229
pelvis. 237, 240
Schweigger-.Seidel tubules, 237
Kihara Biological Institute, 287
KupfFer cells, 193, 248
Kvoto, 287, 330
Lactate, in muscle, 181, 182
Lactic acid, 258
Lagging chromosome, 83, 346, 386
Langerhans" islets, 177; see also Pan-
creas
Lanolin, 383
"Late" mitoses, 193
Latex species, 326
Hevea, 326
Koh saghyz, 326
Leon the Great, 12
Lepidium, 42, 80
ball metaphase, 42
Leukemia. 189, 224, 256, 264
Leukocytes, 176, 184, 186, 187, 189
in dog. 187
eosinophil, 188
monocvtoid, 189
Leukocytosis, 17, 183
colchicine-leukocytosis, 418
Leukopenia, 189,264
Leydig cells, 236
Lieberkiihn glands, 176; see also
Intestine
Ligustrum, 129
Liliaceae, 8, 159
Lilium, 346,413
aneuploids, 346
lilies, 326
lougiflonitn. 346
mixoploidy in anther, 348
Limb
blastema, 242; see also Toes
regeneration of, 242
Linnaeus, 7-8, 9
Liver
cells of, 90
damage. 191
exploded c-metaphase in, 44
fusion of nuclei, 44
hepatectomy, 41, 97
in man, 176, 178
micronuclei, 44
mitoses in, 193,210. 230
mitotic counts of, 378
original studies with, 19
cjuantitative estimate of growth, 376
regenerating cells, 376
regeneration of, 34. 44, 84, 90, 97, 216,
217,236.378
single injections, 57
in vivo studies, 376
Loganberry, 324
Lolium perenne, 323, 348
Lost chromosome, 83
Linnicolchicine, 154. 170
Limdsgaard effect, in muscle, 181
Liiteae, 9
Lycopersicum, esculent inn, 265
Lymph glands, 176
Lymphocytes, 186-88
Lymphoid cells, 261; sre also Lympho-
c)tes
Lymphoid timiors; see Neoplasms
Lyiechinus, imriegatus, 88, 416
M
Maize, 333
cytohistologv, 347
Male gametophvte, 107-20, 386
as pollen. 107-20
as pollen tube, 37, 108, 117
Malformation
in chick embryo, 206-9
in eggs, 377
Subject Index 457
Malignant growth: see Neoplasms
Malonate, iiSl
Mammals, 54, 376, 379
Mammary gland carcinoma; see
Carcinoma
Max haiitia pohinorpJia. 117, 118. 119
diploid gamctophytes induced, 117
Marigold, 326
Marine annelid, 75
Chaetopterus, pergainentiireiis, 75-77,
82
Marine eggs, 87
Mast cells, 188
Materia medica, 1,11, 140
Maturation division, 75
Mechanism of colchicine-mitosis, 391-
422
Medicago, species of: denticulata, 332;
lupuHna, 322: media. 322; sativa,
322
Medical practitioners
Arabian. 3
Babylonian, 2
British, 13-14
Egyptian, 1
French. 13
German, 13
Greek, 2. (w
Hindu, 2-11
medieval, 13
Medicine, 3
early, 11-14
forensic, 176-94
MegacarvcKAtes, 189. 192
Megaloblasts, embryonic, 210
in Addison-Biermer anemia, 210, 21 1
Megaspore mother cells, 110, 386
of embr\o sac, 1 IS
in gametoph\ tes, 110-18
in Tradescautia, 118
Meiosis, 94, 103; see also C-meiosis
chiasmata of, 115
colchicine, 110-18
compared to c-mitosis, 112
in pollen mother cells. 111
treatment and stage, 113, 111
Meiotic metaphase. 386
Melandrium dioecum, \ar. album. 353
Mclaiiophis. diljercntialis, 11 , 90
.Mcl.inthoideae, 8
Mclica, 302
Melittia satyriniforinis Hubner, 31 1
Mercury, action of, on mitosis. 415
Merendera persica, 2, 3, 7
Merostathmokinesis, 86, 400
Mescaline, 405
Metabolic changes
actions of colchicine, 396
Benedict s solution. 131, 132
diastatic activity, 131
dipeptidases, 132
enzyme reaction. 131
growth, 132
metabolic inhibitors, 181
oxygen uptake, 132
plasmolysis, 132
rates, 131
respiration changes, 132
\iscosity correlation, 132
?n vitro, 131
Metabolism
of colchicine, 194-96
of tumors, 260
Metals, 412
-Metamorphosis, 210
Metaphase
arrested, 17, 40, 66, 81
anaphase fewer than, 65
Arbacia. spindle of, 75
ball, 38
bi-, 83
colchicine-, 35-50
colchicine causing arrest at, 24, 25, 26
chromosomes nnoriented at, 30, 31
Cliaetopterus, spindle of, 15-11
clumped, 42, 84
correlated with concentration of
colchicine, 76
disappearance of spindle, 76
distorted star, 38
distributed colchicine. 43
exploded, 38, 40-42
in Allium, 25, 41, 79, 85
in liver of rats, 44
in neuroblasts of grasshopper,
41, 66, 70
in Orthoptero. 41
in pollen tubes, 37
in Siredon, 41
in Triton, 41
in Tri turns. 43
graphic representation of increase in.
30
main types of arrested, 38, 70
multiple star, 41. 66
original statements of arrested, 26-29
oriented arrested, 39-42
percentages of, in root tips of onion, 34
Pernice's observations, 17. 18. 65
photomicrographs of. 37
prophase, 84
stages of, in animal cells. 19
spindle mechanisms and arrested,
81-86
star, 81-83
tissue culture malignant cells
arrested, 17
tissue culture, normal cells, 17
tri-, 83
unoriented, 10
Metastases, treated by colchicine, 263
L-Methioninc, 397
Methowthclidonine. 413
Methyl ciIict, 169, 408
458 Subject Index
Methylcholanthrene, 260, 269, 270
N-Methvlcolcliicamide, 411
Methvlcokhicine: see Deniccokin
()-Methvlethcis, 160
y-Methylphenanthrene, 161
N-Methyl-propyl-colchicamide, 411
Micrasterias tliomasianas. 119, 124
Microchemical tests, 127
Micrococcus oureus. 121-22
Micromiclei, 97, 412
in liver, 237
in tnmor cells, 259
Microscopic tests, 368-70
average diameters, 369
guard cell size, 369
microspores, 369
pollen grains, 369
triploid grains exception, 369
in watermelon, 369
Microspores, 110-15
c-meiosis. 111
c-mitoses, 110
hexaploid, 1 1 1
octo]3loid, 1 1 1
polyploid, 1 14
tetraploid, 11 1
tetraploid monad. 112
luitreated, 1 11
Miscible pool, 197
Mitochondria, 72. 89, 91
shortened outside pseudospindle, 96
Mitoses; see also Mitosis
intestinal, 376
"late," 193
Mitosis: see also Colchicine mitosis
abnormalities spontaneous in
malignant cells, 258
activity in kidney of rat, 239
analysis of colchicine upon. 17
Arbacia eggs in, 74—75
arrest of, 26. 27, 28, 36, 43, 44, 45. 89,
186, 237, 276
arrested, in pancreatic glanil. 177
bipolar nuclear, 31
cell division not synonymous with, 87
cellidar proliferation, 375
chemicals acting upon, 17
chromosomal fdjers of, 67, 81-83
chromosome, without cellidar. 69
chromosome changes like normal
luiclear, 50
colchicine, 21
comparison of colchicine meiosis
and colchicine, 112
continuous fibers of, 67, 81-83
crop-sac of pigeon, 228
cycles of, 68
cytological standards to measiue
chemical action on, 86
discovery of, 188
distributed colchicine, 205, 429-43
thinnal variations, 221
duration of, 216, 217. 218, 221, 236,
246, 259
duration of colchicine, in animal
cells, 48
effect of colchicine on course of, 60
endocrine gland, during poisoning
by carbon tetrachloride, 248
fibers of, 67
fibroblast cidtures showing, 30, 215
glutamate and, 223
graphic representation percentage
of stages of, 30
grasshopper neuroblastic cells in, 71-73
ilial epithelium of male rat.
percentage of, 222
index of, in pregnant guinea pig,
230-32
inhibiting action upon. 31
interphase of, 232
liver, during carbon tetiachloride
poisoning, 248
mechanism to account for colchicine,
391-421
metaphase of, changed bv colchicine,
35-50
methods for culturing pollen to
study, 385
neuroblast metaphases of, 66
nuclear, 24
nimrber of visible, 235
optical anisotrojjic spindle fillers of,
75-76
Pernice's study of, 17, 18
pituitary, 222
pluricentric, 203
pollen tube, 37, 385
jirogressive acciuiiidation of arrested,
'215
prophase, in tissue cidture, 215, 217
prophase of, 31-35
pseudo, 393
repartition of, dining bone repair,
247
return to bipolar, 38, 55, 94-96
reversibility of a colchicine, 91-93
seminal vesicle dose and, 234
Siredon arrested, 48—49
sleep, action, 221
spindle mechanism of, and
colchicine, 65-97
spleen of Siredon arrested first se3n,
42
stage of, treated in neuroblast, 70
stages of, in root tips, 34
stimulation of, 216, 217, 242, 244
study of, 374-75
techni(]ue with animals in siikU of,
374-80
timing upset of, 85
thyroid of guinea pig, 229
topogiaphy of growth, 375
Tradescantia staminal hair cell, 73-74
Subject Index 459
Triton arrested, 4S
Triturus arrested, 48
veritable explosion of, Ifi
\iscosiiv changes at, S9
Mitotic counts, 379
Mitotic index, 238, 378-79
Mitotic poisoning, 379, 391, 392
Mitotic poisons, 257
mitotic stage, 81
Mitotic processes, 10
bv colchicine, 378-79
of kidney tubules, 193
mitotic growth, 193
mitotic stimidation, 193
Mixoploids, 384
Mixoploidy
clones of diploid and tetraploid, 347
diploid and tetraploid ])ollen
mother cells, 348
LoUinu lyerenne, 347
persistence of, 347
Molge, species of: uuninoyatd. 226, 227;
palinata Schneid, 210; jxiIdkiIiix,
210
Monads
formation, 1 12
replace tetrads, 1 12
tetraploid, 112
Monaster expansion, 89
Mono-alcohol, 161
Mono-aldehyde, 162
Monocotyledons, 363
Monocytes, 188
Mononitro-colchicine, 169
Monoploid, 276, 321,333
Monosomic, 357
analysis in Nicotiaiia. 358, 359
9-Monoxime, 163
Monstrous development, in frog, 381-82
Montanae, 9
Mosaic resistance
in tobacco, 284
obtained by transfer, 284
Mucor, 123
Mucosa, 181
Multiple star, 83
Multipolar, 86
Multivalency in polyploid, 308
Muscle, smooth, 181
anaerobic glycolysis, 182, 183
oxidative activity, 182, 183
Muscle, striated, 180
contractility of, 181
Mushroom, 6
Muskmclon, 325
Mustard gas, 91
Mutagens, 124, 275
Mutant
Mutation, 275
in Ddturd stramon'nini . 354
globe, 354
a trisomic, 355
Mycobacter'nuii tuberculosis, 122
Myelin, 246
Myelocytes, 188
Mvoblast cultures, 418
Mvxedema, 229
N
Naphthalene, 400
Naphthalene acetic acid (XAA) , 86, 104,
109, 128
colchicine and, 128
threshold, 101
Narcosis, 111, 180, 396
c-tumor, 110
colchicine and, 402, 403
theory of , 399, 403
Narcotics, 392
Narcotin, 413
National Formulary, 156
National Institute of Genetics, Japan,
332
Natural pohploids, discovery of
in Gossypiinn, spontaneously, 303
in grapes, 324
in Polygonatutn, 385
Necrotic factor in tobacco, 307
polvploidv breeding, 307-9
transfer from T. gluliiiosa, 307
Neoplasms
in abdominal cavity, 377
lymphoid, in man, 264
hniphoid, in mouse, 262
malignant, in horse, 257
malignant, in man, 259
in man, 376-77
Neoplastic cells, 258-60, 378; see also
Cancer, Cancer chemolhcrap\. Car-
cinogenesis
experimental stiuh of, 258
gro^\'th in animals, 194, 255-69
Nephrectomy, unilateral, 237, 238, 241
Nerve cells
colchicine mitosis in regenerating. 246
growth, 210
regeneration, 216
Nervous paralysis, 377
Nessberry, 324'
Neural tissue in chicks, 209
Neuroblasts of grasshopper
bleb formation, 91
Brownian movement, 89
delay in mitosis, 46
destruction of fibers in, 77
effecti\cness of colchicine on, con-
centration, 72
exploded metaphase in, 72
exposure time, 73
inhibition by colchicine, 34
mitochondria obser\ed in, 72
mitotic stage treated, 70, 73
phosphorus, 89
460 Subject Index
Neuroblasts of grasshopper (coiitiitucd)
photomicrographs of, 66
ribo-micieic acid, 89
specific concentrations, 41, 70
spindle inhibition, 68
in study of prophase, 32-33
successi\e changes in, 70
technicjue developed with, 71-73
viscosity changes, 89
in vitro technique, 377
Neuromuscular apparatus, 179
Neuromuscular block, ISO
Neutrons, 2()9
Nicotiana, 254, 288, 346, 347, 357. 362
back-cross segregates. 308
species of: digluta, 307; i^littiuosa. 359:
langsdnrfjii. 356: sauderaea, 356;
tabacuni, 309; tahacuni var. virii,
307
svnthesis of A', digluta. 309
svnthetic tobacco species, 309
trisomies and corolla size, 356
Nicotinamide, 397
Nipple. 226
Nitella mucronata, 119
Nitro-derivatives, 400
Normal interphase. 380
North Clarolina State College. 331
Northern hemisphere, 141, 323
Norway, 322
Nostoc commune, 1 19
Nuclear sap, 202
Nucleic acid, 221
Nucleoli, in neurol^lasts, 70-71
Nucleoprotein metabolism, 197
Nullisomics, 357-58
Number of fruits, triploid, increase, 367
Number of mitoses, 378
Nuptial colors in Rhodcus, 191
Nuts, 279
Oats, 26, 279
Octoploids, 95; see also Aneuploitly,
AiUoploidy, Polyploidy
la\ers in shoot apex, 356
Oedognnium. 1 19
pohploidv in, 124
Oenothera lainarckiana \ar. niiias, 318
Official status of drug, 140
Oil-bearing seeds, 326
Brassica. 326
flax. 326
sesame. 326
Oiomouc, Czechosknakia. 143, 149
fruit development of Colcliinan. 149
Omphalomesenteric vessels, 209
Opatjue hvaline golbulc, 80
Organoleptic tests of drugs, 1 II
Oriental medicine and ih iig ])lants, 6
Orthoptera, 41
Gy rill is assiniilis. 90
Melanoplus diljerentiiilis. 77, 90
Oryzias latipes, 204
Oryza sativa var. indica x ^^^- japonica
autotetraploid and diploid hybrid fer-
tilitv, 321
hybridization, 320
Ovalbimiine, 197
Ovariectomy, 232
Ovary
corpus luteum, 230
follicular cells, 220
of frog, 1 92
germinal epitheliiun. 219
Ovipositor, 191. 226
Oxaloacetic acid, 166
Oxycolchicine, 169; see also Oxydi-
colchicine
Oxydicolchicine, 180, 195
Oxvgen, 223, 224
Oxxtricha. 126
Pairing characteristics, 385
Pairing of chromosomes, 1 13
for classif\ ing polvploids, 283
intergenomal pairing, 293
measure of homologx, 281
quadrivalents, 364
Paleolagus, 12
Pallavacinia spp., 118, 119
Pancreas, exocrine, 193, 210, 230
Langerhans, islets of, 177, 226, 227,
230. 236. 241
late mitoses in. 193
Pancreatic glantl, 177
Papilloma
Shope, in rabbits, 262, 263
\enereal, in man, 263
Paracentrotus. egg, 203, 401
Paramecium
effect of temperature, 374
species of: caiidaliim. 126; niultiiiii-
croniicleatum . 126
in vitro, 374
Parathryoid, 226, 227, 229, 230, 234. 240
Partial inacti\ation spindle, 69
Pasture species. 321
Patulin, 415
Paul of Aeginata, 12
Pelargonium, tiunors in, 266
Peltatin, 261, 413
a-Peltatin. 413
/3-Peltatin, 413
Pelvis; see Kidney
Penicillium notatum. 123
Pentaploids. .354, 382
source of ancuploids, 354
Pepagomeus, 12
Subject Index 461
Peranema, 126
Periclinal chimera, 285
ill apple. 351
in DdtuYd. 350. 356
diploid- 1 etraploid. 351
induced bv colchicine, 348
in Liliuni, 351
in Solan IH71, 351
Periclinal division, 350
Periosteum: see Bone
Petiolar swelling. 106
Petroselium, 42, 47
Peyer's patches, 178
Pharmacognosy of Colclticiun. 140-58
Pharmacology of colchicine, 175-201,
377, 380 '
cellular, .396
in forensic medicine, 176
Pharmacopeia. 1, 140
British. 141
Indian. 141
London. 13
of United States, 141
Pharmacy, 2, 3, 140
Pliase contrast microscope, 67, 77
Phenol. 401
Phenvlhydrazine, 184, 185
Phenylurethane, 105, 392
synergistic action, 105
Phieum. species of: nodosum. 323;
pratense. 322-23
Phlorizin. 419
Phlox. 326
drummoncUi. 364
Phosphorus, 89, 181
Photosynthesis, 397
Phragmoplasts, 73. 89
Phvtohormone. 101, 107, 275
Avena, 106
colchicine not phytohormone, 107
elongation proportional to concen-
tration, 107
Hrli/iiitlius. 106
Lepidium hypocoiyl, 106
Pigeon, crop-sac of, 379
Pigs. 58-59, 381. 382
Pilocarpi!!, 181
Pi sum. 106
potency. 106
swelling, 107
Pisum satwum. action of colchicine and
X-rays on. 267
Pituitar\, 177, 197: see also Hormones
-atlienal slinudation, 193
hormones. 226
mitosis in, 232
prolactin, 226
Planimetric measurements. 378
Plant anatomy, tunica-corpus concept,
348
Plant tumors, 265
Plasmodium, species of: rclirtum. 126:
vivax, 126
Platelets, 176, 189
Plerome, 348
Pleurodeles, deyelopment of, 203
1*1 inv the Elder. 2. 11
Doctrine of .Signatures, 11
Ploidy, defined. 276
Plumide. 284
IMuricellidar animals, 375
Pluricentric mitoses. 203
Plurinucleation, 203
Podagra, 11, 12
Podophvllin. 264,413
Podophyllotoxin, 264, 413, 415
effects of, 416
Podophyllum sp., 264
Poinsettia, 326
Poison. 34, 175, 178
preprophase poison, 35
Polarization midoscope. 67. 71. 75. 76,
77
Polarographic technicjue, 148
Pollen"
cells, 74
for counting chromosomes. 369
grains. 33. 385
mother cells, 369, 386
sterility, 386
tubes, 33, 37, 103
Polycythemia vera, 224, 259
Polxgoiiatum. 385
c-pairs. 1 Ki
distributeil c-mitosis, 347
species of: commutatum, 37, 48: multi-
riorum, 326; pubescens, 108
susceptible to colchicine. 116
Polyploid, classification of. 280-82
intergiading series, 282-92
limits not clearly defined, 292
Polyploids
in Amoeba nucleus, 375
artificial. 26
cell volumes. 381
cells, 93. 95
colchicine not effecti\e in animal,
380
definition of. 276
induction of. in vertebrates, 206
natural. 26
plant tumor cells, 266
principles of breeding, 285, 382
raw. 321
restitution nucleus in plants, 93, 399
series of, 278
species in nature, 279
spermatogenesis, 205
stabilization of, 285
Polyploids, animal, 380-83
Amoeba. 58
Anemia, 58
462 Subject Index
Polyploids, animal {co)i tin tied)
chicken, 58
Drosophila. 58
frogs, 58, 381
pigs, 59, 381,382
rabbits, 59, 381,382
silk^vorms, 58
Triton, 58
Triturus, 58, 97
Xenopus. 58
Polyploids, plant
agricultural species, 279
Brassica, 309-10
Cucurbita, 311-12
forage species, 321-23
forest species, 323-26
fruits. 323-26, 333
Galeopsis, 310
Gossypium, 302-6
Gramineae, 301-2
Xicntiana. 307-9
Ribes. 312
Solanuin, 310-11
Trifolium, 312
vegetables, 323-26
watermelon, 327-31
Polyploidy, criteria for judging, 362-
appearance. 363
bv fruit, 363
microscopic differences, 369
seed, 364
\veight of grain, 366
Pohploidv breeding, principles of
achantages \'s. disachantages, 285
genome transfer, 285
large populations, 283
la\v of optimal numbers. 283
mixoploids. 285
raw polyploids. 282
testing methods, 286
of traiisfer, 284, 293
Polyploidy experimentation
callus tissue, 274
chemical induction, 274
colchicine method, 274-75
heat-shock, 274
history of, 333-34
scope of research, 286-88
Polysaccharides, action on tumors, 261
Polys toma. 119, 125
"Precocious reversion," 73, 93
Pregnancy, 229, 230, 236
Premeiotic stages, 112
Pressor amines, of the adrenals. 397
Pressure, of air, action on mitoses, 225
Pretreatment, 386
Primula keii'cnsis, 278-79
Progesterone, 226, 235
Proiactine, 226, 228, 379
injected with colchicine, 379
Prolactine-thickened crop-sac, 379
Proliferating cells, 385
Propagula, 1 18
Prophase, 31-35, 114, 380; see also
Mitosis
arrested, 64
influence of colchicine, 31-33
Piophase reversal, 50
N-Propvlcolchicairiide, 411
Prostate, 226, 227, 232
ventral, 22()
Protein content, 368
Protoanemomin, 413, 415
Protonema, 118
Protopin, 413
Protozoa. 54, 125
anatomical variation, 126
gross changes, 129
microinjection, 125
role of toxicity and temperature, 125
Psainnicchimis miliaris, 89, 91
birefringence, 91
independence of spindle and mon-
aster, 89
monaster expansion, 89
viscosity changes, 89
Pseudoanaphase, 45, 393
Pseudometaphase ,393
Pseudonuclei in Tubifex eggs, 54
Pseudospindle, 79, 84, 85, 193, 394
achromatic sphere, 84, 85
Psilocybe scinilanceolata, 123
Psychriste, 12
Purdue University, 331
Purine metabolism, 397
Pycnosis, 52, 178, 190, 203, 376
Pyrophosphatase, 261, 397
P-Ouinoue, 118
Rabbit, 191, 381, 382: see also Polyploids,
animal
Racemized colchinol methyl ether, 163
Radiomimetic action, 15, 171
Radiominictic drugs, 392. 415
Radiosensiti\ity, 266
Radish
gigas, 324
Japanese, 324
resistance to disease, 32 1
simimer \ariety, 324
Rana, 210, species of: a<rilis. 203-4; jusca.
210; pipiens, egg of, 191, 202: teni-
poraria, 245
Range species. 321
Raphanobrassica. 279-80, 309
amphiploid, 309
cabbage x ladish, 309
first made in 1826, 309
Karpechenko demonstrated fertile, 309
Raphanus satimts, 309
Subject Index 463
Raspben-v, 324
Rat, 42. 37S
Reco\er\ from colcliiciiie. 379
in AUium. 29. 9li
in animals, 56-58, 9(i-98
in corneal tissues, 97
in li\er. 44
in |)lants. 5(). 94-9(i
])()hploid\ resuitin"^ after, 94-95
principle of re\ersibilitv, 91-91
processes of, 81
reduction in ninnher of nuclei, tl
in sarcoma, 27
after single injection, 57
stages of, in Trilii inn, 95
stages of, in Tri turns, 43
transfer to water, 94
Rectum, 226
Red l)lood cells, 188
diameter of, 188, 189
forming tissues, 376
\oliimes, 381
Red clover, tetraploids, 322
Regeneration, 93, 236-42: see also
Kidnev, Li\er
in amphil)ians, 376
for c-mitosis study, 376
in developing animals, 242—16
and h\pertroph\ , 236-42
inhibition of. 245
of limbs, 242
li\er of rat. 44, 57, 216
of nerve, 246
tail of Xenapus, 242-45
of thvmus, 241
Regenerati\e tissues. 385
Renal artery, ligature of, 40, 238
Reproductive isolation, 292
Resins, 140
Resistance, in plants and animals, 398
bv Colchicuni to colchicine, 107. 398
of golden hamsters, 107, 398
to phytohormone tests, 107
Respiration, 103
cellular. 204
in tumors, 261
Respiratory paralysis, 377
Retention of colchicine in cell, 383
Reticulocytes, 184, 185
Reversible effects of colchicine, 89:
see also Re\ersibilitv
Reversibility, 31
capacity to, 93-91
characteristic important, 91-91
demonstrated, 94
necessary for indudion of poUploidy,
92-93
regeneration of spindle, 94
Reversion to diploidy. 285
Rheumatism, 1, 2, 3
Rhizomes. 381
Rhi/otomi, 2, 3
Rhodeus niuarus, 226, 227
luiptial colors, 191
HI I ()(■() discolor. 109
Ribes
currant and gooseberry combined, 312
meiotic irregularities carried over, 1 16
ne^y species, R. nigrolarin, 312
species of: s^rosstilnria, 312; ?iigruni,
312
Rii)onucleic acid, 89, 397
Ribose, 417
Ribose nucleic acid, 121
Ricine, 206: see also Abrine
induces stro]>hosomy, 206
Ri(iiius. tumors in, 266
Ring A, 161
presence of benzenoid ring, 161
Ring B. 161-67; see also Colchicine
St rue tine
recognized as 7-membered, 166
research on, 161-67
revision of Windaus concept, 166
Ring C, 167, 168, 409
of colchiceine troj^olonoid, 168
comparison with tropolones, 168-69
Dewar's suggestion, 168
enolone properties deri\ed from, 167
Rodents, 376
Root gatherers: see Rhizotomi
Root hairs, 109
ctinnor. 109
not polyploid, 109
Root systems, treated uiih colchicine.
384-85
Root tip, 369, 386
Allium, 19, 25, 27. 35, 41, 49, .55, 78, 79,
83, 84, 85, 96
c-pairs in, 49, 85
c-tumors on, 25. 102-7
colchicine penetrates. 36
correlation region of, and c-mitoses, 55
description of c-mitosis in, 28
distribution of cells in, 55, 79, 95
of onion seedlings, 34
pairs of "skis" in, 51
polyploidy in, 25. 79. 95
tests with, 19, 34
wheat, 90
X-ray on, 105
Root tiimor, 25, 103
Roots, encised, 132, 385
Rudimentary cell plates, 89, 90
Ruuiex acetosa, 132
Russia, 286, 287, 296
Sacrharoinxces cerevisiae, 123
Saccharose, 116
.Saffron, 1
Salaniandra. corneal cells of. 101
Sali\ar\ srlands, 269
464 Subject Index
Sanguiiiarine, 113
Sarcoma, 26, 180
benzopvrene-induced, 261
Crocker, 253, 255, 256
grafted, 26
in rat, 260
Rous, in fowl, 262
treated, 26
imtreated, 26
Sassfifias albidum. 413
Saturated ketone, 163
Saxifrages. 1 1
Scales of liliaceous plants, 381
Schistosonius reflexus, 206
Schwann cells, mitoses of, 246
Schweigger-Seidel tubules; see Kidney
Sea urchin eggs, 392
Seed
aniphiploid weight and size of, 365,
367
of autotetraploid, 326
colchicine from, 144-47, 151, 152, 153,
154, 159
colchicine compounds from, 144-49.
153, 154
Cnlchiciim described, 153
collecting Colchicum, 150-51
criteria for judging polyploid from,
320, 363-67
dehiscence of, 149
effect of pollination on, 367
four X. 32S-29
fruit without, 302, 307, 329
grain weight, 365
markings V)n, 330, 365
mature', 149, .329
in Medicagn. 322
ninnber of, per fruit, 365
ovules appearing as, 321-31
production of, 3x. 332
prc)]3agation of triploid, 328
reduced setting. 371
seed production in rice. 321
seed-producing parts, 5
size of, 365
size correlated with drug production,
152
spring production in CoUliic utii , 3, 7
sugar beet, 332
tctraploid, 328, 330, 363-65
tliitkness of, 328, 365
three X, 328-29, 332-33
treatment with colchicine, 384
variation in colchicine from. 150-52
\olume of tctraploid, 365
watermelon tctraploid, 327-29, 365
yield lower from tctraploid, 284
Seedless fruits, 327
o\iiles appear as seeds, 327-31
term meaningless, 327
Seedling cidture in watermelon. 329
Seedling treatment; see Techniques of
colchicine treatment
Segmental allopolyploid. 277, 281, 282
Self incompatibility, 332
Seminal vesicles, 217, 219, 226, 227, 2,32.
234
Septa. 89
arrested cell plates, 89-90
Serratia marcescens, 261
Serum
homologous, 378
hiunan, 397
Sescjuihydrate crystals, 159
complex crystals, 373
concentrated colchicine solution
deposits, 159
Sex determination in plants, 351
excess of females in hemp, 353
influence of X chromosome, 352-54
methods of determining, 353
and polyploidy, 351-52
role of autosomes, 353
Sex hormones, 230; see also Androgens,
Hormones, Progesterone
Sexual cycle in animals, 376
Shin-Yamato, variety of watermelon, 330
diploid. 3.30
fruits, 330
seeds, 330
tctraploid, 330
triploid, 330
Shoot growth index, 96
effect of colchicine, 96
leaf shoots, 96
Shope papilloma, 262, 263
Siletie otites, 353
female homogametic, 354
male hcterogametic, 353-54
Silk worm. 191
Siredou, 32, 41, 42, 96, 97
duration of c-mitosis in, 48
erythroblastic prophase metaphase. 90
recovery stages in, 45, 56, 97
spleen of, 29, 50
Silanion, 302
artificial polvploid of, 301-3
Skin, growth of, 221, 226, 227, 376
for l)io|)sics, 37(>, 383
Slec|3, action on mitosis, 221
Small intestine of mammal, 378
Snapdragon, 326; see also Antiyyhiuuiii
aftereffects of colchicine, 1 15
pollen germination in, IKi
Sod i tun, 180
Sodium cacodylatc. 17. 29. 397. 411
Sodium diethyldithiocarbamate, 412
Sodiiun methoxidc in methanol, 167
Solatium. 288, 311, 3-'5
crosses with S. tuberosum . 325
hybrids of, 311
interspecific h\l)ridi/ation. 325
Subject Index 465
species of: (inlilxwiczii, 311, 325: nnti-
{loi'iczii X cliococtise, 311, 325:
rluicoeuse. 311, 325; deinissuin.
311: ryliiiiii. 311: luberosuin, 311
transfer of disease resistance, 325
Somatic doubling, 294
Somatic meiosis, 45-46
Somatic reduction, 94
equal or unctiual separations, 42
pseiuioanaphase, 15
relation to meiosis, 45, 94
terminology, 45-46
Somatotropic hormone, 230
Sorbitol, 417
D-Sorl)itol,416
Sorglniin
autotetraploiil sudan grass, 323
segregations, 323
species of: halojiciise, 323: xnilgare var.
sudanense, 323
Spartina toivnsendii, 279
Species Plantarum , 7
Spelt wheat synthesized, 295-97
invested type, 296
Speltoid wlieat, 357
Spermatids, 204-5
Spermatocytes, 204
heads of, 382
Spermatogenesis, 201
Spermatogonia, 204
Spermatozoa, 206, 382
Sphaerechiniis, egg, 202
Spina bifida in chicks, 209
Spinacia, 42, 80
recovery, 95
root tips, 95
Spindle, 65-98, 392, 102, 421: see aha
Deformed spindle, Pseudospindle
acenaphthene. 82
antagonists, 396, 416, 418
in Arbacia. 71-77
arrestetl metaphase, 36, 81
artefacts, 67
as\nmietrical, 94
l)ipolar, 93, 94, 96
birefringence pattern, 75-76
cell plates, 89-90
centromeric, 82
centrosomic, 81, 84
chemical action on, 403-4, 416
chromosomal fibers of, 77
clea\age processes, 53, 87-89
colchicine and, 392-93
continuous fibers of, 87-89
contraction of, 420
cytoplasm and, 65-98
"cvtoplasmatizalion" of, 7 1
damage. 31
deformed, 79
destruction of, 69, 70, 71
disa])pearance of, 76
disengaged from fiber, 36
disturbances classified, 86
ajiolar. 86
Ijipolai', 8()
nudlipolar, 86
unipolar, 86
fibers, 65-67
form of, 78-79
fundameiUal pioljjcni of mcdianism,
395
inacti\ation of, 28
inhibiting actions decreased. 374
inhibition of, 36, 68-69, 399
li]:)oid sohd)ility and. 402
mechanisms of. 81-86
merostathmokinesis. 8(i
metaphase, of Cliaelopterus, 75
microdissection of, 33
mo\emcnt of, 42
narcotics, 401
ncurol)lastic cells. 66
plnagmoplasts, 89-90
physical action, 399-401
physiology of, 394
pluripolar, 204
poisons of, 391-92, 411-16
jjscudo, 79
rate of disappearance, 76
recovery of, 56
retardation studies, 77
reversibility characteristics of, 91-94
"solubiliti/ation" of, 81 .
specificity of, 67, 393
stathmokinesis, 86
submicroscopic structures, 75
successive changes of, 70
svnergists. 396, 416, 418
toxicity, 374
tropokinesis, 86
Tubifex, 53
X-ray, 374
Spiral coiling of chromosomes
chromatin development. 32
excessi\e coilin". 16
major, 32
minor, 32
relational coiling, 46-49
Spirosiyra, 119, 125
Spleen, 17(), 181, 248
Sporogenous tissues, 119
Sporophytic cells, 120
Spring fruiting, 5
Stiuamoirs cells, 376
Staminal hairs, Trcidescantia, 385
process of interjjhasc, 51
technique with, 33, 7,3-75
in vitro study, 73
Star formation: see also C-metaphase
(igures by I'ernice, 18, 39
nudtiple. S3
mull iplc. hi . lllium . 1 1
466 Subject Index
Star formation (continued)
in neiiro])lastic cells, 66, 70, 72
oriented c-metaphase, 36-40
in Triton, 40, 81-83
in Triturus. 40, 43, 81-83
types, 38. 43, 81
Star metaphases, 36, 38, 39, 40, 43, 66,
67,70,71,72,73,81,82,83
mnltiple, 83, 84
Starch. 220
action on mitosis. 221
cliastatic activity and colchicine, 131
digestion of, 131
Stathmokinesis, 67, 86
arrested metaphases, 24
defined, 24
fidl inacti\ation, 86
index of, 224
Steatosis, 191
Steel rye, 319, 366
antotetraploids, 319
in Sweden, 319, 364
Sterile hybrids, 278. 363; see also
Fertilit\
made fertile, 278, 362, 384
Steroid hormones, 418
Stilbestrol, 227
Stilbylamine, 413
Stipa, 302
Stomach epithelium, 409
Stomatal cleyelopment, 128-29
Stress, 177, 190, 193, 248, 262
Striated muscle, 180
Stropharia nierderia. 123
Strophosomy, 207-8
Strychnos arhorea, 413
Siibcompactoid wheat, 357
Sid)microscopic structure, 74, 75, 127,
128
Sidjstances from Colchicuin, 144, 154
B, 144
C, 144, 147, 172
colchicine, 144-47
D, 144-46
deriyati\es and mechanism of
c-mitosis, 404-11
E, 144-46
F (Demecolcin) , 143, 144-47, 154
I, 144-16
Substituted thromosomes, 300
in Xicotiano, 358, 307-8
nidlisomics of wheat, 357
rye for wheat, 300
Sucrose agar media, 386
Sucrose production, 332
percentage, 332
of tetraploids increased, 368
in triploid beets, 332
in watermelon, 368, 417
Sugar beets, 139, 331
anatomical changes, 139
impro\ement, 331
triploid, 331-33
Sugar cane, 279
Sulfanilamide, 418
Sulfhydr\l groups, 403
SidflnihAl poisons, 411
Sidfonamide, 104
Sunlight, 154, 170
effect on colchicine, 154
Super contraction, 85, 113
autonomous, of c-mitosis, 46
cpair contraction, 37, 43, 47
chromosome eyolution, 46-48
maximum contraction, 44, 47
pretreatment of, 48
threshold for, 46
thickness and shortness, 52
Supralethal dose, 34
in rats for maximum arrested
metaphase, 34, 44
Surface changes in eggs, 202
Surinjan, 3, 12
Svalof, 274
chromosome laboratory, 286
experiments, 284
Hilleshog strain of beets, 355
Swedish 'botanists, 274-310
Sweden, 274, 286, 287, 322, 355, 382
Synapsis, 277
chiasmata. 115
of hybridity, 277
meiosis, 112
pairing of chromosomes, 112
Syndrome, adaptation. 192
Synergism, 105, 217, 416
role of colchicine, 105
Synergists, 396, 418
Synsiplion. 8
Tachysterin, 235
Tagetes patula, tumors in, 266
Tail, regeneration of, 242
Taraxacum hohsagliyz, 368
Tautomeric enol system, 160, 168
Tautomerides, 168
Techni(|ucs of colchicine treatment,
373-90
Allium cepa test, 27, 28, 34, 41, 79, 385
in animals, 373-83
Arbacia, 75
birefringence tests. 89
chemical methods of extraction, 153,
154, 159
chromosome studies, 386
corneal cells, 43
effectiveness, 76, 131-32, 383, 404
egg, at second maturation division. 381
embryo sac, 118
embryonic growth, 202-10
Subject Index 467
for exjianding buds, 384
experimental growth, 21 1—49
fibroblast cultures, 30
grafted sarcoma of mouse, 26
neoplastic growth, 25r)-7U
neuroblasts of grasshopper, 32, 33, 66.
70
in plants, 383-86
polarization microscope, 75
pollen, 1 1()
pollen mother cells, 110-13
pollen tube, 37, 108, 383
pohploidv
in animals, 58, 60, 380-83
in plants, 20, 57-58, 384-85
regenerating li\er cells, 44
seedling, 384
Siredon, 45
solutions used, 373-74
staminal hair cell, 73, 385
for stiid\ of mitosis, 374-80
temperature, 374
tissue cidture, 378-79, 385
tissue of tumors, 385
Triton, 40
Triturus, 40, 43
T II hi f ex eggs, 54
Telophase. .50, 65, 94, 117, 222, 376-78
agglutination at, 52
arrest reduces, 28-30
chromosomes of pollen tube. 108
colchicine and, 50
despiralizing stages at, 51
mitotic counts, 378
percentage decreases, 30
recovery in plants and, 94
restitution nucleus via c-telophase, 93
root tip percentages of, 34
in topography of mitotic growth, 375
Temperature, internal, 179
action on toxicitv of colchicine, 194,
203
effect of low, 415
Testis, interstitial cells, 226, 236
Testosterone, 217. 218, 232
propionate, 219, 233, 234
Tetramethoxy-9-methvlphenanthrene,
164
Tetramethoxy lO-methvlphenanthrene,
163
Tetramethoxy-9-phenanthraldehyde, 162
Tetramethoxvphenanthraquinone, 162,
163
Tetramethoxyphenanthrcne-10-
carboxylic acid, 162
4, 5-Tetramethvlene-tropolone, 417
Tetraploid, 276, 282, 283, 382: see also
Autoploidy, Autotetraploid, Poly-
ploid
AUiuiu. 25, 27, 28. 35. 79
alio-, 292-312
appearance of, 363
auto-, 318-19
cell, 95
chimeras, 356
colchicine method to make, 274-75
tliploid h\i)rids as fertile, 362
flower, stem apex, 349
fruit and seed of, 363
in Gossxpium, 304
in grapes. 324
induced in plants, 383-85
kernel weight in rve, 366
leaf of, 363, 368
mitoses of, 97
natural, in cotton, 283
natural tetraploid cell in Polys;o>i(itum.
37
performance of. 319-20
in phlox, 364
physiological features of, 367-68
in plants. 56
in pohploidv breeding, 285
raw, 321
in red clo\er, 368
reiteration of the c-mitosis, 55-56
sex stability in, 351-54
single c-mitosis, 55
in steel rve. 319
in strawljerry, 324
successful genotvpes, 283
in sugar beet, 332-33
superiority of, 320
treatment at anaphase, 70-72
in Triturus. 43
use of. to make triploid, 285, 326,
327, 328
in J'itica, 349
in watermelon, 327-30
Tetraploid wheat, 295
duriuii, 295
emmer, 295
T. persicum, 295-96
T. timopheevi, 298
Thalassemia, 224
Thermodynamic activity, 400, 407
Thiouracil, 238, 248-49
Thorn test, 197
Thousand grain weight in rve, 365
Ihreshold concentrations, 385
Thrombin. 194
Thromljopenia. essential, 189
Thymic cortex, 178
rinnionucleic acids, 53
1 Inmus, regeneration, 241
cells of, destruction, 261
Thvroid, 227. 230. 269
Ihvroidectomy. 249, 288
Thyrotropic hormone, 226, 228, 229, 248
rh\r()xin.238
and kitlncv Injjertrophy, 241
468 Subject Index
Tissue cultures. 105, 196, 21 1, LM5. 216.
219, 223, 257, 374, 379, 3X0, 385, 412
arrested metaphases in, 17, 373
cancer cells, 258
cellular multiplication, 219
chick heart, 418
colchicine derivatives, 41 1-12
fihrohlast cultures, 30, 44, 215-16, 418,
420, 421
hanging drop preparations of, 378
of Heliauthiis, 105
metliods, 17
methylene hlue stain, 91
mitotic stimulation in, 379
rabbit heart, 418
studv of mitosis by, 17, 337, 375
iji vitro, malignant. 17, 257
in vitro, normal, 17, 172, 257
in vivo, malignant, 17, 257
in viiH), normal. 17
Tobacco. 26. 279, 294; see also Nicotianu
Toes, regressive evolution of, 209
Tomato', 104, 325
autotetraploid, 325
Tool for study of growth, 378
Toxicity of colchicine, 3, 94, 96, 121, 195
cimiulati\e, 196
low, in plants, 383
nonspecific toxic reactions, 379
variations in, 194
rradescantia, 32, 69, 73-74, 111. 113. 170-
71, 392, 399, 415; see also Slaminal
hair cell
c-meiosis, 1 15
embryo sac, 117, 118
flower, 109
microspores, 115
pistil, 109
pollen mother cells. 111
polynucleate cells, 115
stem, 106
style, 109
Transformations of chromosomes, 50-52
Treatment with colchicine; see Tech-
niques of colchicine treatment
Triatoma infestans, 205
Tribromo acid, 169
T(//o//n;», 321-22
amphiploid of; T. repens x T. nigres-
cens,iV>
species of; hybridum, 322; pratense,
322; repens, 322
Trimetaphasc, 83
Trimetho\v-3-methvlnaphthalcne-l:2-
dicarboxylic anhydride, 166
Trimethylcolchicinic acid, 160, 40f-5,
408-9
di-benzoylation of, 166
Trimethvlcokhicinic acid methvl
ether, 408
Trinidad, 288
Triploid, 326-33, 381,382
alio-, 326
apples. 333
auto-, 326. 327
fruit bv diploid pollin;ilions, 327
fruits. 333
guava. 333
natural species of. 326
optimal number at, 327
parthenocarpy, 329
propagation of seed for, 328
seed production for. 327
seedless fruits of. 327
source for aneuploids. 354
sugar beets, 331-33
from tetraploid parents. 327
in watermelon, 327-31
Tripneustes escnlentus, 88
Trisomies, 355
Trispecies hybrids, in Gossypiinn, 306
Triticales, 300-301
homology of chromosomes between
rye and wheat, 300
Lebecieft, 300
meiotic irregularity, 300
Meister, 300
Miintzing. 300
Rimpan," 300
stability of, 300
Taylor, 1935, 300
vegetative propagation, 300-301
Triticinae, 362
Triticiim, 95, 277, 287, 288, 354; see also
Tritieales
cell plates, 95
a hexaploid species in, 358
with hybridization, 296
monosomies of, 358
multipolar groups, 95
ludlisomics of, 357-59
oriffin of, without hvbridi/ation. 297
root tips, 403
species of: aestivuiu. 279. 397; dicoc-
coides, 295; dicoeeuni, 297; iiiono-
coceuni, 295, 298; polonicum, 358;
spelta, 295; tinioplieevi, 298
test of spindle poison, 403
Triticuni amphiploids, intergeneric
T. aesliviiin x -^gropyron irlaueiini.
298
T. aestiviiin x --igropxion inter-
medium, 298
T. aestivum x Secale cereale, 300
T. dicoccoides X Aegilops sqnarrosa.
295
T. dicoccum X Aegilops s(juarrosa,
297
T. persiciun x Aegilops sijiiarrosa,
296
Trito7i
arrested metaphases percentage, 40
Subject Index 469
ball iiielaphases, 42
explanation for types of arrest, 82
exploded metaphases of, 41
inhibition of spindle by physical
agents, 399
oriented metaphase, 39, 83
origin of star metaphase, 39, 82, 83
pohploid cells of, 58
reco\er\ and treatment, 40
iinoriented metaphases, 83
vuliraris, 38-40
Trilui us
ball metaphase, 42
bimctaphases, 97
centrosomic body in, 9U
cornea, 97
development, 88, 203
differential counts of mitotic types, 40
distributed c-mitoses, 97
explanation of arrested types, 82, 83
exploded metaphase. 41-42
multiple stars, 83
newlv fertilized eggs, 58
origin of star, 39
reco\er\ figures, 39. 43, 97
species of heJveticus, 88: viridescens,
81. 82, 83, 90
unoriented metaphases, 83-84
Tropokinesis, 67, 86, 400
Tropolone, 15, 171, 415, 417
Trvpafla\ine, 193
Tubifcx. egg, 53, 54, 89, 91, 202
alteration in chromosome structure.
52-53
cytoplasmic \iscosit\. 91
destruction of chromosomes, 42, 53
developing eggs, 54
multiple stars, 39
pseudonuclei, 54
surface changes, 89
Tubuli contorti; see Kidney. con\oluted
tubules
Tumor cells, enlargement of, 266
Tiunors, 255: see also C-tiunors, Neo-
plasms, Neoplastic cells
of colchicine, 268
cure of mice, 261
in man, 267
necrosis of, 260
in plants. 265-66
tiuiior respiration, 397
Tunica in plants, 348
Twin seedling method, 334
cotton, 334
in flax, 334
Gossxpiuni, 334
peppers. 331
u
Ultra violet light. 91. 170
isomerization of colchicine, 170
Llva, 119
zoospores, 125
zygotes. 125
Unipolar, 86
Ignited States of America, 287, 288, 322
fruit impro\ement, 323
State and Federal experiment
stations, 288
Univalents. 1 13
Universitv of California, 287
University of Manchester, 324
University of Oklahoma, 349
Unsaturated ketone, 163
Urechis. 399
Ureter, 237
ligature of. 2.39
Urethane, 265
Uric acid, in goiu, 197
Urine. 194-96
U. S. P. colchicine, 141
U. S. Pharmacopeia, 373
Uterus, 181
inuscle cells of, 226
of rabbit, 217, 218
V
\'accines, 140
Vagina, 226, 227
action of estrogens on, 214
Vnllisneria, 352
\'ascularization, 128
of plant tiuiiors, 258
sclariform vessels. 128
Vaseline-lanoline paste, 377
Vedic texts, 2
\'enom of bee, 104
Veratrine, 14, 413
Vernae, 9
VerticilUum dahline. 123
Vicia faba. 393
\ictoria blue, 420
J' i lira
chromosomes. 349
diploid, 349
larger flowers, 349
pollen mother cells, 349
species of: minor, 348; rosea, 326, 348
tetraploid, 349
\'iridis miuant. 55
\'irus tiunor tissue, 132, 385
Black's original R. strain
\'iscositv, rh\thm of. 74, 420
and c-tiunors, 1 10
cvtoplasm, 110
high, 80, 89
protein dissociation, 110
Vitamin. 275, 368
Bi, 109
C, 324
\'oraiting, symptom of colchicine
poisoning, 178. 179
470 Subject Index
W
Warburg flasks, 377
Warmblooded animals, 1, 374, 379
Warts, 263
W^asa II, 366
Water, 383
Watercress, 324
growth rates reduced, 370
increase of \ itamin C, 324
slower growth, 324
succulence, 324, 370
Watermelon, 327-31
cavity in seeds of, 329
chromosomal types, 327-31
commercial growing, 331
female sterility, 327, 329
genetic marks to distinguish, 329
increase in number of fruits, 329
parthenocarpy in, 329
propagation of seed for, 328
seed for, 328-29
seed production, 331
seedless fruits, 327, 329
special cidtivation of, 329
tetraploid seed parent, 327-31
triploid seed, 327
triploids, 327-31
weight. 329
yield, 329
Waxes, 140
Weed killer, 275
Wheat, 26; see also Triticuin
Wheat seedlings, 104
White blood cells, 176, 184
^Vindaus' formula of colchicine, 171
AVoodland strawberry, cidtivated strains,
323
\Voimd healing, 246
Xanthine-dehydrase, 397
Zanthopterin, 241
A' -chromosome, 352-54
in plants, 351-54
ratios, 353-54
Xeuopiis
amputated tail, 376
laevis, 97, 210, 242, 245
larvae, 376
regeneration of tail, 376
X-ray, 54, 55, 105, 258, 266, 275
crystallographic analysis, 169
V-chromosomc, 352-54
in plants, 352-54
ratios, 353-54
Veast, 120, 121
brewing test, 122
methvlene blue, 123
polyploids, 121
Zea, 354
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