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Full text of "Colchicine in agriculture, medicine, biology, and chemistry"



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 





69 


3 


00 





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 





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 




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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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62 Colchicine 

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tissue cultures. Yale Jour. Biol, and Med. 13:39-49. 1940. 

91. Vaara.ma, \. Morphological and (Vtological stud'es on colchicine-induccil Rihes 
?iigrum. Acta Agralia Fennica. 67:55-92. 1947. Spindle aljnormalities and 
variation in chromosome niniiijer in Rihcs nigrum. Hereditas. 35:136-62. 1949. 

92. ViLTER, V. Inhibition colchicinique de la mitose chez les Mammiferes. C. R. Soc. 
Biol. Paris. 138:60.5-6. 1944. 

93. Wada, B. Lebendbeobachtiuigen iiber die Einwirkimg des Colchicins auf die 
Mitose, insbesondere iiber die Frage der Spindclfigin . C\tologia. 11:93-116. 
1910. Eine neue .\Iethode zin- Lebendbeobachtiuig der .Mitose bei den Trades- 
ca?!t/a-Haarzellen. Cytologia. 13:139-45. 1943. Further studies on the effect 
of colchicine upon the mitosis of the stamen-hair in Tradescanlin. C\tologia. 



I' • 



64 Colchicine 

l&:88-95. ,19,49. The mechanism of mitosis based on studies of the submicio- 
scopic str«c,tuie and of the living state of the Tradescantia cell. Cytologia. 

. . I6:.l-26. ■ vJoO. 

94." WiLSO:?, 6^, .and Cheng, K. Segregation and reduction in somatic tissues. Jour. 

MereO-^OiS-e. 1949. 
^ST" WoKER, H. Phasenspezifische Wirkung des Colchicins auf die ersten Furchung- 
steilungen \on Tiibifex. Rev. Suisse Zool. 50:237-43. 1943. Die Wirkung des 
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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2. Barber, H., and Callan, H. (see Ref. No. 1, Chap. 2. 1943) . 

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Spindle and Cytoplasm 99 

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chromosomes induites. che/, les vcg(?taux, par la colchicine. C. R. Soc. Biol. Paris. 
126:985. 1937. Mc;canisme d'action de la colchicine sur la carvocinese des 
vegetaux. C. R. Soc. Biol. Paris. 128:714. 1938. 

41. Giordano, A., and Grampa, G. Mitosen bci experimenleller Leberregeneration 
unter Colchicin-Wirkung. Zentralbl. Allg. Path. 85:25-29. 1950. 

42. Grampa, G. Azione citoplasmatica della colchicina nel fegato normale e in 
rigenerazione sperimentalc. Studia Ghisleriana. (Studi Medeci Biologici) Ser. 
III. 1:1-10. 1949. Osservazioni col microscopio a contrasta di fase sul condrioma 
delle cellule epatiche in rigenerazione sperimentalc. Boll. Soc. Ital. Biol. Sper. 
27:808-10. 1951. 

43. Guyer, M., and Claus, P. Destructive effects on carcinoma of colchicine followed 
by distilled water. Proc. Soc. Exp. Biol, and Med. 43:272. 1939. 

44. Haas, H. tJber die Beeinflussung des Zellkerns durch Pharmaka. Arch. Exp. Patli. 
197:284-91. 1941. 

45. Hawkes, J. (see Ref. No. 46, Chap. 2. 1912). 

46. Hughes, A. The effect of iodoacetamide upon cell division in tissue cultures of 
the chick embryo, [our. Ro\. Micr. Soc. 69:215-21. 1949. Mitotic cycle. 
.Academic Press, New \ork, 1952. 



700 Colchicine 

47. Inoue, S. a method for measuring small retardations of structure in living cells. 
Exp. Cell Res. 2:513-17. 19.')1. The effect of colchicine on the microscopic and 
submicroscopic structure of the mitotic spindle. Exp. Cell Res. Suppl. 2:305-18. 
1952. 

4S. JAHN. U. (see Ref. No. 50, Chap. 2. 1952) . 

49. Kanter, M. Der Einfluss des Colchicins auf die Zellteilung lebender Amphi- 
bien. Z. Anat. 115:273-8(). 1950. 

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 mi totic 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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59. Havas, L. Is colchicine a "'phvtohormone"? Growth. 2:257-60. 1938. 
Parthenocarpy and accompanyitig hormonal syndromes induced by unrelated 
chemicals. Nature. 157:629-30. 1946. Problems of giowth studied with the 
aid of colchicine in plants and animals. Ann. Soc. Roy. Sci. Med. et Nat. 
Bruxelles. 1:57; cf. Chem. Abst. 42:4676c. 1948. Hormone-mimetic and 
growth effects of colchicine in plants. Exp. Cell Res. Suppl. 1:597-601. 1949. 
Effect of bee-venom on colchicine-induced tumors. Nature. 166:567-68. 1950. 

60. , AND Baldensperger. a. The oncological aspect of the "inimunitv" 

of Colchicum to colchicine. Science. 114:208-10. 1951. 

61. , AND Felfoldy, L. Fasciations and kindred teratisms induced in 

plants bv polvploidogcnic agents. Magvar. Biol. Kut. Muukai. 17:131—40. 
1947. 

62- Hawkes, J. Some effects of the drug colchicine on cell di\ision. Jour. Genet. 
44:11-22. 1942. 

63. Heise, F., and Steenken, \V. Growth and virulence of tubercle bacilli. A 
studv of the effect of vitamin Bj, and B,;, ribona\ine, colchicine, phthiocol and 
th\loquinone. Amer. Rev. Tidjerc. 44:635-36. 1941. 

64. Heitz. E. Moosmutationen 1. Spontane mid durch Colchicin ausgeloste 
pohploide Mutanten dei Aulacomnium androg^'iuim (L.) . Arch. Julius 
Klaus-Stift. 20:119-25. 1945. Uber ■"Colchicinmuster." 8. Jahresber., Schweiz. 
Ges. \'ererbungsforsch. .\rch. Julius Klaus-Stift. (";4) , pp. 497-99. 1948. 

65. J.^KOB, H. Influence de la colchicine sur le dcveloppement de certaines algiies 
d'eau douce. C. R. Acad. Sci. Paris. 230:1203-5. 1950. Action de la colchicine 
sur certaines algues d'eau douce et comparison avec Taction des substances 
metaboliques en provenance d'algues. Rev. Gen. Bot. 58:348-52. 1951. 

66. Jennison, N. The inactivitv of colchicine for bacteria. Jour. Bact. 39:20-21. 
1940. 

67. Kali. to. P. The significance of nuclear quantit\ in the genus Mioaslcyias. 
.\nn. Bot. Soc. Zool. Bot. Eenn. 24:1-122. 1951. 

68. Kartashova, N. Treatment of vegetable cells with colchicine. C. R. Dokl. 
Acad. Sci. URSS 46:372-74. 1945. 

69. Kavser, F., and Besson, S. Action of sulfonamides and colchicine on develop- 
ment oi: pea roots. C. R. .Soc. Biol. Paris. 142:84-86. 1948. 

70. King, C. Changes of carbohydrates of germinating wheat seeds in manganese 
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 
X-rays on plant tissue. Torrey Bot. Club Bull. 72:563-74. 1945. Action of 
colchicine on cell division in human cancer, animal and plant tissues. Ann. 
N. Y. Acad. Sci. 51:1365-1406. 1951. 

86. , AND Lein, J. The effect of various growth substances on the 

number and the length of roots of Allium cepa. Amer. Jour. Bot. 28:163-68. 
1941. 

87. Loo, T., AND Tang, Y. Growth stimulation bv manganese sulphate, indole- 
3-acetic acid, and colchicine in the seed germination and early growth of 
several cultivated plants. Amer. Jour. Bot. 32:106-14. 1945. 

■""Tft 88. Mairoij;, F. Studicn an colchiciniericn Pflanzen. Protoplasma. 37:445-521. 
1943. 

89. , AND Weber, F. Matricaria cliamnmilla durch Colcliizinierung ohne 

Strahlenbliiten. Phyton. 2:271-75. 1950. 

90. Mangenot, G. Substances mitoclasiques et cellules vegetales. £tat actuel 
de la cjuestion d'apres les travaux publics juscjua la fin de 1940. Rev. Cvtol. 
et Cytophysiol. Veg. 5:169-264. 1941. Action de la colchicine sur les racines 
d'Allium cepa. Hermann and Cie., Paris. 120 pp. 1942. 

91. Mariin, G. Action de la colchicine sur les tissus de topinamliour culti\e /;; 
vitro. Rev. Cytol. et Cytophysiol. Veg. 8:1-34. 1945. 

92. Martinez, L. Influence of various alkaloids on the growth of wheat seedlings. 
Farmacognosia. Madrid. 9:231-46. 1949. 

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 
the pollen grains of some Ephedra Spp. Proc. Nat. Inst. Sci. India. 12:333-40. 
1946. Colchicine effect and the production of abnormal spermatozoids in the 
prothalli of Dryopteris subpubesceus (Bl.) C. Chr. and C.ouiopteris prolifera 
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 
after colchicine treatment. Naturwiss. 31:570. 1943. 



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. 

101. XiCKEEE, L. Effect of certain jjlant hormones and colchicine on the growth 
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 
et chimicjues sur les plantides de hie. Re\ . Can. Biol. 7:35-159. 1948. 

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- 
cosis. Mecanisme de la Xarcose. Colloques Internat. Centre Xat. Recherche 
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- 
chicine hehanticlde CV/»»M/)/5-planten. Erfelijkheid in Praktijk. 4:171-73. 1939. 

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. 
^ Suppl. Abst. 680-81. 1949. 

144. Vandendries, R., and Gavaudan, P. Action de la colchicine sur quelqucs orga- 
nismes inferieurs. C. R. Acad. Sci. Paris. 208:1675-77. 1939. 

Inderwalle, R. Observation sur Taction de la colchicine et autres sub- 
stances mitoinhibitrices sur quelqucs champignons phytopathogenes. Bull. 
Soc. Roy. Bot. Belg. 72:63-67. 1939. 
146. Vietez, E. Palynological observations on some Spanish honeys. Torrey Bot. 
Club Bull. 77:495-502. 1950. 
. 147. Wada, B. Lebendbeobachtungen iiber die Einuirkung des Colchicins auf die 
V Mitose, insbesondere iiber die Frage der Spindelfigur. Cytologia. 11:93-116. 

1940. 
.148. Walker^ R. The effect of colchicine on microspore mother cells and micro- 
spores of Tradescantia paludosa. Amer. Jour. Bot. 25:280-85. 1938. The 



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. 

149. Walker, A., and Youmans, G. Growth of bacteria in media containing col- 
chicine. Proc. Soc. Exp. Biol, and Med. 44:271-73. 1940. 

150. Wang. F. Effects of auxin, colchicine and certain amino acids on the germi- 
nation of Lotus cortiiciildlus pollen. Biociiem. Hull. China. 38:1-3. 1914. 

151. Weber. F. Spaltotlnungsapparat-anomalien colchicinierter TnidcscautiaAAat- 
ter. Protoplasma. 37:556-65. 1943. 

152. Weichsee, G. Polyploidie, veranlasst durch chemische Mittel, insl)Csondere 
Colchicinwirkung l)ci Lef>uminosen. Zuchter. 12:25-32. 1940. 

133. Weissenbock, K. Studien an colchizinierten Pfianzen. I. Anatomisdie I'nter- 
suchungen. Phyton. 1:282-300. 1949. 

154. Werner, G. Untersuchungen fiber die Moglichkeit der Erzeugung polyploider 
Kultiupfianzen durch Colchicinbehandlung. Zuchter. 11:51-71. 1940. 

155. Wevland, H. The action of chemicals on plants and its significance in medi- 
cine. Z. Krebsforsch. 56:148-64. 1948. 

156. WiTKUS, E., AND Berger, C. Induced vascular ditlerentiation. Torrey Bot. 
Club Bull. 77:301-5. 1950. 

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. 
Bot. and Zool. .Syokubuta Oyobi Dobuta. 8:1709-14. 1940. 

159. Zambruno, D. .Vzione della colchicina. della narcotina, e dell'androstendione 
sulla moltiplicazione delle Staplixlococcus aureus. Giorn. Batt. hnmul. 
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 




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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. 
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[147] 



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 



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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. 

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23. Hooper. E., and King, K. The international standardization of Cohhicuni 
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33. Ki.EiN, G., and PoLLAiiF, G. Microchemical detection of alkaloids in i)lants. 
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156 Colchicine 

34. KoLDA, J. Excretion of chugs in the millv II. Bioh Listv. 12:236-67. 1926. 

35. KoLTHOFF. I. Tlie dissociation constants, sohiliilitv piocluct and titration of 
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36. KuHN, A., AND ScHAFER^ G. Distribution of plant constituents in the capillary 
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37. Laungy. L. Sensitiveness of the general method for extracting alkaloids from 
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38. Lazurevskii, G., and Maslennikova, V. Investigation of cokliicine-contain- 
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39. LiPTAK, p. Localization of alkaloids in the seed of Colrhicuni autinnuale L. 
Pharm. Monatsh. 8:125-26. 1927. 

40. Loudon. J., and Spearman, J. The solubilitv of colchicine in water. Research. 
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41. Lyons, A. Assav of Colcliicum corm. Amer. Druggist. Feb. Pharm. Jour. 
82:270. 1909. 

42. Mack, H., and Finn, E. A calorimetric method for llic estimation of colchicine 
in pharmaceutical preparations. Jour. Amer. Pliarm. Assoc. 39:532-34. 1950. 

43. Martini, A. Contribution to the microchemistry of colchicine and atropen. 
Anales Asoc. Quim. Argentina. 31:62. 1943. 

44. Mascre, M., and Dfysson, G. Action mitoclasicjue de la desmethylcolchicine, 
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234:2480-82. 1952. 

45. Mehra, p., and Khoshoo, T. Observations on .some colchicinc-containing 
plants. Jour. Pharm. and Pharmacol. 3:486-96. 1951. 

46. Mokrantza. M. A new reagent for the identification of various alkaloids. Bull. 
Soc. Chim. Ray. Yougoslav.' 3:171-76. 1932. 

47. Morrison, J. Preliminary examination of the crystal structures of colchiceine 
and its copper salt. (Univ. Glasgow.) Acta Cryst. 4:69-70. 1951. 

48. MuHLEMANN, H., AND ToBLER, R. Chioma togiaphisch-titrimctrische .Alkaloidge- 
haltbestimmung von Tinctura Colchici. Ph. H. V. und Semen Cokhici Ph. 
H. V. Pharm. Acta Helv. 21:34-46. 1946. 

49. Nakayama, K. Habit and culture of CukJiicuin as the pharmaceutical plant. 
Agr. and Hort. Tokyo. 24:639-12. 1949. 

50. National Formulary, 8th ed. Amer. Pharm. Assoc. W'ashington, 1). C. 1946. 

51. Niemann, E. Exj^eriments on the use of flowers of Colchicuni autmunale in 
place of semen colchici. Pharm. Acta Helv. 8:92-107. 1933. 

52. North, E., and Beal, G. The preparation, properties and uses of silicoduo- 
tungstic acid. Jour. Amer. Pharm. Assoc. 13:889-98; 1001-9. 1924. 

53. OsoL. A., AND Farrar, G. U. S. Dispensatorv, 24th ed. |. B. Lippincott, Phila- 
delphia, Pa. 1947. 

54. Parthasarathy, N. An Indian source for colchicine. Curr. Sci. Bangalore. 
10:446. 1941. 

55. Paschkis, H. Phaimakologische Untersuchungen iiber Colchicin. Med. Jahrb. 
Vienna. Pp. 257-58. 1883! 

56. Perrot. E. Une plante nouvelle a colchicine, le lofout hlacee saharienne. C. R. 
Acad. Sci. Paris. 202:1088-89. 1936. 

57. RoBERC. M. Occurrence and distribution of saponins in seed drugs, .\rcli. Pharm. 
275:328-36. 1937. 

58. RojAHN, C, AND Herzoc, H. Decrease in content of alkaloidal salt solutions 
and pharmaceutical tinciures in sun and ultra violet light. Pharm. Zentralhalle. 
73:401-10. 1932. 

59. Rosenthaler, L. Microchemical behavior of the official alkaloids. Amer. Jour. 
Pharm. 101:821-29. 1929. Economic drug testing. XII. Pharm. Ztg. 76:288. 
I93I. 

60. Salgues, R. Influence of fertilizer on the yield and composition of some cul- 
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61. Santavy, F. Polarograficke stanoveni kolchicinu a kolchiceinu. Zvlast. Otisk 
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z ocunu jescnnino. Chem. Listv. Rocnick. 42:177-80. 1948. Sur la variabilite 



Sources of f he Drug 7 57 

de la tcneur en colchicine des seinences de colchique. Phaim. Acta Helv. 
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62. , AND lit ciiMCEK, J. Siu la variabilite de la lencur en colchicine des 

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63. . AND Rkichstein, T. Alkaloide der Herbstzeitlosenzwiebeln wahrend 

deren Entwicklung: Substanzen der Herbzeillose unci ihre Derivale. XXV. 
Pharm. Acta Helv. 27:71-76. 1952. 

64. . et al. Siihslances oi Colchicum autunuuilc u.nd iheir i\eri\AU\cs. XXI. 

Isolation of substances from the conns. Ann. Pharm. Franc. 9:50-59; cf. 
Chem. Abst. 45:4343a. 4888a. 1951. 

65. ScHi'ULER. H. Spectral and phvsiochemical properties of colchicine. C. R. .\cad. 
Sci. Paris. 210:490-93. 1940. 

66. Self, P., and Corfield, C. Determination of colchicine in Colcliicuin corm and 
seed and the official Colchicum preparations. Quart. Jour. Pharm. and Phar- 
macol. 5:347-56. 1932. 

67. Seifert, R. Determination of colchicine in the drug and tincture. Deut. .\poth. 
Ztg. 58:77-78. 1943. 

68. Stefanoff. B. (see Ref. Xo. 41, Chap. 1. 1926) . 

69. SuzLKA, O., and Savvak, K. a study on the tetraploid Datura stianionium L. 
induced bv colchicine. Jap. Jour. Pharmacog. 4:14-17. 1951. 

70. Taran, E. Chemical study of some alkaloid-bearing plants. I\'. Alkaloids of 
Caucasian Colchicum speciosum Stev. Farmatsiya. No. 9-10, 38-40. 1940. 

71. Traub, H. Colchicine poisoning in relation to HememcaUis and some other 
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72. Uffflie, O. The determination of colchicine in Colchicum seed. Pharm. 
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73. I'mnev, J. Notes on Colchicum seeds. Pharm. Jour. 95:393. 1915. 

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86:342-72. 1920. 

ADDITIONAL REFERENCES FOR TABLES 5.2 AND 5.3 
NOT CITED ABOVE 

82. Beer, A. A.: C. R. Dokl. Acad. Sci. IRSS. 69:369. 1949. Chem. .\bst. 4 1:2178 
g. 1950. 

83. Bellet, M. p. .\nn. Pharm. Franc. 1():S1. 1952. 

81. Geiger, P. L. Ann. Chem. Pharm. (later, Liebigs .Vnn.) 7:274. 1833. 

85. Houde, A.: C. R. Acad. Sci. Paris. 98:1442. 1884. 

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URSS. 87:227. 19,52. 

87. Masinova, v., and Santavy, F. Chem. Listy. In press. 

88. Oberlin. L. Ann. Chim. Phvs. [3] 50:108. 1857. 



758 Colchicine 

89. Pelletier, p. J., and Caventou, J. Ann. Chira. Phys. 14:69. 1820. 

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75:3854. 1953. 

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In press. 

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101. AND Talas, M. Chem. Listy. 47:232. 1953. 

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103. SuBBARATNAM, A. V. Sci. lud. Rcs. 11:446. 1952. 
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105. Weizmann, a. Bull. Research -Council Israel. 2:21. 1952. 

106. Zeisel, S. Monath. fiir Chem. 7:557. 1886. 



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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52. Grewe, R., and WulFj W. Die Umwandlung des Colchicins dinch Soinicnlicht. 
Chem. Ber. 84:621-25. 1951. 

53. Santavv, F. Sid)stan/en der Herbstzeitlose imd ihre Derl\ate. XXII. Photo- 
chemisclie Produkte des Colchicins und einige seiner Derivate. Coll. Czech. 
Chem. C;omm. 16:665-75. 1951. 

54. Boekelheide, V., and Pennington, F. C. Coumarins as intermediates in the 
s\nthesis of colchicine analogs. Join. .\mer. Chem. Soc. 74:1558-62. 1952. 

55. .\nderson, A. G., and CiREEF, H. F. Synthesis of dimelhvl 6.7,8,9. -tetrahydro- 
5H-c\cloheptahcnzene-5-acetate-6-pr(>pionate. }our. Amer. C4iem. Soc. 74:5203-4. 
1952.' 

56. Ginsberg, D., and Paim'o. R. Colchicine studies. I. Synthesis and reactions of 
2-arvlrTr/ohept-2-enones. Jour. Amer. Chem. Soc. 75:1094-97. 1953. 

57. Koo, j., AND Hartwei.i,, ). L. Synthesis of 2:3:4-trimethoxybenw)suberene and 
2:3:4:-trimetho\vbenzosuberancarboxvlic acids and esters, [our. Amer. Chem. 
Soc, 75:1625-28.' 1953. 

58. Tarbell. D. S., Hirschi.er. H. R., and Hall, T. J. Syntheses in the thiochrom- 
anone field, jour, .\nier. Chem. Soc. 75:1985-87. 1953. 

59. GuTscHE, C. b., AND Seetc:man. K. L. Preliminary experiments on the synthesis 
of colchicine: a method for s\nlhesising ring B. )our. Amer. Clicm. Soc. 
75:2579-84. 1953. 

60. Wada, B. The eflect of chemicals on mitosis studied in Tradcsriniti/i cells /» 
x'ix'o. I. p-.\cet\laminotropolone. Cytologia. 17:14-34. 1952. 

61. Raffauf, R. ¥.. Farrfn, .\. L., and I'llvot, G. E. C:"-Labeled cokhitine dc 
rivatives. Jour. Amer. Chem. Soc. 75:2576-78. 1953. 

62. Horowitz, R. M.. and I'i.lyot, G. E. Desinethylcolchicine, a constituent of 
U.S. P. colchicine. Science. 115:216. 19,52. 

63. Bellet, p. Le colchicoside. I. Ann. Pharm. Franc. 10:81-88. 1952. 

64. . ,\miard, G., Pesfz, M., and Petit, A. Sur le colchicoside. II. Syn- 

these partielle et constitution. Ann. Pharm. Franc. 10:211-16. 1952. 

65. AND Regnier, P. Colchicoside et colchicine. III. Sur ([uelques 

singularites de pouvoir rotatoire. .\nn. Pharm. Franc. 10:340-44. 1952. 

66. Santavv. F.. and Reichstein. T. .\lkaloide der Herbst/eitlosen/w iebeln [Colchi- 
nun tiuluiinialc L.) wahiend deren Entu icklung. Sul)stan/en der Hei bstzeitlose 
inul ihre Derivate. (25. Mitteilung) Pharm. Acta Helv. 27:71-76. 1952. 

67. -, C.ERNOCH. M., Malinskv, J., Lang, B., and Zajickova, .\. Isolement 

des substances des bulhes des differentes especes du genre Colchkiue. Sub- 
stances tirees du Clolchicjue et letirs deri\es. (21e Commmiicatioii) .\nn. 
Pharm. Franc. 9:50-59. 1951. 

(i8. . Substanzen der Herbstzeitlose und ihre Derivate. XX\11. Beitrag 

/in- Konstitntion der Substanz F. Coll. Czech. Chem. Comm. 16:(i7()-8S. 1951. 



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 

•,\ 



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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 






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D 







D 




-D 


JI 


u 


0) 


>- 


u 


■•- 


-C 


^ 




ID 






o 


M- 


>. 


C 


_0 





^ 




CO 


OJ 


-D 



Q. 


iq 


s 





K 


5 




_^ 




"5 


(-> 


_D1 




•4- 


(U 

It 


Ll_ 




• — 


0) 



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 

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 . 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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Pharmacology 799 

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31. Haismann, W. t^ber den Einfluss der Temperatur auf die Inkuhationszeit 
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32. Havas, L. Influence of colchicine on the sexually induced colour change of 
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34. Hellman, L. Production of acute goutv arthritis l)y adreno-corlicotrophin. 
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35 Jacobj, C. Pliarmakoligische I'ntcrsuc.hiuig iibcr das Colchicum-gift. Arch. 
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38. Klein, H. Zur pathologische Anatomic der Alarmreaktion nach Kerngiften. 
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39. Lavam, F., Aschkenasy, A., and Mouzon, M. Intoxication aigue par la col- 
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40. Lambers, K. uber Organveranderungen bei chronischer Colchicin-\ergiftung. 
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41. Landoet, R. Uber die Wirkimg der Colchicins auf das normale und Icukiimische 
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42. Lebeond. C. p., and .Segal, G. Action de la colchicine sur la surrenale et les 
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43. Lecomte, J. .\ction de la colchicine et des poisons radiomimc'-tiques sur le 
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44. Leibholz. Ein Fall von Colchicin-Vergiftung und einige Bemerkungen iiber 
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45. Levjn. M. H., Fred, L., and Bassett, S. H. Metabolic studies in i;()ui. Jour. 
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46. Levine, H. The effect of colchicine on the adrenal cortex. Jour. Lai). Clin. 
Med. 38:921. 1951. 

47. Lris. F. Contribution a I'ctude des reactions cellulaires pro\o(pu'Cs par la 
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reactions et lesions celhdaires piosociuces par la (oUhitine. Ar(b. Int. Nh'd. 
Exp. 11:811-901. 19.36. 



200 Colchicine 

48. LoicQ, R. Recherches sur les effets de la colcliicine sui la coagulabilitc du 
sang. Arch. Int. Med. Exp. 12:371. 1936. 

49. LoRTHioiR. p. Hvperlentocvtose experimentale et glycoregulation. C. R. Soc. 
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50. Manx. H. Die Einwirkung von Colchicin und Sexualhormonen auf den 
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51. McKracken, |., Owen, P., and Pratt, J. Gout: still a forgotten disease. Jour. 
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52. McPhail, M. K., and Wilbur, K. M. The stimulating action of colchicine 
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Absence of potentiation of gonadotropin and steroid function in mammals 
by colchicine. Endocrinology. 35:196-97. 1944. 

53. MiszrRSKi. B., and Doljan.ski, L. Effect of colchicine on the rat li\er. .\mer. 
Jour. Anat. 85:523-45. 1949. 

54. MuGLER, A. Action de la colchicine sin- les accidents allergiques. Ann. Med. 

51:495-505. 1950. 

55. , AND HAUS^VAED, R. A propos de I'efficacitc de la colchicine sur le 

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histaminii|uc. Bull. Mem. Soc. Med. Hop. Paris. 1952. 

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57. PaschivIS, K. E., Cantarow, A., Walkling, A. A., and BovlE;, D. Adrenal corti- 
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338. 1950. 

58. Ravmond-Hamet. Sur uue proprictc i^livsiologiciuc noiuelle de la colchicine. 
C. R. Soc. Biol. Paris. 118:1292-95. 1935. 

59. Rehbein, M. Pharmakologische Untersuchimgen iiber die Darmwirkung des 
Colchicins. Disser. Freiburg, i. B. 1917. 

60. Robin.so\, W. D., Conn, J. W., Block. W. D., Lot is, L. H., and Ratz, J. Role 
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61. RossBACH, M. J. Die phvsiologischen Wirkungcn des Colchicins. .\rch. Ges. 
Phvsiol. 12:.308-25. 1876.' 

62. Rossi, S. Steatosi parenchimali da colchicina. Dillcrcnza di sensiljilita dci due 
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63. Sanno, \. Uber den Einfluss der Tcmperatur auf die Giftempfindlichkeit des 
Frosches. \'ersuche mit Atoxvla und Colchicin. .\rch. Exp. Path. 65:325-36. 
1911. 

64. Santaw, F. Les variations sanguines au coins de I'intoxicatiou par la col- 
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65. , and Kadlec;, K. Glucose du sang chez le chien intoxiiiuc par la col- 
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6G. Schmidt, F., and Mugler, .'\. Action de la colchicine sur la tension artcrielle 
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67. Sc;hroff, K. D. v. Iniersiuhungen iiber die /\\iel)el der Zeitlose und \'er- 
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68. Seed, L., Slaughter, D. P., and Limarzi, L. R. Effect of colchicine on human 
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70. Stetten, De Witt, Jr. The pool of miscible uric acid in normal and gouty 
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1 950. 



Pharmacology 201 

71. Tatsumi. J. Effect of colchicine and epinephrine on the erythrocvlc diameter. 
Min. Meci. Akad. Kioto. 2:1070-80. 1940. 

72. X'AccARr, F.. and Rossanda, M. Blood glvcosis dining the alarm-reaction. Natine. 
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74. \'n«ERT, C. Precis de toxicologic. Paris. 1907. 

75. \'oLLMER, H. Tiergrosse und Enipfindlichkeit gegen Hvdrochinon luid col- 
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7(>. \\ Ai ASZEK. E. J., Kelsev. F. E., and Geilin'O. E. M. K. Biosynthesis and iso- 
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(ACTH) and colchicine. Jour. Lab. Clin. Med.' 34:1766. 1949. 

79. , Hunt, H. D., Cohn, C., Robinson. AV. D., and Dufe. L F. ACTH and 

colchicine in the clinical treatment of acute gouty arthritis. Physiological 
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80. , AND Cohn. C. The role of the pituitary adrenocorticotrophic hor- 
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Conf. The Blakiston Co., Philadelphia. 1950.' 



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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.5. Bretscher, .\. Reduktion der Zehenzahl bei Xenopus-I ar\en nacli lokalei 
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6. Brock, N., Drickrey, H.. and Herken. H. Uber Kerngiftc luul Cxtoplasma- 
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7. BcsHNELL, R. J. Some effects of colchicine on the early development of the 
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154:150. 1944. 

9. CoEOMBO, G. L'azione della colchicina sulla sviluppo embrionalc di Rana 
agilis. Boll. Soc. Ital. Biol. Sper. 20:657-58. 1945. 

10. Gabriel, M. L. Production of strophosomy in the chick embr\o l)\ local 
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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- 
mals. Kungl. Svenska Vetenskapakad. Handl. IV. Ser. 1:1-11. 1950. Kol- 
chizininduzierte Heteroploidie beim Sch\vein. Kungl. Svenska Vetenskapakad. 
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- 
ture of the cortical layer of fertilized and parthenogenetic sea urchin eggs. 
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- 

handelten Froschkeimen. .Arb. Ung. Biol. Forsch. Inst. (Tihanv) . 13:516-58. 
1941. 

44. AVoKER, H. Phasenspezifische AV'irkung des Colchicins auf die ersten Furchung- 
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Colchicins auf Furchimgsmitosen und Entwickhmgleistungen des Tubifex- 
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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. 







0.28 
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 



41 


5 



35 




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. 








H 


• 








70. 


o 

o 

8 


• 




\ 








60- 


O 
o 
o 


8 

o 
o 




• 


• \ 

• \ 

• \ 













\& 






• > 


V 






so- 





^v 




V 8 


• 


\ 













\^, 


<k ! 


•\ 






40- 
30- 




o 


o 

o 
o 




-x 


t 










o 

o 




X 


• 


\- 










o 


o \ 




V 


20- 








o 







o 




o N 

8 


• 
• 


^ 


lO 






r- 


1 


1 


1 






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 
. 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 


. 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 





0.2 



















II 



2 

0.3 

1 





1 

2 

0.5 





A 



I 



5 
2 
1 
2 



0.3 

1 

2 

1 





II 

17 

7 

14 

4 




B 



I 



1 
1 









6 

9.5 







II 



4 


6 
5 
10 











* 

0.5 







II 



6 
3 

2 
1 



5 

18 
5 




* 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 





4.5 


3 


43.5 


13.5 


0.5 


57.5 


8 


43 


9 


1.5 


53.5 


14 


18 
18 


4 







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 


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 >. - 

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O IV 3 

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■ - OJ O 
-^ w 

4, 0) O 

^ c 

O m 



"0 



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lU 

c 



3 

a 
o 

c 

V 



•5 2 

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-a 

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<ll 



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-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 





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 








4. . . 


81 





185 


81 


9 


164 


'-1 

1 


1 





5. . . . 


31 


1 


86 


15 


14 


12 





9 





9 


2 





8 


27 


8 


10 











12. . . . 


7 


1 


7 


27 


22 


8 





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 










REFERE 


:NCES 





Allen, E., and Greadick, R. N. Ovogenesis during sexual maturity. The first 
stage of mitosis in the germinal epithelium, as shown bv the colchicine tech- 
nique. Anat. Rec. 69:191-95. 1937. 

. Smith, M., and Gardner, W. U. Accentuation of the gro\vth effect of 

theelin on genital tissues by arrest of mitosis with colchicine. Anat. Rec. 67: 
Suppl. 1:49. 1936. Accentuation of the growth effect of theelin on genital 
tissues of the ovariectomized mouse by arrest of mitosis with colchicine. Amer. 
Jour. Anat. 61:321-42. 1937. A short test for ovarian follicular hoiuione and 
other estrogens. Endocrinolog)'. 21:412-13. 1937. 

, , and Reynolds, S. R. M. Hyperplasia of uterine muscle, as 

studied bv the colchicine method. Proc. Soc. Exp. Biol, and Med. 37:257-59. 
1937. 

-, Thomas, T. B., Wilson. J. 



G., and Hession. D. Differential grouih in 
the time of ovui;ition in rats treated with 



8. 

9. 
10. 



the ovaries and genital tract near 

colchicine. Amer. Jour. Anat. 72:291-337. 1943. 

Astaldi, G., Bernardelli, E., and Rebaudo, G. Research on the proliferation 

activity of ervihroblasts at low atmospheric pressure. Experieniia. 8:117-19. 

1952. La proliferation de lervthroblaste en depression. Le Sang. 23:293-310. 

1952. 

and Macri. C. La valutazione dellaltivita proliferativa dclle cellule 

midollari. Studio di un "test statmocinetico." Haematologica. 33:1-46. 1949. 

, AND . New criteria for the evaluation of the bone-marrow cells 

mitotic activitv. Le Sang. 21:378-82. 1950. 



AND Di Guglielmo, L. 



L'effetto dei raghi roentgen sulfattivita 



proliferativa degli eritroblasti studiati ncl midollo osseo umano in cidlura. 

Haematologica. 35:867. 1950. 

Bair, F. Uber das \'orkommen \ou Mitoscn im vorder 

dcr Hypophyse. Acta Neerl. Morph. 3:97-128. 1939. 

Baker^ D. D., and Baillif, R. N. Role of capsule in surrenal regeneration 

studied witli aid of colchicine. Proc. Soc. Exp. Biol, and Med. 10:117-21. 1939. 



der- und /w ischcnlappen 



250 Colchicine 

11. Bastenie, p. Detection dc I'hormone thvieotrope dans les urines. Methodc ct 
resultats. Arch. Int. Med. Exp. 14:111-22. 1939. 

12. , AND ZvLBER.szAC^ C. Mlse cn evidence des stinndations hcjrmonales par 

la colchicine. I. Detection de stimulation thyroidienne par I'extrait ante- 
livpo])hysaire. C. R. Soc. Biol. Paris. 126:446. 1937. II. Detection de Taction 
stimidatrice dii propionate de testosterone stir les vesicules seminales. C. R. 
Soc. Biol. Paris. 126:891. 1937. III. Action de I'extrait antc-hypophysaire sur 
I'appareil genital du cobaye impubere. C. R. Soc. Biol. Paris. 126:1282. 1937. 
IV. Doses croissantes de propionate de testosterone sur I'appareil genital du 
cobave impuliere. C. R. Soc. Biol. Paris. 126:1283. 1937. V. Stimulation de la 
parathyroide. C. R. Soc. Biol. Paris. 127:882. 1938. Mise en evidence de slinui- 
lations hormonales par la mcthode colchicinicjue de Diisiin. .\rcli. Int. Met!. 
Exp. 13:183-203. 1938. Influence de la tachysterine irradice. (AT 10 de Holtz) 
sur la parathyroide de la rate. C. R. Soc. Biol. Paris. 132:95-96. 1939. 

13. Bernhard, \V. Regenerationshemmung iind Auslosung epithclialcr W'ucherun- 
gen diiich Colchicin am Schwanz \C)n Ranal.arven. Re\ . Suisse /ool. 51:713- 
57. 1947. 

14. Berrian, J. H., AND DoRNFELD^ E. J. Cellular proliferation in the germinal 
epithelium of immature rat ovaries. An in vitro method for the study of mitotic 
rate. Jour. Exp. Zool. 115:493-512. 1950. The effects of ribomuleotides on 
mitosis in the germinal epithelium of immature rat o\aries cultuied iri vitro. 
Jour. Exp. Zool. 115:513-20. 1950. 

15. BiMES, C. Mitoses dans le myometre chez la femellc du coliave h\perfollicu- 
linisee. C. R. Assoc. Anat. 34:48-55. 1947. 

16. BoRDONARO. F. La correlazione ipofiso-tiroidca ncl latto a tiattamento tioura- 
cilo. Studio citofunzionale a me/zo del metodo cokhicinico. Re\. "LO-ipedalc 
Magg." 35 (6) Giugno, 1947. 

17. BoRCHETTi, v., AND Parini, F. Fiattuie sperimentale studiate con 1 aiuto dc un 
metodo colchicinico. Med. Sper. Arch. Ital. 8:665-84. 1941. 

18. Bretschneider, L. H., and Duvvene de \Vit, J. J. Histophvsiologische .Vnalyse 
der sexuallendokrinen Organisation des Bitteriingweibcheus. (liliodciis aina- 
rus). Z. Zellforsch. 31:227-334. 1941. 

19. Brues, a. M. The effect of colchicine on regenerating lixcr. (Pioc. Plivsiol. 
Soc.) Jour. Physiol. 86:63-64. 1936. 

20. , AND Cohen. A. Effects of colcliicine aiul related sul)stances on cell divi- 
sion. Biochem. Jour. 30:1363-68. 193(). 

21. . AND Marble, B. B. An analvsis of mitosis in li\er restoration, four. 

Exp. Med. 65:15. 1937. 

22. , AND Jackson, E. B. Nuclear abnormalities resulting from inhibition ol 

mitosis by colchicine and other sul)staiucs. Amci'. Jour. Cancer. 30:501-11. 1937. 

23. Bucher. O. Zur \\'irkiing einiger Mitoscgifle auf die Cewebekultur. Le Sang. 
21:382-89. 1950. Le role de la culture des tissus in x'ilro dans I'ctude des poi- 
sons de la mitose. Mem. Soc. Vaudoise Sci. Nat. 10:245-70. 1951. 

24. BrLLOiCH, W. S. Mitotic activity in the adult female mouse Miis init.s(ithis 
L. A study of its relation to the oestrus cycle in normal and al)nurmal condi- 
tions. Phil. Trans. Roy. Soc. B 231:435. 1946. Epidermal tliickness following 
oestrone injections in the mouse. Nature. 159:101-2. 1947. The action of 
colchicine in arresting epidermal mitosis. Jour. Exp. Biol. 26:287-91. 1949. 
The mitogenic actions of starch and oestrone on the epidermis of the adult 
mouse. Jour. Endocrin. 6:350-61. 1950. Epidermal mitotic ;uti\it\ in the 
adult female mouse. Jour. Endocrin. 6:340-19. 1950. St i ess and epidermal 
mitotic acti\ity. I. The effects of the adienal liormoiies. Join. Endocrin. 
8:265-74. 1952. 

25. , AND Van Oordt, J. The mitogenic actions of testosterone propionate 

and of oestrone on the epidermis of the adult male mouse. .\cta Endocrin. 
C;openhague. 4:291-305. 1950. 

26. , and Eisa, E. A. The effect of a graded series of restricted diets on 

epidermal mitotic activitv in the mouse. Brit. Jour. Cancer. 4:321-28. 1950. 

27. , AND JOHN.soN, .\I. Epidermal mitotic activitv and oxvgen tension. Na- 

luie. 167:488. 1951. 



Experimental Growth in Animals 251 

28. lURtAU. y. L'action de loestrone ct de la progesterone sur la conic uterine 
de la lapine, etudiee par la nuHhoilc a la colchicine. C. R. Soc. Biol. Paris. 
130:933-36. 1939. 

29. lit RKHART. Z. E. Colchicine reactions in ventral prostate of castrated male rats 
follo\\ing androgenic treatment. Proc. Soc. Exp. Biol. 40:137-39. 1939. .\ studv 
of the effects of androgenic substances in the rat by the aid of colchicine. 
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- 
cation par le tetrachlorure de carbone. poison cirrhogene, mises en evidence 
par la niethode stathmocinetique (colchicinique) de Dustin. .Arch. Int. Med. 
Exp. 14:15-22. 1939. Les glandes endocrines an cours de la grossesse. £tade 
cvto-phvsiologi([ue faite a I'aide de la reaction colchicinicjue (stathmocinetique) 
de Dustin. .\rch. Int. Med. Exp. 11:123-35. 1939. .Application de la methode 
colchicinque a Tetude du diabrtc alloxaniquc clicz le lat. Rev. Beige Path. 
18:32.3-32. 1947. 

34. , and Pellegrini. G. F. L'effetto colchicinico nel "rene endocrino" di 

Sche. Atti. .Soc. Ital. Path. 1:408. 1949. 

.35. DWncona, U. A'erifica del poliploidismo delle cellule cpaliche dei mamnufeji 
nelle cariocinesi provocate sperimcntalmente. .\rcli. Ital. .\nat. Emlirvol. 47: 
253-86. 1942. 

36. Delcoirt. R. fitude de la regeneration iles nerfs peripheriques par la reaction 
stathmocinetique. Acta BreV. Neerl. Phvsiol. 9:241. 1938. Contribution a 
letude de la formation des bandes de Bungner-Ranvier par la reaction stath- 
mocinetique de Dustin. Arch. Int. Med. Exp. 13:1-13. 1940. 

37. Desclin. L. .\ propos de l'action androgenique de la progesterone. C. R. .Soc. 
Biol. Paris. 132:4.3-45. 1939. H\pophvse et parathvroides. Les parathyroides 
apres hvpophvsectomie chez le rat blanc. Bull. Acad. Rov. Med. Belg. Me. Ser. 
8:427-38. 1943. 

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. 
1 95 1 . 

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] 



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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"') 





•. ' •■• •' 


* 


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•' 


.* .*! 


♦ 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. 

REFERENCES 

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13. BRiicKE, E. T. v., and Hijber, E. v. tiber die erfolgreiche Behandhmg emer 
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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. 

21. DusTiN, A. P. Recherches d'histologie normale et experimentale .stir Ic ih\imis 
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colchicine snr le sarcome grefle. t\i5e Crocker, de la sonris. Bull. Acad. Rov. Med. 
Belg. 14:487-502. 1934. L'action de la colchicine snr les tnmenrs malignes. 
Leeuwenhoeck A'er. 55e Conf. Colchicine et cancer. Gaz. Hop. Paris. 41:10 pp. 
193S. 

25. — . AM1 C.REGOiRE, C. Contri l)u t ion a rcliide des poisons car\oclasiqiies snr 

les tnmenrs animales. I. Action dn cacodvlate de Xa et de la trvpaflavine snr 
le sarcome grefle, type Crocker, de la sonris. Bull. Acad. Ro\. Med. Belg. 
13:585-92. 1933. 

2f). CiRAMTA. G., AND DusTiN, P., Jr. Analvsc. par la colchicine, des effets radiomi- 
metic[nes de I'acide 4-amino-ptero\lgintanric|ne (aminoptcrine) . Re\. Beige 
Path. 22:115-25. 1952. Iiripiego associato di aniinoi)tcrina e colchicina e 
anomalie nncleari: ricerche sperimentali snll'intestino del topo. Tnmori. 
39:63-71. 1953. 

27. Gri,f.n, W. J., Jr.. and Lushbaugh, C. C. Histopathologic stndv of the mode of 
inhibition of cellnlar proliferation bv methane. Effect of nrethane on Walker 
rat carcinoma 256. Cancer Res. 9:199-209. 1949. 

28. GuicHARD, A., Brette, R., and Philii^pe. L. P. Essai de traiiemeni dc tlenx 
cas de leiicemie aigne par la colchicine intr;unedullaire. Le Sang. 17:247-49. 
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- 
cine, "phytocarcinomata" and plant hormones. Nature. 140:191-92. 1937. 

32. HiRsciiEELD. J. W., Tennant. R... and Ot'GHTERSON, A. W . The effect of col- 
chicine and X-rav on transplantable mammary carcinoma in mice. Yale Jour. 
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- 
let) . Librairie J. B. Bailliere et Fils. Paris. 1927. 

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 
trois groupes") . Carvologia. 4:98-109. 1951. On the mechanism of the 
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. 
17:75-77. 1942. 

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- 
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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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70. Shear. M. J. Chemical treatment of tumors. IX. Reactions of mice with 
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polysaccharide. Jour. Nat. Cancer Inst. 4:461-76. 1944. 

71. Skipper, H. E., Chapman, J. B., and Bell, M. The antileukemic action of 
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1951. 

72. SoiACOLU, T., AND CoNSTANTiNESCO, M., AND D. Actiou de la colchicuie sur les 
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130:1148-50. 1939. 

73. Tennant, R., and Liebow. A. Actions of colchicine and ethylcarbvlamme on 
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74. Thomas, P. T. Experimental imitation of tinnour conditions. Nature. 156: 
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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- 
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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. 

REFERENCES 

1. Akerman, A. Swedish Seed Association. Annual Report. 1950. S\eri;^. Utsadesf. 
Tidskr. 61:124-91. 1951. 

2. Anderson, E. Introgressive hvlnidi/ation. John Wilev and Sons, Xew York. 
1949. 

3. Andres, J. Sojas tetraploides oijtcnidas por tratamienta ton coUhicina. I'niv. 
Buenos Aires Inst. Genet. 2:95-102. 1944. 

4. Atwood, S. Cvtogenetics and lirecdine; of foraojc crops. Academic Press, Xew 
York. Vol. 1. 1947. 

5. . AND Brewbaker, J. Multiple oppositional alleles in autoploid white 

clo\er. Genetics. 35:653. 1950. 

6. Bates, G. Polyploidy induced by colchicine and its economic possibilities. 
Nature. 144:315-16. 1939. 

7. Beal. J. Induced chromosomal changes and tlieir signihcance in growth and 
deyelopment. Airier. Nat. 76:239-52. 1942. 

8. Beaslev, J. The production of polyploids in r.o55Yp/(;»(. Jour. Hercd. 31:39-48. 
1940. 

9. Bell, G. Inyestigations in the Triticinae. I. Colchicine techniques for chromo- 
some doubling in interspecific and iritergenene hybridization. Joui. Agr. Sci. 
London. 40:9-18. 1950. 

10. Belling. T- Production of triploid and tetraploid plants. Jour. Hercd. l(i: 
463-66. 1925. 



The Experimental Polyploids 289 

11. Blakeslee, a. Deboublemeiu clii iiombre de chromosomes chez les plantes par 
iraitcMneiit chimi(]ue. C. R. Acad. Sci. Paris. 205:476-79. 1937. The present 
and potential scr\ice of chemistry to plant breeding. Amer. |our. lioi. 26: 
163-72. 1939. 

12. , AND AvKRV, A. Methods ot inducing doid)ling of cliromosomes in 

plants. Jonr. Hered. 28:393-411. 1937. 

13. , et ol. Induction of periclinal chimeras in Datum stramonium by col- 
chicine treatment. Science. 89:402. 1939. 

I 1. HRA(.no. M. When tlie chromosomes are doubled. Clokhicine and polyploidy. 

Norsk. Hagetid. 67:27-28. 1951. 
15. Rrown, M. Polyploids and anenploids deriyed from species hybrids in Gos- 

s\l>iiim. Hereditas Snppl. Vol. Pp. 543-44. 1949. 
1(). ClAPi LLETTi. C. La Colchicina ed i poliploidi. Saggiatore. 2:293-306. 1941. 

17. Clausen, R. Mosaic resistance ransferred from wild tobacco to cnltiyated 
varieties through science of genetics. Calif. Agr. 3(7) :7, 16. 1949. 

18. Clausen, J., et al. Experimental studies on the nature of species. II. Plant 
e\olntion through amphiploidv and autoploidv with examples from the 
Madiinae. Carnegie Inst. Wash! I'nbl. 564. Pp.' 174, 1945. 

19. Crane, M., and Lewis, D. Genetical studies in pears. Jour. Genet. 43:31-43. 
1942. 

20. CuA, L. A newly devised colchicine method for inducing pohploidv in rice. 
Bot. Gaz. 112:327-29. 1951. 

21. Darlington, C. The fruit, the seed, and the soil. ()li\er and Boyd, Edinburgh. 
1949. 

22. Darrow, G. Polyploidy in fruit improyement. Sci. Monthly. 70:211-19. 1950. 
Breeding of small fruits in the United States. Sci. Monthly. 75:288-97. 1952. 

23. Dermen, H. Ontogeny of tissues in stem and leaf of cytochimeral apples. Amer. 
[our. Bot. 38:753-60. 1951. Polyploidy in the apple. Jour. Hered. 43:7-8. 
1952. 

24. DoBZHANSKV, \\ Gcuctics and the origin of species. Columbia Uni\. Press, 
New York. 446 pp. 1941. 

25. DoR.ST, J. Neue Wege auf dem Gebiete der Pflanzen/uchtung besondcrs in Hol- 
land. Schweiz. Landiv. Mh. 29:411-27. 1951. 

26. Di'ssEAU, A., AND Fardv, a. Hybrides amphidiploides de Nicotiaua obtenus 
par Taction de la colchicine. Rey. C.ytol. et Cytophysiol. Veg. 7:24-44. 1944. 

27. EiGSTi, O. Research with colchicine in retrospect. The Biologist. 22:143-51. 
1941. 

28. , and Dustin, p., Jr. {sec Ref. No. 16. Chap. 1) . 

29. , and Tennev, B. Colchicine — a report on experiments. I'niv. Okla. 

Press, Norman, Okla. 40 pp. 1912. 

30. Emsweller, S. Polyploidy in Liliiim !())iiilfh)ntin. Amer. Join. Bot. 36:135-44. 
1949. 

31. , and Ruttle, M. Induced pohploidv in floi iculture. Amer. Nat. 75: 

310-26. 1941. 

32. Frandsen, K. lakttagelser over polyploidie Former av nogle kidturplanter. 
Tidsskrift f. Planteavl. 49:445-96. ' 1945. 

33. Gaulden, M., and Carlson, J. Cvtokigical effects of colchicine on the grass- 
hopper neuroblast /;; I'iiro, with special reference to the origin of the spindle. 
Exp. Cell Res. 2:416-33. 1951. 

34. GooDsi'EED. T. El tabaco y otras especies del genero Nicotiaua. Bol. Fac. Ag- 
ron. Vet. Buenos Aires. No. 22. 1942. 

35. , AND Bradley, M. Amphidiploidy. Bot. Rev. 8:271-316. 1942. 

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. 

62. Ruttle, M., .\nd Nebel, B. Cytogenetic results with colchicine. Biol. Zentrall:)!. 
59:79-87. 1939. 

63. Sacharov, v., et al. Autotetraploidv in different varieties of Inickwheat. C. R. 
Dokl. Acad. Sci. URSS. 46:79-82. 1945. 

64. Sears, E. Amphidiploids in the Triticinae induced by colchicine. Jour. Hered. 
30:38-43. 1939. Amphidiploids in the seven-chromo.some Triticinae. Mo. Agr. 
Exp. Sta. Bull. 336:1-46. Columbia, Mo. 1941. Chromosome pairing and 
fertility in hybrids and amphidiploids in the Triticinae. Mo. Agr. Exp. Sta. 
Bull. 337. Pp. 1-20. Clolumbia, Mo., 1911. The cytology and genetics of the 
wheats and their relatives. In Achaiues in genetics. 2:239-70. Academic Press, 
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. 

()(). SrhBiiiNS. G. 1 vpcs of pohploids. /// Advances in genetics. Vol. 1. Acad. Press, 
Inc., New York. 1917. \ariation and e\olnti()n in plants. C'.olnml)ia l'ni\. 
Press, New York. (543 pp. 19r)(l. 

(57. .Stephens, S. The internal niedianisni of speciation in Cusixpnun. Uui. Rc\. 
16:115-49. 1950. 

()S. Trai'B, H. C'.olcliicineindiiced Hciiicrocallls pohploids and their hiccding be- 
haviour. Plant Life. 7:S3-11(5. 1951. 

()9. 1 1'RESSON, G. Kromosomfordobling och \axtf()radling. ^Veibull'^ III. .Xrbok 
for Vaxtforadling och \'axiodling. 41:16-23. 1946. 

70. X'AviLOV, X. Genetics in the USSR. Chron. Bot. 5:14-15. 1939. 

71. \Va[>a, B. (see Ref. No. 43. Chap. 1). 

7L'. ^\'^I.LE^slEK, S. The ne^vest fad, colchicine and its origin. Chron. Boi. 5:15-17. 
1939. Methods for producing Iriticales. Jour. Hered. 38:167-73. 1947. 

73. Wexelsen, H. Polvploidiforedling. En Oversikt. Forskning Fors. Landbruk. 
Oslo. 1:287-310. 1950. 

74. Zhebrak, a. New amphidiploid species of \vheat and their signifuance for 
selection and e\()lulion. .Amer. Nat. SO:271-79. 1946. 

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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82. NoRDENSKiOLD, H. {see Ref. No. 55, Chap. 11) . 

83. Oka, H. The improvement of Nieotiana by means of polyploidy. .\gr. and 
Hort. Japan. 16:2001-2. 1941. 

84. Partiiasarathy, N., and Kedharnath, S. (see Ref. No. 57, Chap. 11). 

85. Pe,\rson, O., et al. Notes on species crosses in Cucurbita. Proc. Amer. Soc. Hort. 
Sci. 57:310-22. 1951. 

86. Perak. J. Tritieum durum tetraploide obtenido por colchicina. Ann. Inst. 
Fitolech. Santa Clalalina. 2:7-8. 1940. 

87. Pksoea, V. Survey of jjlanl breeding. Dept. .\gr. Res. Inst. Finland. Z. 
Pllanzens. 29:282-87. 1951. 

88. Peto, F., AND BoVES, J. Hybridi/ciuon oi Tritieum Aud Igropxrou. \ I. huhucd 
fertility in vernal ennner x •■'• gl'iu<um. Can. |our. Res. Sec. C. Bot. Sci. IS: 
230-39. 1940. 

89. , AND YoUNfi, G. Colchicine and the produdion of new iNjjes of forage 

crops. Nature. 149:641. 1942. 
90. Poi'i'E, W. Rpt. 5th Western Wiie.ii Conl. l.S.D.A. Washington. 5:82. 1950. 



316 Colchicine 

91. Ramanujam, S. An interspecific hvl)rid in Sesa)tiunis S. orioUuJe x ^- prostra- 
tiim KeVi. Curr. Sci. Bangalore. 1 1 :426-2.S. 1942. 

92. , AND Deshmukh, M. Cokhicine-intluced polyploidv in crop planls. III. 

Oleiferons Brassicae. Indian Jour. Genet, and Plant Breeding. 5:63-81. 1945. 

93. , .\ND Srimvasachar, D. Cytogenetic in\estigations in the genus Brassica 

and the artificial synthesis of B. jiincea. Indian Jour. Genet, and Plant Breed- 
ing. 3:73-88. 1943'. 

94. Raw. A. Intcrgeneric h\i)ridization. A preliminary note of inyestigations on 
the use of colchicine in inducing fertility. Jour. Dept. Agr. Victoria. 37:50-52. 
1939. 

95. Richmond, T. (see Ref. No. 61, Chap. 11) . 

96. RuDORF. W. Die Bedeutung der Pohploidie fiir die Eyolution und die Pflan- 
zenziichtung. Angew. Bot. 25:92-101. 1943. Neue Beobachtinigen an Bastarden 
zwischen Phaseolus vulgaris L. imd Phaseolus nntlliflnruiu Lam. 9th Internal. 
Cong. Genet. No. 128. Bellagio, Italy. 1953. 

97. RuTTLE, M., AND Nebel, B. (see Ref. No. 62, Chap. II) . 

98. Sachs, L. Reproductiye isolation in Triliritiv. 9th Internat. Cong. Genet. 
No. 286. Bellagio, Italy. 1953. 

99. ScHRocK, O. Beohachtungen an einem Bastard zwischen Lir/erne imcl Gelljklee 
und seiner Nachkommenschaft. Zuchter. 15:4-10. 1943. 21:109-10. 1951. 

100. Sears, E. (see Ref. No. 64, Chap. 11). 

101. SiMONET, M. Production damphidiploi'des fertiles et stables par intercroise- 
ments d'especes rendues aulotctraploides apres traitements colchiciniques. 
C. R. Acad. Agr. France. 33:121-23. 1947. £tude cytogcnctitiue de I'amphidi- 
ploide Aegilops ventricosa x Triliruni dicoccoides. 9th Internat. Cong. Genet. 
No. 306. Bellagio, Italy, 1953. 

102. , AND Fardv, a. Comportement c\ togencticjue d'lui liybride amphidi- 

ploide fertile Xicotiaua tahacinii L. yar. purpurea Anast. X .V. sxli'cslris Speg. 
et Comes, obtenii apres traitements a la colchicine. C. R. Acad. Sci. Paris. 
215:378. 1942. 

103. Smith. H. Induction of polyploidy in Nicotiana species and species hybrids by 
treatment with colchicine. Jour. Hered. 30:290-306. 1939. Pohploidy in 
Nicotiana. Amer. Nat. 75:307-9. 1941. The dcyelopment of mor|jh()logically 
distinct and genetically isolated populations by interspecific hybridization and 
selection. 9th Internat. Cong. Genet. No. 139. Bellagio, Italy. 1953. 

104. Smith, L. Cytology and genetics of barley. Bot. Rey. 17:1-355. 1951. 

105. Stebbins, G. ' (seeKei. No. 66, Cihap. II). Hereditas Suppl. Pp. 461-85. 1949. 

106. Stephens, S. I. Colchicine produced jjohploids in Goss\piiitn. Jour. Genet. 
44:272-295. 1942. II. Join-. Genet. 46:.303-12. 1945. Meiosis of a triple 
species hybrid in Cossypium. Nature. 153:82-83. 1944. Genome analysis in 
amphidiploids. |our. Hered. 40:102-4. 1947. The cytogenetics of speciation in 
Gossypiuiu. I. Selectiye elimination of the donor parent genotype in inter- 
specific backcrosses. Genetics. 31:(J27-37. 1949. (See Ref. No. 67, Chap. II). 

107. Swaminathan, M. Notes on induced polyploids in the tuber-bearing SoUniuni 
species and their crossabilit\ ^viih S'. tuhryositni. Amer. Potato Jour. 28:472-89. 
1951. 

108. Thomi'son, W., ('/ al. The artificial synthesis of a 42-chromosome species re- 
sembling common wheat. Can. Jour. Res. Sec. C. Bot. Sci. 21:134-44. 1943. 

109. Toxoi'EUS, H. Preliminary account in a new amphidiploid: Solamim arlificiale. 
Genetica. 24:93-6. 1947. ' 

110. Unrau, J. The use of monosomes aiul nullisomes in cMogehetic studies of 
common wheat. Sci. Agr. 30:66-89. 1950. 

111. Valleau,, W. The genetics of iiu)saic resistance in Xicoliana gluliiiosa. Joiu". 
Agr. Res. 78:77-79. 1949. Breeding tobacco for disease resistance. Econ. Bot. 
6:69-102. 1952. 

112. Vaarama, a. Inheritance of morphological characters and fertility in the 
progeny of Rubus idaeiis x 'neliciis. 9ih Internat. Cong. Genet. No. 130. 
Bellagio, Italy. 1953. 

113. Warmke, H., and Blakeslee, A. Induction of simple and multiple pohploidy 
in Nicntinna by colchicine treatment. Jour. Hered. 30:419-32. 1939. 



The Amphiploids 3J7 

111. Wf.llensiek, S. Methods for producing Triticales. Jour. Hered. 38:167-73. 1947. 

11.5. WuiTAKFR. T.. AND BoHN, G. The taxonomy, genetics, production and uses of 
the cidtivatcd species of Cucurhita. Econ. Bot. 4:.52-Sl. 1950. 

I l(i. ^■AKl'WA, K. On allopoKploids obtained from Ix Brassira chiucusis L. x ^^ 
limssica uapits L. Jap. Jour. Genet. 19:229-34. 1943. 

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_ 




'- 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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336 Colchicine 

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39. Darrow, G. (see Ref. No. 22, Chap. 11). 

40. Dawson, R. Cinchona polyploids. Lloydia. ll:Si-S5. 1948. 

41. Decxjux, L., et al. Rrsultats |)relimiiiaires en \ue d'elutlier Taction de la col- 
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42. DtRMKN, H. Colchicine, polvploidv and technicjue. Bot. Re\. 6:599-635. 1940. 
Inducing polvploidv in jjeach varieties. Jour. Hered. 38:77-82. Periclinal 
c\t<)< himcras and histogenesis in craid)errv. Amer. Jour. Bot. 3t:."2-13. 1917. 
Pohploidy in the apple. Jour. Hered. 43:7-8. 1952. 

43. , AND Bain, H. Periclinal and total polyploid\ in cranberries induced 

liy colchicine. Proc. .\mer. .Soc. Hort. Sci. 38:400. 1941. .\ general cvtological 
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44. , AND Darrow, Ci. Colcliicine-induced tetraploid and Kiploid straw- 

I)erries. Proc. Amer. .Soc. Hort. Sci. 36:300-301. 1938. 

45. , AND Scott, D. A note on natural and colchicine-induced |)()l\j)loi(l\ in 

peaches. Proc. Amer. .Soc: Hort. .Sci. 36:299. 1938. 

46. DoBZHANSKY, T. (scc Ref. No. 24, Chap. 11). 

47. Doio, J. Experiments with colchicine. Prof. Card. 1:310. 1949. 

48. DoRSEV. E. Chromosome doubling in the cereals. Jour. Hered. 30:393-95. 1939. 

49. DouwES, H. Colchicine treatment of young cotton seedlings as a means of 
inducing polvploidv. Jour. Genet. 51:7-25. 1952. 

5(!. DiissEAii. A. Etlecls of telraploidy in sorghum. C. R. Acad. Sci. Paris. 221: 
11,5-16. 1945. 

51. Ek.sii. O., and Dlistin, P., Jr. (see Ref. No. 16, Chap. 1) . 

52. , AND Taylor, H. The induction of polvploidv in l'lil<)\ b\ coUliicine. 

Proc. Okla. Acad. Sci. 22:120-22. 1942. 

53. , AND Tenney, B. (see Ref. No. 29. Chap. 11) . 

54. Emsweller, S. (see Ref. No. 30, Chap. 11). Recent developments in lily 
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55. . AND BRn:RLEV , P. Colchicine-induced tetraploidv in Liliinn. Jour. 

Hered. 31:22.3-.30. 1910. 



The Autoploids 337 

3(i. . AMI LuMsniN. D. (ice Ref. No. 31. Chap. 12) . 

.-,7. AM) RiTiiK, M. (see Ref. No. 31. Chap. 11). 

5S. AM) SiKWARr, R. Diploid and tetraploid pollen mother eells in lilv 

iliinieras Proc. .\niei. Soc. Hoit. Sci. 57:414-1S. 19.')!. 
5<). Ernoiu). L. Le boutuiage chez la helteiave. I'iil>l. I BAH dust. Beige pour 

IWmclioiation de la Betterave) , Tiilemont, Belgium. 12:55. 194-1. Lauto- 

pohl^loidie Expeiimcntale chez la betterave. Cellule. 3:363-430. 1946. 
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94:144-45. 1941. 

62. Frahm-Lklivf.ld, J. Polvploidii bij planten door chemische verl)nulMigen. 
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63. Frandskn. K. Nve iagtagelser over tetraploid og iliploid Foderbede. Forelobig 
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Iagtagelser over polvploide Former av nogle Kultur])lanter. lidsskr. f. Plant- 
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Berctn. NJF's Kong. Oslo. 508-27. 1948. 

64. FiKisHiMA. E. On the intergeneric Fj hybrid between liiassicd (innuitu Braun 
and Raphanus salivus L. Jap. Jour. Genet. 18:202-3. 1942. 

65. FuRiSATO. K. Polvploid jjlanls produced bv colchicine. Bot. and Zool. 8:130,3- 
11. 1940. 

66. FrriKAiTi, S. Tetraploid .Asiatic cotton plants induced l)\ the colchunie treat- 
ment. Bot. and Zool. 8:597-601. 1910. 

67. Gabafv. G. Experiments on colchicine and acenaphthene treatment of the 
cucumber for the production of pohploids. C. R. Dokl. Ac:id. Sci. I'RSS. 
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68. Glotox-, V. {see Ref. No. 39, Clhap. 12) . 

69 GoiiBiNSKij, J. A tetraploid form of Ociuiini ((mum Sims, expernnentally 
produced. C:."R. Dokl. .\cad. Sci. URSS. 15:261-62. 1937. 
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71. (.RAMixii.. 1.. Axn Oliien. E. De tetraploida applenas utm tjando 1 \axt- 
I'oradlingarbetet. Balsgard. Sweden. 1952. 

72. G\f)Ri-i\. B. letraploid paprika. Acta Biologica, Pars Bot. (Szeged). 5:30-38. 
1939. Die Colchicinmethode zur Erzeugung pohploitler PHan/en. /uchter. 
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73. Harlam). S. Personal connnunication. 1953. 

74. Hartmair. \'. Fine kinistlich erzeugte fertile tetraploide Melone. Bodenkultur. 
\ieinia. 4:142-44. 19.50. 

75. Hi.tiir. A. Clolchicine-indiiced tetraploich in Oenotlicra. Proc. Intliana .\cad. 
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76. HririN(.\. G. Colchicine and l)rceding of forest trees. Tectona. 39:392-94. 
I94<.*. 

77. Hill. H., AND Mkvkrs, M. Isolation of diploid and tetraploid clones from 
mixaploid plants of rve grass, produced bv treatment of germinating seeds 
with colchicine. |oin. Hered. 35:359-61. 1944. 

78. HiRAVosHt. 1. Studies on artificial pohploids of foiest trees. Boi. and Zool. 
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1 950. 

79. HoFMFvrR, J. Fhe use of ct)lchicine in horticulture, with special reference 
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80. HosoDA. T. (Fertilitv of ami)hidiploids between limssica and Rdplianus) . 
.\gr. and Hort. lokvo. 21:515. (On llie dimension of Fi seeds in crosses among 



70 



338 Colchicine 

nrassica, Siuaph and Ral>h(imts) . A^i . and Hoit. Tokyo. 21:316. 1946. On 
the fertilitv of niijiluiiius-Brassira and Brassua-Raphanus obtained by colchi- 
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obtained bv inter-specitic and inter-generic crosses aiiiong Bmssica, Sinafiis and 
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-81. Howard, H. The size of seeds. in diploid and autotetraploid Brasska oleracca L. 
Joirr. Genet. 38:325-40. 1939. The effect of pohploidv and hvbriditv on seed 
size in crosses between Bvassica cJiiucnsis, B. carhuitu. aniphidiploid B. chiuen- 
sis-carinata, and autotetraploid B. chinensis. Jour. Genet. 43:105-19. 1942. 
Autotetraploid green watercress. Jour. Hort. Sci. 27:273-77. 1952. 

82. Hunter, A., and Danielsson. B. Induced polvploid\ in horticultural crops. 
Progress Report 1934-1948. Div. Hort., Central Exp. Farm. Ottawa, Canada. 
I'p.' 40-47. 1949. 

83. HvDE, B. Forsythia pf)l\ploids. Jour. Arnold Arb. 32:155-56. 1951. 

84. Inoue, V. Colchicine-induced tetraploid in Chinese cabbage, Brassica jjckiiioi- 
sis Rupr. Jap. Jour. Genet. 15:318-19. Tetraploid melons from colchicine 
treatments. III. Bot. and Zool. 7:1879-82. The results of colchicine treatment 
on melon. Bot. and Zool. 7:793-94. 1939. 

85. , and Abe, S. Tetraploid melons from colchicine treatments, jour. Hort. 

Assoc. Japan. 10:109-19. 1939. 

86. Janaki-Ammal. E. Personal comnuuiication. 1953. 

87. Jaretskv, R., and ,Sc:henk, G. \'ersuthe mit Accnaphten luul Colchicin an 
Gramineen und Leguminosenkeimlingcn. Jahrb. W'iss. Bot. 99:13-19. 1940. 

88. Jensen, H., and Le\an, A. Cokliicine-induced tetraploidv in Seijuoid a^igaiitea. 
Hereditas. 27:220-24. 1941. 

89. JoHNSSON, H. On the C„ and C, generations in .Ihiiis i^lutiiiosn. Hereditas. 
'36:205-19. 1950. 

90. , AND Ekli'NDH, C:. Colchicinbehandling som mcthotl \id \a\tf()radling 

a\ hhtiad. Svensk. Papperstid. Medd. 43:3.55-60. 1910. 

91. Johnson, I., and Sass, J. Tetraploid) in Alelilolus alba induced h\ colchicine. 
Proc. Iowa Acad. .Sci. 49:254. 1942. .Self and cross-fertility relationships and 
cMologv of autotetraploid sweet clover, Mclilotus alba. four. .\mer. Soc. Agron. 
36:21-1-27. 1944. 

92. JuLEN, G. Investigations on diploid, triploid and tetraploid lucerne. Hereditas. 
30:567-82. 1944. Clover and timothy ijreeding: breeding of red and Alsike 
clover. Hereditas Suppl. Pp. 44-45. 1949. 

93. Kasi'aravan, A. (see Ref. Xo. 54, Chap. 12) . 

94. Kedharnath, S., and pARrnASARAXH^, \. Varietal dilierences in il.e bleeding 
behaviour of colchicine-induced autotetraploids of chilli. Cajislcuin ainiiniin I>. 
Indian Jour. Genet, and Plant Breeding. 10:14-20. 1950. 

95. Kkhr, a. (see Ref. Xo. 55, Chap. 12) . 

96. Kiellander, C. Demonstration of the Conifer Department. Hereditas Su|)pl. 
Pp. 54-55. 1949. 

97. Kihara, H. Triploid watermelons. Proc. Amer. Soc. Hort. Sci. 58:217-30. 1952. 

98. . , and Kishimoto, E.- Erzeugung polvploider Indivithicn durch Colchicin 

bei Celosia cristata. Agr. and Hort. ' 13:262.3-28. 1938. 

99. , AND KoNDO, X. Studies on ainphidiploids of Aetrilaps cauduta X ^^■ 

uiiibellata induced i)v colchicine. Kihara lust. Biol. Res. Seiken Ziho. 2:24-42. 

1943. 
IOC. ; AND XiSHivAMA, I. An application of sterility of autotriploids to the 

breeding of seedless watermelons. Kihara Inst. Biol. Res. Seiken Zilio. 3:92-103. 

1917. 
101. , AND VA.M.\sinrA, K. A prcliminar\ in\estigation for the foiniaiioii 

of tetraploid watermelons. Kihara Inst. Biol. Res. Seiken Ziho. 3:89-92. 1947. 

102. KoNDO, X. (iee Ref. Xo. .59, Chap. 12) . 

103. KoNDo, V. (An induction of pohploid rice-plants bv treatment uiili (okhi- 
cine) . Agr. and Hort. 17:209-11. 1942. 

104. KosTOFF, D. Irregidaritv in the mitosis and polvploidv induced b\ colchicine 
and acenaphthene. C. R. Dokl. Acad. Sci. IRS.S. 19 (3) :197-99. 1938. 



The Autoploids 339 

105. KRisHNASWA-\n . N'.. <'l III. .\n aiiiotctiaploid in the pearl iiiillci. ( im. Sci. 

I'.): 252-53. 1950. 
10(). Kri'g. C, AM) (.AK\ AMio. A. 1 he genetics of Cojjcii. \o\. 1:127-58. 1951. 

Atadcmic Press. Inc., New ^()^k. 
107. ki MAR, L. Indiiclion of pohploidy in crop planis. Cuir. Sd. Uanoalore. 11: 

112-13. 1942. --^ comparative study of autotctraploid and diploid i\pes in 

mung. Proc. Indian .\cad. Sci. Sect'. B 21:2fi6-6«. 1945. 
lOS. l.AMM, R. Notes on octoploid Solamiiu suuac. Hereditas. 29:193-95. 1943. 

Self-incompatil)ilit\ in lAcopcysicon jxnin'liniiiiii Mill. Hereditas. 36:509-11. 

1950. 
109. La.ngham, D. Fertile tetraploids of sesame, Srsiiininn iiuliniin Loeu, induced 

hv colchicine. Science. 96:204-5. 1942. 
lie. I.ApiN, V. (see Ref. No. 62, Chap. 12) . 

111. I.ARSF.N, P. The aspects of pohploidy in the genus Solanum. 11. Production 
of dry matter, rate of photos\ntliesis and respiration, aiul de\eloi)incnt of leaf 
area in some diploid, autotctraploid and amphidi|)loid Sf)lanunis. K. Danske 
Vidensk. .Selsk. Riol. Mcdd. 18:1-51. 1943. 

112. l.Arri.N, G. Spontane mid indu/ierte Polvploidie hei Rehen. Zuchter. 12:22.5- 
31. 1940. 

113. Le\.a\, A. Tetraploid\ and ocioploiiU inducetl Iin Kilchitine in dii)loid Petunia. 
Hereditas. 25:109-31. 1939. Framstallning av tetraploid rodklover. Sverig. 
rtsadcsf. Tidskr. 50:115-24. 1940. Plant breeding by the induction of 
pohploidv and some results in clover. Hereditas. 28:245-46. 1942. The re- 
s|i()nse of some flax strains to tetraploich. Hereditas. 28:245-46. 1942. Polv- 
ploidiforadlingens Xuvarande Lage. S\erig. Utsadesf. lidskr. Pp. 109-43. 1945. 
.\ haploid sugar beet after colchicine treatment. Hereditas. 31:399—410. 1945. 
Polvploidv in flax, sugar beets and timothv. Hereditas Suppl. \'ol. V\). 46-47. 
1949. 

11 1. , .WD Olsson, P. On the decreased tendenc\ to bolting in teira]}l()ids of 

mangels and sugar iieets. Hereditas. 30:253-54. 1944 

115. Lewis, D., and Modlibowska, I. Genetical studies in pears. I\ . i'ollcn tube 
growth and incompatibilitv. Jour. Genet. 43:211-22. 1943. 

116. I.iiTLK. r. Tetraploidv in .A)itinliitiii»i niajiis induced bv sanguinarine hvdro- 
chloride Science 96:l.S8-89 1942. 

117. LtTK()\, A. Mass pioduction of tetraploid flax ]jlants 1)\ colchicine treatment. 
C:. R. Dokl. Acad. Sci. I'RSS. 22:175-79. 1939. 

118. .Mai.hotra. S., and MilHra, p. Colchicine-induccd autotetraploid .hiyenioue 
inexicnna. Proc. 31st Indian Sci. Cong, .\ssoc. Pt. 111. P. 92. 1944. 

119. Martens, P., et al. Obtention par la colchicine, dc bettera\es sucrieres tri- 
ploides et tetraploi'des. Publ. 1B.\B (Inst. Beige pour IWmelioration de la 
Betterave) , Tirlemont. Belgium. 12:251-56. 1944. 

120. Masima. I. Studies on the tetra])loid flax induced bv colchicine. Cvtologia. 
12:460-68. 1942. C.hiomosome pairing in autotetraploid plants. .\ review. La 
Kiomosome. 3-4:148-62. 1947. 

121. MAtsuMiiRA, S. Induzierte Haploitlie und .Autotetraploidie bei .Aei^ilops o-oata 
L. Bot. Mag. Tokvo. 54:404-13. 1940. 

122. , et fil. Genetische und /\tologische rnleisuchungen bei Iieta-.\ntn. II. 

Cokhicinindu/ierte polvploide Pllan/en und ihre Nachkommen Betii vulgaris. 
Kihara Inst. Biol. Res. Seiken /iho. 1:16-23. 1942. 

123. Meheqi'ist, G. Role of genetics in floriculture, (ienetics. 24:75. 1949. 

124. .Mehra, p. Colchicine-induced pohploids in Moiiiordica eharautia and Hyosrya- 
luus niger. Proc. 32nd Indian Sci. Cong. Assoc. Pt. 111. Pp. !('->. 1915. 

125. Menoes, .\. Polyploid cottons obtained thiough use of colchicine. I. (Atological 
observations in octoploid Gossypium hirsutuni. Bot. Gaz. 102:287-91. 1940. 
Observacoes citologicas em Cofl'ea. XI. Metodos de tratamento pela colchicina. 
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126. Mexdes, I,. ln\estigacoes preliminares solne a du|)licacao do ninncro de 
cromosomios da Seiingueira pela acao da colchicina. Inst. .Agio. Norte. Bol. 

Ic-cn. 7:1-60. 1916. 



340 Colchicine 

127. MizusHiMA. U. (On tin- studv of alio- and autopolvploids induced in lirassica, 
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128. MoL, W. i)F. Colchicine-Proeven met enige .Sr/Y/rt-Soorten tei Verkryging van 
polyploide Vormen. Natuiivet. Tijdschi. Anisteniani. 32 pp. 19r>0. 

129. , AM) Westendorff. W. Morphologische und cytologische Abweichungen 

bei BeUevallia usw. duicli Colchicin, souie der theoietische und praktische 
Wert der Cokhicin-Behandlung ini Vergleich /u dein Wertc andeier Mittle. 
Cellule. 4«:261-7(i. 1940. 

130. Morgan, D., and Rapi'LEVE, R. Twin and triplet pepper seedlings. A study of 
polvemhrvonv in Capsicum frutescens. Jour. Hered. 41:91-95. 19.")0. 

131. MiENDi.FR. M.. ano SciiWAMTZ, F. tjber einen Ertrags- mid Dunginigsversuch 
mit diploidcn und autotctraploiden Mimchener Bicrrettich. /ucliter. 14:137- 

40. 1942. 

132. MiJNTZiNC. A. Various demonstrations. A. Autotetra]>l<)Klv in barlev and rye, 
C. Material of rvewheat (Triticale) . Hereditas Suppl. Pp. 48-.50. 1949. Cyto- 
genetic properties and practical value of tetraploid rye. Hereditas. 37:1-84. 

1951. 
133. AND RuNQuisr. E. Note on some colchicine-iuduccd polyploids. Heredi- 
tas. 25:491-95. 1939. 

134. Murray, M. Colchicine-induced tetraploids in dioecious and monoecious species 
of the Amaranthaceae. Jour. Hered. 31:477-85. 1910. 

135. Myers. ^V. Colchiciiic-'indiiced tctrapioidv in perennial ryegrass (Luliuni 
perenne) . Jour. Hered. 30:499-504. 1939. Cytology and genetics of forage 
grasses. Bot. Rev. 13:319-421. 1947. 

136. kAKAjiMA. G. The tctra])loid plant Cnrchorus capsularis and Abnlilon avij 
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The tetraploid plants in Ouamoclit laisccl bv the colchicine method. Jap. Jour, 
Genet. 25:81-90. 1950. 

137. Nakatomi, S. Induced polvploidy in Asiatic varieties of cotton phmt bv col 
chicine treatment. Proc. Crop. .Sci. Soc. Japan. 12:16-20. 1940. 

138. Navashin, M.. and Gfrassimova, H. Production of tetraploid rubber-vielding 
plant. Taraxacum koksa<^li\z'Kod\n. Aud its practical l)earing. C. R. Dokl. Acad. 
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139. Xf.godi, G. Poliploidi da colchiciiia in licJlis pociiiiis. B. annua. Jtilnylinium 
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140. Newcomer, E. A colcliicine-induced homozygous tomato obtained through 
doubling clonal haploids. Proc. Amer. ,Soc. Hort. Sci. 38:610-12. 1940. A col- 
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cabbage and some comparisons with its diploid progenitors. J. Elisha Mitchell 
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141. NiESSON-EiiEE. H. Framstallning af skogstrad med okat kromosomtal och okat 
\erkesprodiicsiou. Svensk Pappeistidning. Nr. 2. 1938. 

142. NiLSsoN. F. Fetraploidi hos •paroiiplaiitan franikallad med hjalp av colchicin. 
Sverig Pomol. Fiir. Arsskr. 41:103-7. 1940. Polyploids in Riiies. Fiagaria. 
Rapimnus and Lactnca. Hereditas Suppl. Pp. 34-35. 1949. .Some experi- 
ments with tetraploid tomatoes. Hereditas. 36:181-202. 1950. 

1 13. AND .Andfrsson. E. {see Ref. No. 79, Chap. 12) . 

111. NiSHiYAMA. 1. Polyploid plants induced bv the colchicine method. Jap. Bot. 
and Zool. 6:74-76. 1938. Studies on artificial polyploid plants. I. Production 
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1939. Studies on artificial polyploid i^lants. I\'. Comparative studies on lX-4\ 
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115. . AND Kawakami. K. Studies on artificial pohi)l()id i)laiits. 1\. Yield 

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The Autoploids 341 

14(i. . AM) Matsi BAVAsm, C;. (see Ref. No. 54, Cliap. 11). 

147. , .\M) Mk(,1'RC), T. .Studies on artificial pohploid |)laius. X. A pieliiniiiarv 

cultivation of the tetiaploid radish "Minovonbai-daikon' in Hokkaido. Kihara 
Insl. Biol. Res. Sciken /.iho. :kI2.'{-24. 1947. 

I IS. . et (il. .Studies on artificial polyploid plants. II. The effect of colchicine 

on t^erniinating seeds. Bot. and /.ool. 8:47-32. H*'M). 

149. Nociir, V. Studies on the pohploidv in spinach. Jap. Jour. C.enci. 19:106-7. 
1943. 

l.")0. . A\i> Suc.AWARA, T. On the autopolvploids in Fagoj>\runi and Helianlliiis. 

Jap. Jour. Genet. IS: 117-18. 1942. 

I.")l. . et al. Studies on the pohploidv in Xicotiatni induced hy the treatment 

with colchicine. I. General obser\ations on the autotetraploid of Xicoliaiia 
rustica and iV. tabncum. Jap. Jour. Bot. 10:309-19. 1939. Studies on the poly- 
ploich in Xirntiand inducecl I)n the treatment \\iih colchicine. II. Cirowth rate 
and chemical anahsis of dijjloid and its autotetraploid in Xicotiaini rustica and 
.\. tahacum. Jap. Jour. Bot. 10:343-64. 1949. 

152. NoRDKNSKioLD, H. S\ uthesis of Phleinn pratense L. from P. iii)dosuin L. 
Hereditas. 3.5:190-202. 1949. 

1.53. \\(.RKN, A. The i^enesis of some Scandina\ian species of Cafnmagrostis. Heredi- 
tas. .32:131-38. 1916. 

154. Oki.MA. K.. AM) Oka. H. On the fertility of autotetraploid\. Bot. and /ool. 
8:1196-98. 1940. 

155. OiMO, H. Breeding new tetraploid grape \arieties. I'roc. Amer. Soc. Hort. Sci. 
41:22,5-27. 1942. 

156. Oi.ssoN, G. Auto- and allo-pohploicK in the i^cniis Bitissira. Hereditas Suppl. 
I'p. 47-48. 1949. 

157. , AND Ri'Fii.r, B. Spontaneous crossing between diploid and letraj)loid 

Siiuipis alba. Hereditas. 34:351-65. 1948. 

158. 0\<). r. Imestigations on the ])rc)duction of poh|)loids of l)aile\ and other 
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I5'.< OoMKN. H. Polyploidy in canna. Genetics. 24:333-38. 1949. 
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161. I'AKinASARATHV, \.. AM) Kkoharnath, S. (scc Ref. No. 57. Chap. 11). 

162. Pkrak, J. Triticuni (liintm tetraploide obtenido por colchicina. .Ann. Inst. 
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163. Peto, F., and Bovks. J. (see Ref. No. 58. Chap. 11). 

164. . AND HiLi.. K. Ciolchicine treatments of sugar beets and the \ielding 

capacity of the resulting pobploids. Proc. Amer. .Soc. Sugar Beet lechn. 
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165. — , AND '^'oi'Nr,, G. Colchicine and the production of ne^v types of forage 

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166. Pn naar, R. C.Mogcnelic stiuh of the genus Era<^roslis. Ihesis: l.il)rar\ rni\. 
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167. P1R.SCHL1:, K. Wasserkulturversuche mit polvploiden Pfianzen. I, II. Biol. 
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.\l)stamm. \'ererb. Lehre. 1942. 

168. Ramanujam, S., and Df.sh.mukm. \I. Colchicine induced pohploidv in crop 
plants, ill. Indian Jour. Genet, and Plant Breeding. 5:63-81. 1945. 

169. , AND Josiii, .\. Colchicine-induced pohploich in crop plants. I. Indian 

[our. .\gr. Sci. 11:835-49. 1941. Inleispec ilic In l)ridi/alion in Xitoliaiia. In- 
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170. Randolph, L. .\n evaluation of induced poh|jloidy as a method of l)rceding 
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171. R\|an, S., et al. Breakdown of telraploidv in colchic ine-iTiduced autotetra- 
|)l()id Eruca satii'a I. am. Indian Jour. (lenet. and PlaiU Breeding. 10:43-55. 
1950. 

172. Rasmi'sson, J., and Lfaan, A. Tetraploid sugar l)cets from colchicine treat- 
ments. Hereditas. 25:97-102. 1939. 



342 Colchicine 

173. RicHHARiA, R., AM) I'KRSAi. D. Tetiaploid til (Sesaiiiiini orieutaU- L.) from col- 
chicine treatment. Cliur. Sci. 9:542. 1940. 

174. Ross, H. Die Vererhung der Immiuiitat gegen das \'irns X in tetraploidem 
Snlnnum acatile. 9th Internat. Cong. Genet. No. 273. Bellagio, Italv. 1953. 

175. RurJORF, W. (see Ref. No. 96, Chap. 12) . 

176. RuTTLE, M., AND Nebel, B. (see Ref. No. 62, Chajx 11). 

177 Rm!I\ V Tetraploid Solaninn rybiuii Juz et Biik. jnoduced bv colchicine 
treatment. C. R. Doivl. Acad. Sci. URSS. 27 (2) : 151-54. 1940. 

178. Saharov, v., et al. High fertility of buckwheat tctraploids ()l)tained bv means 
of colchicine treatment. Nature. 154:613. 1944. 

179. Saito, K. Studies on inducing polyploid flower plants and their utilization. 
HI. Jour. Hort. .Assoc, japan." 19:195-99. 1950. 

180. Salomon, E. .S«)i,^/n/;/( siidcniese Staph, tetraploide oijtenido por colchicina. 
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181. Sampavo, T., and Castro, D. Colchicine-induced tetraploidy in Luzula [nu- 
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182. Sando. \V. a coicliicine-induced tetraploid in huckwlicat. Join. Hered. 30: 
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183. ScuENK, G. Versuche zur Erzeugiuig einer polvploiden Mciitlui pipoita durch 
Colchicin und Acanaphten. Die Deutsche Heilfianze, Schriftenreihe. No. 8. 
1911. 

184. ScHiLDr, R., AND .AkERBERGER E. Sludicr over tetraploid och diploid rag vid 
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185. Sc:Fn,ossER. L. Polyploidie in der Zuckcrriilien/iichlung. Dent. Landwirt. Gcsell. 
Milt. (i7:551-52. 1952. 

186. ScHNACK. B. Variaciones producidas en Salvia sjjiriuleiis por la accion de la 
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1S7. ScHNELi., L. The indiution of polyploidy in ]"m(a rosea. .\mcr. Join'. Bat. 
28:.5s. 1941. 

188. Sc:hroc.k, O. (see Ref. No. 99, Chap. 12) . 

189. ScHWANiTZ, F. Untersuclumgen an polyploiden I'llan/en. VHI. Zuchter. 20: 
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190. Scott, D. Cvtological stuiHes on pohploids derived from tetraploid Fragaria 
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191. Sengbusch, R. I'olyploider Roggen. Zuchter. 12:185-89. 1940. i'ohploidc 
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192. SirvEVf.iN, I. Production of tetraploids in Lolitiin bv treating germinatuig seeds 
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193. SuiERiss, O. Polyploids in the genus Citeuiuis. join. Hered. 33:144-52. 1942. 

194. Siii.MAMURA, T. Exi^eriments of inducing tetraploid in tomatoes by means of 
colchicine, jap. (our. Genet. 14:304-8. 1938. 

19.-). AND KoBA.'s ASHi, T. Studies on artificial polyploid plants of Sesa»iu))t 

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196. SnEMOTOMAi, N. Ihc artificially produced pol\i)loids in Clnxsaiilhennim 
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197. SiMONET, M. De rol)tention de varietes polvploides a grandes fieurs apres 
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198. SiNoro, v., AND Sato, D. Colchicine polyploids in Faoopyi iiin. Bol. aiul Zool. 
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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. 
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71:r)l-()(i. C;oni])aiati\e "growth rates of diploid and aiilotetraploid Stijxi lepkla. 

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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. 
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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- 
lat dutch Polvploidisierung. Ber. Deutsch. Bot. Cies. 59:296-301. 1911. 

209. SvvAMiNATHAN, M. (scc Ref. No. 107, Chap. 12) . 

210. lAiiAiA, H., AND Ki'Rn AMA, H. On the chromosome doubling induced hv col- 
chicine in rice plant. Jap. jour. Genet. 24:90-93. 1949. 

211. Tanc;, p., AM) Loo, W. Pohploidy in soybean, pea. wheat, and rice, induced 
by colchicine treatment. Science. 91:222. 1940. 

212. 'Eakknaka, \. Notes on c\tological observations in Colchicuni with reference 
to auto-to\icosis and sterility. C'.vtologia. 16:95-99. 1950. 

213. 1 AM)ON, S. Colchicine indiued polyploidy in spin;ich. Curr. Sci. 19:6(). 1950. 
Colchicine-induced polyploidy in Brasska olcracca var. botrytis L. Sci. and 
Culture. 16:483-84. 1951. 

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. 

Proc. Amer. Soc. Hort. Sci. 36:641-44. 1939. 
21(). Iominaga. Y. tJber die kiinstlich erzeugten Tetraplonien von Gossypium. Jap. 

four. Genet. 21:60-61. 1946. Morphologische und c\ tologische I'ntersuchungen 

tiber den amphidiploide Gattiuigsbastard Heterolxipjuis (ncnarius x Kalinicris 

iiicisa. Jap. Jour. Genet. 21:83. 1946. 

217. looLE, M., ANt) Bamford, R. The formation of cli|)l()id plants from haploid 
pcjjpers. Jour. Hered. 36:67-70. 1945. 

218. 1 oxoi'Ki's, H. SoltniiDii (ntificinlc and its breeding \alue. Natiuirw. rijdschr. 
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Hibiscus sabdarifja and Hibiscus cannabinus L. Genetica. 24:330-32. 1948. 

21!;. iRAi'B, H. (see Ref. No. 68, Chap. II) . 

220. 1 I'RFssoN, G. (see Ref. No. 69, Chap. 11). 

221. , et al. Demonstrations in the experimental gaiden. Bot. Genet. Inst. 

Roy. .\gr. Coll., I jjsala, Sweden. Hereditas Suppl. \'oi. Pp. 60-()2. 1919. 

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Kihara Inst. Biol. Res. Seiken Ziho. 3:125-4 1. 1917. 

223. \'aarama, a. Cr\piic ])ol\ploich and xariation of chromosome nund)cr in 
Ribes nigrum. Natuie. 1()2:782. 1948. 

221. Warmke, H. Polyploidy investigations. Annual report of director of depart 
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106:316-24. 1945. 

225. W'tDDi.F. C. Two colchicine-induced pohploids of the greenhouse chr\santhe- 
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Inbrid of Calenchda — its F., popid;ition :ind ils telr;ipl()id. Proc. \mcr. Soc. 
Hon. Sci. 39:393-9(i. 1941. 



344 Colchicine 

22(i. Weichsel, G. Polyploidie, veranlasst duich chemische Mittel, insbesondeie 
Colchicinwirkuns; hci Legiiminosen. Z.uchter. 12:25-32. 1940. 

227. Weissemsock, K. Stiidicn an colchiziiiiciten Pflanzen. I. .\natomische Ihiter- 
suduuigen. Phvtoii. l:2S2-300. 1919. Studien an colchiziniei ten Pflanzen. II. 
Phvton. 2:134-52. 1950. 

228. Wexelsen, H. Polvploidifoiedlino. En oveisikt. Foisknins' Fors. Landbruk. 

Oslo. 1:287-310. 1950. 

229. YAMAfiUTi, Y. Inheritance of autoletraploid I'lnliiltKii. Jaj). jour. Genet. 
Suppl. Vol. 1:119-20. 1949. 

230. Yamasaki. M., et al. Polyploid kowliang (Aiidrupogon sorghuiii) indiued by 
colchicine. Agr. and Hon. 15:641-46. 1940. 

231. Yamashita, K. Cotton i)lants treated with colchicine. Jap. Jour. Genet. 16: 
267-70. 1940. 

232. Zhebrak, a., and Rzai:v. M. (see ReL No. 119. Chap. 12) . 

233. ZiuRBiN, A. (see Ref. No. 120, Chap. 12). 

ADDITIONAL REFERENCES 

234. Atwood, S., Axn Grin. 1'. Cytogenetics of alfalfa. Bibliog. Genetica. 14:133- 
88. 1951. . . 

235. FuRUSATO, K. 'Flie tetraploid watermelon, Kalio, raised by the colchicme 
method. Rpt. Kihara Inst, for Biol. Res. Kyoto. Seiken Ziho. 5:131-,32. 19.52. 

236. JosEFSSjN. A. Tetraploida royor, foriidling och Forsok yid Syeriges Utsades- 
forening. Syerig. IJtsadesf. Tidskr. 3:165-80. 1953. 

237. Matsiimira, S. Improyement of sugar beets by means of triploidy. Science- 
,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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360 Colchicine 

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sex ratio in the progenv of the autotetrai)loid hemp. Kihara Inst. Biol. Res. 
Seiken Zilio. 3:145-51. 1947. 

35. Olmo, H. (see Ref. No. 155, Chap. 13) . 

36. Olsson, G., and Rufelt, B. (see Ref. No. 157. Chap. 13) . 

37. O'Mara. J. Cvtogenetic studies in Triticale I. Genetics. 25:401-8. 1940. 

38. Ono, T. Polyploidy and sex determination in Mclaudrium. I. Colchicine- 
induced polyploids of Melandrium album. Bot. Mag. Tokyo. 53:549-56. 1939. 
Polvploidv and sex determination in Melandrium. II. The effect of polyploidy 
upon sex' in M. album. Bot. Mag. lOkvo. 54:225-30. 1940. Polvploidy and 
sex determination in Melandrium. III. Intersex in M. (dhum. Bot. Mag. 
Tokyo. 54:348-56. 1940. The effects of polyploidy upon morphological and 
physiological characters in Pisum satii'um. Bot. and Zool. 8:1265-74. 1940. 
.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- 
chicine. Bot. Gaz. 106:483-88. 1945. 

42. Satina, S. Periclinal chimeras in Datura in relation to development and struc- 
ture (a) of stvle and stigma (b) of calvx and corolla. Amer. Jour. Bot. 31: 
493-502. 1944. Periclinal chimeras in Datura in relation to the development 
and structure of the ovule. Amer. Jour. Bot. 32:72-81. 1945. 

43. , et al. Demonstration of the three germ layers in the shoot apex of 

Datura b\ means of induced polvploidv in periclinal chimeras. Amer. Jour. 
Bot. 27:895-905. 1940. 

44. , AND Blakeslee, a. Periclinal ciiimeras in Datura stramonium in rela- 
tion to development of leaf and flower. Amer. Jour. Bot. 28:862-71. 1941. 

45. Sawyer, M. A colchicine-induced chimera in a Datura hybrid, 2n for one 
species and 4n for another. Amer. Jour. Bot. 36:802. 1949. 

46. Sears, E. NuUisomics in Triticum vulij;are. Genetics. 26:167-68. 1941. Cvto- 
genetic studies with polvploid species of wheat. II. Additional chromosomal 
aberrations in Triticum vulgare. Genetics. 29:232-46. 1944. The sphaero- 
coccum gene in wheat. Rec. Genet. Soc. Amer. 15:65-66. 1946. The cytology 
and genetics of the wheals and their relatives. //( Ad\anccs in genetics. 
2:239-70. Academic Press, inc.. New York. 1948. 

47. , AND Rodenhister, H. Nullisoniic analysis of stem rust reaction in 

'irili(uiit inilixare var. Timstein. Rec. Genet. Soc. Amer. 16:50-51. 1947. 



The Aneuploids 361 

48. Smith, H. Effects of genome I)alaiuc. pohploidy, and single extra chromosomes 
on size in Nicotlaua. Genetics. 28:227-36. Studies on induced hetcioploids on 
Nicotiana. Amer. Jour. Bot. 30:121-.'30. 1943. 

49. Smith, L. (see Ref. No. 200, Chap. 13). 

50. Takenaka, Y. Relation between the genome and gigantism and iis bearin<'^ on 
plant breeding. Jap. Jour. Genet. 18:155-56. 1942. 

51. L.\R.\u, J. (see Ref. \o. 110, Chap. 12) . 

52. Vaarama, a. (see Ref. No. 223, Chap. 13) . 

53. AVarmkf, H. .\ new method for determining the sex hetero/vgote in species 
with morphologically undifferentiated sex chromosomes, and its application to 
Silene otites. Genetics. 27:174. 1942. Sex determination and sex balance in 
Melandrium. .Amer. Jour. Bot. 33:648-60. 1946. 

54. , and Blakeslee, \. Sex mechanism in polyploids of Melandr'nun. 

Science. 89:391-92. 1939. Effect of polvploidv upon sex mechanism in dioecious 
plants. Genetics. 24:88-89. 1939. 1 he establishment of a 4n-dioccious race in 
Melandrium. Amer. Jour. Bot. 27:751-62. 1940. 

55. Westergaard, .M. Studies on cvtologv and sex determination in polxploid forms 
oi Melandrium album. Dansk. Bot. Arkiv. 10:3-131. 1940. 



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. 

REFERENCES — SECTION A 

1. Ashley, J. X., and Harris. J. O. Purification of colchicine by chromatogra- 
phy. Jour. Chem. Soc. P. 677. 1944. 

2. AsTALDi, G., AND Mauri. C. La valutazione deH'attivita proliferativa delle 
cellule miodlari. Studio di un "test stathmocinetico." Haematologica. 33:1-46. 
1949. New criteria for the evaluation of the bone-marrow cells mitotic acti\ity. 
LeSang. 21:378-82. 1950. 

3. , Bernardelli. E., and Rondanelli, E. La colchicine dans letude de la 

proliferation des cellides hemopoicticjues de I'embrvon. Rev. Beige Path. 
21:406-13. 1952. 

4. Beams, H. W., and Evans, T. C. Some effects of colchicine upon the first cleav- 
age in Arbacia punctulata. Biol. Bull. 79:188-98. 1940. 



Techniques of Colchicine Treatment 387 

5. Beatt\', R. a. Heteroploiih in mammals. Animal breeding Abst. 19:283-92. 

1951. 

6. AND FiSHBERG, M. Spontaneous and induced triploidv in pie-implantation 

mouse eggs. Nature. 1()3:S07. 1919. Polvploidv in rabbits." .Nature. H)6:238. 
1950. Heteroploidy in mammals. III. Induciion ot tetraploidv in pre-implan- 
tation mouse eggs. Jour. Genet. 50:47-79. 1952. 

7. Berrian, J. H., AND DoRNFELD, E. J. Cellular proliferation in the germinal 
epithelium of immature rat ovaries. An in xntro method for the study of 
mitotic rate. }our. Exp. Zool. 115:493-512. 1950. The effects of ribonucleotides 
on mitosis in the germinal cpithelimn of innnature rat ovaries cultured in 
vitro. Jour. Exp. Zool. 115:513-20. 1950. 

8. BouRG. R., AND DusTiN, P., Jr. Lc traitemeut des papillomes \ilvaries par 
I'appliaition locale de colchicine. Presse Med. 43:578. 1945. 

9. Branch, C. E., Fo(;(;, L. C, and Ui.lvot, G. E. Colchicine and cokhicine-like 
compounds as chemolherapeutic agents. Acta Unio Internat. Cancrum. 6:439- 
47. 1949. 

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 
in I'itro dans IV'tude des poisons de la mitose. Mc>m. Soc. \'audoise Sci. Nat. 
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. 
Phil. Trans. Rov. Soc. B: 231-435. 1946. The diurnal cvcles and iheir relation 
to waking and sleeping. Ihid. 135:212-33. 191S. 1 he effects of experi- 
mentally induced rest and exercise on the epidermal mitotic activity of the 
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. 

58. Wii.EiAMS, W. L., Stein. K. P., 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. 

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- 
chicine. Jour. Hered. 29:3-9. 1938. 

21. Pearson, O., et al. Notes on species crosses in Cucurbita. Proc. Amer. Soc. 
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