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A HANDBOOK OF PLANT
TISSUE CULTURE

BY

PHILIP R. WHITE, A.B., Ph.D.

THE JAQUES CATTELL PRESS
LANCASTER, PENNSYLVANIA

1943

Copyright, 1943, by
The Jaques Cattell Press

PRINTED IN U. S. A.

THE SCIENCE PRESS PRINTING COMPANY

LANCASTER, PENNSYLVANIA

DEDICATION

to Henry K. White, to whom I owe a certain
curiosity about the workings of nature and whose
first lessons in scientific thinking and procedure
are the foundation upon which all of my work has
since been built,

to Mary P. White, to whom I owe patience and
perseverance and what humility I may claim, to
carry through the tasks at hand,

to Caroline D. White, whose inspiration and
constant help have supported me at many a diffi- «
cult pass, and

to little Christopher John and Jonathan Peter
for whom, after all, all things in heaven and earth
are done,

this little volume is humbly dedicated.

"Entia non sunt multiplicand! praeter

necessitatem"

Wm. of Occam, 1270-1349.

PEEFACE

The writing of any book, and particularly one
in a new and special field, should, in these days of
stress and in the presence of an already stagger-
ing plethora of publication, be a matter for very
serious consideration. Books are written to be
read, and unless one has at least a potential public
one should hesitate long and seriously about bring-
ing out a new volume. Yet the history of Science
has been from the first a history of methods, either
experimental methods or methods of thought.
When, therefore, a new method or a new applica-
tion of older methods is developed, it behooves
those who are responsible for its development to
consider carefully when it has progressed far
enough beyond adolescence to warrant a formal
introduction in society.

Tissue Culture as a branch of animal biology
has long since passed that stage and has received
extended attention in the handbooks of Fischer,
Erdmann, Willmer, Ephrussi, Parker, and others.
Many of these handbooks carry titles conveying
the impression that they treat the subject in its
entirety. Yet it is in vain that one searches their
pages for any but the briefest possible treatment
of the cultivation of plant tissues. One might

vi Preface

assume that the animal biologists had a strict
monopoly on the method. This is, of course, not
at all a fair picture. It is with the conviction that
Tissue Culture is, in fact, the domain of all biolo-
gists and that Plant Tissue Culture has, in the last
decade, developed to the point where it deserves
and even demands consideration as a separate,
valid, useful, and promising, if not yet fully ma-
ture, discipline, that I have undertaken the prepa-
ration of this little volume.

Having decided that the time is ripe for publi-
cation in a new field, an author must then deter-
mine the scope of his subject, the type of reader
he wishes to reach, and the degree of detail which
it is valuable to present. I have planned this book
as an aid to those who may actually make use of
the technique — students, investigators in other
fields for whom the methods presented may be
useful in the solution of their own problems, and
perhaps a very few who may approach the subject
for its own sake. With this in mind, I have tried
to keep the presentation as simple and concise as
seemed compatible with completeness and lucidity
and have used, wherever possible, photographs
and drawings in place of long descriptions. I
have not attempted an inclusive survey of the field
in all its past and present ramifications but have
concentrated on those matters which may suggest
to the student new fields of conquest and provide
him with the basic information and the techniques

Preface vii

necessary to an intelligent approach to those fields.
Most of the techniques described are those de-
veloped and perfected in my own laboratory, but
I have tried also to provide a working outline of
other techniques and methods of approach.

One does not prepare a book, even a small one,
alone. I should like especially to acknowledge
the help of Professor Edmund Sinnott of Yale,
Professor George Avery and Miss Bethe Ander-
son of Connecticut College, Dr. Esther Carpenter
of Smith College, Dr. and Mrs. Warren Lewis of
the Carnegie Institution, Dr. Frank Thone of Sci-
ence Service, and Harriet Butler Bunker of Dum-
merston, Vermont, all of whom examined the early
plan, read and criticized the preliminary drafts
of the manuscript, made many helpful criticisms
and suggestions, and gave me their unstinted en-
couragement throughout the task. I should like
also to express my gratitude to Professor Gottlieb
Haberlandt, Dr. Ross Harrison, Dr. Alexis Carrel,
Dr. and Mrs. Warren Lewis, Dr. Walter Kotte,
and Dr. Roger Gautheret for permission to use
their portraits. These will, I hope, add greatly
to the living quality of the book. Dr. Gautheret
in particular deserves my deepest gratitude for
the difficulties and perhaps danger which he over-
came in sending me his picture and biography out
of war-ridden Paris. I sincerely regret that Dr.
W. J. Robbins preferred to have his portrait
omitted from the series. The illustrations, with

viii Preface

the exception of these portraits and of figures 1,
2, 3, 4, 14, 15, 35, 39, and 64, are all from my own
laboratory and were photographed or drawn by
Mr. Julian A. Carlile whose competent help has
certainly greatly enhanced whatever value the
book may have. My sincere thanks are due him
and other members of the technical staff of the
Rockefeller Institute for their unfailing coopera-
tion. I should like to thank Dr. James Bonner
for the photographs used in figure 35 and John
Wiley and Sons for permission to republish figure
22, the original of which I furnished them in 1937.
Pictures taken from periodicals and from older
books carry their acknowledgments in the text.
The bibliography at the end of the book is intended
to cover the major contributions in the field of
Plant Tissue Cultures and to contain in addition
some collateral material from related fields suffi-
cient to suggest to the reader possible interrela-
tions. Corrections, suggestions, and criticisms
will, of course, be welcomed.

With this brief introduction, I turn my offspring
over to the good graces of a critical and exacting,
but I trust understanding, public.

Princeton, New Jersey,
September, 1942.

TABLE OF CONTENTS

PAGE

Chapter I. Introduction 1

Chapter II. The History of Plant Tissue Culture 19

Chapter III. The Living Materials 43

Chapter IV. The Laboratory 67

Chapter V. Nutrients 90

Chapter VI. How Cultures Are Started 117

Chapter VII. Culture Techniques 131

Chapter VIII. Growth Measurements and Their

Interpretation 154

Chapter IX. Tissue Culture and the Study of
Problems in the Pathology and General Physi-
ology of Plants 183

Chapter X. Morphogenesis 209

Bibliography 227

Index 262

,

13

LIST OF ILLUSTRATIONS

PAGE
Fig. 1. Polarity in regeneration of cut tubers of Corydalis

solida 6

Fig. 2. Eetention of specific characteristics by grafts of

fruits of three varieties of gourds 13

Fig. 3. Eetention of specific characteristics by grafts be-
tween two species of tadpoles 14

Fig. 4. Production of lens by grafted belly epithelium of a

toad, under induction from the eye-cup of a frog 15

Fig. 5. Gottlieb Haberlandt 16

Fig. 6. Ross G. Harrison 23

Fig. 7. Warren H. Lewis 24

Fig. 8. Margaret R. (Mrs. Warren H.) Lewis 25

Fig. 9. Alexis Carrel 26

Fig. 10. W. Kotte 37

Fig. 11. R. J. Gautheret 38

Fig. 12. Philip R. White 39

Fig. 13. Roots of tomato, white clover, and red clover 40

Fig. 14. Section of seed of Peperomia hispidula 46

Fig. 15. Undifferentiated embryo of Peperomia hispidula 47

Fig. 16. Leaves regenerated on root of dandelion 51

Fig. 17. Cultures of sunflower tissues 52

Fig. 18. Cultures of Nicotiana callus 53

Fig. 19. Cultures of bacteria-free crown-gall tissues of sun-
flower 53

Fig. 20. Young embryo of Portulaca oleracea 54

Fig. 21. Root cap on root of white clover 61

Fig. 22. Root-hairs on growing excised tomato root 62

Fig. 23. Root-hairs on growing excised buckwheat root 63

Fig. 24. Growing excised roots of tomato and buckwheat 64

Fig. 25. Plan of a laboratory suite 68

Fig. 26. Week by week increment rates of excised roots over

eight years 78

xi

xii List of Illustrations

PAGE

Fig. 27. Diagram of special pierced slide for hanging-drop

cultures 80

Fig. 28. Implements commonly used in plant tissue culture

work 85

Fig. 29. Equipment for preparing and transferring cultures 86

Fig. 30. Procedure of cutting roots within flasks 87

Fig. 31. Distributing nutrient to flasks 87

Fig. 32. Method of obtaining sterile adventitious roots 88

Fig. 33. Excising adventitious roots from cuttings 88

Fig. 34. Method of obtaining uniform stocks of root tips 113

Fig. 35. Bean pod segments used in testing wound stimulants 114

Fig. 36. Eemoving blocks of cambium from large trees 115

Fig. 37. Procedure in making cultures of cambium 115

Fig. 38. Cambium culture of Salix caproea 116

Fig. 39. Surface of cambial cultures of Abies pectinata 127

Fig. 40. Measuring a root within the culture flask 128

Fig. 41. Sample pages from a book of records 129

Fig. 42. Equipment used in making hanging-drop cultures 130

Fig. 43. Average daily increment rates of 1000 roots during

one week in vitro 141

Fig. 44. Effects of temperature on growth rates of tomato

roots 145

Fig. 45. Example of a line graph recording daily march of

increments of excised roots 174

Fig. 46. Example of a histogram recording effects of nutrient

variables on growth 175

Fig. 47. Example of an isopleth diagram showing the effects

of two simultaneous variables 176

Fig. 48. Tomato root showing gross effect of indole acetic

acid on growth 179

Fig. 49. Effects of iron concentration on root growth 180

Fig. 50. Effects of iron concentration on root growth 181

Fig. 51. Detail of the effects of indole acetic acid on cells of

an excised root 182

Fig. 52. Graph showing effects of indole acetic acid on growth

rates 187

Fig. 53. Graph of rates of water secretion (root pressure) in

excised tomato roots 191

List of Illustrations xiii

PAGE

Fig. 54. Developmental cycle of plastids in cells of Stellaria

media 193

Fig. 55. Eegion of maturation of excised tomato root 195

Fig. 56. Manometer for measuring root pressure 196

Fig. 57. Manometer assembly, detail 197

Fig. 58. A battery of manometers with attached pressure

equipment 198

Fig. 59. Titration of distribution of aucuba-mosaic virus in

a tomato root 201

Fig. 60. Tyrosinase distribution in an excised tomato root 205

Fig. 61. Hyperhydric enlargement of cells of seed primordia

of Antirrhinum 206

Fig. 62. Meristematic cells in Nicotiana callus culture 207

Fig. 63. Scalariform cells in Nicotiana callus culture _ 208

Fig. 64. In vitro grafts of cambium cultures 213

Fig. 65. Cross section of a Nicotiana callus culture 214

Fig. 66. Detail from figure 65 215

Fig. 67. Disorganized vascular tissue from a sunflower tumor

tissue culture 216

Fig. 68. Formation of stem growing point on a Nicotiana

callus culture 223

Fig. 69. Section showing Nicotiana callus cultivated in a rab-
bit 's eye 224

Fig. 70. Detail from figure 69 225

Fig. 71. Effect of aeration on differentiation of a Nicotiana

callus culture 226

"Man kann sich also folgende zwei Vorstellungen von der Ursache der
organischen Erscheinungen, des Wachstum usw. machen : Erstens die
Ursache liegt in der Totalitat des Organismus. . . . Die andere Erklar-
ungsweise ist die : Das Wachstum geschieht nicht durch eine im ganzen
Organismus begriindete Kraft, sondern jeder einzelne Elementarteil
besitzt eine selbstandige Kraft, ein selbstandiges Leben. . . .

"Wir haben gesehen, dass alle Organismen aus wesentlich gleichen
Teilen, namlich aus Zellen zusammengesetzt sind, dass diese Zellen nach
wesentlich denselben Gesetzen sich bilden und wachsen, dass also diese
Prozesse iiberall auch durch dieselben Krafte hervorgebracht werden
miissen. Finden wir nun, dass einzelne dieser Elementarteile, die sich
von den iibrigen nicht unterscheiden, sich vom Organismus lostrennen
und selbstandig weiter wachsen konnen, so konnen wir daraus schliessen,
dass auch jeder der iibrigen Elementarteile, jede Zelle fur sich schon
die Kraft besitzt, neue Molekiile anzuziehen und zu wachsen, dass also
jeder Elementarteile eine eigentiimliche Kraft, ein selbstandiges Leben
besitzt vermoge dessen er selbstandig sich zu entwickeln imstande ware,
wenn ihm bloss die ausseren Bedingungen dargeboten wiirden, unter
welchen er im Organismus steht. Solche selbstandig, getrennt vom
Organismus wachsende Zellen sind z. B. die Eier der Tiere. . . . Bei
niederen Pflanzen kann sich jede beliebige Zelle von der Pflanze lostren-
nen und dann selbstandig weiter wachsen. Hier bestehen also ganze
Pflanzen aus Zellen, deren selbstandiges Leben unmittelbar nachweisen
lasst. Da nun alle Zellen nach denselben Gesetzen wachsen, also nicht
in einem Falle der Grund des Wachstum in der Zelle selbst, im andern
Falle im ganzen Organismus liegen kann, da sich ferner nachweisen
lasst, dass einzelne, von den iibrigen in der Art des Wachstums nicht
verschiedene Zellen selbstandig sich entwickeln, so miissen wir iiber-
haupt den Zellen ein selbstandiges Leben zuschreiben. . . . Dass nicht
wirklich jede einzelne Zelle, wenn sie von einem Organismus getrennt
wird, weiter wachst, ist gegen diese Theorie so wenig ein Einwurf, als
es ein Einwurf gegen das selbstandige Leben einer Biene ist, wenn sie
getrennt von ihrem Schwarm auf die Dauer nicht fortbestehen kann."

"One can thus construct the following two hypotheses concerning the
origin of organic phenomena such as growth : either this origin is a
function of the organism as a whole, or growth does not take place by
means of any force residing in the entire organism but each elementary
part possesses an individual force, a separate life.

"We have seen that all organisms consist of essentially like parts, the
cells ; that these cells are formed and grow according to essentially the
same laws ; that these processes are thus everywhere the result of the
same forces. If, therefore, we find that some of these elementary parts,
which do not differ from others, are capable of being separated from the
organism and of continuing to grow independently, we can conclude
that each of the other elementary parts, each cell, must possess the
capacity to gather new molecules to itself and to grow, that therefore
each cell possesses a particular force, an independent life, as a result of
which it too would be capable of developing independently if only there
be provided the external conditions under which it exists in the organ-
ism. The eggs of animals are in fact such cells, capable of living sepa-
rated from the organism. Among the lower plants any cell can be
separated from the plant and continue to grow. Thus, entire plants
may consist of cells whose capacity for independent life can be clearly
demonstrated. Now, since all cells grow according to the same laws so
that it is not possible that in one case the cause of growth lies in the
cell itself while in another case it lies in the entire organism, and since
moreover it can be demonstrated that single cells whose growth does not
differ from that of other cells can develop independently, we must there-
fore ascribe an independent life to the cell as such. That not every cell,
when separated from the organism does in fact grow is no more an argu-
ment against this theory than is the fact that a bee soon dies when
separated from its swarm, a valid argument against the individual life
of the bee."

TnEODORE Schwann. 1839, 29. pp. 188-190.

Chapter I
INTRODUCTION

In the whole gamut of human thought, there
is perhaps no question that crops up more fre-
quently in one form or another than that which
asks ''By what means do the myriads of different
forms and processes which we see in the Universe
around us come into being, what controls them
within normal metes and bounds, and how does
it come about that those 'normal' bounds are occa-
sionally transcended, so often with disastrous
results? What is it that brings about and main-
tains the extraordinary harmony which we usually
find in Nature, yet from time to time alters that
harmony by what appear to us to be transgres-
sions ! " If we knew the full answers to the mul-
titudinous variants of this question, there would
be very little left worth inquiring about.

For this question does have a multitude of
variants. It is asked at all sorts of different
levels of human experience. At the cosmic level
it becomes the question of creation itself. The
psychologist asks it at the level of human thought

2 Plant Tissue Culture

and behavior. The sociologist asks it at the level
of races and nations, tribes, sects, and societies.
The chemist asks it at the level of "substances"
and the physicist at the level of material bodies
great or small, be they galaxies or particles of
light. The geneticist and taxonomist ask it con-
cerning the segregation and delimitation of char-
acters of related organisms. With regard to
single organisms, we ask ' ' How is it that the size,
form, texture, rate of development, function of the
various parts are determined and limited? "Why
does an anterior limb-bud give rise to a hand while
a posterior limb-bud gives rise to a foot? Why
does a cell of a given type, when chance places it
in one region of the body, become an element of
the glandular epithelium of the kidney, while in
another part of the body the same type of cell
appears as a constituent of the iris? Why does
the sub-epidermal cell of a foliar leaf become a
center of food synthesis, while in a floral leaf it
becomes a megaspore-mother-cell with a repro-
ductive function? Why do the cells surrounding
a wound in a "normal" individual take part in
an orderly granulation, closing the wound, form-
ing well-limited scar tissue, organizing a harmon-
ious replacement of the injured regions, while in
an individual carrying a cancer, even at some dis-
tant part of the body, cells of the same region may

Introduction 3

be detonated into an explosive, disordered, dis-
harmonious, and ultimately self -destroying over-
growth?" The factors which control these char-
acteristics of size, form, function, and rate of
development of living organisms, which maintain
at all times the proper balance among their parts,
and which, when disturbed, result in creatures of
abnormal type, constitute the materials of that
important branch of biological science which we
call ' ' morphogenesis. ' '

Science progresses in five main steps. First,
there is the observation of more or less evident
facts, their codification and analysis. Second,
there is the formulation of ideas and principles
based on these facts and their arrangement into
working hypotheses. Third, there is the develop-
ment of techniques for the testing of these hy-
potheses. Fourth, there is the acquiring through
use of these techniques of pertinent but less evi-
dent information, the verification, modification,
and refinement of these hypotheses, until they
themselves become facts. And fifth, there is the
integration of these latterly acquired larger facts
into the general picture. In the study of living
organisms, one of the early observed "facts" was
the ubiquity of those units which we call cells.
Information about cells, acquired by general ob-
servation, very early led to the formulation of the

4 Plant Tissue Culture

fundamental hypothesis of the so-called ''cell
theory," promulgated a century ago in the words
which are set at the head of this chapter (Schwann,
1839, 29* that all cells are essentially elementary
organisms, theoretically alike and capable of au-
tonomous existence. But there is an alternative
hypothesis which also rests upon a considerable
mass of observed facts, namely, that somewhere
in the developmental history of an organism its
constituent cells cease to be totipotent, by segrega-
tion and loss of certain functions. No mechanism
is known by which such segregation or loss can
take place, yet it so often appears to have taken
place that this alternative hypothesis has today
many adherents.

If all cells of an organism are essentially alike
and, within the genetic pattern, totipotent, then
the differences in behavior of cells of a given type
in different situations in the body must result
from the interrelations of these cells with their
environments and with other cells in the organism.
It should be possible to restore suppressed func-
tions by isolating the cell from those external in-
fluences which were responsible for the suppres-
sion. If, on the other hand, there has been a true
segregation and loss of function so that the cells
are, in the mature organism, no longer totipotent,

* Italic numbers refer to bibliography.

Introduction 5

then no modification of a given cell-line 's environ-
ment could hope to restore the lost functions. It
is clear that a decision between these two alterna-
tive hypotheses is essential to any valid notion of
the origin of form and function. And it is also
clear that one of the most promising techniques
for arriving at such a decision lies in the segrega-
tion of cells, tissues, and organs from the associ-
ated members of the body and their maintenance
and study as isolated units, under as nearly opti-
mal and as fully controlled conditions as possible.
The attempt to reduce an organism to its constitu-
ent cells and to study these cells as elementary
organisms is thus a project of fundamental impor-
tance in the solution of basic biological questions.
Schleiden (1838, 27) and Schwann (1839, 29),
who formulated the concept of cellular totipotency,
and Virchow (1858, 433), who popularized that
concept, made no attempt to put it to experimental
test. A half century elapsed between its formu-
lation and the first well-organized experimental
study of the question. In the 1870 's, several
workers attacked the problem, no one with more
lucidity and patience than Vochting. Vochting
chose two methods of approach. First, he dis-
sected plants into smaller and smaller fragments,
studying the phenomena of polarity in these f ra,
ments. He found "polarity" to be a chara

6 Plant Tissue Culture

istic of every fragment, irrespective of size, and
hence by implication of the individual cells (1878,
329, 1884, 329) . Thus, the distal portion of a stem
or piece of stem always produced leaves and the
proximal portion roots, but whether a given cell

Fig. 1. Tubers of Corydalis solida, cut and allowed to regen-
erate. The tissues at the center of the tuber form leaves (left),
roots (center), or merely wound callus (right), depending on the
spatial orientation of the cut, showing the dependence of morpho-
genetic expression on fortuitous external factors. (From Goebel,
K. 1908. Einleitung in die experimentelle Morphologie der
Pflanzen. 220, fig. Ill, 293.)

Introduction 7

produced leaves or roots depended on that cell's
fortuitous position, whether distal to or proximal
to the nearest cut surface or uninjured growing
point. If a three-foot piece of willow was left
intact, the distal foot of stem would produce only
roots, but if a six-inch piece was isolated from the
distal end of such a stem either by removal or
merely by severing the bark, the proximal three
inches of this piece would produce leaves instead
of roots. Later observations by von Goebel ( 1908,
293) (Fig. 1) Schwanitz (1935, 323) and others led
to similar conclusions. Here the morphogenetic
pattern was a function of the "organism as a
whole" (considering here the autonomous frag-
ment as the organism), of the relationships of cell
to cell within the particular tissue continuum.
The cell appeared to be totipotent, its actual ex-
pressed function being a resultant of the in-
fluences coming from outside. Vochting's second
approach involved the age-old method of grafting
(Fig. 2) or transplantation (1892, 330, 1894, 331)
and merely confirmed what had been known from
time immemorial, that no matter what the environ-
ment or host into which a scion was transplanted,
it always developed in a given pattern which was
fixed by the species from which it came. Not only
was the developmental pattern fixed, but the
physiological behavior was also fixed by the spe-

8 Plant Tissue Culture

cies and could not be altered by contact with or
even dependence on the tissues of another species
(Vochting, 1894, 331). The transplantation ex-
periments demonstrated the restrictions which
species places on the potencies of a cell. The dis-
section experiments demonstrated the essential
totipotency of the cell within these species limita-
tions. Similar conclusions may be drawn from
experiments with animal materials (Figs. 3, 4).

To carry this method of dissection to its logical
end, we would have to go to the cells themselves,
as we have already said. Implicit in Schleiden
and Schwann's concept, subjected to a preliminary
pragmatic test by Vochting, this idea seems never-
theless not to have been explicitly formulated as
a basis for planning experiments with either plant
or animal materials until much later. In the early
years of the present century, Haberlandt (1902,
98) (Fig. 5) set forth the principle frankly and
lucidly in the words that I have placed at the head
of Chapter II. He then proceeded methodically
to fill the gap in our knowledge, the existence of
which he had called to our attention. He set out
to grow cells and small groups of cells in suitable
nutrients and to study their behavior. The dis-
cipline which Haberlandt thus outlined is that
which, in the four decades since his paper, has
gradually crystallized into what we know today

Introduction 9

as "tissue cultures." (See Harrison, 1928, 18).
(See also references 1-41.)

But it is not always given to one individual
both to formulate the broad outlines of a new tech-
nique and to put that technique into actual prac-
tice. Haberlandt was faced at the start with a
series of major difficulties inherent in plant
anatomy and morphogenesis which it has actually
taken workers a third of a century to learn to
overcome. These difficulties are chiefly three. In
the first place, most cells of a plant are not bathed
in any free, complete nutrient medium. The
xylem sap lacks many of the organic constituents
needed to maintain life. The phloem sap is in
direct contact with only a very few specialized
cells. Most plant cells must obtain a large part
of their nutriment by diffusion through neighbor-
ing cells. There is no "natural" nutrient which
can be extracted from a plant and used for the
cultivation of its cells and for subsequent analysis.
In the second place, most plant cells, with rare
exceptions, are surrounded by a rigid pellicle.
This prevents the cells from seeking and engulfing
food, thus greatly restricting the forms of food
which they can use. It prevents their adhering
satisfactorily to any solid substratum. And, since
excision involves rupturing this pellicle and expos-
ing the protoplast naked to the surrounding

10 Plant Tissue Culture

medium in a way that seldom occurs in nature, it
greatly increases the shock which any cell must
inevitably suffer in the process of removing it
from the body. In the third place, "growth," in
the plant, is normally restricted to a few special-
ized regions of the body, leaving all other parts in
an essentially inert condition, as far as capacity
for continued cell multiplication is concerned, and
greatly restricting the experimenter in his choice
of materials. As a result of these difficulties,
although the problem has been investigated by a
considerable number of workers, more than 30
years elapsed between Haberlandt's formulation
of the tissue culture concept and the first really
successful experiments with plant tissue cultures.
Fortunately for the development of the field as
a whole, however, animal tissues present none of
the above-named difficulties, at least to anything
like a comparable degree. In the first place, ani-
mal cells throughout the body are regularly bathed
in one or both of two characteristic and essentially
complete nutrient fluids, the blood and lymph.
These can be easily removed from the body in
large quantity. They serve as basic "natural"
nutrients for animal cells in which such cells can
be immersed without serious shock. They can be
analyzed at leisure to determine which of their
constituents are truly essential. In the second

Introduction 11

place, animal cells are for the most part sur-
rounded by a tough but mobile pellicle in place of
the rigid pellicle of the plant cell. Animal cells
can therefore be removed from the body with a
minimum of shock. Anyone who has worked with
both types will appreciate this difference. Ani-
mal cells adhere well to a variety of solid sub-
strata. They are capable of autonomous move-
ment and phagocytosis, so that they can be fed on
relatively complex nutrient materials which they
themselves transform into constituents which can
pass readily into the protoplasts. And finally,
"growth" of the animal body regularly takes
place in all parts of the body and, in the sense of
replacement, throughout the life of the animal.
There is thus comparatively little obvious restric-
tion placed on the experimenter in choosing favor-
able materials for cultivation. These facts have
largely affected the progress of tissue cultures
in the two living kingdoms and are responsible for
the early success in the animal field. Plant tissue
cultures have only within the last half decade
reached anything like a comparable position.

SUMMAKY

The question of the origin of form and function
in our Universe is one of the most fundamental
problems to the solution of which man has set his

12 Plant Tissue Culture

intellect. As regards living organisms, this prob-
lem embraces everything that we include under
the name of "morphogenesis." Since the cell is
theoretically the ultimate unit of structure in liv-
ing organisms, the question of the origin of form
and function in living beings may be stated thus :
"Are the factors which determine these charac-
teristics external to the individual cell (in which
case the cell is totipotent but its particular expres-
sion comes about through a potentially reversible
suppression of some of its potencies due to its
spatial association with other cells), or are they
internal, in the individual cell (in which case the
cell is obviously not totipotent but has lost some
of its potencies at some point in the sequence of
events which constitute its temporal association
with other cells)?" One method of deciding be-
tween these two possibilities lies in the isolation
and cultivation of individual cells as separate
units or elementary organisms. The technique
of isolating and studying cells in this way has, in
the last four decades, become established as the
technique of "tissue culture."

Introduction

13

Fig. 2. Ovary segments of three varieties of gourds (a fruits
jaunes, above; poire verte, center; a fruits blancs, below) grafted
together. Each variety retains its specific characteristics, although
in intimate anatomical and physiological contact with the tissues of
other varieties. The portion a fruits blancs must obtain all its
nutriment through the tissues of two other varieties, yet remains
unaffected by this fact. The dependence of morphogenetic capac-
ity on strictly hereditary internal factors is clearly shown. (From
Carriere, E. A. 1875. Eev. Horticole 1875 : 14-16, 278.)

14

/'hint Tissue Cult/ire

**Msl8&

Pig. 3. Segments of larvae of Bana palustris (left) and R.
sylvatica (right) having differenl pigmentation, grafted together
in the early embryonic stages. Each retains its own specific char-
acteristics in intimate anatomical and physiological contacl with the
other. Al the right, progressive stages in development. At the
upper left, histological detail of the region of union between the
two. Compare with Fig. 2. (From Earrison, R. G. L904. Ark.
in ik. An.-ii. 63: 35 L49, US.)

Introduction

15

Fig. 4. Besults of grafting belly epithelium from a toad em-
bryo (Bufo vulgaris) onto the eye region of an embryo of a frog
(Eaiia esoulenta). Here the grafted epithelium has formed, due to
its spatial relationship to the eye-cup of the host, a lens. Since
eyes are never normally present in the belly of the toad, this result
shows that belly epithelium possesses a potency (the lens-forming
potency) which, while never normally expressed, can be evoked by
suitable circumstances. The species characteristics of the transplant
(melanotic pigmentation) remain determined by the source, but the
morphological characteristics have been determined by the host. At
the right, cross section of entire head region. At the left, detail
of the right-hand eye showing the transplant. (From Filatow, D.
1925. Arch. entw. mech. Org. 105: 475-482, 402.)

16

Plant Tissue Culture

^6.

Pig. 5. Gottlieb Haberlandt, 1854 . Professor Emeritus,

Pflanzenphysiologisches Enstitul der Qniversitat, Berlin Dahlem,
Germany. Firsl to formulate specifically the concepl of tissue
cult ures.

"Es sind meines Wissens nach bisher noch keine planmassig angelegten
Versuche gemacht worden, isolierte vegetative Zellen von holier ent-
wickelten Pflanzen in geeigneten Nahrlosungen zu kultivieren. Und doch
miissten die Ergebnisse solcher Kulturversuche manches interessante
Streiflicht auf die Eigenschaften und Fahigkeiten werfen die die Zelle
als Elementarorganismus in sich birgt : sie miissten Aufschliisse bringen
fiber die Wechselbeziehungen und gegenseitigen Beeinflussungen denen
die Zellen innerhalb des vielzelligen Gesamtorganismus ausgesetzt sind."

"There has been, so far as I know, up to the present, no planned at-
tempt to cultivate the vegetative cells of higher plants in suitable nutri-
ents. Yet the results of such attempts should cast many interesting
sidelights on the peculiarities and capacities which the cell, as an ele-
mentary organism, possesses : they should make possible conclusions as
to the interrelations and reciprocal influences to which the cell is sub-
jected within the multicellular organism."

G. Habeblandt. 1902, 98. p. 69.

Chapter II

THE HISTORY OF PLANT TISSUE
CULTURE

The science of plant tissue cultures has had a
long but tenuous history. It would be difficult to
assign an exact date for the "first step" in its
development. Certainly, as we have seen, the
idea of cultivating fragments of plants was im-
plicit in much that was done in the 19th century.
Rechinger, in 1893, 108, seems to have been the
first to investigate experimentally the "minimum
limit" of divisibility of plant parts, using isolated
buds of Populus nigra, Fraxinus Ornus, roots of
Beta vulgaris, Brassica rapa, sections of stems of
Pothos celatocaulis, Coleus arifolia, and many
other materials. He concluded that pieces less
than 20 mm. thick would only rarely regenerate
entire plants and that some vascular tissue must
be included in the fragments cultivated. But in
this work he used no nutrient solutions, "grow-
ing" his cultures on sand moistened with tap
water. These were scarcely "tissue cultures" in
any real sense.

20 Plant Tissue Culture

A decade later, as we have seen, the real prob-
lem was clearly formulated for the first time by
Haberlandt (1902,98) (Fig. 5). Where Rechinger
had sought to determine the limits of divisibility
of plant materials, Haberlandt boldly took his
point of departure directly from the cell theory
and assumed that down to the cell level there were
no limits of divisibility. He, therefore, chose to
work with single cells, an aim which we now look
upon as an elusive and as yet unattained ideal.
Further, Haberlandt fully appreciated the impor-
tance of the photosynthetic process to all living
material and consequently conceived the idea that,
since most plants contain green cells, by cultivat-
ing these particular cells the necessity for pro-
viding carbohydrate in the nutrient would be
eliminated and the problem of technique simpli-
fied. In this he apparently lost sight of the fact
that green cells are, for the most part, relatively
mature and highly differentiated cells which have,
to a great extent, lost their meristematic functions.
This ''simplification" led him to work with pali-
sade cells of Lamium, pith cells from the petioles
of Eichhomia crassipes, glandular hairs of Pul-
monaria, stamen hairs of Tradescantia, stinging
hairs of Urtica, stomatal guard cells of Ornitho-
galum, and many other similar materials. The
results were, without exception, disappointing

The History of Plant Tissue Culture 21

and the attempt to attack the problem directly was
regretfully abandoned for another decade with the
remark that

' ' Jedenf alls diirf te die Methode ' • At any rate, the method of

der Ziichtung isolierter Pflan- cultivating isolated plant cells

zenzellen in Nahrlosungen ver- in nutrient solution should make

schiedene wichtige Probleme von possible the experimental study

einer neuen Seite her der ex- of many important problems

perimentellen Bearbeitung zug- from a new point of view."

anglich machen." (1902, 98)

Haberlandt turned to an indirect approach, that
of the study of wound healing (1913, 294, 1914,
295, 1919, 245, 296, 1920, 297, 1921, 354, 1922, 355),
and has never returned to the original problem.

It was during this unfruitful first decade of the
20th century that the sister science of animal tis-
sue cultures saw its initiation and greatest devel-
opment. Harrison (Fig. 6) in 1907 succeeded in
settling an old controversy by just the methods
suggested by Haberlandt, when he cultivated the
neuroblast of the frog in clotted lymph (1907, 131,
1908, 132, 1910, 414). He followed the presenta-
tion of his experimental results with a brilliant
expose of the possibilities of this method of ap-
proach. Two years later Burrows studied with
Harrison, subsequently joined Carrel, and to-
gether these two established the present-day
method of cultivating excised animal tissues in a
nutrient made up of blood plasma and embryo

22 Plant Tissue Culture

juice or their equivalents (Burrows, 1910, 117,
118, 1911, 119; Carrel, 1911, 120, 1912, 121-125,
1913, 190; Carrel and Burrows, 1910, 128). The
method outlined in 1913 by Carrel (Fig. 9) has
changed only in minor details since that date.

Carrel and Burrows ' method of cultivating ani-
mal cells in a blood-plasma embryo-juice nutrient,
while relatively simple and easily duplicable, has
not led to any exact knowledge of the nutrient
requirements of such cells and tissues. Blood
plasma and embryo juice are organic complexes
of unknown constitution, and such attempts as
have been made to elucidate their makeup have not
been especially successful (Baker, 1929, 233, 1933,
183; Baker and Carrel, 1925, 195, 1926, 184, 185,
1927, 186, 1928, 187-189 ; Carrel and Baker, 1926,
191 ; Ebeling, 1921, 192 ; Fischer and Demuth, 1928,
194). Nor have the attempts at a synthesis of a
nutrient, led by Dr. and Mrs. Warren H. Lewis
(Figs. 7, 8) in this country and Dr. A. H. Drew in
England been much more successful in eliminating
the unknowns (Baker, 1936, 234; Drew, 1922, 145,
1923, 146 ; M. E. Lewis, 1916, 148 ; M. R. and W. H.
Lewis, 1911, 149-151, 1912, 152, 153 ; W. H. Lewis,
1921, 154, 1923, 155, 1929, 156; Vogelaar and
Erlichmann, 1933, 162, 1934, 163, 164, 1936, 235,
1937, 236, 1939, 165). The cultivation of animal
tissues is still an empirical science.

The History of Plant Tissue Culture 23

Fig. 6. Boss G. Harrison, 1870- . Professor Emeritus, Os-
born Zoological Laboratory, Yale University, New Haven, Connecti-
cut. First to carry out successful cultures of excised animal tissues.

'24

Phi lit Tissue Culture

Pig. 7. Warren II. Lewis, L870- . Emeritus Associate, The
Carnegie [institution of Washington. One of the first to investi-
gate the behavior of animal tissue cultures in media nt' known
constitution.

The History of Plant Tissue Culture 25

'

-■

WjLa>-i\ Qj-utx- \jYfij«-2> v^^-^-s

Fig. 8. Margaret E. (Mrs. Warren H.) Lewis, 1881- . Re-
search Associate, The Carnegie Institution of Washington. Investi-
gator of animal tissue cultures, particularly of malignant tissues.

26

Plant Tissue Culture

QlJU^^^

Fig. 9. Alexis Carrel, L873- . Member Emeritus, The
Rockefeller Institute for Medical Research, New Fork City. First
to develop ;i method of maintaining animal tissues in culture for
unlimited periods.

The History of Plant Tissue Culture 27

Attempts were, of course, made to duplicate
Carrel's and Lewis' brilliant results, with plant
tissues, by use of some "natural" nutrient com-
parable to embryo juice. All such attempts, with
one possible exception, have consistently failed.
Schmucker, in 1929, 110, reported that he had suc-
cessfully grown individual leaf-mesophyll cells of
Bocconia in a fluid prepared from a filtered ex-
tract of Bocconia leaves. Details were appar-
ently never published, the work has not been
verified by any later worker, and the result is so
at variance with all other recorded experiments
that its correctness is to be doubted. In the hands
of other workers, all preparations of tissue juices,
xylem sap, phloem exudate, liquid endosperm, etc.,
have given evidence of toxicity (Prat, 1927, 451;
Bobbins, 1922, 177 ; Bobbins and V. B. White, 1937,
178* ) . This has, of course, forced workers in the
plant tissue culture field to resort to the much
more tedious, difficult, and uncertain, but at the
same time when successful more satisfying
method of developing step by step an essentially
synthetic medium.

The profound influence of Haberlandt's wide
reputation determined the subsequent orientation

* van Overbeek, Conklin and Blakeslee have reported successful
use of coconut milk as a nutrient for Datura embryos, too late for
the reference to be included in the bibliography of this volume.
(Science 94: 350-351, 1941).

28 Plant Tissue Culture

of the majority of investigations concerned with
the cultivation of plant tissues for a quarter of a
century and, while animal tissue culture studies
were making rapid strides, plant studies were for
the most part wandering in what we now look
upon as blind alleys. Smith in 1907, 325, Bobilioff-
Preisser in 1917, 90, Lamprecht in 1918, 102, 103,
Thielman in 1924, 111, and 1925, 112, 113, Borger,
92, and Czech, 93, in 1926, Kunkel, 101, and Prat,
451, in 1927, tJlehla, 115, and Kemmer, 99, in 1928,
Pfeiffer, 107, and Scheitterer, 109, in 1931, five of
them students of Haberlandt, all attempted to grow
cells of mature type. The materials studied in-
cluded leaf fragments of Peperomia, Bryophyllum,
Kalanchoe, Crassula, Sempervivum, Allium, Poly-
podium, Aspidium, Dicranum, Begonia, Triticum,
Pisum, Tradescantia, etc., epidermis of Viola,
Thunbergia, Rlioeo, stem tissues of Solanum, Bras-
sica, Saintpaulia, Coleus, Daucus, young ovaries
of Cucurbita, pith cells of Symphoricarpus, and a
variety of other plant parts. All gave equally
unsatisfactory results. Kiister, in 1928, 20, con-
sidered the problem hopeless ("aussichtslos")
and asked if perhaps the assumption that cells are
totipotent might not itself be false. There was,
indeed, much evidence from a variety of sources
which appeared to be contrary to this assumption.
Not the least indicative of these evidences came

The History of Plant Tissue Culture 29

from the field of animal tissue cultures. Fibro-
blasts, when grown in culture, not only always
maintained their characters as fibroblasts, but
retained growth rates which were characteristic
of the particular tissues or organs from which
they had been excised (Parker, 1931, 421, 1932,
422-424, 1933, 425, 426). Epithelial cells also con-
tinued true to type (Ebeling, 1924, 389, 1925, 390;
Ebeling and Fischer, 1922, 391 ; Fischer, 1922, 403,
404, 1929, 408). These results conveyed the im-
pression that somewhere in the ontogeny of the
individual animal the potential capacities of the
cells had been segregated, only "fibroblast charac-
ters" being retained by fibroblasts and "epithe-
lium characters" by epithelium. This impression
has been so emphasized by much recent biological
work that the older concept of the cell as an " ele-
mentary organism" and the plant or animal as a
"society of cells" has fallen into disrepute and
has, to a considerable extent, given way to the
modern concept of the "organism as a whole."
The cell has come to be looked upon as a more or
less fortuitous structural element without any
fundamental importance except as one means out
of several by which the integrations of the whole
may be attained.

There nevertheless remains much indisputable
evidence which seems to point to the importance

30 Plant Tissue Culture

of the cell as a physiological entity and to its fun-
damental totipotency. The whole gene mecha-
nism, as exemplified in somatic cells, seems to
lose its meaning without this concept. The regen-
eration of leaves and roots from tissues which are
neither leaf nor root points to the existence of
capacities which are called forth only by excep-
tional circumstances. Vochting's dissection ex-
periments suggest a similar explanation. Spe-
mann and his followers have obtained in their
transplantation experiments striking support of
this concept, for, while a piece of epithelium of a
melanotic amphibian is always melanotic (species
specific) in whatever host it may be grown (Fig.
3), it may form tail, or mouth, or eyelid, depending
on the position in the body in which it is placed
(Fig. 4) (Harrison, 1904, 413; Filatow, 1925, 402;
Spemann, 1936, 431; Schotte, 1932, 430). Some
transformations such as, for example, that from
fibroblast to macrophage are also known to take
place in animal tissue cultures (Ebeling and
Fischer, 1922, 391; Fischer, 1925, 406, 1926, 407).
There thus still seems to be much evidence to sup-
port Haberlandt's original concept and to lend
support to the hope that, while excised plant cells
have not yet been grown in vitro with the facility
which Haberlandt appears to have anticipated,
they will eventually be so grown. Such is the

The History of Plant Tissue Culture 31

basis for the continued study of the subject of
"plant tissue cultures."

Haberlandt's influence, while tending to orient
the subject in a fundamentally unfruitful direction,
nevertheless did keep interest in the field alive.
Moreover, Haberlandt was a sound enough scien-
tist to see that there were other possible orienta-
tions and to encourage them even though he did
not recognize their full value. In 1922 one of his
students, Kotte, 51, 52, (Fig. 10) made the first
important experimental contribution to the sub-
ject. Kotte chose to work not with single, green,
mature cells, but with colorless meristems, excised
root tips. His motivation in this choice, although
not very clearly formulated, appears to have been
similar to that which oriented the present writer's
choice of material a decade later (White, 1931, 32,
1932, 89), namely, their meristematic character,
their ease of excision and consequent relative
freedom from trauma, and the considerable
variety of their normal physiological responses.
Kotte succeeded in cultivating excised root tips
of pea and maize in a variety of nutrients. These
all contained the salts of a dilute Knop solution.
To these were added various combinations of glu-
cose, peptone, asparagin, alanine, glycine, Liebig's
meat extract, a pepsin-diastase digest of pea
seeds, etc. The most satisfactory results, in

32 Plant Tissue Culture

which normal development and response were
maintained for considerable periods, were ob-
tained in a solution containing, besides the salts,
1 per cent dextrose plus 5 g. Liebig's meat extract
per liter. The work was well planned, excellently
carried out, and, as we can now see, of consider-
able promise, but no subcultures were attempted,
Haberlandt apparently overlooked its importance,
and no attempt seems to have been made to follow
it up. By one of those coincidences which often
crop up in science, Bobbins in the United States,
following the leadership of Jacques Loeb (Rob-
bins, 1939, 220; Loeb, 1915, 302, 1916, 23, 303,
1917, 304, 305, 1918, 306, 1924, 24) and Louis
Knudson (Knudson, 1916, 168, 1919, 100 ; Knudson
and Smith, 1919, 169), simultaneously and inde-
pendently carried out similar work with compar-
able results (Robbins, 1922, 57). His method dif-
fered from Kotte's chiefly in the use of yeast
extract in place of Liebig's meat extract. Rob-
bins (1922, 177) and his colleague, Maneval
(Robbins and Maneval, 1923, 59, 1924, 258) carried
their work somewhat further than did Kotte and,
by using subcultures, succeeded in maintaining
cultures for some 20 weeks. But in every series,
there set in a gradual diminution in growth rate
and all cultures were ultimately lost. The results
seemed to suggest that some unknown material

The History of Plant Tissue Culture 33

not present in the nutrient might be essential for
continued growth. The work was dropped and
was not resumed by Bobbins for another decade.
Somewhat analogous studies were made by Cham-
bers in 1923, 43, 44, Mayer in 1929, 138, Heidt in

1931, 50, and Malyschev in 1932, 53, 54.

The ideas originally outlined by Haberlandt and
given their first really encouraging investigation
by Kotte and Robbins, passed then to other hands
and have seen considerable fruition since 1930.
Progress since that date has been largely under
the leadership of two workers, in France and in
the United States. Gautheret (Fig. 11), a stu-
dent of Guilliermond and disciple of Molliard,
carried out a great many investigations in the field
of "plant tissue cultures" in the years 1930-1940.
Studies similar to those of Kotte with excised root
tips were without marked success (Gautheret,

1932, 48, 1933, 49, 95, 96, 1935, 15, 1937, 242, 1939,
97). But simultaneous studies using cambium
from large trees were much more promising. He
succeded in growing cambium tissues for con-
siderable periods (Gautheret, 1934, 67, 1935, 15,
1937, 68, 1938, 69, 70, 1939, 71, 72, 1940, 73), in pro-
ducing in vitro grafts (1935, 15, 1937, 16), and in
learning a great deal about the nutrient require-
ments of such tissues. With the work of his com-
patriot Nobecourt (Nobecourt, 1937, 74, 1938, 75,

34 Plant Tissue Culture

76, 1939, 77; Nobecourt and Dusseau, 1938, 311),
and with White's (Fig. 12) independent work in
America, these studies have led to the first really
successful prolonged cultivation of undifferenti-
ated plant tissues (White, 1939, 78, 333) . At about
the same time that Gautheret's work was initiated,
the author of this volume, under the influence of Dr.
and Mrs. Warren H. Lewis and of the late Profes-
sor Duncan S. Johnson, began at the Johns Hopkins
University an intensive study of the whole prob-
lem of plant tissue cultures. The methods of ap-
proach finally adopted as most promising, al-
though developed independently, came to resem-
ble superficially those of Kotte and Bobbins on the
one hand and of Gautheret on the other (White,
1931, 32, 1932, 63, 89, 1933, 64, 116, 141, 142).
These studies led in 1934 to the first prolonged
cultivation of excised roots (White, 1934, 65), to
the development of satisfactory synthetic culture
media in 1937 (White, 1937, 141, 143, 144, 180, 181,
231, 1939, 182, 1940, 172, 173, 232), and to the pro-
longed cultivation of undifferentiated plant tis-
sues in 1939 (White, 1939, 78). The work of Bon-
ner (Bonner, 1937, 198, 1938, 199, 1940, 201; Bon-
ner and Addicott, 1937, 42), Fiedler (1936, 45),
Galligar (1934, 46, 1936, 166, 1938, 167, 1939, 47),
Marotto (1935, 55, 1936, 56), Loo and Loo (1935,
216, 1936, 217), and others, and recent studies of

The History of Plant Tissue Culture 35

Bobbins and his coworkers on excised roots (Rob-
bins and Bartley, 1937, 170, 222, 223; Robbins,
Bartley, and V. B. White, 1936, 58; Robbins and
Schmidt, 1938, 60, 1939, 61, 224, 225 ; Robbins and
V. B. White, 1936, 62) stem largely from these
studies of 1930-1934. It is this work since 1930
which forms the basis for the present volume.
The field is still too new to be frozen into definite
patterns. This account merely attempts to bring
it as nearly up to date as possible and to make the
present techniques available to students of the
field.

Summary

The history of plant tissue culture has been a
long and tenuous one. It may be conveniently
divided into four periods: 1834-1900 — The con-
cept of cellular totipotency and its demonstration
by cultivation of single cells was implicit in the
fundamentals of the "cell theory" but was neither
clearly expressed as a distinct concept nor sub-
jected to experimental test during this period.
1900-1922 — The idea of tissue cultures was out-
lined for the first time in its broad aspects by
Haberlandt in 1902 and was first put into practice,
with animal tissues, by Harrison in 1907 and by
Burrows and Carrel in 1910. Efforts aimed at
the establishment of similar cultures of plant tis-
sues were fruitless until 1922 when Kotte and Rob-

36 Plant Tissue Culture

bins independently cultivated excised root tips for
brief periods. 1922-1934 — The method as out-
lined by Kotte and Robbins was still far from
fully successful, and the period from 1922 to 1934
was again one of largely unsuccessful attempts in
a great many different directions. 1934—1939 —
In 1934 White and Gautheret gave a new impetus
and orientation to the subject, White by demon-
strating the possibility of growing isolated roots
for unlimited periods in vitro, Gautheret by dem-
onstrating the possibility of obtaining in vitro
growth of fragments of cambium. The period
1934 to 1939 was one of rapid expansion of the
field, of perfecting the nutrients and culture con-
ditions, and of gradual establishment of the tech-
nique on a firm basis. This was capped in 1939
by White's, Nobecourt's, and Gautheret 's indepen-
dent demonstration of the possibility of growing
undifferentiated tissues for unlimited periods and
by White's demonstration of experimental control
of differentiation in such cultures.

* William J. Robbins, 1890- . Director, The New York Botanical
Garden, Bronx Park, New York City. Simultaneously and independently
from Kotte, cultivated excised root tips for the first time for limited
periods in artificial media. Student of the vitamin requirements of
plants. The author regrets that Dr. Robbins has requested that his
portrait be omitted from this series.

The History of Plant Tissue Culture 37

/

tf

Fig. 10. Walter Kotte, . Reg. Bot., HauptsteUe fur

PflanzenschutZj Augustenberg, Post Grotzingen, Baden, Germany.
Simultaneously and independently from Bobbins* cultivated excised
root tips for the first time for limited periods in artificial media.

:;s

Plant Tissue Culture

Fig. II. Roger J. Gautheret, L910- . Charge de eours,
Faculty ilrs Sciences, Paris, Prance. First to cultivate, bide
pendently from White, undifferentiated planl tissues in vitro for
unlimited periods, student of the nutrition and behavior of ex-
cised planl tissues, especially cambium.

The History of Plant Tissue Culture 39

Fig. 12. Philip R. White, 1901- . Associate, The Rocke-
feller Institute for Medical Research, Princeton, N. J. First to
cultivate root tips and, independently from Gautheret, undiffer-
entiated plant tissues in vitro for unlimited periods. Student of
the nutrition and morphogenesis of excised plant tissues.

40

Plant Tissue Culture

Pig. 13. Roots of tomato (left), white clover (center), and red
clover (right) grown in culture for ten or more passages. (From
White, I". R. 1938. Am. .). Hot. 25: 352, tig. 10, 66.)

42 Plant Tissue Culture

"AXka 8i «f &>v Tavra. <£Xoios fiiXcw prjrpa 6a a ?xet P-MrPav- iravra 8'
opoLoptpfj. nal ra Tovraiy 8e in vpoTtpa ical if dv Tavra, vypov U 4>\of/
aapf apxai yap avrai. irXrjv el ns \eyoi t&s twv proixeiuiv Swapeis,
avrai 8e KOival irdvrusv. 17 pev ovv crixrla Kai 77 6\rj <pixjis b> rovrois.

"Again there are the things of which such parts (root, stem, leaf)
are composed, namely, bark, wood, and core (in the case of those plants
which have it), and these are all 'composed of like parts.' Further,
there are the things which are even prior to these, from which they are
derived — sap, fibre, veins, flesh ; for these are elementary substances —
unless one should prefer to call them the active principles of the ele-
ments ; and they are common to all parts of the plant. Thus, the
essence and entire material of plants consists in these."

Theophrastus. Enquiry into plants. I. II. I.
Translation by Sir Arthur Hort. Loeb Classical
Library. G. P. Putnam's Sons. 1916, +5 3.

Chapter III
THE LIVING MATERIALS

In the chapter on the history of plant tissue cul-
ture, data were presented on a long series of at-
tempts to grow excised plant tissues. Most of
these attempts failed ; a few were successful. If
plant tissues are classified according to their ob-
served usefulness as culture material, it becomes
immediately obvious that all of the tissues which
have given promise of being grown satisfactorily,
with the single exception of Schmucker's uncor-
roborated report (1929, 110) on leaf-mesophyll
cells, have been meristematic tissues. With this
single exception, all "mature" tissues have failed.
This is not to say that they are all incapable of
growth. But available evidence seems to indicate
that, when a meristematic function is resumed by
cells at any point in the plant body, as in the case
of healing internal wounds, these cells are for the
most part not truly mature (Schilling, 1915, 321,
1923, 322 ; Jaeger, 1928, 298) . The medullary ray
parenchyma, phloem parenchyma, pith cells in
general, pericycle and endodermis, and cortical
parenchyma are known to retain their meriste-

44 Plant Tissue Culture

matic capacity throughout most of the life of the
plant (tTlehla, 1928, 115; MacDougal, 1926, 307;
MacDougal and Shreve, 1924, 445; Sinnott and
Bloch, 1941, 324). Epidermal tissue does so ordi-
narily for somewhat shorter periods (Nay lor,
1931, 308 ; Naylor and Johnson, 1937, 309 ; Naylor
and Sperry, 1938, 310), while such cells as tri-
chomes, guard cells, xylem fibers, etc., retain this
capacity for only very brief periods (Haberlandt,
1913, 294, 1914, 295, 1919, 245, 296, 1920, 297, 1921,
354), because of mechanical modifications if for no
other reason. It is probable that means may ulti-
mately be found for obtaining cultures from most
living, nucleated cells. But the initial work in any
discipline is quite properly concentrated on the
easier materials available and the less promising
ones may well await the establishment of more
adequate techniques. Kecent work in this field
has, therefore, been concentrated on the meri-
stems.

Plant meristems can, for the most part, be clas-
sified in three main groups, leaving a rather large
residue of those more difficult to classify.

The fertilized egg is, of course, the meristematic
cell par excellence, since there can be no question
about its fundamental totipotency. It is, how-
ever, an extremely delicate "organism," (Fig.
20) normally hedged around by all sorts of protec-

The Living Materials 45

tions against mechanical and chemical shocks,
provided with very special and obscure nutrition,
and requiring considerable manipulation for its
excision. Hannig (1904, 81), Stingl (1907, 85),
Dietrich (1924, 79), Essenbeck u. Suessenguth
(1925, 80), White (1932, 89), Tukey (1933, 86,
1934, 87, 1938, 328), Werckmeister (1934, 88),
LaRue (1936, 82), LaRue and Avery (1938, 83),
Razdorskii (1938, 84), and others have grown ex-
cised embryos of more or less advanced stages of
development, but egg cells have apparently not
yet been cultured. It is probable that the difficul-
ties involved can be overcome. Indeed, Tukey 's
results with older embryos of stone-fruits indicate
considerable progress. However, since the culti-
vation of such embryos, as practiced, results in
entire plants which differ in no wise from plants
obtained from viable seed by the more usual meth-
ods, such cultures are likely to prove of value only
in special cases. Noteworthy among these are the
fairly numerous cases where viable embryos are
initiated but where, for one reason or another,
endosperm is not developed so that the embryos
die of starvation. Such was the case with Tukey 's
hybrid peaches (1933, 86) and with the Musa semi-
nifera studied by White (1928, 332). Similar
"physiologically sterile" plants have been re-
ported in the literature on numerous occasions.

46

Plant Tissue Culture

The embryo itself, even up to a fairly late stage
of development, is largely or entirely made up of
undifferentiated cells. Indeed, in the orchids,
Peperomias, and many other plants there is little
or no morphological differentiation evident in the

Fig. 14. Longitudinal section of seed of Peperomia hispidula
showing the mature but completely undifferentiated, few-celled
embryo. (From Johnson, D. S. 1914. Am. J. Bot. 1: 396, fig.
100, 444.)

The Living Materials 47

embryo at the time the seeds are "ripe" (Johnson,
1912, 444). (Figs. 14, 15.) It is, therefore, pos-
sible to cut an embryo into bits and grow any one
of these fragments. But, as Smith (1907, 325),
Molliard (1921, 106), LaRue (1936, 82, 104), Dau-
phine (1929, 94), and others have shown, even in
the embryo there are some regions having a
greater proliferative capacity than others, an ob-
servation which agrees with that established in
animal embryology. Cotyledons will usually re-
generate easily (Molliard, 1921, 106; Behre, 1929,
275). Roots can be counted on to grow as roots,
but means of inducing them to develop leaves
have, with rare exceptions (especially among the
Compositae) (Fig. 16), not yet been found.
Hypocotyl is in general still more refractory,

E<

Fig. 15. Detail of endosperm and embryo of Peperomia hispi-
dula. This is an ideal type of material for the study of embryo
differentiation after germination. (From Johnson, D. S. 1914.
Am. J. Bot. 1: 396, fig. 110, 444.)

48 Plant Tissue Culture

while the epicotyl and plumule tend to form roots
and develop into normal plants without serious
difficulty. Thus, even in the embryo there is the
beginning of an apparent loss of totipotency, a
segregation of function which is marked in the
root, somewhat less in hypocotyl and cotyledon,
and lacking in epicotyl and plumule.

As the embryo grows into a plant, there is a pro-
gressive maturation of some tissues so that, ex-
cept for "juvenile" cells like the medullary ray
parenchyma scattered through the plant body,
there are segregated certain meristematic re-
gions which carry on the function of prolifera-
tive growth (Priestley, 1928, 314; Priestley and
Swingle, 1929, 316 ; Jaeger, 1928, 298) . These re-
gions are: the terminal growing points of roots
and stems, the lateral meristems designated as
cambiums and phellogens, and intercalary meri-
stems in the procambial region, at the base of the
internodes in grasses and lianes and at the base
of some leaves or, as in Camptosorus, near the
tips of leaves.

The intercalary meristems require considerable
manipulation for their excision and therefore suf-
fer rather severe injury. Scheitterer (1931, 109)
had slight success with the intercalary meristem
of grasses, and none of those of lianes have been
cultivated although they should be fairly easy to

The Living Materials 49

work with. The only intercalary meristem which
has been successfully grown is that from the pro-
cambial region of tobacco (White, 1939, 78). If
the procambial tissues of fairly succulent plants
such as tobacco or squash are carefully excised
under aseptic conditions (see later) and placed on
a nutrient substratum, proliferation will occur,
the newly formed callus tissue can be removed
from adherent non-proliferating medullary ele-
ments, and a rapidly growing "pure-line" callus
culture will result which can be propagated in
series indefinitely. This is one of the most im-
portant types of culture obtained to date and will
be discussed in more detail later (Fig. 18).

If, moreover, the cambium is taken from peren-
nial plants having secondary thickening, and for
this purpose trees of fairly good size offer fewer
mechanical difficulties than do smaller plants, and
is placed on a nutrient, a similar proliferation
occurs, the product of which can be treated in the
same manner (Gautheret, 1934, 67, 1935, 15, 1937,
68, 1938, 69, 70, 1939, 71, 72, 1940, 73). When es-
tablished in culture, it behaves much as do cul-
tures derived from procambium. Gautheret has
been especially successful with this type of cul-
ture and has made a number of very interesting
experiments therewith (Figs. 38, 64). The cul-
tures of callus from carrot root, carried out by

50 Plant Tissue Culture

Gautheret and Nobecourt (Nobecourt, 1937, 74,
1938, 75, 76, 1939, 77), may be considered as "cam-
bium" cultures altbougb grown from root cam-
bium rather than from stem cambium.

Terminal meristems are of two types — stem tips
and root tips. The plumule taken from the em-
bryo is a stem tip and cultures of this region,
taken from no matter what age of plant, behave
in much the same way. They differentiate into
normal organs ; if they become successfully estab-
lished, they develop roots and subsequently be-
have as entire plants. As grown to date, they
thus offer little or no advantage for physiological
studies over seedlings derived in the usual way.
Hence, although excised stem tips have been
grown with some success by Robbins (1922, 57),
White (1933, 116), Avery and LaRue (1938, 237),
Behre (1929, 275), and others, such cultures have
not proved of great value to the tissue culture
field and have not been pushed very far.

Root tips, on the other hand, represent one of
the most important types of material for tissue
or organ cultures. If the tips of very young roots
of any sort, from ungerminated embryos, seed-
lings, old root systems, or adventitious roots pro-
duced on cuttings, are obtained in an aseptic con-
dition and are excised and placed in nutrient solu-
tion or on a nutrient substratum, they will, if con-

The Living Materials

51

Fig. 16. Root of dandelion grown in culture. Callus normally
forms on roots of many composites on both distal and proximal
ends of cut or injured segments (see paired nodules at the left).
Leaves frequently develop from these calluses (upper left). This
is in shari> contrast to the behavior of callus on plants which
proliferate only at the distal ends of segments.

.)!'

Plant Tissue Culture

Pig. 17. Tissues from sunflower. Left-hand column, fragments
immediately after excision. Right-hand column, the same after two
weeks mi nutrient agar. Top: wedge-shaped segment taken from
.•in old stem, containing (left) cortex, (center) vascular tissue in
eluding cambium, and ( righl i pith. Second row: transverse seg-
ment from procambial region where nil parts are approximately
equally meristematic. Third row: transverse segment from some
wlint older stem. Proliferation is largely from the cambial ring.
Bottom: transverse segmenl from an old stem. (From White.
I\ l,\. and Braun, A. C. 1942. (•.•nicer Research 2: fig. :'.. Si

The Living Materials

53

Fig. 18. Culture of undifferentiated callus from the hybrid,
Nicotiana langsdorffiix X. glauea maintained for many passages in
vitro on nutrient agar. (From White, P. E. 1989. Am. J. Bot.
26: 60, fig. 3, 78.)

Fig. 19. Cultures of bacteria free crown-gall tissue from sun-
flower, grown in vitro on nutrient agar.

54

Plant Tissue Cult are

I
\

.»

s

\

v

v

\ ' Pi ■ • •' •

- --*% V

\

\

Pig. 20. Young embryo of Portulaca oleracea with remnants of
the embryo sac still adhering. If embryos of this and still younger
stages can be grown, much may be learned of the factors involved
in early embryo development. ( Prom White, P. K. L932. Arch,
exp. Zellf. 1 1 : 610, fig. s, 89. |

The Living Materials 55

ditions are right, grow normally. They main-
tain for the most part their normal morphology,
branching and extending in a regular way. The
details of this morphology are characteristic for
different species (Figs. 13, 21-24). There is,
for example, a very marked and wide difference
in degree of apical dominance (White, 1938, 66).
In the mustard, Brassica nigra, apical dominance
is extremely weak; as fast as branches are laid
down, they grow with a vigor equal to that of the
parent root so that an open network of rootlets
is formed without definite outlines. Roots of
clover, Trifolium repens (Fig. 13), on the other
hand have a very marked apical dominance so
that, even though many branches may be laid
down, their increment is greatly retarded as com-
pared with that of the original root and the
growth habit is that of a well denned and very
attenuate cone. The habits are thus roughly com-
parable to those of the white oak (Quercus alba),
on the one hand, and the white fir (Abies lasio-
carpa), on the other.

Such roots can be excised with a minimum of
injury since the cross sectional area is small in
proportion to the volume of even a comparatively
short piece. They are meristematic for a consid-
erable length and consequently can be counted on
to grow with fair rapidity. They are naturally

56 Plant Tissue Culture

immersed in a nutrient substratum (the soil solu-
tion), the essential feature of which is a mobile
liquid solution so that placing them in a liquid
nutrient does not involve any marked change in
their environment, provided the nutrient is suffi-
ciently aerated in some way (Cannon, 1932, 240;
Zimmerman, 1930, 270; Steward, Berry, and
Broyer, 1936, 262; Hoagland and Broyer, 1936,
246). This substratum is, in nature, a fairly com-
plex solution of salts, humic acids, and other sim-
ple organic compounds. The major problem in
duplicating the environment of a root is in pro-
viding the equivalent of the organic materials
ordinarily supplied by the parent plant, and a
glance at the anatomy of a rapidly growing root
shows that these materials, coming from the
phloem, must travel for considerable distances by
diffusion through other cells before they can reach
the dividing cells themselves. It might, therefore,
be anticipated that these organic materials would
prove to be relatively simple substances, as indeed
they have (see later). The problem of growing
excised roots should, therefore, both from a me-
chanical and a physiological point of view, be rela-
tively simple. Kotte's (1922, 51, 52) and Robbins'
(1922, 57) early work supported this supposition.
Excised root tips have, in fact, served as the ex-
perimental material par excellence for the pre-

The Living Materials 57

liminary work in developing a tissue and organ
culture technique, and practically all of the fun-
damental steps in that development have been
made first with root tips and only later applied
to other tissues. Such roots are, in fact, very
sensitive to differences in cultural conditions such
as temperature, pH (White, 1932, 63), osmotic
value (Bonner and Addicott, 1937, 42), concentra-
tion and proportion of nutrient salts (White, 1933,
141, 1937, 143; Bonner and Addicott, 1937, 42),
carbohydrate supply (White, 1932, 63, 1940, 172,
173), etc.; this sensitivity manifests itself either
in change of growth habit (qualitative) or change
in growth rate (quantitative). Growth rate can
be measured quite accurately by any of a number
of methods (see later), so that it is possible to
follow the effects of relatively small differences
in environmental conditions and to establish opti-
mal ranges for these conditions.

There remains a variety of meristematic re-
gions which are more difficult to classify. The
entire young flower, and particularly the ovary,
is at first meristematic. This function is retained
in some tissues for a long time. Wehnelt (1927,
230) and subsequently Bonner and English (1938,
342) have taken advantage of this fact in using
the inner surface of bean pods as test-pieces for
a study of the growth stimulating properties of

58 Plant Tissue Culture

certain plant extracts (Fig. 35). White (1932,
89) and Moebius (1922, 105) have attempted to
grow immature ovules and Tukey (1937, 114) and
Borger (1926, 92) attempted to grow excised
placental tissue without marked success. There
are, in additon to these meristematic regions, cells
scattered throughout the plant body, such as the
medullary ray cells, the "xylem parenchyma"
cells, the phloem companion cells, which retain a
characteristic meristematic appearance long after
the neighboring cells have "matured," and are
capable of resuming growth when freed from the
inhibitory effects of the neighboring tissues. Cells
of the pith, of the cortex, of the epidermis, and
other tissues may also, under special and at pres-
ent little understood conditions, resume a meriste-
matic function (Reiche, 1924, 369; Okado, 1920,
312; Wilhelm, 1930, 338; Winkler, 1902, 339; Sin-
nott and Bloch, 1941, 324; Stingl, 1909, 326), but
until a great deal more is known about them than
at present they cannot be looked upon as satisfac-
tory material for plant tissue cultures.

The Living Materials 59

Summary

Examination of the history of plant tissue cul-
tures shows that only those tissues have so far
been successfully cultivated which possess a fun-
damentally meristematic character and do not
have to undergo any major degree of de-differen-
tiation. Plant meristems are, for the most part,
of three main types. Apical meristems, particu-
larly root tips, have been cultivated extensively
and most of the details of technique have been
developed through their study. Intercalary meri-
stems such as cambium have also been successfully
grown, as undifferentiated masses or with their
differentiation experimentally controlled. Egg-
cells and embryos, insofar as they have been
grown in culture, have given rise to complete
plants. Such cultures can be of importance in the
study of the origin of form and function only
insofar as the degree and direction of their dif-
ferentiation can be controlled. Other tissues,
such as young ovaries, placental tissues, etc., have
been studied to a lesser degree and have in some
cases proved useful for special purposes.

The Living Materials

61

Fig. 21. White-clover root grown in vitro, showing the massive
root-cap commonly formed on the roots of legumes. (From White,
P. E, 1938. Am. J. Bot. 25: 352, fig. 11a, 66.)

t\'2

Plant Tissue Culture

Fig.

Roo1 hairs from an excised tomato rool grown in vitro.

mts. L938. ."•'J, fig. 19.)

i Prom SeifriZj W., The Physiology of 1

The Li ring Materials

63

Fig. 23. Root hairs on roots of buckwheat grown in vitro.
(From White, P. R. 1938. Am. J. Bot. 25: 349, fig. 4, 66.)

64

Plant Tissue Culture

pIQ. 24. Roo1 of tomato (left) and buckwheal (right).
White, P. R. L938. Am. J. Bot. 25: 349, fig. 2, 66.)

( From

66 Plant Tissue Culture

"(My) room was generally hung round with Guts, stomachs, bladders,
preparations of parts and drawings. I had sand furnaces, Calots, Glasses
and all sorts of Chymical Implements. . . . Here I and my Associates
often dined upon the same table as our dogs lay upon. I often prepared
the pulvis fulminans and sometimes surprized the whole College with a
sudden explosion. I cur'd a lad once of an ague with it, by fright. In
my own Elaboratory I made large quantitys of sal volatile oleosum,
Tinctura Metallorum, Elixir Proprietatis and such matters as would
serve to put into our Drink."

Diary of Mr. Wm. Stukeley, 1707, describing his
rooms at Cambridge where he and his fellow stu-
dent, Mr. Stephen Hales, planned and carried out
some of the first real experiments in plant and
animal physiology. US.

Chapter IV
THE LABORATORY

The description of a laboratory given above is
presented as an example, not an ideal ! The gen-
eral requirements for a plant tissue culture lab-
oratory are the same as those for any detailed
micro-culture work: 1, facilities for the prepara-
tion, sterilization, and storage of nutrients and
for the cleansing of used equipment; 2, a place
for the aseptic manipulation of tissue masses; 3,
facilities for the maintenance of cultures under
carefully controlled conditions; 4, facilities for
examination and study of cultures in whatever
ways may be desired, and 5, a place for the assem-
blage and filing of records. It will seldom be
possible to carry on all these procedures in a
single room with any degree of effectiveness, but
the degree of complexity introduced into the or-
ganization of the laboratory will depend on the
particular needs and the facilities available for
any given work. The ideal organization would
allow a separate room for each of the above pro-
cedures— a media room, a transfer room, one or
more culture rooms, a laboratory, and an office.
(Compare Parker, 1938, 26.) (Fig. 25.)

68

Plant Tissue Culture

The media room. The media room will ordi-
narily be used for preparing and sterilizing of
nutrients and equipment and probably also for the
storage of extra supplies. If possible, it should
be reserved for the preparation of nutrients and
equipment for tissue cultures only, since sharing
it with bacteriologists, mycologists, etc., involves
definite risks of contaminations, both biological
and chemical. There will be required: a good
stove, an autoclave, a dry-sterilization oven, a
refrigerator, a sink, a water still, tables and cup-

rhell Mu

QDesk
I , OFFICE

Files ,

ff 1 Table

Microscope*

o

/

w\

~] 1 Shelv

LAVATORY,

Table
Cupcoa rds & Bookcases \-~ '

,.^~

Cupbd. Shelt

Table '

7=A ul"e \

Et |° \

Cupbdi 1 Shelfft

CULTURE RM.

Fig. 25. Plan for a suite of rooms designed particularly for use
in the study of plant tissue cultures.

The Laboratory 69

boards, and possibly an Arnold sterilizer. Gas,
vacuum for use in filtering, compressed air for use
in drying pipettes, etc., and extra water connec-
tions and electric outlets should be provided. The
stove, autoclave, and oven require no special com-
ment. The sink should be of acid-proof vitreous
material with vitreous piping, or should be kept
thoroughly paraffined since a great deal of wash-
ing in chromic acid cleaning solution will have to
be done therein. It should be fairly large, with
the faucets placed high enough to escape possible
splashing from the sink. It has been found con-
venient to place a broad shelf above the sink wide
enough to take two 20-liter Pyrex carboys, one for
single-distilled water, the other for double-dis-
tilled water, and to drain graduates from a rack
below the shelf. There should also be a draining
rack for flasks and miscellaneous glassware. If
distilled water is available as a laboratory service,
it can be piped above the sink and replace the
carboy of single-distilled water. Such a supply
should not, however, be relied upon for water for
making up solutions or for the final rinsing of
glassware. For these purposes, water should al-
ways be re-distilled in a special still installed in
the media room. Where a laboratory supply of
distilled water is not available, it will be necessary
to have two stills, either separate or built in tan-

70 Plant Tissue Culture

dem. The best procedure appears to be to start
with ''tap water" from a spring or well (city
water frequently contains chlorine which may
pass over in the still and reappear in the product
unless the condenser is properly vented). This
should be treated with a softener and filter to re-
move the greater amount of dissolved salts, oil
from pumps, particulate matter, and other prod-
ucts which might interfere with the efficiency of
the first distillation. It should then be distilled
with an ordinary well-vented still and should
finally be passed into a second still, re-distilled,
and the final product collected in Pyrex contain-
ers. A block tin still of the "Precision" type, if
properly handled, is quite satisfactory for the
final distillation for all ordinary purposes. A
Pyrex still can also be used. Distillation in
quartz, silica, silver, or other special types of
stills will seldom be necessary. Special precau-
tions to insure removal of traces of particular ele-
ments may have to be taken in special cases — thus,
Pyrex can, of course, not be used for storage of
water to be used in any critical experiments on
the behavior of tissues towards boron, rubber
tubing or stoppers should not be used in experi-
ments with zinc or sulfur, and so on (Anion, 1938,
133; Arnon and Stout, 1939, 134, 135; Stout and
Arnon, 1939, 140; Hoagland and Arnon, 1941,

The Laboratory 71

137). With these points in mind, a satisfactory
supply of water can be prepared.

Salts must be accurately weighed. With the
exception of the sugar (see later) which can be
weighed on a coarse torsion balance in the media
room, all weighings should be carried out in the
laboratory room on high grade quantitative bal-
ances.

The actual preparation of media will be dis-
cussed in a separate chapter and the glassware
required will be listed there.

Glassware must be kept scrupulously clean
(Richards, 1936, 452). Carrel (see Parker, 1938,
26) boils his flasks in soap solution, rinses thor-
oughly with water and then with 95 per cent alco-
hol, and allows them to dry, after which they are
dry-sterilized. Vogelaar and Erlichmann (1939,
457) recommend washing in a sodium pyrophos-
phate solution. In this laboratory, it has been
customary to wash all glassware after use in a
sulfuric acid-potassium dichromate solution, made
up without added water. The cleaning solution
is kept in 3-gallon glazed crocks, such a crock
being placed in one corner of the vitreous or well-
paraffined sink. The flasks to be washed are first
emptied and, if they contain any solid matter, this
is rinsed out (Step 1 in the cleaning routine).
They are then placed in the acid and allowed to

72 Plant Tissue Culture

stand for a few minutes (Step 2). For ordinary
routine washing of flasks used only for liquid
nutrients, the time required to transfer 50 flasks
from the draining table to the rinsing container
(Step 4 in the washing routine) is usually long
enough for flasks to stand in the acid. Flasks
which have contained solid residues likely to stick
to the walls should be left in acid over night.
After standing the requisite time, the flasks are
removed from the acid, using linemen's rubber
gloves, are drained thoroughly into the crock, and
are then placed on 18" X 24" enamel trays for
further draining (Step 3). While they are drain-
ing, a second batch of flasks can be immersed in
the crock of acid. After thorough draining on
the trays, the flasks are placed under running
warm water for a few minutes (Step 4). It is
important that all traces of bichromate be re-
moved (Richards, 1936, 452). They are then
rinsed thoroughly at least five times in running
tap water, twice in single-distilled water, and
finally once in double-distilled water, after which
they are inverted in paper-lined metal trays and
set aside to dry. Excess cleaning solution from
the draining trays can be poured back into the
crock at intervals. The acid should be replaced
by fresh whenever it turns green and ceases to
remove pencil markings on flasks without rub-

The Laboratory 73

bing. Do not pour exhausted solution down the
sink! It can be emptied in some waste place
where it will do no serious damage.

Nutrients will generally be made up in 4-liter,
10-liter, or 20-liter serum bottles (see later). A
supply of graduates of various sizes, 125, 250,
500, and 1000 ml. Erlenmeyer flasks, 500, 1000, and
3000 ml. balloon flasks, test tubes, pipettes, etc.,
will be required for the preparation of different
nutrients and should be kept in the media room.
Both absorbent and non-absorbent cotton, a good
grade of cheesecloth, heavy paper for wrapping
glassware for dry-sterilization, cord, stones for
sharpening instruments, etc., should also be pro-
vided.

Cotton plugs for flasks should be of non-ab-
sorbent cotton and should be wrapped in cheese-
cloth. This reduces the danger of plugs sticking
to the mouths of the flasks, prevents loose fibers
from falling into the flasks, and helps to preserve
the form of the plugs. Plugs prepared in this
way can be used repeatedly. It has been found
best to cover each plugged flask with an inverted
50 ml. beaker to keep out dust and water of con-
densation, else molds are likely to grow in the
plugs and ultimately penetrate to the nutrient
(Figs. 29-31). (See Tukey, 1934,57). Metal trays
13" X 21" X 3^" (Fig. 31) are of the proper dimen-

74 Plant Tissue Culture

sions to hold forty 125 ml. Erlenmeyer flasks, and
three such trays of flasks can be placed simul-
taneously in a 20-inch autoclave. Shelves should
be provided of proper depth to hold these trays
where nutrient can be stored after sterilization.

The transfer room. This is perhaps the most
important room in the laboratory. A good trans-
fer room is absolutely essential for satisfactory
tissue culture work. For very simple manipula-
tions, a low bacteriologist's hood may suffice, but
for any extensive work a special room is essential,
and, since the conditions required differ somewhat
from those in a pathological laboratory, it should,
if possible, be reserved for this work alone. Rob-
bins, and Lewis use plywood chambers about
4'6" X 5'6" X 7'6", provided with air filters, elec-
tricity, and gas. The ideal transfer room should
be an inside room, without windows. Windows
always leak, no matter how well built, so that a
marked increase in contaminations in a room pro-
vided with them always occurs in windy weather.
Light from windows is also very irregular. The
temperature drop across a window, moreover, sets
up convection currents within the room. These
annoyances are eliminated by using an inside
room. It should have no special heating unit,
since such a unit is also a source of convection
currents. A well designed air-conditioning outfit

The Laboratory 75

with proper filters and correctly placed outlets
will provide adequate aeration and temperature
control. A room about 8 X 10 ft. with 8 ft. ceiling
is an excellent size. The lighting should be dif-
fuse, preferably through a broad skylight supple-
mented by properly placed indirect lamps. Cup-
boards with tight, sliding doors may be built into
the walls, but there should be no fixed furniture
in the room and all wall surfaces, doors, etc.,
should be designed so as to reduce to a minimum
the possibility of dust accumulating. Outlets for
electricity, compressed air, vacuum, and water
should be provided. There should be a sink built
into a flush hood at one end of the room. A
movable table and stools should be provided. The
walls and ceiling should be of washable tile or
enamel and, as Carrel (see Parker, 1938, 26) has
pointed out, a dark gray color is best since it
reduces reflections and the mental distractions
which lighter-colored surfaces always impose.
Provision should be made for washing the air of
the room with a series of water sprays or steam
jets so as to remove any spores which may get in
when the room is opened.

A transfer room for plant tissue cultures should
not contain gas outlets. Gas is extremely toxic to
plant materials and unburned gas may easily be
occluded in the culture flasks if the mouths are

76 Plant Tissue Culture

flamed (Saeger, 1933, 261). Cotton plugs char,
and bits of charred cotton falling into flasks of
nutrient may vitiate the results of nutrition
experiments. Cotton itself contains significant
quantities of thiamin (Schopfer et Rytz, 1937,
226). With rare exceptions, the custom of flam-
ing the mouths of tubes and plugs is a pernicious
hocus-pocus. Flasks which are properly handled
and plugged and are capped to keep out dust and
water of condensation will remain sterile for
months without flaming. Instruments can be ade-
quately sterilized in boiling water. If needles for
bacteriological studies must be flamed, this can be
done quite satisfactorily in a small alcohol flame
without the hazards involved in the use of gas.
The use of gas around a plant tissue culture labo-
ratory should be reduced to a minimum.

The culture rooms. The details of culture
rooms, laboratory, and office will, of course, be
modified to meet the needs and facilities of indi-
vidual workers. Facilities for accurate control
of temperature, illumination, and humidity, and
experimental comparison of different levels of
these factors are desirable. Since plant tissue
cultures are extremely sensitive to temperature
fluctuations (White, 1932, 63, 1937, 265, 266) and
have an optimum temperature only slightly above
room temperature (about 27°-30° C), it is impor-

The Laboratory 11

tant that even the room used for routine stock
cultures be air-conditioned, with faciilties for
cooling in extremely hot weather. Cultures main-
tained in an ordinary laboratory room without
such control show a marked seasonal variation in
growth rate (Fig. 26) (White, 1937, 265) and
may, in extreme weather, suffer serious injury.
The experimental culture room should be pro-
vided with plenty of table space or, alternatively,
with table-height wall benches, since most experi-
mental cultures must be examined at frequent
intervals and must be easily accessible. Stock
cultures can be kept on shelves, since they do not
need to be handled.

The laboratory. Like the culture room, the
laboratory must be adapted for the particular
type of problem in view. A complete laboratory
should contain facilities for chemical analyses, a
good hood, balances, drying ovens, equipment for
preparation of sections and slides and their ex-
amination, equipment for special types of experi-
ments such as respiration apparatus, facilities for
glass-blowing, etc. Gas outlets for use in connec-
tion with plant tissue culture work should, where
possible, be restricted to this room and the media
room. There should, of course, be adequate cup-
board space for chemicals.

The office. Every man has his own ideas of

78

Plant Tissue Culture

3annno a3d ww

The Laboratory 79

how an office should be arranged, so that that need
not be treated in detail here.

What has been outlined above is approximately
an ideal suite. Most setups will, of course, fall
short of this ideal, but that need not detract from
the quality of the work done so long as the prob-
lems attacked are restricted to those for which
the available equipment is adequate. Good work
has been done in many a garret. But it must be
remembered that the garret is usually the birth-
place of sciences, not their adult milieu. Jacques
Loeb's tumblers and finger bowls would hardly
have sufficed for the determination of particle
shape of virus molecules.

Most of the equipment used in preparing and
maintaining plant tissue cultures — glassware, im-
plements, etc. — are the same as those available
in any good microbiological laboratory, so that
they need only be enumerated. There are, how-
ever, a few items not so likely to be found in the
average laboratory.

Glassware. Graduates, pipettes, Erlenmeyer
flasks, balloon flasks, beakers, Stender dishes,
watch glasses, test tubes, hollow ground slides,
cover slips of various sizes and shapes, burettes,
etc., are, of course, stock necessities. Most cul-
tures of both roots and callus can best be main-
tained in 125 ml. Erlenmeyer flasks. These can

80

Plant Tissue Culture

be handled far more easily than test tubes (which
require special sloped racks), they present a
larger space in which to work when dissections
are to be performed on the cultures, they will
stand on an ordinary table without rolling, and
they are easily cleaned. They do occupy consid-
erable space. Special flasks such as Carrel flasks

f b C D E

=c*=

?:1

f.j.r.1

/

*•■ .

\

(.

o

)

A

!■*--

*-7mm»
■ 1 5mm

*-►•!.

i

i i

i

! 1

-75mm

— -j

Zbmn

Fig. 27. Diagram of a special, pierced slide found particularly
satisfactory for hanging-drop cultures of plant tissues. A, tissue
fragment. B, drop of nutrient. C, square 22 x 22 mm. No. 1 cover
glass on which culture is made. D, small 7x7 mm. No. 0 cover
(square or round) held on lower surface of nutrient by capillarity
providing a flat lower as well as upper surface to the drop. E,
square 22 x 22 mm. No. 2 cover glass closing the bottom of the
chamber.

The Laboratory 81

appear not to have any considerable advantages
for plant tissue cultures. If test tubes are to be
used — as may be desirable in some experiments —
the 1" X 6" size is the most useful (Fig. 29).

Two special pieces of glassware have been found
extremely useful. The first is the Maximow em-
bryological watch glass (Maximow, 1925, 420).
This has a cemented plate-glass bottom which
offers an optically flat surface far superior to the
pressed type in common use, especially for fine
dissections under the binocular. Those with the
beveled wall to the cavity are more easily cleaned
than those with a straight up-and-down wall.
This same property of optical flatness is often like-
wise important in the slides used for hanging-
drop cultures. This requirement may be met in
several ways. The glass Van Tiegham cell can be
used. Dr. Warren Lewis places low brass rings
dipped in paraffin on ordinary slides and cements
the inverted cover slips to these. A still better
method is to use special 3 mm. thick slides, each
of which is pierced with a 10-12 mm. hole. Short
pieces of thin (1 mm.) slides are cemented to both
top and bottom of these, at both ends, leaving a
space 25 mm. long in the center, surrounding the
hole, free. A 22 mm. square No. 2 cover slip is
fastened with vaseline to the bottom of the slide
in this space, forming a circular chamber with a

82 Plant Tissue Culture

thin flat bottom. The cover slip carrying the cul-
ture is then inverted over the chamber. This
chamber is optically flat. It can be opened either
by removal of the culture or, if access to the cul-
ture is desired without disturbing it, as for micro-
manipulation, the chamber can be opened from
below by sliding out the first cover glass. Because
of the cemented-on strips at the ends of the slides,
they can be stacked one on top of another or used
freely on the microscope platform without danger
of deranging the cultures. Pierced slides for this
purpose can be obtained for about 30 cents apiece
(Fig. 27).

A special burette for filling flasks has been
found useful. This will be discussed in the chap-
ter on the preparation of nutrients.

Implements. Scalpels, scissors, needles, for-
ceps, platinum loops, etc., are standard equipment.
For severing roots in the flasks, Bobbins uses a
long-handled scalpel made by spot-welding a chip
of razor blade to a wire held in a transfer loop
handle. A surgeon's nasal scissors of the Gruene-
wald or Heymann type is, for many types of dis-
section, still more satisfactory. It is of just the
right length to reach to the bottom of a 125 ml.
Erlenmeyer flask, does not require a wide space
for manipulation as do iridectomy scissors,
will cut cleanly even against considerable resis-

The Laboratory 83

tance (a point of importance in operating on
woody cultures), and is generally very efficient.
For working in very large flasks, cystoscopic
scissors may be desirable, but will be needed only
in special cases. Scalpels have, in general, been
found not very satisfactory (Fig. 28).

As has been pointed out elsewhere, it is not
desirable to have gas in the transfer room. Most
implements are best sterilized by boiling. This
may be done in a surgeon's sterilizing pan or on
an ordinary hot plate. A very satisfactory
method is to place a one-liter stainless steel beaker
of distilled water, heated with a Westinghouse
immersion heater such as is sold to traveling sales-
men for heating shaving water, on the operating
table. The instruments may be kept immersed in
this when not in actual use. In making transfers
usually two needles or loops are employed, one
being kept in the boiling water at all times. With
this precaution, bacterial or fungus contamina-
tions are almost never carried from one flask to
another (Figs. 29, 30).

For flaming flasks, tubes, or needles in special
cases as, for example, when it is necessary to pour
nutrient from one flask to another, an alcohol lamp
may be provided.

84 Plant Tissue Culture

Summary

Ideal facilities for plant tissue culture work
should include a media room for the preparation
of nutrients and equipment, an operating and
transfer room for the preparation of cultures, one
or more culture rooms for the maintenance of cul-
tures under accurately controlled and systemati-
cally manipulated environmental conditions, a
laboratory for the study and analysis of nutrients
and cultures and for chemical and physical tests,
and an office for the study and filing of experi-
mental records.

Little special equipment is required other than
that to be found in any good microbiological
laboratory.

The Lab orator//

85

Fig. 28. Implements commonly used in plant tissue culture
work. From left to right : spiral loop used for transferring large
callus cultures intact; platinum spatula for transferring small cul-
tures; loop for transferring small roots, callus cultures, and sus-
pensions of bacteria, yeasts, etc.; hooked wire used by Bobbins to
transfer small pieces of roots; loop used in transferring root seg-
ments a centimeter or more in length; two types of scalpels with
replaceable blades, suitable for dissecting callus cultures in situ
and two smaller scalpels with fixed blades ; a surgeon 's ear-curette
used for making initial isolations of procambial and other meriste-
matic tissues from stems, galls, etc. ; glass spatula used for grinding
tissues and making inoculations to leaves in the study of viruses in
plant tissues; a surgeon's ear-and-throat scissor of a length suitable
for cutting up root cultures in a 125-ml. Erlenmeyer flask. Above,
two dissecting needles suitable for fine dissections, and two types
of forceps.

86

Plant Tissue Culture

■j.

iJC

DC

if

CO

—

- 1 :.

The Laboratory

87

Fig. 30. Procedure employed in cutting roots within their flasks,
preparatory to transferring them to fresh nutrient.

Fig. 31. Distributing nutrient to flasks. The tray to the opera-
tor's left contains empty flasks which are filled from a semi-auto-
matic burette and transferred to the tray to the right, to be plugged,
capped, and sterilized.

88

Plant Tissue Culture

Fig. 32. Method of obtaining sterile adventitious roots from cut-
tings of tomato. Cuttings are suspended inside a glass cylinder
lined with constantly wot blotting paper until roots develop.

Pig. 33. Method id' excising adventitious roots from cuttings,

for inoculation into sterile nutrient.

Chapter V
NUTRIENTS

Unlike animal tissue cultures, plant tissue cul-
tures have from the beginning been carried out in
nutrients which are wholly or in major part syn-
thetic. This is, of course, of great practical as
well as theoretical importance. Nevertheless, it
has come about as a matter of necessity rather
than by deliberate intent. Animal tissue culture
nutrients are for the most part made up of com-
plex organic materials — embryo extract (Carrel,
1913, 190), blood plasma or serum (Harrison,
1907, 131; Erlichman, 1935, 147), peptic digests
of fibrin (Baker, 1933, 183; Baker and Carrel,
1926, 184, 185, 1928, 187-189; Carrel and Baker,
1926, 191 ; Ebeling, 1921, 192, 1924, 193 ; Fischer
and Demuth, 1928, 194), tissue broth and the like
with, it is true, salt mixtures such as Ringer's
(1886, 160), Tyrode's (1910, 161), Locke's (1895,
157-159), or other solutions (Drew, 1922, 145,
1924, 146 ; Lewis, M. R., 1916, 148 ; Lewis, M. R.,
and Lewis, W. H., 1911, 149-151, 1912, 152, 153;
Lewis, W. H., 1921, 154, 1923, 155, 1929, 156;
Vogelaar and Erlichman, 1933, 162, 1934, 163, 164,

Nutrients 91

1936, 235, 1939, 165), added, but in which these
synthetic adjuncts play only a secondary and ill-
defined role. Little is really known about the ex-
act constitution of such nutrients and, although
they are quite satisfactory for their primary pur-
pose of sustaining growth of isolated members,
their preparation is largely a matter of rule-of-
thumb. The successful use of such complexes by
the animal tissue culturists antedated by many
years the development of satisfactory plant tissue
culture media. Attempts were, of course, made to
utilize similar media for plant tissues but, as we
have already seen, these attempts all failed, with
the exception of the single unconfirmed piece of
work by Schmucker (1929, 110). This failure has
had as one consequence the forcing of investiga-
tion into a different channel and the slow, tedious
building up of a mass of information sufficient to
permit the designing of nutrients made up of well-
defined products of synthetic origin in place of the
ill-defined organic broths and juices.

Plant tissue culture nutrients contain four cate-
gories of materials. These are, first, water ; sec-
ond, inorganic salts; third, organic constituents;
and sometimes, fourth, coagulating agents.

Water. Since water makes up approximately
97 per cent of the mass of all nutrients, while some
other ingredients are required in amounts repre-

92 Plant Tissue Culture

sented by ten-thousandths of a per cent, the purity
of the water used is of prime importance and can-
not be overemphasized (White, 1932, 63, 1933,
141). ''Tap water" is, of course, worse than use-
less in any critical studies on nutrition. Many
laboratory supplies of "distilled water" are like-
wise worthless, because of significant amounts of
impurities of many sorts. These may range from
traces of lead from improperly soldered joints in
faucets to distillable petroleum-oil fractions com-
ing from the pumps used in wells or pumping
plants from which the ' * tap water ' ' is derived. In
order to insure a truly pure supply, water should
be first run through a softener and filter (see
Chapter III), then distilled at least twice, and
finally stored in vessels of hard glass or Pyrex.
Carboys can be covered with inverted beakers to
keep out dust or can be plugged with wads of clean
cotton enclosed in well-boiled and rinsed cheese-
cloth. Rubber stoppers should not be used, and
care must be taken that water does not come in
contact with cotton plugs.

Salts. Plant tissues require for satisfactory
growth at least eleven elements besides oxygen,
hydrogen, and carbon. These are calcium, potas-
sium, magnesium, nitrogen, sulfur, phosphorus,
iron, manganese, zinc, boron, and iodine. While
not essential, sodium and chlorine are to be con-

Nutrients 93

sidered as not undesirable additions. These ele-
ments are supplied in the form of inorganic salts
(White, 1933, 141, 1937, 142, 143, 1938, 144 ; Berthe-
lot, 1934, 136; Bobbins, V. B. White, et al., 1936,
139; Bonner and Addicott, 1937, 42). Because of
the extremely minute quantities of some of these
elements which are required and because of the
toxic effects which very small traces of other ele-
ments may exert, it is essential that the salts used
be of the greatest possible purity. For stock
nutrients, the "C.P. Grade" products of most
reliable manufacturers are satisfactory, but for
critical studies it may be necessary to prepare
specially purified salts. Methods of purification
for particular purposes, by recrystallization or by
other means, have been developed and are to be
found in the literature of plant nutrition (see
Arnon, 1938, 133 ; Anion and Stout, 1939, 134, 135 ;
Stout and Arnon, 1939, 140). Detailed procedures
for any given problem will have to be chosen with
the particular exigencies of that special problem
in mind. Procedures used in the preparation of
nutrients will be considered later.

Organic materials. The organic materials re-
quired for the nutrition of plant tissue cultures
fall into four main groups : carbohydrates, vita-
mins and hormones, amino-acids and other simple
nitrogenous substances, and organic complexes of
the tissue broth type.

94 Plant Tissue Culture

Carbohydrates. Carbohydrates make up from
0.5 per cent to 10 per cent of the mass of nutrients.
They serve two main purposes: as sources of
energy for the maintenance of the various living
processes in the growing tissues, and as osmotic
agents. Apparently the first of these, as energy
source, is their major role, for nutrient solutions
which have proved satisfactory for plant tissues,
in marked contrast to animal tissue culture nutri-
ents, are seldom isotonic with the tissues them-
selves. The osmotic value of the nutrient seldom
exceeds 2 atmospheres for a tissue having an
"osmotic value" of 5 to 10 atmospheres (Thiel-
man u. Berzin, 1927, 263). This discrepancy may
be more apparent than real, since the "osmotic
value" usually given for plant tissues is the
plasmolytic value and may be greatly in excess of
the true osmotic value of the protoplasm itself
(Bennet-Clark, Greenwood and Barker, 1936, 238;
Mason and Phillis, 1939, 446). That the major
role of the carbohydrate is nutritional rather than
osmotic is further indicated by the fact that spe-
cific carbohydrates, not carbohydrate per se, are
required. Glucose and fructose have been em-
ployed in some media, particularly those used for
the cultivation of tissues of monocotyledonous
plants, but with results which have not been en-
tirely satisfactory (Kotte, 1922, 51, 52; Robbins,

Nutrients 95

1922, 57 ; Robbins and Maneval, 1923, 59 ; Robbins
and V. B. White, 1937, 178; Malyschev, 1933, 53,
54; White, 1932, 63, 1940, 172, 173). Mannose,
galactose, etc., have also been tested. There is
some evidence that galactose may be quite toxic
(White, 1940, 173). This unsatisfactory behavior
of the monosaccharides may be due in part to the
difficulty in purifying them adequately (Robbins
and Bartley, 1937, 170; Robbins and Schmidt,
1939, 61). All commercial preparations of mono-
saccharides, even the best ' ' C.P. Grade, ' ' contain
varying amounts of impurities which may be in-
jurious. They are also relatively unstable to heat
and cannot be autoclaved with impunity (Thiel-
man, 1938, 171). Fortunately, the much more
easily purified and more stable disaccharide, su-
crose, is a completely satisfactory carbohydrate for
all plant tissues which have so far been grown suc-
cessfully (White, 1934, et seq., 65, 66, 78, 172, 173;
Fiedler, 1936, 45; Robbins and Bartley, 1937, 222;
Robbins and Schmidt, 1938, 60; Thielman, 1938,
171). Although maltose, brown sugar (Robbins
and Schmidt, 1938, 60, 1939, 224), and even dextrin
(White, 1940, 173) have given excellent results in
some experiments, their beneficial effects above
those of sucrose are to be attributed to non-car-
bohydrate impurities. There is to date no evi-
dence that any sugar other than sucrose needs

96 Plant Tissue Culture

to be employed. Sucrose crystallizes easily so
that even the commercial sugar to be bought in
every grocery store is usually as pure or purer
than most "C.P. Grade" chemicals, though per-
haps less reliable. The use of "C.P. Grade" is
to be recommended for all critical studies, as a
precaution. "C.P. Grade" sucrose usually con-
tains some calcium and magnesium, but the quan-
tities of these two elements required for normal
growth of plant tissues are so high that the traces
likely to come from the sugar are too small to be
of probable significance. Its content of copper
and zinc may possibly be significant. It does not
appear to contain significant quantities of any of
the vitamins known to be important for the activ-
ity of plant tissues. (Compare Ebeling, 1914,
271, 1936, 174; Lewis, 1922, 175.)

Vitamins and hormones. Four oligodynamic
organic substances have been reported to be im-
portant for the growth of excised plant tissues.
Fortunately, all four of these substances are avail-
able as synthetic products of high purity.

The only universally necessary member of this
group seems to be thiamin (vitamin Bx) (Bonner,
1937, 198, 199; Robbins and Bartley, 1937, 222,
223 ; White, 1937, 231 ; Gautheret, 1938, 69 ; Nobe-
court, 1938, 75). Thiamin or its precursors have
been shown to be essential for, or at least highly
beneficial to, the growth of a great variety of plant

Nutrients 97

parts. In some cases apparently either the thia-
zole portion of the thiamin (Robbins and Bartley,

1937, 223) or both thiazole and pyrimidine (Bon-
ner, 1938, 199, 1940, 201; Bonner and Buckman,

1938, 239) may be substituted for thiamin itself,
and there appears to be some evidence that the
thiazole can be replaced by certain complex sub-
stituted pyridines, but no plant tissues appear to
be able to maintain themselves for long without
some source of thiamin activity. In many types of
tissues, this requirement becomes evident in com-
plete failure of the cultures if thiamin or thiazole
is not included in the nutrient (Bonner, 1937, 198,

1938, 199; Robbins and Bartley, 1937, 222, 223;
White, 1937, 231, 1939, 182 ; Bonner and Devirian,

1939, 205). Here the tissues are clearly unable to
synthesize thiamin. In other cases, no growth
failure may occur in the absence of an external
source of thiamin, but thiamin may be recovered
in increasing quantities from cultures grown for
long periods, showing that the vitamin has been
synthesized by the tissues in quantities sufficient
to supply their growth requirements (Bonner and
Devirian, 1939, 205; Nobecourt, 1940, 252; Mc-
Clary, 1940, 248).

Nicotinic acid appears to be equally important
for a somewhat smaller number of plants such as
legumes (Bonner, 1938, 200; Addicott and Bonner,

98 Plant Tissue Culture

1938, 197 ; Bonner and Axtman, 1937, 203 ; Bonner
and Devirian, 1939, 205). These show a marked
failure in the absence of a significant external
source of nicotinic acid. Since no means of esti-
mating nicotinic acid concentrations in small quan-
tities of plant tissues is yet available, it is not yet
possible to determine if those plant members
which do not require an external source of nico-
tinic acid may synthesize it in quantities sufficient
to satisfy their needs. It is, therefore, not pos-
sible as yet to say if all plant tissues require nico-
tinic acid or not. It is only clear that most tissues
do not require an external source and that a few
tissues de require such a source.

Pyridoxine (vitamin B6) is also clearly bene-
ficial to many plant tissues. It has been reported
to be necessary for satisfactory growth of roots
of tomato (Robbins and Schmidt, 1939, 61, 224, 225 ;
Bobbins, 1940, 202 ; Day 1941, 208 ; Bonner, 1940,
201). The evidence in support of this report is
not conclusive and has been opposed by other evi-
dence (White, 1940, 232). Certainly many plant
tissues can dispense with all external sources of
this vitamin. As with nicotinic acid, there is no
evidence either for or against its synthesis by
plant tissues.

Indole acetic acid has been reported to benefit
the growth of roots of maize (Geiger-Huber, 1936,

Nutrients 99

214; Geiger-Huber u. Burlet, 1936, 215; Thielman
u. Pelece, 1940, 227; Duhamet, 1939, 209), callus
cultures from poplar and carrot (Gautheret, 1937,
211, 1939, 212, 1940, 213 ; Nobecourt, 1937, 74, 1938,
75, 1939, 77), and some other types of plant issue
cultures (Fiedler, 1938, 45; Thimann and Skoog,
1940, 454) . It has also been shown to be produced
by some roots (Thimann, 1936, 228; Nagao, 1936,
249, 1937, 250, 1938, 251 ; van Overbeek, 1939, 253
254 ; van Overbeek and Bonner, 1938, 255) . There
is no evidence that it is necessary for such growth
and considerable evidence to show that it may be
highly injurious. Ascorbic acid, biotin, and other
vitamins may likewise play an as yet undeter-
mined role in the development of plant tissues.
(Kogl u. Haagen-Smit, 1936, 358; Bonner and
Bonner, 1938, 204 ; Duhamet, 1939, 209).

Organic nitrogen. Amino-acids. Many plant
tissues require, in addition to water, salts, carbo-
hydrate, and thiamin or other vitamin, some
source of organic nitrogen. This may be supplied
in a variety of ways. Many plant tissues will
thrive on a nutrient in which this need is met by
the single amino-acid glycine (glycocoll) (White,
1939, 182). This is obtainable as a synthetic prod-
uct of high purity and is to be recommended wher-
ever its use will give satisfactory results. In
other cases, it may be necessary to supply a more

100 Plant Tissue Culture

complex source in the form of a mixture of a
number of amino-acids (White, 1935, 179, 1937,
181 ; Bonner and Addicott, 1937, 42) . In that case,
the synthetic acids should be used wherever pos-
sible, or where they are not available amino-acids
of the highest possible purity should be obtained.
This must be emphasized because amino-acids of
"natural" origin are likely to be themselves mix-
tures. Thus, even the best commercial samples of
leucine are known to contain considerable and
highly significant amounts of methionine (Mueller,
1935,447).

Organic complexes. All tissue culture media
contain accessory organic (non-carbohydrate) ma-
terial of some sort. The early nutrients included
yeast extract (Robbins, 1922, 177; White, 1932,
63, 1934, 65, 1937, 180 ; Fiedler, 1936, 45), Liebig's
beef extract (Kotte, 1922, 51, 52; White, 1932, 89),
peptones (Robbins, 1922, 177), fibrin digests
( White, 1932, 89 ) , etc. ( See also Haberlandt, 1902,
98 ; Loo and Loo, 1935, 216, 1936, 217) . The use of
thiamin, nicotinic acid, pyridoxine, the amino-
acids, etc., is the outcome of a long and pains-
taking study of these complexes and in most cases
permits us to dispense with the more complex
materials entirely. This has not always proved
true, however, and it is frequently desirable, when
a new tissue or a member taken from a previously

Nutrients 101

unstudied species of plant is under investigation,
to employ a complex source of organic adjuncts
and only gradually to approach the simpler media.
While peptone or fibrin proteose preparations
(Fischer and Demuth, 1928, 194; Baker, 1933, 183)
may sometimes be used with success, the complex
of most uniform usefulness is yeast extract.
Brewer's yeast is apparently superior to baker's
yeast and the well known "Brewer's Yeast — Har-
ris" has proved very satisfactory for this pur-
pose. The standard procedure has been to weigh
out 10 grams of dry "Brewer's Yeast — Harris"
into a liter of distilled water, boil for one-half
hour, then centrifuge, decant the supernatant,
make up to one liter, divide into 50 ml. aliquots in
Pyrex test tubes, and freeze at - 15° C. Ten ml.
of this concentrate is sufficient to make one liter
of complete nutrient (White, 1937, 180) (see
later). In the frozen state this will keep indefi-
nitely without deterioration. Fibrin digest may
be prepared in a similarly concentrated form by
use of pepsin or by bacterial digestion (Fischer
and Demuth, 1928, 194).

Coagulants. While most tissues grow satisfac-
torily in liquid media, there are some circum-
stances under which it is useful to provide a semi-
solid substratum (see Bobbins, 1922, 57; Kotte,
1922, 51, 52; trlehla, 1928, 115; White, 1933, 64,

102 Plant Tissue Culture

1939, 78, 333 ; Fiedler, 1936, 45 ; Gautheret, 1935,
15, 1937, 68). It is, of course, desirable to keep the
medium as nearly as possible of the same chemical
constitution as are the liquid media. Two com-
mon coagulants are available. Gelatin has been
used by some workers. This requires a concen-
tration of 10-20 per cent to maintain a satisfac-
tory consistency, which introduces not only a con-
siderable risk of chemical contamination but also
a marked adsorption factor. Agar, on the other
hand, will give satisfactory consistency at 0.75
per cent. Commercial agar is seldom free of
soluble contaminants and must be thoroughly
leached. Take 60 g. of finely shredded agar (the
powdered forms are likely to contain a contami-
nating "filler"), add 2 to 3 liters of distilled water,
and set aside in a cool room. Change the water 3
times daily for a week. After thorough leaching,
make up to the required volume with nutrient
solution, melt, filter through cotton, and distribute
to flasks or test tubes. These can then be auto-
claved and set aside to cool.

The preparation of nutrients. While there is
already a considerable number of nutrients in use
having slightly different constitutions, the pro-
cedures used in their preparation will scarcely
vary greatly in any important details. A stock
example can, therefore, be given which can be con-

Nutrients 103

sidered typical. The nutrient used in this labora-
tory for all routine cultures at present (1942) has
the following constitution:

Salts Mg. /liter Mols.

MgSO*

360.0

3x10-*

Ca(NOs)2

200.0

1.2 x 10s

Na2SO«

200.0

1.4 x 10^

KN03

80.0

8x10-*

KC1

65.0

9 x 10-4

NaH-PCX • H20

16.5

9X10"5

Fe2(S01)3

2.5

6xl0-«

MnS04

4.5

3.7 x 10-8

ZnSO,

1.5

9.4 x 10^

H3B03

1.5

1.2 x 10^

KI

0.75

4.5 x lO^7

Sucrose

2000.0

6 x 10-s

Glycine

3.0

4xl0-«

Nicotinic acid

0.5

4x10^

Pyridoxine

0.1

4.9 x 10"7

Thiamin

0.1

2.3 x 10 7

Osmotic value n - 1.8 atm.

Since this nutrient is used in great quantity and
must be made up at frequent intervals, it is not
efficient to weigh out the separate constituents for
each new set of nutrient. In practice only the
sucrose and the iron are weighed each time, the
first because any stock solution containing sucrose
would ferment unless sterilized and kept sterile,
the second because in solution Fe2(S04)3 rapidly
oxidizes and precipitates. All the other constitu-
ents are taken from concentrated stock solutions
which will keep indefinitely.

The inorganic stock solution is made up as fol-

104 Plant Tissue Culture

lows: 20 g. Ca(N03)2, 20 g. Na2S04, 6.5 g. KC1,
8 g. KN03, 1.65 g. NaH2P04, 0.45 g. MnS04, 0.15 g.
ZnS04, 0.15 g. H3B03, and 0.075 g. KI are dis-
solved together in 8 liters of distilled water. 36
g. of MgS04 are dissolved in 2 liters of distilled
water. The two solutions are then mixed slowly,
stirring constantly. Some insoluble precipitate
may form in the bottom of the bottle. The result-
ing solution is stored in a dark bottle to prevent
growth of algae. Ten liters of this solution are
sufficient to make 100 liters of nutrient, taking 100
ml. for each liter of nutrient required.

The stock solution of accessory organic mate-
rials is made up as follows: Three grams of
glycine, 500 mg. of nicotinic acid, 100 mg. of
pyridoxine, and 100 mg. of thiamin are dissolved
together in 1 liter of distilled water. This is
pipetted into Pyrex test tubes in 10 ml. aliquots
and stored in the icebox at - 5° C. A liter of this
stock will make 1000 liters of nutrient, taking 1

* For callus only.

**"Gelose" — presumably means agar, but is not so specified,
15, 72.

t Extract of 100 mg. dried brewer's yeast (Harris).

t Gautheret says merely : ' ' Knop solution, i strength, ' ' 72. Since
there are many different formulae in use for ' ' Knop solution, ' ' this
is indefinite. The concentrations given above correspond to a "i
strength solution" of the "dilute formula" given by Palladin, 449,
for use with young plants. The solution for mature plants is given
with a concentration 5 times that of the "dilute" solution and 10
times the concentration given in this table.

§ Gautheret says: "Take 10 drops of Berthelot's solution to each
liter of nutrient," 72. Assuming 20 drops = 1 ml., Berthelot's, 136,
formula would give approximately the concentrations shown in this
table.

Nutrients

105

TABLE 1

Nutrients commonly used for cultivation of plant tissues

White

Bob-
bins

Bonner

Gautheret

<B

d to
3 to

us

®4

.4

+3

«i

®*

00 -

■N

e*

©i

^3

■M

l>

*H

?>

So

.S

od

•i-t

3

Boots: tomato
flower, etc.
Callus: tobacc

oots: radish,
mustard, bu
etc., 66

o
03

s

o

+3

on
O

o

o

08

a

o

03

o
o

OS

of
s

CD

O

O

73
O

o

oots: wheat,
etc., 15

O

r— t
93

03

■4-3

o

M

03
«

00

03

fc

m

«

m

«

M

m

G

o

Grams per liter

Sucrose

Dextrose

Agar

Glycine

Yeast

Vit. B1

Cystein-HCl

I.A.A

Nic. acid

Vit. B6

Ca(N03)2 ...

CaS04

MgS04

KN03

KC1

KH2P04

Na2S04

NaH2P04

KI

Fe2(S04)s ...

Fe-tart

MnS04

ZnS04

H3BOa

NiCL.

CoCl2

Ti2(S04)3 ...

CuS04

G1S04

H2S04

20

7.5*

20

20 20 20 20

20
15**

20
15**

Milligrams per liter

3
0.1

0.5
0.1

200

360

80

65

200

16.5

0.75

2.5

4.5
1.5
1.5

lOOt
0.1

100

35

80

16.5

0.75

2.5

4.5
1.5
1.5

0.2

0.2
333

40
63
42
60

2.5

0.1
0.1

0.1

0.5
0.1

170

21

85
61
20

1.5

0.1

675

170

21

85
61
20

1.5

0.1

170

21
85
61
20

1.5

10

285*

72t"

72*
72*
72 1

20 1
0.4$

1

10

0.01

285*

72 *
72*
72*
72*

20 *
6.4$

20
13**

1

10

0.01

285*
0.2

72*
72*

72*
72*

20 *

0.4$

0.2$

0.02$

0.01$

0.01$

0.8$

0.01$

0.02$

0.0004$

106 Plant Tissue Culture

ml. for each liter of nutrient required. A com-
parison of this nutrient with a number of others
in common use is given in the accompanying table.

A convenient experimental series includes 200
cultures requiring 10 liters of nutrient. Each cul-
ture receives 50 ml. of solution. To prepare
nutrient for such a series, 200 g. of sucrose are
dissolved in 2 liters of double-distilled water. In
1 liter of water are dissolved 25 mg. of Fe2(S04)3-
These are mixed, 1 liter of stock salt solution and
10 ml. of glycine-thiamin solution are added, and
the whole is made up to 10 liters. This is then
distributed to the culture flasks in 50 ml. aliquots,
using a special semi-automatic measuring burette
(Fig. 31) which greatly facilitates rapid ma-
nipulation. For experimental nutrients (as dis-
tinct from stock nutrients) where each liter differs
from each other one in constitution, it is best to
use graduates instead of burettes for this distri-
bution. After all the flasks are correctly charged
with nutrient, they are plugged with cheesecloth-
covered non-absorbent cotton plugs, capped with
inverted 50 ml. beakers, and sterilized.

Sterilization. Most nutrients used for plant tis-
sue cultures contain sucrose and are unaffected by
autoclaving. The usual routine is to sterilize for
20 minutes at 18-20 lbs. pressure. It has been
found convenient to place flasks of nutrient as soon
as filled in galvanized iron trays 13" X 21" X 3£".

Nutrients 107

Each tray holds forty 125 ml. Erlenmeyer flasks
and three trays (120 flasks) can be sterilized at
once in a 20-inch autoclave. They can then be con-
veniently handled and stored without removing
from the trays. While autoclaving is satisfac-
tory for most nutrients likely to be used in plant
tissue culture work, those containing dextrose are
somewhat thermolabile and are better sterilized
either in the Arnold type of sterilizer or by filtra-
tion through Seitz filters or Berkefeld candles.
These methods are all so thoroughly standardized
in general microbiological practice that they need
not be described here.

Storage. Once sterilized, the stock nutrients
will keep indefinitely aside from the gradual con-
centration which results from evaporation. Be-
cause of this last factor, it is best not to use nutri-
ents that are more than a month old unless they
have been stored in a moist room.

Special nutrients. The details presented above
apply specifically to the standard nutrient used
for the maintenance of all stock cultures in this
laboratory. This standard nutrient is employed
in most nutrition experiments as a basis of com-
parison designated as the control. Experiments
involving the stimulatory or depressant effects of
increased concentrations of standard ingredients
or of materials not present in this nutrient can be
set up merely by adding the required materials

108

Plant Tissue Culture

Example 1

To study effects of various concentrations and combinations of
thiamin and vitamin Be

November, 1939

6

Final solution contains
in addition to salts and
sucrose, the following
per liter

Stock solution = 320 g. sucrose,
1600 ml. salt solution, 10 mg.
Fe2(S04)s in 8 liters H20

Glycine solution = 300 mg. in
1 liter H20

Thiamin solution = 100 mg. in
1 liter H20

Pyridoxine solution = 100 mg.
in 1 liter H20

Water

Milliliters per liter

1

2

3.0 mg. glycine

0 1 mg. thiamin (Ba)

500

< <

< «

< i

500

< i

( i

1 1

500

< i

1 1
t (

500

1 1

1 1
1 1

10

10

10

< i

1 1
« i

10

t i

1 1

1
1

1

< <

« <

1

« <

< <

< i

10

30

10

3

1

30

10

3

1

30

10

3

1

490
499

3
4

5
6

1.0 mg. pyridoxine (B„)

3.0 mg. glycine + 0.1 mg. B2*

+ 3.0 mg. B9

3 0 mg. + 1.0 " ' '

490
489

460
480

7

glycine + 0.3 ' ' " ..

487

g

+ 0.1 " "

489

9
10

+ 3.0 mg. Be

0.1 mg. +1.0 " "

469
489

11

thiamin + 0.3 ' ' ' '

496

12

+ 0.1 " "

498

13
14

3 mg. + 3.0 mg. B„

glycine + 1.0 " ' '

459
479

15

+ 0.1 mg. +0.3 " "

486

16

thiamin +0.1 " "

488

Control.

Nutrients

109

Example 2

To study effects of various concentrations of three iron salts

May, 1940

o

CO "

d

d a t3

2 ro

.

•rt S fl

60

II

CO -+3

•i|l.s

bi)'-1 00

a

o

d
o

•t-t

sq

11 O

o 8 ' r

II

oK

gw

©

© ° f^.2

O DO 0> a.

co a g

II 5 d

d o.S

CO

3 CD

■rt a» 2

"S o o fl
5 += ^ "*"
° d § _r *
50 o 55 .a .2

CD .rH _T* *"3

d TS w re l.
Eh re .ft 53 ft

o 9

■H+>B

S re -a

A 50 -fl

o . +»

m "3 &

M .8
oo >L

*" «5 rt

GGrH 60

o

CO n

OH

» — -*3

pHrH

Oi iH

« •
O bo

ga

°d

^a

W «0

u

CD

CO

]

VTillilite

rs per liter

1

500

500

2

0.6xl0-« MFe2(S04)3

< <

5

495

3

3.6 '

< < <

< <

17

483

4

6.0 «

< < <

< <

50

450

5

36.0 «

< <<

1 1

167

333

6

60.0 '

< < <

1 1

500

7

0.6 «

« Fe(N03)3 • 9H20

500

5

495

8

3.0 '

t t <

1 1

17

483

9

6.0 '

< < «

1 1

50

450

10

36.0 '

« < <

1 1

167

333

11

60.0 <

i i t

1 1

500

12

0.6 '

' FeCl3 • 6H20

500

5

495

13

3.6 «

i it

< t

17

483

14

6.0 '

( 1 1

1 1

50

450

15

36.0 <

t it

1 1

167

333

16

60.0 '

i 1 1

1 1

500

110

Plant Tissue Culture

Oi
CO
OS

^3

J.
si

s

■to
s

•«»

■»-

s
o

o

Si
3

•ia^Ai

53

Ol Ol O) O Oi
OO 00 05 O <35
CO GO CO CO CO

<3> -

OO ~
CO

IS

OOOOOOOOOOOOO
1—I1— 1 1— li— 11— 1 1— 1 1— li— It— li— 1 1— 1 1— t 1— 1

0 0

iH T-l

ja^X x ui 'Sra ggt

Eoa£H

OOOOOOOOOOOO
1— It— 1 1— 1 1— li— 1 1— 1 1-1 1— li— 1 1— 1 r- li— 1

000

rtrlH

aa^ix x ui 'Sui OSI

fosuz

OOOOOOOOOOO
1— 1 1— 1 1— (i— 1 1— 1 1— 1 1— li— li— 1 1— It— 1

OOOO

I— 1 TH T— 1 T— 1

ld%l\ x ui 'Sin 0^^

OOOOOOOOOO
1— IriHHrii- 1 i—l 1— 1 1— t i—l

OOOOO
1— ) rH 1— 1 1— 1 r-l

ja;il x ui 'Stn OSS

OOOOOOOOO
1— li— It— li— li— li— It— 1 1— It— 1

OOOOOO

T-l T— 1 T— 1 T— 1 T— 1 T— 1

J9;H x ui '3 SS'I
'OcFHS

OOOOOOOO
t— It— It— It— It— It— It— It— 1

OOOOOOO

T-l T— 1 I— 1 T— 1 T— 1 T-l T— 1

jo^iX x UT *3 S"9
IDS

OOOOOOO IOOOOOOO
t— It— It-It— It— It— It— 1 it— It— It-It— It— It— It— 1

EONM

OOOOOO

T-i T— 1 T— 1 I—l T— 1 T— 1

OOOOOOOO
t— Ii-Ht— It— It— It— It— It— 1

ja^ x UT '3 S'S

0 0 0 0 0
1-1 i-l iH r-l iH

OOOOOOOOOO
T— It— It— It— It-Hi— It— It— It— It— 1

aa^il x ui -3 oi
B(80tf)*Q

OOOO
rH t-l t-H 1— 1

OOOOOOOOOOO
tHlHt— li-Hi- lt-li— It— It-It— 1 1 — 1

"l"1 00T ui 'Slu 01

mUTBiqj,

i-H 1— 1 O if- li-Hr-li- IrHt-liHi-lt-li-i' ■ iH
t— 1

iom x ui -Sin 008
9UI0ix£)

00 :ooooooooooo :o

riH 1— It— li— It— It— It— It— It— li— It— It— 1 : rH

0*H 8J9*H 8 UT 's 0o 8
esoiong

O : O

B^OT^ UOI^n[OS l^UI^

♦

b

*-
c

y

o4

m

a
.=

>

.=

g

•2

E-

c

s

N
ft

a

T*

C

X

19

*

'c
■J.

1

re

PC

a

)

Glyc. and thiamin
KNOa and KCl ...

'ON

< —

03

w

Tt

ir

) tc

> t>

• oc

c

=

r*

e

x

t

>c to

Nutrients 111

either before or after sterilization. Experiments
involving omission or reduction in concentration
of the constituents of this nutrient itself (salts,
sugar, thiamin, etc.) require the setting up of
special nutrients. While this approach is familiar
to all physiologists and experienced workers may
have their own methods of preparing such nutri-
ents, it seems desirable to outline briefly the
simplified procedures developed here. Three ex-
amples will be given.

Each of the three sets of experiments outlined
above involves a comparison of 16 different solu-
tions, each provided in a quantity of one liter.
Since 50 ml. of nutrients are used for each culture
(see later), this provides 20 replications in each
nutrient. By employing condensed records of this
sort and simplified procedures, two men working
together can easily make up nutrient for 15 or 20
sets of cultures in a half day, which is about the
maximum that can be satisfactorily inoculated
and measured in a day. Sets of 16 have been
found most practical.

Nutrients containing special ingredients not dis-
cussed here may require special treatment which
the individual worker will have to adapt to his
particular needs.

Summary

Nutrients for plant tissue cultures are for the
most part made up of definitely known synthetic

112 Plant Tissue Culture

or highly purified ingredients. These include
salts of various kinds (chlorides, nitrates, sulfates,
phosphates, and iodides of calcium, magnesium,
potassium, sodium, iron, manganese, zinc, and
boron) ; carbohydrate (especially sucrose) ; or-
ganic nitrogenous materials (amino-acids, tissue
digest peptides, peptones, etc.) ; hormones and
vitamins (thiamin, nicotinic acid, vitamin B6,
indole acetic acid, etc.) ; and sometimes coagu-
lating agents (agar-agar, gelatine, etc.).

Standard procedures for the preparation of
nutrients are described.

Nutrients

L13

Pig. 34. At the top, a healthy excised tomato root at the end of
a one-week passage. In the second row a similar root lias been
divided into eight pieces to be used in setting up a stock of com-
parable root tips for experimental use. The ninth, apical piece was
discarded. Third row, eight root segments similar to those shown
above after a second week's cultivation in fresh nutrient. At the
bottom are 32 selected root tips, all derived under comparable con-
ditions from the single apex used as inoculum two passages earlier.

114

Plant Tissue Culture

Fig. 35. Bean pod segments used as test material for plant
wound stimulants. Above: segments from the centers of five
selected pods, each divided into six pieces so thai each piece con
tains tlic depression lefl by a single ovule. Center, left: ;i drop of
the solution under investigation is placed in each depression and the
series placed in ;i moisl chamber; right: :i similar scries 48 hours
later showing ;i small intumescence ;it the site of each drop of
liquid. Below, top row: sections through ;i control series treated
with distilled water; bottom row: ;i similar series treated with :i
solution of ' ' traumatin. " (From Bonner, J., and English, .1.
L938. Planl Physiol. 13: 335, fig. 1. •

Nutrients

115

PlG. 36. Removal of blocks containing undisturbed cambium
from large trees. The surface bark is shaved off, four deep in-
cisions made, well into the sap wood, and the block removed with a
chisel and transferred to a sterile Petri dish in which it can be
carried to the laboratory.

Fig. 37. In the laboratory the block is broken open carefully at
the cambium level and fragments of tissue removed for cultivation
on nutrient agar.

116

Plant Tissue Culture

Fig. 38. <','iinlii.-il culture of Salix caproea after four months in
vitro. (From Gautheret, l>*. J. l!»:'>7. La culture des tissus
vegetaux. Actualites scientifiques el industrielles 554, plate 2, 16.)

Chapter VI
HOW CULTURES ARE STARTED

As we have already seen, the two types of plant
material which have so far proved most useful in
plant tissue culture work have been, first, root
tips and, second, cambium or procambium. Most
of the following discussion will apply to these
types.

Seedling roots. Root tips are perhaps sub-
jected in nature to more environmental vicissi-
tudes than are any other types of meristems.
They are, therefore, relatively resistant to injury.
Nevertheless, once a root is growing either in the
soil or in a culture medium, if it is not already
aseptic it cannot be rendered so by any but rather
drastic means which may well influence its subse-
quent behavior. It is, therefore, important that
roots for culture be obtained wherever possible
from sterile seedlings and, moreover, it is highly
desirable that the seeds themselves be rendered
aseptic with as little chemical interference as pos-
sible in order that consideration of potential after-
effects need not enter into the interpretation of
results. In many cases, aseptic seeds can be ob-

118 Plant Tissue Culture

tained without any chemical treatment whatever.
This is true of all seeds which, when ripe, are en-
closed in fleshy, woody, or membranaceous fruits
(tomato, apple, squash, bean, tobacco, etc.).
Sound, clean fruits of these plants are opened by
breaking or tearing in such a way that no instru-
ment penetrates to the seed cavity. The seeds
themselves are then lifted out with sterile needles
or forceps, placed on slips of sterile paper in Petri
dishes, and allowed to dry under conditions which
will preclude the entry of contaminants from out-
side. These seeds can then be used for starting
cultures without further treatment (White, 1934,
65).

Seeds contained in very small fruits, in drupes
or achenes, or the seeds of Gymnosperms cannot
be obtained in a uniformly aseptic condition by
mechanical manipulation alone. These must
therefore be surface-sterilized. Where ultra-
violet light is available, this may sometimes serve
as a sterilizing agent, but it is rather uncertain
since spores may easily become lodged in crevices
in the seed surface which are protected from illu-
mination even if the seeds are constantly shaken.
The most satisfactory result is to be obtained with
bromine gas (Klein u. Kisser, 1924, 299; Burlet,
1936, 207). Seeds are shaken for 5 to 10 minutes
in a bottle containing a few ml. of 1 per cent

How Cultures Are Started 119

bromine-water. They are then spread out on
sterile filter paper in Petri dishes. The bromine
quickly penetrates all crevices in the seed coats,
killing most if not all contaminants, yet evaporates
completely leaving no traces to affect the seed-
lings. In spite of its obvious advantages, bromine
must be handled with great care, under a hood, so
that some workers prefer to use other reagents.
A solution of calcium hypochlorite containing 1
per cent chlorine will give fairly satisfactory re-
sults (Wilson, 1915, 455; White, 1932, 63) if seeds
are shaken therein for 15 to 30 minutes, then
washed in sterile distilled water and placed in
Petri dishes. The time required for complete
sterilization will vary considerably with the
species of seed, as will the amount of injury to the
seedling which usually results. Each species must
be studied specially. Dakin solution (Parker,
1938, 26) is also good. Mercuric chloride, 85 per
cent alcohol, "Uspulin," and other disinfectants
have been used by particular workers. The meth-
ods of using these reagents are standard biological
procedures and need not be presented in detail.

Once sterilized, the seeds may be placed for
germination either directly in flasks or tubes of
nutrient or in Petri dishes on filter paper mois-
tened with sterile water or on agar. If sown on
a semi-solid substratum such as agar, several

120 Plant Tissue Culture

seeds can be placed in each receptacle since any
contaminant which may be present on one seed
will not easily spread to another. If placed di-
rectly in a nutrient solution, only a single seed
should be put in each container. The flasks, Petri
dishes, or test tubes containing the seeds should
be set aside in darkness until the seedling roots
have attained a length of 10 to 15 mm. The roots
are then severed with scissors or scalpel at a
length of 3 mm. or more and transferred to the
nutrient in which they are to be grown.

Adventitious roots. While most cultures can
best be started from seedling roots, conditions
occasionally arise which cannot be fulfilled by such
materials. This has been notably true of certain
virus "cultures" in which no method of inocu-
lating virus into roots growing in vitro has yet
been found (White, 1934, 438). It is then neces-
sary to resort to adventitious roots coming from
cuttings of infected plants (see later). With
plants like tomato which root easily, this presents
no great difficulty. Cuttings are made from rap-
idly growing plants having a lush growth and the
leaves are removed, using a sharp clean scalpel.
The cuttings are then suspended in large flasks or
crocks containing a little water in the bottom and
lined with moist blotting paper so as to provide a
saturated atmosphere (Fig. 32). These are set in

How Cultures Are Started 121

darkness or in dim light until adventitious roots
emerge. The cuttings are removed in a sterile
room, the roots carefully severed and placed
one by one in flasks of nutrient. (Fig. 33). Using
this simple procedure, many roots will, of course,
be contaminated with molds or bacteria and
must be discarded. Some, however, will regu-
larly remain sterile. These can then be made to
serve as the origins for clones of sterile root cul-
tures each carrying the virus of the disease with
which the original cutting was infected. With
slower-rooting cuttings, greater precautions
against contamination will be necessary, and the
surfaces will have to be sterilized by some one of
the methods described for the sterilization of
seeds. Whatever sterilizing agent is used, it
must be removed by thorough washing with sterile
distilled water. The containers must also be ster-
ile and all manipulations must be carried out
aseptically. A high yield of sound aseptic root-
lets cannot be expected under these exacting con-
ditions, but they can be obtained with some
patience.

Cambium. Theoretically the cambium layer,
since it continues to grow throughout the life of
the plant, for hundreds and even thousands of
years in some cases, should be excellent material
for cultivation. Practically, however, it offers of

122 Plant Tissue Culture

all the major meristematic regions except the
medullary rays perhaps most difficulties in the
way of isolating fragments aseptically without too
great injury (White, 1931, 32). Most of the work
carried out to date on the cultivation of cambium
in vitro has been done by Gautheret (Gautheret,
1934, 67, 1935, 15, 1937, 68, 1938, 69, 70, 1939, 71,
72, 212, 1940, 73; Gioelli, 1937, 291, 1938, 292).

To isolate a block of cambium, all six faces of
the block must be cut, and this must be done with-
out carrying contaminating spores from the sur-
face of the plant onto the excised fragment. To
perform this operation on the cambium of small
woody stems would be almost impossible. It can,
however, be successfully carried out on large trees.
A piece of bark an inch or more square is cut out
with a sharp knife or chisel, being careful to cut
well into the sap wood (Fig. 36). Gautheret per-
formed this operation with carefully sterilized
instruments after surface-sterilizing the tree
trunk (Gautheret, 1935, 15), but these precautions
are not necessary at this stage. The block is
placed in a clean container and transferred to the
laboratory. There, under aseptic conditions, it is
split at the cambium level in such a way that
neither instruments nor possibly contaminated
tissue surfaces come in contact with the center of
the exposed surface. Pieces of cambium are then

How Cultures Are Started 123

carefully cut or torn out of this sterile surface
using sterile instruments and are transferred to
nutrient solution, preferably to a semi-solid sub-
stratum (Fig. 37). Such fragments should be
quite sterile. Cultures of this sort have been pre-
pared from willow, poplar, beech, oak, pine, and
other trees. Gautheret's most successful cultures
were of cambium from Salix capraea (Gautheret,
1935, 15, 1937, 68, 1938, 69 ) . The growth obtained
is of a loose, fungoid character and in general de-
velops rather slowly (Figs. 38, 39).

Procambial strands. The procedure to be used
in isolating procambial strands for cultivation is
somewhat similar to that employed with cambium
(White, 1939, 78). This tissue can, of course, be
most easily removed from stems of succulent indi-
viduals. Rapidly growing tips of tobacco, squash,
sunflower, and similar plants make excellent mate-
rial. Terminal pieces about 8 to 10 inches long
are severed and the leaves carefully removed with
a sharp scalpel or razor, leaving as little stub as
possible. Under aseptic conditions, the epidermis
of the distal 1 or 2 inches is stripped off, begin-
ning from the proximal end and taking care that
the exposed surfaces are not touched by unsterile
instruments or epidermis fragments. When the
terminal portion is completely exposed, the grow-
ing point is grasped with sterile forceps and bent

124 Plant Tissue Culture

sharply sidewise so that the stem breaks. The
break will take place in the region of active growth
where the cells possess a maximum of turgor with
a minimum of cell-wall deposit. Slices about 0.5
mm. thick are made across the stem in or near this
region and the pieces transferred to nutrient agar
or to very shallow layers of liquid nutrient. Pro-
liferation from very young tissues will take place
in all parts, while from older tissues only the re-
gion of the provascular strands will proliferate
(Fig. 17). Tubercles of white or green callus are
formed on the uppermost cut surface. It usually
requires 10 days or 2 weeks for these to attain a
sufficient size to justify further division. These
tubercles can then be separated from the underly-
ing non-proliferative tissue, or the culture can be
simply divided into a number of fragments con-
taining both growing and non-proliferative mate-
rial, and the pieces transferred to fresh nutrient.
Cultures from young carrot roots, potato tubers,
kohl-rabi, and other fleshy, only partially differ-
entiated members can be made in a similar fash-
ion. A similar technique is used in making cul-
tures from secondary crown-gall tumors (White
and Braun, 1941, 335, 1942, 336, 337).

Miscellaneous materials. The excision of inter-
calary meristems such as those of grasses requires
much the same technique as that for the procam-

How Cultures Are Started 125

bial strands. Medullary rays, the multiple peri-
cycle, and certain phelloderm cambiums should in
theory be fairly satisfactory experimental mate-
rial, albeit difficult to isolate. They have appar-
ently not been studied to date. There are in
plants, of course, many other regions which show
meristematic activity. Very young seed pri-
mordia are made up entirely of meristematic cells
and are easily removed with little danger of con-
tamination and a minimum of trauma (White,
1932, 89 ; Moebius, 1928, 105; Molliard, 1921, 106).
All of the tissues of young ovaries are likewise
meristematic as are some of those of more mature
ovaries (Tukey, 1937, 114). Wehnelt (1937, 230)
and subsequently Bonner and English (1938, 342)
have taken advantage of this fact and used tissue
from the inner surface of bean pods as material for
the study of proliferation. Bonner has had some
success with tissue cultures of this material (Bon-
ner, 1936, Pi) (Fig. 35). The meristematic centers
in the leaves of Begonia, Bryopliyllum (Naylor,
1931, 308),Saintpaulia (Naylor and Johnson, 1937,
309), Chlorophytum (Naylor and Sperry, 1938,
310), Camptosorus, etc., and the stipular gemmae
of Marattia should theoretically be excellent mate-
rial with which to work but have not been studied
as yet. Many "mature" regions may also be
stimulated to renewed activity by proximity to a

126 Plant Tissue Culture

wound (Haberlandt, 1913, 294, 1914, 295, 1919, 245,
296, 1921, 354, 1922, 355; Reiche, 1924, 369;
Wehnelt, 1937, 230; Bonner and English, 1938,
342; Sinnott and Bloch, 1941, 324) or by the in-
jection of such substances as the gall-inducing
secretions of certain insects (Kiister, 1911, 300).
Only the easiest types of materials have, up to the
present, been carefully studied. The field open to
future investigation is obviously very large.

Summary

Cultures may be started from seeds, from cut-
tings, or from excised fragments. In any case,
the final result must be a relatively uninjured,
aseptic mass of tissue, and the procedures for
obtaining such masses, either with or without the
intervention of chemical disinfectants, are of the
greatest importance. These procedures for ob-
taining aseptic seeds, adventitious roots, etc., and
for dissecting cambium and procambial tissues
are described and figured. Procedures for han-
dling other miscellaneous meristematic tissues are
also presented.

Hoir Cultures Are Started

127

Pig. 39. Two views of the surface of eambial cultures of Abies
pectinata after 8 days (below) and one month (above) in vitro.
(From Gautheret, E. J. 1935. Eecherches sur la culture des tissus
vegetaux. (These) 215, plate XV, 15.)

128

Plant Tissue Culture

Pig. 40. Bow root cultures are measured. The flask containing
the culture is held in the Lef1 hand. The righl hand holds a flexible
celluloid rule which is placed under the flask and is bent to a curva
ture approximating that of the root. With the zero poinl under the
base of the root, ;i very accurate estimate can be made of the root's
length. In expert hands the error of such measurements "will seldom
exceed 3 or l per cenl of the true length.

How Cultures Are Started

129

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130

Plant Tissue Culture

"> ^

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Ll

Fig. 42. Equipment used in making hanging-drop cultures and
other cultures of small masses of tissue. Top: wire rack for clear
'2'2 ■ l'l' mm. square cover glasses. .Middle row, from left to right:
1 X i! inch slides provided with three sizes of hollow-ground cavities;
concentrically grooved depression slide: heavy slide with deep flal
bottomed molded cavity; pierced slide i see Pig. '27); slide with
ylass ring (Y;in Tiegham cell); slide with removable, ground,
pierced plate, without and with suspended culture. Bottom vow,

left to right: small embryological watch glass with round bottomed,

molded cavity; Mnximow einbryological watch glass with tl;it pl.-ito-

glass bottom; two sizes of chemical watch glasses in Petri dishes,
used as moist chambers, as employed by Pell for tissue and organ

cultures.

Chapter VII
CULTURE TECHNIQUES

Tissue cultures, whether of plant or animal
materials, have for the most part been carried out
in three general classes of containers, each class
being especially adapted to a particular type of
problem. These are, first, cultures in flasks or
special vessels of one type or another adapted to
the study of relatively large masses of tissue
under conditions which involve considerable ex-
perimental manipulation (Carrel, 1912, 125, 1923,
126, 127; Maximow, 1925, 420; Fell, 1928, 392;
Bobbins, 1922, 57; White, 1934, 65; Bonner and
Addicott, 1937, 42; Parker, 1934, 427, 1936, 428,
429) ; second, cultures in test tubes, likewise
adapted to the study of masses of tissues but in
general less suitable to their manipulation (Kotte,
1922, 51, 52 ; Malyschev, 1932, 53, 54 ; Fiedler, 1936,
45; Gey, 1933, 129) ; and, third, hanging-drop cul-
tures adapted to the minute study and observation
of very small pieces of tissues (Harrison, 1907,
131 ; Burrows, 1910, 117 ; Carrel, 1911, 120 ; Lewis
and Lewis, 1911, 149 ; Chambers, 1923, 43, 1924,
44; White, 1932, 89). By far the most important

132 Plant Tissue Culture

of these categories in the preliminary attack upon
most problems is the first, since for most materials
it possesses, of all the methods of approach, the
greatest reliability and flexibility.

Bonner (1940, 201) carries root cultures in Petri
dishes, but these seem to the writer of this volume
to offer no significant advantages over flasks. Cer-
tainly they must be handled with much greater care
to avoid slopping, and would seem much more likely
to become contaminated. Fell (1928 et seq., 392-
401) uses, for animal tissue cultures, shallow ves-
sels of the "salt-cellar" or embryological watch
glass type placed in Petri dishes acting as moist
chambers. These may have distinct advantages
for cultures of small volume such as callus cul-
tures but cannot be used for roots.

The preparation of stocks. In any piece of
scientific work, one of the prime requisites if sig-
nificant results are to be obtained is an adequate
supply of uniform, duplicable material with which
to work. The tissue culture technique has in this
respect one great advantage over most other cul-
ture practices — that it permits the building up of
vegetatively derived clones, all members derived
by division of a single ancestral fragment so that
all members have exactly the same genetic consti-
tution. The question of genetic uniformity and
stability is entirely eliminated. One might expect

Culture Techniques 133

that under these circumstances all members of such
a clone could be counted on to behave exactly alike
under a given set of conditions. This is generally
true of animal tissue cultures (Ebeling, 1921,
456; Parker, 1932, 422, 423, 1938, 26) and cultures
of undifferentiated plant materials (White, 1939,
78). It does not, however, appear to be the case
with cultures of excised roots (Bobbins and Bart-
ley, 1937, 170; Bobbins and Schmidt, 1938, 60,
1939, 61; Bobbins and V. B. White, 1936, 62;
Delarge, 1938, 280, 1939, 281). Sister cultures,
placed under supposedly identical conditions, may
behave in markedly different manners. This is
one of the most disconcerting phenomena encoun-
tered in the field of plant tissue cultures. In
mycological and bacteriological studies, the inocu-
lation of two fragments of mycelium or two bac-
terial cells can be counted upon to give rise to
colonies of like appearance, aside from the rather
infrequent occurrence of saltations. Why is this
not equally true of root segments of higher plants?
The difference is probably more apparent than
real. When a fragment of fungus mycelium is
transplanted, it branches rapidly to form a discoid
or spherical colony, the outline of which is delim-
ited by many hundreds of branches, each of which
has its own particular increment rate. The col-
ony derived from a bacterial transplant or a frag-

134 Plant Tissue Culture

ment of animal tissue likewise consists of hun-
dreds of cells which behave as individuals. "What
we observe in measuring such a colony is the inte-
grated behavior of many individuals in which
whatever variations exist in the behavior of any
one individual disappear in the final average for
the colony. It is not at all certain that the individ-
uals in such a colony have behaved alike. In fol-
lowing the behavior of a root culture, on the other
hand, one is dealing with a single branch behaving
as an individual, so that the full measure of vari-
ability is immediately evident. It is only by taking
the average of a large number of such individual
cultures that one can get a picture truly comparable
to that of the single fungus, bacterial or fibroblast
colony. Since it is patently impossible to deal
with even hundreds of cultures at a time in abso-
lutely uniform conditions of environment, the indi-
vidual variability comes into unavoidable promi-
nence.

Part of this variability doubtless arises as a
result of slight and unrecognized differences in
the environmental conditions to which individual
flasks are subjected. Anyone who has studied, for
example, the differences in atmospheric humidity
in different parts of a single greenhouse bench
knows how difficult it is to maintain uniform con-
ditions even a few inches apart. Not all variables

Culture Techniques 135

are known. Such sources of error can be reduced
by careful study but can never be completely
eliminated.

Much of the variability, however, probably
arises from differences in age, state of nutrition,
degree of established dominance, extent of ex-
cision trauma, and similar characteristics of the
separate explants. The effects of these differ-
ences can be greatly reduced by careful selection
of the individual fragments used in each experi-
ment. The importance of adequate uniform
stocks from which to make this selection is thus
obvious.

It is clear, for example, that, if a rapidly grow-
ing root culture containing one terminal growing
point and 18 branches none of which has yet ac-
quired an individual dominance is divided into 19
pieces each containing a single apical meristem,
these pieces will be far from alike. The terminal
meristem will be of much greater diameter than
the others and of quite a different past history.
It will suffer far less from the shock of excision,
since its traumatized surface will be far less in
proportion to its volume than for the laterals. It
will recover more quickly and will show a much
greater growth in the first few days after excision.
Experience has shown that this difference can be
somewhat reduced by cutting the main root above

136 Plant Tissue Culture

and below each branch instead of severing the
branch itself, in such a way that a "heel" of older
tissue is left attached to each branch. The differ-
ence is still more reduced if only branches which
have already acquired their full individuality or
"dominance" are used. With tomato this domi-
nance usually develops after the branch has
reached a length of about 20 mm. If branches of
this sort are severed, they will generally show no
more retardation on excision than will apical
meristems and can be considered comparable
thereto. It is, therefore, advantageous in prepar-
ing a stock from which to select root tips, to have
as high a proportion of well established branches
as possible. Experience has shown that, if a
piece of old tissue carrying several branch initials
but from which the apex has been removed is cul-
tured for a time, a considerable proportion of
these initials will establish themselves with a com-
parable degree of dominance. Acquaintance with
these facts has given rise to a standard experi-
mental procedure.

Experience has shown that, in root cultures in
flasks, satisfactorily reliable results cannot be
counted on if a given experiment contains less
than 20 replications under each experimental con-
dition complex (see later). If, for example, it is
desired to compare four sets of conditions among

Culture Techniques 137

one another and with a control, the experiment
will require a minimum of 100 cultures. The
routine procedure is as follows :

Ten rapidly growing, uniform cultures of clonal
ancestry and each about 100 mm. long are selected.
The terminal segment of each to a length of 3-4
mm. is removed and discarded. The remainder is
divided into 10 to 15 mm. segments and each seg-
ment is placed in a flask of fresh nutrient and
allowed to grow for a week. Each will develop
from 3 or 4 to a dozen branches, and of these an
average of 4 or 5 on each piece will have estab-
lished comparable growth rates and full domi-
nance. There should thus be available 300 or more
uniform, well established root tips. These are care-
fully severed at a length of 10-15 mm. and from
them 100 tips of as uniform character as possible
are chosen for use in the actual experiment (Fig.
34). There is thus available a supply of tips
which are all root apexes, of uniform size, age,
nutrition, and degree of trauma, and which have
all come from comparable root pieces of clonal
relationship. The individual variability is thus
reduced to a minimum. This does not mean that
all cultures will behave exactly alike, even under
as nearly like conditions as we are able to set up —
there still remains a considerable residual vari-
ability that is as yet unexplained. But when a

138 Plant Tissue Culture

sufficient number of replications is provided, the
results can be counted on to be, under like condi-
tions, similar from one test to another and, after
properly correcting for regular seasonal differ-
ences (see later), they will give comparable results
from year to year and from season to season.

The setting up of stocks for all cultures involves
essentially the same procedure. The prime requi-
sites are a considerably greater amount of avail-
able material than is at all likely to be needed so
that a selection is possible, all material to be of
clonal origin. Callus cultures grow much more
slowly than do root cultures, so that a greater
time, usually about a month, must be allowed
between the setting up of stocks and their actual
utilization.

Inoculation. The procedure in making inocula-
tions is comparatively simple. The whole pro-
cedure is carried out in the transfer room. Stock
cultures from which tips or callus fragments are
to be taken are placed in a tray to the left of the
operator. The instruments are in the sterilizing
beaker or pan, in front of him (Fig. 30). Unin-
oculated flasks of nutrient are placed in trays to
his right and a box for discarded caps stands at
his left. The glass caps are removed from both
sets of flasks. A flask containing a stock culture
is grasped in the left hand, tilted to reduce the

Culture Techniques 139

horizontal exposure of the opening, and the cotton
plug is removed with the little finger and heel of
the right hand and retained. The operating scis-
sors or scalpel is taken out of the boiling water
with the right hand, and all satisfactory appearing
root tips or callus fragments on the stock culture
are severed and a mental note made of their num-
ber. The plug is replaced, the scissors returned
to the boiling water, and the flask returned to the
tray. Root pieces longer than 15 mm. become dif-
ficult to pick up. Pieces shorter than 10 mm. are
likely to suffer considerable shock from excision
and to respond poorly after transfer. While
fragments as small as 0.1 mm. in length have been
grown successfully (White, 1932, 89), experience
has shown that those from 10 to 15 mm. long offer
the most satisfactory balance between these vari-
ous characteristics. The process of excision is
repeated until all available tips have been severed.
Since a mental note has been made of the total
number of root tips severed and the number re-
quired for the experiment planned is known, it is
then possible to judge how drastically the avail-
able pieces can be culled and still leave a sufficient
number.

After all tips have been severed, the actual in-
oculation can be begun. A stock flask is grasped
in the left hand and an uninoculated one is placed

140 Plant Tissue Culture

on the table in front of the operator. The plug is
removed from the first, and a sound, rapidly grow-
ing root tip is selected and picked up on a long
chromel-wire loop. Usually two loops are em-
ployed alternately so that one is always in the
sterilizing beaker while the other one is in use.
The stock flask is plugged and set on the table, the
uninoculated flask is taken up in the left hand, its
plug removed with the right hand which still holds
the transfer loop, and the culture fragment is in-
serted, the flask plugged and returned to its tray.
The loop is returned to the boiling water and a
fresh one taken up. The process is then repeated
with another flask. In choosing tips for inocula-
tion, it will be noted that occasionally a root which
does not visibly differ from the others will never-
theless sink to the bottom of the flask instead of
floating at the surface. Such tips should not be
used, as experience has shown that they are regu-
larly retarded in their development. Such re-
tarded tips will sometimes recover completely, but
they cannot be relied on to do so. What the cause
of this condition is is not known, although it ap-
pears to be some obscure characteristic of the
general "state of health" of the stock culture
before excision.

The above description applies to the setting up
of root cultures. The procedure for callus cul-

Culture Techniques

141

tures is similar except that, for dividing the tissue
mass, a small, sharp scalpel gives better results
than do the scissors.

Growth. Under ideal conditions, root cultures
set up as described above will begin to grow almost
immediately and will increase 3 to 4 mm. in length
in the first 24 hours. (For methods of measuring
growth, see later.) This increment rate acceler-
ates up to about the 5th day when average rates
of 10 to 15 mm. per day are common (occasional
roots may grow as much as 80 mm. per day for
single 24-hour periods), after which the growth
rate again decreases rapidly (Fig. 43). This re-
tardation indicates that the 5th or 6th day is the

12 3 4 5 6 7

Days

Fig. 43. Typical increment rates of tomato roots (average of

1000 cultures) on each day of a single one-week passage. The

maximum increment rate is reached on the fifth day.

142 Plant Tissue Culture

best time for subculturing. A one-week culture
period has been chosen as best for routine work.
The average growth rate throughout the year, of
cultures grown in the laboratory without special
temperature control, is about 7 to 8 mm. per cul-
ture per day, being greater than this in summer
and less in winter (Fig. 26). Cultures begin to
branch on about the 3rd day and may have as
many as 30 branches by the end of a week. Some
of the decrease in linear increment after the 5th
day may be due to distribution of material into the
branches instead of its concentration in a single
axis. However, if, instead of using tips as inocula,
non-apical segments bearing branch primordia
such as are employed as stock cultures are used,
the branches, although at first somewhat retarded,
will when established increase at rates character-
istic for the particular species and for the nutrient
used, but independent of the number of branches
developing on the culture. This behavior makes
it clear that the rate of growth of a branch (and
presumably of the main axis as well) is not deter-
mined by the number of branches present and that
the retardation of growth rate after the 5th day
is not entirely due to diversion of nutrient mate-
rials to newly formed branches.

While this laboratory has found a one-week cul-
ture period to be best adapted to the problems

Culture Techniques 143

under consideration here, Bobbins and his col-
leagues have chosen a much longer period, usually
from a month to two months. This has certain
definite advantages as well as disadvantages.
Robbins uses dry-weight as a measure of growth.
By this method cultures are examined only at the
end of the passage, when they must be sacrificed
(see later). A two-month period thus requires
only an eighth as much work as do eight one-week
periods. The long period permits tissues to ma-
ture and might, in theory, give time for morpho-
logical changes to become evident that would
never appear in cultures maintained for repeated
short periods. (Compare Parker, 1936, 428, 429;
Fischer u. Parker, 1929, 411.) It permits the
accumulation of large masses of tissue so that the
percentage differential between the final size in
two different nutrients may be much greater than
after a shorter period. These appear to be ad-
vantages. On the other hand, the long period
permits the study of far fewer cultures in a given
space of time, tying up both laboratory space and
glassware. Contrary to expectation, morphologi-
cal changes which might be anticipated have not
actually appeared. Moreover, the large masses
of tissue accumulated in a long period may be
largely inert carbohydrate — cell-wall materials,
starch, etc. There is some evidence that such cul-

144 Plant Tissue Culture

tures may be built up by a recurrent cyclic re-
utilization of materials leached out of the older
inactive or even dead tissues. The increment
figures obtained at the end of a long period may
thus give no true measure of the level of meta-
bolic activity of the tissue involved. It is doubt-
ful whether the advantages of a long culture
period are not greatly outweighed by its disad-
vantages.

Culture conditions required. The requisite
characteristics of the nutrient have been discussed
elsewhere. Cultures are apparently for the most
part relatively insensitive to light and can be
placed either in darkness or in diffuse light
(Robbins and Maneval, 1924, 258; White, 1932, 63,
1937, 265; Malyschev, 1932, 53, 54; Gautheret,
1935, 15). They should never be exposed for long
periods to direct light as they are extremely sen-
sitive to high temperature and to temperature
changes and could easily become overheated
(White, 1932, 63, 1937, 265) . It is highly desirable
to have the temperature of the culture chamber
and transfer room rather accurately controlled.
Root tips of wheat have an optimum temperature
of about 27° (White, 1932, 63), tomato nearer 30°
(White, 1937, 265). The range between optimum
and maximum is, however, very narrow and a 5°
rise above these temperatures, if maintained for

Culture Techniques

145

very long, will kill the cultures (Fig. 44). It is
because of this extreme sensitivity to high tem-
peratures that cultures, when maintained at
"room temperature," fluctuate so much more
widely in growth rate in summer than in winter.
Cultures will, on the other hand, endure low tem-
peratures— down to + 5° C. — for long periods

o

Q_
Ld

tr

3
O

a.

I5h

10

o= Growth rates at the indicated temperatures.

x = Growth rates at 22', of cultures previously
held for one week at the indicated
temperatures.

x— x-x

10

20 30

DEGREES C.

Fig. 44. Average increment rates of isolated tomato roots main-
tained for one week at a series of controlled temperatures ranging
from 5° to 40° C. (open circles) and then all transferred for a sec-
ond week (crosses) to a temperature of about 22° C. All except
those maintained at 35° and 40° show an immediate reversion to the
growth rate typical for 22°. (From White, P. R. 1937. Plant
Physiol. 12 : 773, fig. 1, 266.)

146 Plant Tissue Culture

without apparent injury and, while growing very
slowly at such temperatures, will resume a normal
growth rate within a few days when removed to
higher temperatures (White, 1937, 266). Aside
from these two requirements, of maintained tem-
perature level and protection from direct insola-
tion, together with protection from traces of gases
or volatile materials, the environmental require-
ments of plant tissue cultures are not especially
exacting.

Flask cultures of the type described above have
great flexibility and permit considerable freedom
of manipulation. They can be handled easily and
do not readily become contaminated. They do,
however, occupy a good deal of space, the nutrient
evaporates rapidly with consequent alteration in
its solute concentration so that the cultures must be
transferred at frequent intervals, and they require
large volumes of nutrient. Conditions may some-
times arise under which these objectionable fac-
tors outweigh the difficulties of manipulation in
small space so that it is desirable to grow cultures
in test tubes, either on agar or in liquid nutrient.
The size of the tube to be used will depend on the
requirements of the particular experiment. Gey
uses a special 15 X 100 mm. tube for " roller tube"
cultivation of animal tissue cultures (Gey, 1933,
129; Gey and Gey, 1936, 130). This method has

Culture Techniques 147

not been tried with plant tissue cultures but de-
serves attention. Lewis uses ordinary 15 X 150
mm. test tubes for the same purpose (Lewis, 1935,
419). Callus cultures have sometimes been grown
in 2 ml. of nutrient in 12 X 100 mm. serum tubes,
but such cultures must, of course, be removed from
the tubes for any kind of manipulation, even for
division in order to make transfers. Tubes 25
X 150 mm. without lip and charged with 10 ml.
of nutrient, have proved to be a fairly satisfactory
compromise between the advantages and disad-
vantages of the test tube and the Erlenmeyer flask.
They are shallow enough and have wide enough
mouths that considerable dissection and other
manipulation can be practiced on the cultures
without removing them from the tube. The liquid
surface is still small enough in proportion to the
volume to reduce evaporation to a considerable
extent, yet the depth of liquid is small enough to
permit some degree of aeration without special
precautions. Such tubes are especially well
adapted for routine callus cultures on agar. Eoot
cultures in tubes must be watched to see that the
tips do not turn upward when the culture reaches
a length greater than the diameter of the tube.
Otherwise, with continued growth the tip may be
forced out into the air where it will no longer grow
satisfactorily.

148 Plant Tissue Culture

Flask cultures and tube cultures are suitable for
experiments in which gross increase and habit of
growth are the features of chief interest and in
which, when further details are required, they can
be obtained by removing the cultures from the
original containers where, for example, they are
to be fixed and sectioned. These are, in general,
characteristics of the initial phases in the develop-
ment of any field of investigation. But these
methods do not permit the observer to follow the
behavior of individual cells except under condi-
tions which entail a more or less serious disruption
of the continuity of growth. Plant tissue cells do
not migrate along a solid substratum (Chambers,
1923, 43 ; Pf eiffer, 1933, 450 ; Scheitterer, 1931, 109,
and Gautheret, 1937, 282, notwithstanding), since
the cellulose pellicle interferes with the adhesive
qualities of the cells. It is, therefore, not possible
to use flasks of the Carrel-flask type for micro-
scopic examination under growing conditions.
For this purpose, hanging-drop cultures (see
Parker, 1938, 26), in spite of numerous objection-
able characteristics, appear to be best. This type
of culture is second only to the flask type in im-
portance in carrying out plant tissue cultures.

In the typical hanging-drop culture a drop of
nutrient, either liquid or semi-solid, is placed on a
cover glass, the cover is inverted over some form

Culture Techniques 149

of depression slide so as to seal the drop into a
chamber which will reduce evaporation from the
drop, and the culture is then incubated. This
technique permits many modifications which have
been described in more or less detail in other man-
uals of microbiological methods.

Slides. A variety of slides have been developed
for use with hanging drops (Fig. 42). The sim-
plest of these are the hollow ground slides used
by the protozoologists. The curvature of these
depressions provides only a very poor optical
medium through which to illuminate cultures for
observation by transmitted light. Slides with a
deep hot-pressed well with "flat" bottom instead
of the ground depression are somewhat better in
this respect. Low ' ' Van Tiegham ' ' rings of glass,
or the brass rings used by Dr. and Mrs. Warren
Lewis (Parker, 1938, 26), cemented to ordinary
flat slides, provide still better optical properties
but are somewhat clumsy to handle. The pierced
slide described in Chapter IV (Fig. 27), in spite
of its fragility, offers the best, most easily handled,
most widely useful type of slide for hanging-drop
cultures. While the optical properties of this
slide are excellent, the curvature of the surface of
the drop may cause some trouble. This can be
obviated by floating a 5 X 5 mm. sq. piece of No. 0
cover on the surface of the drop over the culture.

150 Plant Tissue Culture

Capillary forces will hold this piece firmly in place
while the 2.5 mm. distance from center to margin
will still permit a free interchange of gases be-
tween the liquid and the air in the chamber.

Cover glasses. Standard No. 1 cover glasses
are usually used for hanging-drop cultures.
These should be thoroughly cleaned, preferably in
cleaning solution, rinsed, dipped in a 50-50 ether-
absolute alcohol mixture which is then burned off,
and stacked for use in a wire rack. Covers of
mica are frequently employed, but their optical
properties are seldom satisfactory and they some-
times contain soluble impurities which may be
injurious. All covers now available have objec-
tionable features. Cover glasses are usually made
of "soft" glass, are quite soluble, and tend to
rapidly alkalinize the nutrient drop. In animal
tissue cultures, where the optimal pH is about 7.6
and where the tendency of the tissue metabolism
is to render the solution more acid, this alkaliniz-
ing tendency of soft glass is not objectionable —
especially as animal tissue culture media are
mostly highly buffered. But with plant cultures
which are made in unbuffered solutions having an
optimal pH of about 5.4, and in which a reaction
above 6.0 may be very injurious, this tendency
may be quite serious in its effects. Pyrex covers,
if available, would obviate this difficulty, but these

Culture Techniques 151

have not yet been successfully manufactured.
Quartz covers are much too expensive for routine
work. Some progress has been made towards
solving this problem by dipping glass covers in
chemically and physiologically inert plastics such
as ethyl-methacrylate (du Pont), "Clarite," etc.
This method has given considerable promise, but
a reliable technique for obtaining duplicable re-
sults has not yet been perfected. At present, this
seems, however, to be the best lead.

Methods. Hanging-drop cultures, of course, re-
quire very minute amounts of both tissues and
nutrients. Methods for their preparation are
described in great detail in Parker's (1938, 26)
recent handbook. The nutrients can be sterilized
in either small flasks or test tubes. Sterile pi-
pettes with sterile rubber bulbs must be provided.
The material to be cultured is cut up into pieces
of the required size — root tips 0.5 mm. or less in
length (Chambers, 1923, 43, 1924, 44; White,
1932, 89), bits of callus, intercalary meristems
(Scheitterer, 1931, 109), seed primordia (White,
1932, 89), small bud centers (White, 1933, 116),
etc. — and is placed in sterile nutrient in watch
glasses which will be set at the operator's left.
Instruments, covers, and slides are placed in
front. The slides are first prepared with a small
drop of vaseline on each side of the depression

152 Plant Tissue Culture

or hole. A clean cover is grasped between the
thumb and middle finger of the left hand (the form
of the rack used facilitates this) and, while it is
held horizontal, a drop of nutrient solution is
placed on the middle of the cover. This is spread
around in a uniform thin layer about 8 mm. in
diameter. The fragment of tissue is then placed
in the center of the drop, the cover is inverted
with a quick, smooth motion so as not to throw off
the drop of liquid, and is pressed down over the
depression of a slide. The slide is set to the
operator's right and the process repeated. After
a sufficient series is completed, the covers are
ringed with hot paraffin or vaseline, using a small
brush, so as to seal them. They are then ready for
observation or incubation.

Hanging-drop cultures should be transferred to
fresh nutrient at least every 3rd or 4th day. If
regular depression slides are used, this will neces-
sitate removing the cover carrying the culture, a
process which requires great care (Parker, 1938,
26). If special pierced slides are used, it is only
necessary to slip out the lower cover, draw off the
nutrient with a clean pipette, replace it with a
fresh drop, and insert a clean cover in place of
the one removed.

Culture Techniques 153

Summary

Cultures are maintained either in flasks, tubes,
or hanging drops. Most of the preliminary stud-
ies, particularly on problems of nutrition, have
been carried out in Erlenmeyer flasks. These are
large enough to permit a good deal of manipula-
tion of cultures and to provide a large volume of
nutrient which is only slowly altered by the metab-
olism of the tissues. Test tubes require much less
space and nutrient but permit only a very limited
amount of manipulation. Neither method permits
microscopic examination of cultures. A technique
for such examination is provided in the various
sorts of hanging-drop cultures which are impor-
tant for this reason, although they require much
more work and do not provide the stable nutrient
conditions necessary for satisfactory study of
nutritional problems. Since each of these three
techniques has its own special advantages for par-
ticular types of problem, the best technique for
any given problem will have to be chosen with
these characteristics in mind.

Chapter VIII

GROWTH MEASUREMENTS AND THEIR
INTERPRETATION

The chief aims of plant tissue cultures are, of
course, first, the setting up of a group of condi-
tions which shall adequately duplicate those under
which the organ, tissue, or cell lives in nature, in
such a manner that its behavior therein is quanti-
tatively and qualitatively "normal," that is simi-
lar to what it would have been in its native
environment ; and, second, the study of the quanti-
tative and qualitative changes which take place
in its behavior when single elements of these con-
ditions are altered. This gives us a measure of
the importance and function of each character-
istic of the environment in the economy of the
organ, tissue, or cell under investigation, permits
us to sort out the significant from the merely for-
tuitous elements of behavior, thus simplifying our
picture of the real organism, and should lead to
a better understanding of behavior itself.

Qualitative differences cannot easily be repre-
sented except by descriptive — hence subjective —
means. These must inevitably fall short of the

Growth Measurements 155

ideal requirements of scientific presentation.
True science rests upon quantitative representa-
tions. And quantitative changes can be precisely
represented in terms of measurements. It is thus
natural that measurement should play a dominant
role in tissue culture studies and that the accuracy
and reliability of measurement should be a major
concern of the student of this subject.

Living organisms, particularly those which, like
most plant materials, are incapable of autonomous
movement, give evidence of their reactions to
changes in environment chiefly by changes in char-
acter or velocity of growth. And growth comes
into evidence chiefly through increase either in
mass or in complexity of some one or more parts
of the living material or its products. Increase
can be measured in a variety of ways, depending
on the nature of the material to be measured.

Methods. We are, of course, primarily inter-
ested in tissue cultures because they are living
materials. Increase of living material, as dis-
tinct from non-living secretions, is therefore of
the greatest importance. These living materials
are chiefly proteins. The carbohydrates and fats
and the nitrogenous materials of non-protein
character are largely constituents which can be
removed without destroying the organism's ca-
pacity to live. This is also true of some of the

156 Plant Tissue Culture

proteins themselves, but, if we set aside all con-
stituents of an organism except protein and fol-
low the increase or decrease in this one constitu-
ent, we have about as good a measure of the
increase or decrease in living substance under a
given set of conditions as is at present available.
This can be done by aliquot determinations of non-
soluble amino-nitrogen. Mueller recognized this
when he chose this criterion as the only acceptable
one in the measurement of growth of diphtheria
bacilli under the influence of different nutrient
complexes (Mueller, 1935, 176).

The actual measurement of amino-nitrogen,
while slow and tedious, is not especially difficult.
It could be applied to tissue culture practice as a
routine procedure, if desired. It has, however,
one very serious drawback which, together with
its tediousness, has prevented its coming into
common practice. This is the fact that it requires
the destruction of the material measured. In
studying living, growing organisms, this is a seri-
ous drawback, since it prevents the making of sub-
cultures from examples which prove to be espe-
cially favorable. The best that can be done is to
make such measurements on a series of aliquots,
using sufficient numbers so that the results will
truly represent the characteristics of the whole
series. This method easily becomes cumbersome
or unreliable, or both.

Growth Measurements 157

Increase in protein nitrogen is the best measure
of increase in living material available. Increase
in carbohydrate may also give a measure of the
activity of the protoplasm. It is, however, a cumu-
lative or catalytic process, since any given unit of
protoplasm may produce 1 or 10 or 100 units of
carbohydrate without itself necessarily either in-
creasing or decreasing in amount. Estimation of
dry weight has been frequently used as a measure
of increase of the total material in a culture. If
samples for such determination are taken when
the cultures are growing actively, this may give a
fairly accurate indication of protoplasmic activ-
ity. But, if they are taken in what we term the
"differentiating" stage in development, there may
easily be a rapid increase in dry weight along
with an actual decrease in living material. Dry
weight determinations are tedious and time-con-
suming, although less so than amino-nitrogen
determinations. They can be made with a fair
degree of accuracy, the error in good hands prob-
ably amounting to about 5 per cent plus or minus
the true value. This method of recording increase
has been used in plant tissue culture studies to
some extent, especially by Robbins and his col-
leagues (Robbins, 1922, 57, 177; Robbins and
Maneval, 1923, 59, 1924, 258; Robbins and Bartley,
1937, 170, 222, 223; Robbins and Schmidt, 1938,

158 Plant Tissue Culture

60, 1939, 61, 224), by Gautheret (1939, 285) and
by Malyschev (1932, 53, 54). Like ammo-nitro-
gen determinations, however, it involves destruc-
tion of the specimens studied, discontinuity of
observations, and the use of aliquots.

There is, however, a third method which does
not involve destruction of the tissue, permits con-
secutive measurements, and in practiced hands is
at least as accurate as is the measurement of dry
weights. That is the measurement of area in-
creases. In studying animal tissue cultures, it has
long been the practice to project the image of a
culture on paper at hourly, daily, or weekly inter-
vals, making tracings of the outline, to measure
the areas of these tracings with a planimeter, and
from the results to construct curves represent-
ing the growth increments (Ebeling, 1921, 456;
Parker, 1938, 26). It is recognized that this
method may err in making no distinction between
cell migration and cell increase and in taking no
account of changes in thickness of the cultures.
The first objection does not apply to the use of this
method for plant cultures, since no migration
takes place from plant tissues. The second objec-
tion is valid when the areas of callus cultures are
under investigation, since these ordinarily develop
as somewhat flattened hemispheres of tissue.

When applied to root cultures, however, a modi-

Growth Measurements 159

fication of this method, using linear rather than
area increments as a criterion of growth, has
furnished the most generally satisfactory mea-
sure of increase yet devised. When grown in
culture, roots do not undergo secondary thicken-
ing (White, 1934, 65). As a result, they ordinarily
take up a diameter characteristic of the species
and of the culture medium within a very few milli-
meters of the growing point and retain this diam-
eter throughout the length of even very old cul-
tures (White, 1932, 63, 1933, 64, 141, 1934, 65).
The cross sectional area is thus a constant and
can be ignored in comparing measurements, so
that length alone becomes an accurate measure of
volume and weight. Fiedler (1936, 45) has verfied
the fact that the weights and linear increments of
root cultures bear a constant relation to one
another, by parallel measurements and weighings
of a series of cultures of different length. This
statement, of course, does not apply to branches
at the time of or shortly after their initiation
(these always have a smaller diameter than do
fully established roots), but it is a well established
fact that once a branch has acquired its full indi-
viduality or "dominance" (discussed elsewhere)
it acquires this characteristic diameter. Measure-
ments which record only the linear increment of
the central axis will thus give an accurate record

160 Plant Tissue Culture

of the increase, so long as the culture does not
branch. If the branches formed are ignored, this
measurement will then be somewhat beloiv the true
value as measured by dry-weight or ammo-nitro-
gen methods, but will never, on the average, exceed
the true value. It will be in error in proportion to
the number and importance of the branches pres-
ent. Since normally growing roots seldom pro-
duce branches in great number or of great length,
the error introduced in this way will seldom be
great. Thus, for example, one series of 20 cultures
studied in June, 1939, in the control nutrient, had
a total linear increment of the main axes of 7449
mm. in a week's time. There were 492 branches
(an unusually large number) with a total length
of 2804 mm. Since the cross sectional area of the
branches would average about one-fifth that of the
main axes, the ratio volume of branches : volume

2804 560

of main axes would be about — ^— : 7450 = „.^q =

1:13 = 7.5%. Ignoring the branches thus intro-
duces an error of -7.5 per cent into the record.
Similarly, an experimental series in which potas-
sium was omitted from the nutrient (April, 1939)
gave a linear axial increment of 1654 mm., an
increment in branches of 214 mm., and an error of

9.14. 4.2

^F : 1654 = tSt = 1:38 = 2.6%. An error of
5 1654

- 2.6 per cent is less than that likely to be intro-

Growth Measurements 161

duced into dry weight measurements by hygro-
scopic absorption of moisture and is less than
errors likely to arise from inaccuracies of mea-
surements (see below), so that it is not important.
Moreover, this error is always a negative one, the
measurements will (aside from errors of manipu-
lation) always be minimal ones, with the result
that every experiment recorded in this way is
slightly weighted against the experimenter. This
has thus furnished a very satisfactory criterion
of growth.

The usefulness of any method depends, of
course, largely on its simplicity, accuracy, and
degree of reliability. The method used in making
measurements is extremely simple (Fig. 40). The
flask containing the culture to be measured is held
in the left hand. A flexible celluloid rule is used
to make the measurement. If the culture is very
short (under 30 mm.), it can be caused to float
against the glass so that both ends touch and the
ruler is laid alongside. For cultures more than
30 mm. long, particularly if they have curved
greatly, it is best to place the rule below the flask
and bend it to approximate the curve of the root.
One must be careful to look through the glass
downward (parallel to the straight component of
the sides) rather than sidewise (radially to the
curved component) so that the curvatures will not

162 Plant Tissue Culture

distort and enlarge the apparent image. By this
method, measurements can be made with sur-
prising accuracy. The accuracy of measurement
has been determined in two ways. In the first
place, a series of 10 roots varying in length from
18 to 110 mm. was measured. The flasks were
shuffled so that the order of measurements was
changed. The roots were remeasured and the
process repeated seven times. The measurements
were then subjected to statistical analysis. The
results are shown in Table 2. They indicate that
measurements made by an observer on a single
set of roots will vary from measurement to mea-
surement by somewhat less than 2 per cent but
give no indication of the deviation from the true
length.

In the second place, a series of 20 cultures was
measured in the routine way by two different ob-
servers and the results recorded. The roots were
then removed from the nutrient, placed in a vessel
of water, and stretched out along a ruler in such
a way that an accurate measure of the true length
could be obtained. These last measurements were
then compared with the measurements of the two
observers made by the standard method, giving a
basis for expressing the average error of the
method in the hands of these observers. (Ob-
server A was an experienced worker; observer B

Growth Measurements

163

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164 Plant Tissue Culture

had much less experience with the method.) The
results were :

Root Tru* A's B's A, B,

length measure- measure- errQr errQr

mm. ments ments

1

129

135

115

+ 6

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2

158

165

135

+ 7

-23

3

37

38

28

+ 1

- 9

4

175

188

150

+ 13

-25

5

140

144

130

+ 4

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6

58

58

56

0

- 2

7

71

75

68

+ 4

- 3

8

173

182

150

+ 9

-23

9

178

184

165

+ 6

-13

10

162

170

135

+ 8

-32

11

161

172

145

+ 11

-16

12

162

174

150

+ 12

-12

13

147

157

130

+ 10

-17

14

156

167

137

+ 11

-19

15

154

170

150

+ 16

- 4

16

170

182

160

+ 12

-10

17

168

180

150

+ 12

-18

18

66

70

70

+ 4

- 4

19

114

118

115

+ 4

- 1

20

141

148

122

+ 7

-19

Average 131 144 123 + 7.9 -13.3

Error as per cent of true value = +6.0 - 10.2

In this series the experienced observer tended
to overestimate the length of cultures by about
6 per cent, which is just about enough to compen-
sate for the systematic error of - 7.5 per cent due
to the presence of unmeasured branches (see
above), while the less experienced observer tended
to underestimate the lengths by a considerably
greater margin. In experienced hands, this
method will give results of an accuracy and relia-

Growth Measurements 165

bility very nearly equal to that obtainable in
making dry weight determinations and has the
great advantage that it can be practiced rapidly
and without removing the cultures from their
flasks. Repeated measurements can be made on
each growing culture at any desired interval,
growth curves can be set up, and subcultures can
be made from those roots which show, over an
extended period, any desired type of behavior.

Replications. As we have already seen, root
and callus cultures, since they are biological mate-
rials, show a considerable degree of variability.
In the series presented in the last table, the lengths
at the end of a week in the control nutrient varied
between 37 and 178 mm., with an average of 131
mm. This is fairly typical of root cultures. It is
obvious that materials of this degree of variability
cannot be treated as individuals but must be
treated statistically. This being the case, it is
important to decide first how many replications
are necessary in order that significant results can
be obtained and, second, how wide a difference
must a given experimental variable produce in a
series of the chosen number of cultures before that
effect can be considered as being due to this vari-
able and not to chance. Decision on the first point
involves a compromise between the ideal of an
infinite number on which to base averages and the

166 Plant Tissue Culture

number which can be practicably handled in a
laboratory with restricted space and facilities and
limited time. This laboratory has chosen 20 as
the standard number of cultures used in setting
up averages, and each experiment is generally re-
peated several times. The second point can be
settled by a statistical analysis of actual results
using this standard number. Some examples are
given in Table 3.

In this group of experiments, in each of which
20 cultures grown under one set of conditions are
compared with 20 cultures grown either under a
different set of conditions or else under the same
conditions but on different dates, differences up
to 12 per cent between the two series (Nos. 1, 2,
3, 4, and 10) are without statistical significance;
differences of 24 and 25 per cent (Nos. 5 and 8)
are of doubtful significance (odds of less than
20 : 1 against the results being due to chance) ;
while differences of 31 per cent and over (Nos. 6,
7, and 9) are highly significant (odds of more than
20:1 against the results being due to chance).
This analysis indicates that in experiments car-
ried out in this way and using 20 replications in
each experimental complex differences of 30 per
cent and over can, in general, be relied on to indi-
cate a true difference in behavior and not merely
fortuitous differences due to the variability of the

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168 Plant Tissue Culture

material. This, then, has been used as a basis in
interpreting results of experiments of this sort.
Records. Each worker will have his own prefer-
ences as to records, which he will adapt to his own
particular problems. This laboratory's records,
as regards root cultures, may be worth presenting.
All records are kept in 5 X 7£" notebooks contain-
ing about 120 pages, cross-ruled at \" intervals.
This book is of a size that will slip into a coat
pocket (Fig. 41). The rulings permit the organi-
zation of the page into both horizontal and vertical
columns and are close enough together so that a
great deal of information can be placed on a single
page. Three sample pages will suffice to indicate
the ways in which such pages can be organized.
The three sample pages indicate clearly the way
in which routine numerical records are kept. In
addition, photographs are taken of typical roots
in each important experiment chosen to represent
as nearly as possible the average length and
growth habit of those grown in each solution or
environmental complex. Thus, in Example 2,
root No. 136 was set aside for photographing since
it most nearly approximated the average of the
group. The actual figures were:

Average increment = 24.8 mm.

No. 136 = 42—18 = 24 mm.

Growth Measurements 169

Average number of branches = 2.3

No. 136 = 2

Average length of longest branch = 5 mm.

No. 136 = 5 mm.

From these records and from any qualitative notes
which may be taken, a picture of the effects of any
particular experimental variable can be built up.
Presentation of results. It is seldom if ever
necessary or desirable to present the numerical
data in an experiment in toto. The results can
usually be presented better in the form of either
histograms where only average results for total
passages are desired or curves where temporal
trends are to be given. Where a number of simul-
taneous experiments are to be compared, the re-
sults can be presented in absolute units — mm. or
mg. or per cent nitrogen. Where experiments
carried out on different tissues or at different
times, particularly at different times of year, are
to be compared, this is not possible, since the ef-
fects of varietal differences or of uncontrolled
seasonal variables in the environment may have
as great or greater effects on growth rates than
do the experimental variables. Since all cultures
react on the average in approximately the same
way and to the same extent to most variables in
the environment, this difficulty can be obviated by
setting up a control in each series, treating this
control always as the norm, and comparing all

170

Plant Tissue Culture

Example 1

Stock

cultures

51

Passag

s 326

1939

Passage

327

No.

Derived
from*

July
25

August
1

No.

Derived
from

Aug.
1

August
8

1

1

15t

68 4- 9-13J

53$

1

1

17

85 4- 6-18

68

2

2

16

90 + 11- 6

74

2

2

18

83 4- 4-10

65

3

«

16

53 +5

37

3

4

16

M ||

0

4

4

19

108 4-19-30

89

4

a

19

90 + 11-16

71

5

5

17

134 4-24- 8

117

5

5

17

85 + 10- 8

68

6

«

16

105 4-15- 9

89

6

6

17

105 + 20- 5

88

7

8

17

25 4- 6-60

60

7

7

16

50+ 6-37

37

8

9

18

66 4- 8-52

52

8

u

15

75 + 10-18

60

9

10

16

98 4-15- 8

82

9

«

15

52+ 7-32

37

10

u

19

127 4-20-13

108

10

u

14

45+ 3-10

31

11

11

17

95 4-13-23

78

11

8

16

115 + 16- 7

99

12

12

16

85 4- 3- 7

69

12

u

17

95+ 2- 5

78

13

M

16

110 4-16-12

94

13

«

16

70 M ||

54

14

«

14

85 4-10- 8

71

14

u

16

67+ 6-25

51

15

(I

15

88 4- 6- 3

73

15

9

18

51+ 4-15M||

33

16

14

18

115 4-24-11

97

16

10

18

136 + 20-20

118

17

16

18

108 4-27- 8

90

17

11

15

86 + 20-16

71

18

18

17

50 4- 7-14

33

18

12

19

110 + 10- 5

91

19

19

16

4-5-43

43

19

13

18

145 + 12-20

122

20

20

16

33 4- 4- 7

17

20

14

19

129 + 20-16

110

21

tt

15

33 4- 5-10

18

21

15

18

132 + 22-19

114

22

21

17

87 4- 9-26

70

22

16

17

75 + 15-20

58

23

22

17

20 S ||

3

23

17

18

65

47

24

23

17

72 4-10-29

55

24

19

17

75+ 8-22

58

25

24

16

107 4- 8- 6

91

25

22

17

87 + 15-17

20

T

otal inci

ement

= 1663 mm.

1704 mm.

!ean inci
otes:

./cult./d

ay = 9.5 mm.

9.7 mm.

* A record is kept of the genealogy of each culture. Thus, Nos. 2 and 3 of passage
326 were derived from No. 2 of passage 325, Nos. 11, 12, 13, and 14 of passage 327 were
taken as tips of branches of No. 8 of passage 326, etc. Thus, the history of any culture
can be traced at will.

t The length of each culture is recorded as soon after transfer as possible.

t At the end of the passage (one week), the length of the main axis, the number of
brandies, and the length of the longest branch are recorded. Thus, 68 + 9-13 means
that the culture had attained a total length of 68 mm. and had formed 9 branches the
longest of which was 13 mm. long. 53 + 5 indicates a total length of 53 mm. with only
a single branch 5 mm. long. +5-43 indicates that the main axis had not grown at all
but that there were 5 brandies the longest of which was 43 mm. long. In this case the
length of the longest branch is used in calculating increments.

§ These figures represent tin' increase during a week's growth, that is, the difference
between 15 mm. initial length and 68 mm. final length.

|| "M" indicates contamination with mold, "B" contamination with bacteria, and
"S ' ' indicates that the culture had sunk to the bottom of the flask.

Growth Measurements

171

cm

CO
<M

cm

co
bo

t8
in

73
(33
PM

T3
M
cS

T3
d
eS

in

O

o

^3
CO

o

d
Ti

CO

d

o

(4

d

«

d
o
e

a

d

•l-H

I — I
S3

O

o

15

OS

CO
O

00

<o

lO

■*

10IOOMrtOOOHH«SrtHN«^NHO«

CO
I'

t» OS t- 00

co m NMNioH^

1 1

cq CO

1

CO

+ +

+

CO 00

CM

+

| I I I I

CO CO ■* *0 CO CO

+ + + + + +
CO tJH 00 00 00 <M
CO CO ■* CO ■* ■*

I

CO

CO

CO

^ffit»MWIOO(SCqHMNHHNW«Nt-M

I I I I I

CO CO CO CO CM lO

++++++

CO CO ■* OS CO t>

ic irst-coojiooscoeo
I I I I I I I I I

-*-tiexico',!*lirac?cic\i coco
++++++++ ++

00 CO iH i-H CO t- CO CO 00 "* CO t£

«33gSS SSSSS^SSSw coco^

inCOO^NCOCOOCOr-ICOeoeO^^^eoeOCOCO

in co co io io
II II I

CXI CM CO CO IO CO

+ + + + + +

TjH t-

■* CO

CO O CM CO

CO CO "* TjH

IC K3t>MNIOtOMN
I | I I I I I I I

Tt<-^CM in CO CO CO CO CO

+ + + + + + + + +

th oo wtuntoNaH

■<*l <M

CO ■* CO CO CO CO ""#

lnffiHin©TfooMHCDt-t:o>2,»»l>ir5S
cococoiom w tjh comco ^ ^ co cm

,1111 I I III I I I I

CO CO CO CO CO W CO COTJH CO ^ *0 CO CO CO CO

+ + + + + + + ++ + + + + +± +

•apura piooaa o_m -lupung

a a

CO
CO

o

CO

©

CO

"V,

co

t-<

d

o

II

O

o

in

CM

a

d

o

a

in
CO

in^koiocMTH»OTtHinco^ioiCTtH-*<Micio>n^

sssssssss^ssggsssjsss

00
00

CMTHCO^CO^CD^^iOCvlCMlOCOCOCOTtHCMCMlO

-sssssssssssssssssss

o

_,^-,^.rf(in!OKCCffiOH(MC<3^K)CONCCiaO

o

d

o

°i r-i

"*

CJD

fl

00 d

-* C3

-<J

d

. ©

►»«

c3 ,

^"S

>.s

»H !h

d -tJ

-u d

3 a

\CB

. -u

^. a^

-a£

oo o

• o

tH

<M p,

11 -M

■+J -^

.^ d d

"J © q)

co g a

05 CO

^H J-l

O O

pi d

• r-t .rH

. I 1 l 1

t~t 71 7t

O -^ -l^>

d o o

.H+»+'

(I) CO CO

60 &D CJO

r: .. -"t

»H M >h

CO CO CO

> t» >

«<

172

Plant Tissue Culture

Example 3

Koot-pressures balanced against compressed air of measured

■nrp.ssnrp. Vfl.lllAfl

25

pressure values

Set up June 15, 1937

Date

Days

Time

Hours

Height

Remarks

June

15

0

14: 00

0

75

16: 00

2'

75

16

1

8: 30

18'30"

100

12: 00

22'

106

16: 00

26'

117

1 atm. pressure applied

16: 10

26'10"

120

o < < tt i '

16: 40

26'40"

120

17

2

8: 30

42'30"

148

Q C t ( ( ( <

11: 00

45'

164

4 < < < < <'

12: 00

46'

164

13:30

47'30"

176

5 " " "

15: 30

49'30"

172*

c 1 1 a i (

17: 30

51'30"

172

18

3

8: 30

66'30"

176

Pressure removed

11: 30

69'

197

* Balance between ex-

12: 00

70'

210

ternal and internal

15: 00

73'

234

pressure (or leakage

17: 00

75'

244

point) was reached

19

4

8: 30

90'30"

360

in this experiment at

12: 00

94'

398

5 atm. or 75 lbs. per

20

5

sq. inch.

21

6

9: 00

139'

604

Top of manometer
reached

other cultures within the series with the control,
on a percentage basis. The percentage results
can then be compared from one experiment to
another, which could not be done with the absolute
values. Thus, in two series (White, 1940, 232) on
the effects of vitamin B<$ on two different strains
of roots, one strain in an experiment using 1.0
ppm. vitamin B6 as the only organic accessory

Growth Measurements 173

substance and completed on July 13, 1939, gave a
numerical index of 58.6 mm., the other strain, in a
similar experiment completed on December 7,
1939, gave a numerical index of only 21.8 mm.,
but the percentage values for the two were 60 and
62, respectively. While the use of percentages
may be objected to on the grounds that it involves
a subjective choice of a standard, it is the only
way in which non-simultaneous experiments can
be compared and has proved very satisfactory as
a method of presenting results. Three examples
of graphical representations will serve to show
the types found most useful (Figs. 45^7).

Interpretation of results. While the results
themselves must, wherever possible, be presented
in numerical form with a definite idea of the de-
gree of accuracy and significance which can be
attached to them, the interpretation of results is
the duty of the observer. It is necessary for him
to take into account not merely the numerical data
but also many qualitative features of the results
which cannot be set down in numerical form. In-
terpretation involves the integration of many
sorts of information which only the person who
actually handles the cultures can have available.
How this is to be done is a matter of personal
inclination and cannot be presented in any hand-
book. Yet it is in this, just as much as in the

174

Plant Tissue Culture

16

14

o = Control with yeast
- A=5-amino-acid mixture
» = Control wi hout yeast

<

Q

<r
u
o.

u

3

D
O

a:

u
a.

PASSAGES
! I 2

0 b 10 15

DAYS
Fig. 45. A line graph recording the average daily increment
rates of three series of cultures, 20 roots in each series, maintained
for 14 days (two passages), each in a different nutrient.

Growth Measurements

175

Controls
Fig. 46. A histogram recording the average weekly increment
rates of 6 sets of cultures grown in as many different nutrients
through 5 successive passages, each of one week's duration. The
results are expressed as percentage of the increment rate in the con-
trol nutrient containing salts, carbohydrate, and yeast extract.
Each vertical column represents the average of 20 cultures for a
single week. The solid dots connected by solid lines represent the
averages for all five passages (5 weeks) in each solution. (From
White, P. R. 1939. Plant Physiol. 14: 531, fig. 4, 182.)

176

Plant Tissue Culture

5 ,_ *> >

ro

<o

o e

20:0

1980.2 100: 1 -

Osmotic values in atmospheres

0,45 1,5 4.5

1_>

19.4: 0.6 30: 1

>- 182 10:1

15:5 3:1 -

FlG. 47. An isopleth diagram (three-dimensional graph) show-
ing the approximate average increment rates (indicated by isopleth
lines) obtained when tomato roots were grown in solutions in which
the concentrations of sucrose and of sodium chloride were simul-
taneously varied in a regular way so as to obtain a graded series of
ratios (indicated on the ordinates) and a simultaneous graded series
of osmotic values (indicated on the abscissae). This type of dia-
gram, which may also be built into the form of a solid graph like
a relief map, is extremely useful when it is desired to record the
effects of the interaction of two simultaneous variables. (From
White, P. R. 1942. Plant Physiol. 17 : 159, fig. 5, 37.)

Growth Measurements 177

recording of data, that the scientist shows his true
calibre. It does not seem amiss, therefore, to
emphasize the importance as well as the difficulty
of this phase of any experimental project.

Summary

The significance of any piece of scientific work
rests primarily on the ability to express results
in quantitative terms and the reliability of these
terms themselves. The measurement of cultures
and the interpretation and evaluation of such mea-
surements are, thus, matters of prime importance.
Plant tissue cultures may be measured by deter-
mining protein nitrogen, dry weight, respiration
intensity, area, volume, and doubtless other char-
acteristics. The first three methods require the
destruction of the tissue studied. With callus
cultures none of these methods is fully satisfac-
tory and a subjective criterion can scarcely be
avoided. With root cultures linear increment has
been found to be a satisfactory criterion of growth
which is easily and accurately obtained, yet does
not involve destruction of the tissue studied.
Measurement of the central axis alone, ignoring
all branches, is in error by about 5 to 8 per cent
as compared to dry weights. Linear measure-
ments can be made with a mean error of 6 to 8 per
cent, an accuracy comparable to that obtainable

178 Plant Tissue Culture

in making dry weight determinations. In order
to reduce the random error, each experiment
should include at least 20 replications in each
environmental complex, and with this number a
difference of at least 30 per cent of the control
value must be produced by any given variable
before that variable can safely be assumed to be
responsible for the difference. Records are kept
in standard notebooks of a size to be slipped into
the coat pocket. Final results may be presented
as tables, graphs, histograms, or models. In order
that experiments carried out at different times
may be compared, it is desirable to express results
in terms of percentages of a control maintained
under as nearly as possible standardized con-
ditions.

Growth Measurements

179

Fig. 48. Tip of excised tomato root grown for one week in a
complete nutrient to which were added 3 x 10~G g. of indole acetic
acid per ml. of nutrient. Failure of the axis to elongate has
crowded the branch-primordia which are usually scattered over
many centimeters of root into a few millimeters giving the appear-
ance of a "rooting" response, x 25.

ISO

Plant Tissue Cull me

Pig. \'.k Excised tomato roots grown in solutions containing, in
addition to other essentia] constituents, the following concentrations
of Fe.,(S04)3, reading from Left to right: none, 0.6, L.8, 6.0, 18.0,
;mil 60.0 • Ki " M. Photograph was taken after cultivation for one
week. (From White, P. E. 1942. Planl Physiol. 17: L58, fig. 4,
37.)

Growth Measurements

181

Fig. 50. Excised tomato roots grown in solutions containing,
from left to right, the following concentrations of Fe2(S04)3: none,
0.675, 1.35, 2.025, 2.7, 3.375, 4.05, 4.725, 5.4, 6.077, 6.75, 7.425, 8.1,
8.775, 9.45, and 10.125 x 10"6 M. The linear increments of roots
grown in iron concentrations from 1.35 to 5.4 x lO"*3 M are greater
than at higher concentrations, but the branches are short and the
general habit poor. Experience has shown that roots grow more
satisfactorily and for longer periods if the iron concentration is
from 6.0 to 7.0 x 10-6 M. The extreme sensitivity of these roots to
slight variations in concentration of ions which are present in as
minute amounts as is iron is one of the striking characteristics of
such cultures.

1S12

Plant Tissue Culture

Fig. 51. Surface view of the region of elongation of a tomato
root grown in a solution containing indole acetic acid, showing the
scattered necrotic lesions resulting from plasmoptysis (explosion)
of many cortical cells. Compare with Fig. 56 wliich shows a com-
parable region of :i root grown in ;i nutrient Lacking indole acetic
acid, x 150.

Chapter IX

TISSUE CULTURE AND THE STUDY OF

PROBLEMS IN THE PATHOLOGY

AND GENERAL PHYSIOLOGY

OF PLANTS

The history of any biological method covers, in
general, three phases which may or may not be
simultaneous. These are: the development and
perfection of basic techniques ; the application of
those techniques to already recognized problems ;
and the formulation of new and unforeseen appli-
cations. Harrison (1907, 131) began with the sec-
ond phase in this series, having before him a defi-
nite problem, the elucidation of the origin of nerve
fibrils, before he devised the tissue culture tech-
nique. Having solved that particular problem by
relatively simple methods, he found no further
incentive to perfect or expand the technique and
turned to other problems amenable to other tech-
niques. Carrel (1911 et seq., 120), on the other
hand, without a specific problem but with a less
formulated concept of a vast field of problems,
took this technique and perfected it to a very high
degree. The application to new but specific prob-

184 Plant Tissue Culture

lems again passed to a third set of workers, where
Fell used it in studying bone-phosphatase activity
(Fell, 1928 et seq., 392-401), Warburg in studying
tumor metabolism (Warburg, 1923, 272, 1926, 273 ;
Warburg and Kubowitz, 1927, 274), Rivers in
growing vaccines (Rivers, Haagen, and Mucken-
fuss, 1929, 440, 441; Rivers and Ward, 1935,
442), etc.

The plant tissue culture idea began with a
theory rather than with a specific problem.
Haberlandt's theory (1902, 98) gave rise to a
technique in the hands of Gautheret (1932 et seq.,
48), White (1934 et seq., 65), and others. Being
of much more recent development than the animal
tissue culture technique, it has no such great
literature of applications, yet it has already found
a variety of fields of usefulness and will doubtless
find many more in the future.

The applications of the tissue culture technique
fall for the most part in three fields : in physiology,
in pathology, and in morphogenesis.

Physiology

Gross reactions. As we have seen elsewhere,
that aspect of a culture's reaction to its environ-
ment which can be most easily recorded in quanti-
tative terms is its increase in mass. Its altera-
tions in form come second, while in the third rank

General Physiology of Plants 185

in ease of investigation, but by no means third in
importance, are its changes in rate of activity as
evinced in terms of enzymatic digestion, respira-
tion, photosynthesis (if any), etc.

Mass increase has been used in measuring cul-
tures' behavior towards nutrient variables, vita-
mins, temperature and light changes, and so on.
In this way it has been demonstrated, for example,
that, whereas the absolute concentrations of va-
rious ions or molecules which give optimal growth
rates differ widely for different elements — 0.05 M
for sucrose (Bobbins, 1922, 57; Bonner and Addi-
cott, 1937, 42; White, 1932, 63), 0.02 M for calcium
and magnesium (White, 1932, 141), 0.000,002 M
for iron (Figs. 49, 50) (White, 1932, 141 ; Robbins
and Schmidt, 1938, 60), and 0.000,000,3 M for thia-
min (Robbins and Bartley, 1937, 222 ; Bonner, 1937,
198; White, 1937, 231) — the range around these
optima which will still permit normal growth is
in almost all cases of about the same magnitude,
from about 0.5 to 5.0 times the absolute optimal
value (White, in press). The capacity of the
tissue to adjust its behavior vis a vis these widely
different substances appears to be about the same,
irrespective of the substance under investigation.
These powers of adjustment may be enhanced or
reduced according to the existing conditions as
regards other variables in the environment with

186 Plant Tissue Culture

which they have no apparent connection. Thus,
cultures grown in a glycine-thiamin nutrient give
results which, at their best, are superior to the best
results obtained in a yeast nutrient, yet in a gly-
cine-thiamin nutrient they appear to be far more
sensitive to temperature changes than in a yeast
nutrient, so that at suboptimal or supraoptimal
temperatures the relations between growth rates
in these two nutrients may be just reversed
(White, unpublished). The presence of a large
number of substances of various character in the
yeast extract seems to "buffer" the cultures
against a too great sensitivity to other limiting
factors as compared to the few substances in
the synthetic (glycine-thiamin) solution. Perhaps
this is one reason for past failures to obtain satis-
factory growth of animal tissues in simple nutri-
ents. Studies of this sort bid fair to give us
considerably better insight into the functions and
interactions of the various nutrient molecules and
ions as well as other environmental variables in
the plant's economy.

Qualitative changes. A tissue 's or organ 's reac-
tion to nutritional variables may, of course, be
studied qualitatively as well as quantitatively.
The reaction to indole acetic acid and its homo-
logues serves as a striking but by no means iso-
lated example. A graded series of concentrations

General Physiology of Plants

187

of indole acetic acid added to a basic nutrient will
give quantitative results which may be expressed
in a fairly uniform curve (Fig. 52) (Fiedler, 1936,
45; Geiger-Huber, 1936, 214; Geiger-Huber u.
Burlet, 1936, 215; Duhamet, 1939, 209; Bonner

10" l2 to-'4

GRAMS- INDOLE-ACETlC-ACID PER ML NUTRIENT

Fig. 52. Graph showing the relative rates of increment of ex-
cised tomato roots grown for one week in a complete nutrient to
which had been added (from left to right) decreasing concentrations
of indole acetic acid. While 10'12 and 10"1S g. of indole acetic acid
per ml. of nutrient gave results which might indicate a stimulative
effect, the improvement over the control without indole acetic acid
was less than the probable error of the experiment and hence of
doubtful significance.

188 Plant Tissue Culture

and Koepfli, 1939, 343; White, unpublished).
But it is also possible to observe, at concen-
trations just exceeding the threshold level for
growth retardation, a characteristic shorten-
ing and thickening of the root axis, resulting in a
crowding of the branch primordia into a relatively
small space (Fig. 48) (Leonian and Lilly, 1937,
219). This gives an apparent but not real increase
in the number of branches and simulates a ' ' root-
ing" response, although in point of fact a rapidly
growing culture of the same age, grown in a nutri-
ent lacking indole acetic acid, would normally
form just as many branches but scattered over
from 10 to 20 times as great a length of root. Root
cultures deprived of potassium continue to grow
in length with unimpaired vigor but become pro-
gressively more and more slender and fail to
branch, as if all the potassium present in the orig-
inal explant was being retained in the apical meri-
stem so that none was available for the formation
of branch primordia. Omission of iodine from the
nutrient likewise leaves the culture quantitatively
unimpaired but results in a spasmodic growth, the
tips dying after 3 or 4 days and a series of new
branches developing, giving a picture quite differ-
ent from that produced by potassium deficiency
(White, in press).
Changes in activity. As has already been

General Physiology of Plants 189

pointed out, changes in mass and in form are only
secondary symptoms of the real activity of the
tissue and must be interpreted with some caution.
The activity of the tissue can be studied in a some-
what more direct fashion by examining its capaci-
ties for fermentation or more especially its re-
spiratory activity. This has been done, to date,
in only a single piece of work, carried out on callus
cultures of Salix capraea by Plantefol (1938, 256,
257) in co-operation with Gautheret. Both callus
cultures and root cultures, because of their rela-
tively reduced dimensions which permit a com-
paratively easy gas interchange between culture
and nutrient, because of their freedom from
trauma, and because of their high growth rates,
should provide ideal material for study of proc-
esses of this sort (Warburg, 1923, 272, 1926, 273).

Nothing has been done in the study of photo-
synthetic activity using plant tissue cultures.
Roots normally do not possess such activity and
that of callus cultures is probably low so that they
do not appear likely to prove very useful in this
respect.

While these are some of the obvious tissue ac-
tivities which suggest themselves as possible sub-
jects of investigation, there certainly exist less
obvious but no less important ones. One of these
turned up unexpectedly in the form of a glandular

190 Plant Tissue Culture

function in root tissues. Root cultures, although
immersed in nutrient fluid, nevertheless form well
differentiated vascular strands (Fig. 55). The
existence of such strands suggested the possibility
that they might be serving some real function, and
means were devised which made possible the
demonstration of the existence of a unidirectional
flow of liquid through these immersed organs
(White, 1936, 264). Further study showed that
this flow was not continuous but had a diurnal
rhythm and that it was maintained even when
opposed by very great external pressures (Fig.
53) (White, 1938, 267-269). The existence of a
rhythmic secretion was verified by Grossenbacher
(1938, 243, 1939, 244), and the unidirectional
character of the flow and the magnitude of the
force were verified by Rosene (1937, 259, 1941,
260). A correlative diurnal rhythm in respira-
tion rate has likewise been demonstrated in grow-
ing excised roots (White, 1942, 37, 38). The tech-
niques required for study of this phenomenon are
far from simple, so that investigations thereon
have been intensive rather than extensive (Figs.
56-58). The problem is, however, an impor-
tant one in its bearing on the question of water
movement in plants and of glandular activity in
general. It serves as an excellent example of the
unforeseen applications which a new technique
may find.

General Physiology of Plants

191

Hours
20 30 40 50

70 80

500 1

_L

0

10

20 30

40
Hours

50

60

70 80

Fig. 53. Graph showing the rates of water secretion of two
similar tomato roots over a period of 3$ days, one (above) against
uniform (atmospheric) pressure, the other (below) against imposed
pressures up to 90 lbs./sq. in. The diurnal rhythm with which
secretion proceeds is a striking characteristic of the behavior of
tissues of this sort. (From White, P. R. 1938. Am. J. Bot. 25:
226, fig. 6, 267.)

192 Plant Tissue Culture

Tropisms. Kotte (1922, 52) and Fiedler (1936,
45) have both made cursory observations on geo-
tropic response of roots grown in an agar sub-
stratum and report normal curvature. It has
been the experience of this laboratory that roots
floated on or near the surface of a liquid sub-
stratum show no such response. Two possible
explanations of this difference have suggested
themselves — either that the positive chemotropic
response to the oxygen gradient in a liquid me-
dium is more powerful than the geotropic response
and masks the latter (an explanation which should
be equally applicable to a semi-solid substratum)
or that the absence of a fixed support or fulcrum
from which to bend makes it impossible for a root
to retain a unilateral orientation. The positive
reaction on agar seems to support the latter ex-
planation, yet, once a root has grown long enough
in a liquid nutrient to curve around the flask some-
what, it has built up a bilateral symmetry which
should make a reaction to gravity possible. This
does not take place. Yet in experiments in which
roots were fixed in a constantly aerated medium
lacking an oxygen gradient, the response reap-
peared (White, unpublished). A clear explana-
tion is yet to be obtained. This example should
suffice to show that cultures of this sort are capa-
ble of serving as excellent material for certain
types of tropistic studies.

General Physiology of Plants

193

Cellular reactions. While the use of hanging-
drop cultures adapted to continuous observation
of single cells has so far been little developed as
applied to plant tissue cultures, there seems to be
no a priori reason why questions of the reactions
of individual cells should not be followed by means
of such a technique. This has, indeed, been done
in a number of scattered instances. The gradual
resorption of starch from the plastids in hanging-
drop cultures of Stellaria media has been studied
in this way (Fig. 54) (White, 1933, 116). The
localization of tyrosinase in the plastids of root-
hairs and cortical cells has been demonstrated by

Fig. 54. A typical developmental cycle of individual cells in a
culture of the stem tip of Stellaria media maintained for two weeks
in a hanging drop of nutrient, showing the progressive increase and
then decrease in size of the plastids as the cell grows, reaches
maturity, passes into senility, and finally dies. (From White, P. R.
1933. Protoplasma 9 : 110, fig. 19, 116.)

194 Plant Tissue Culture

growing root cultures in a nutrient containing 5
ppm. of tyrosine, a concentration which permits
normal growth (Fig. 60) (White, unpublished).
The behavior of localized root cells towards indole
acetic acid with the formation of scattered necrotic
lesions produced by the bursting of individual cells
(plasmoptysis) has likewise been studied (Fig.
51) (White, unpublished). The tendency towards
differentiation of scalariform cells in the interior
of callus cultures (White, 1939, 78) may be looked
upon as an example of cellular response to a par-
ticular environmental complex, in this case one of
low available oxygen content (Figs. 62, 63, 65, 66).
Cyclosis can be observed very satisfactorily in the
large " callus-blasen " to be found on the surfaces
of callus cultures (Fig. 61) (White, 1932, 89),
which should facilitate an accurate study of the
effects of environmental variables on this process.
These serve merely to point out some few places
at which physiological processes have been or can
be approached by means of tissue cultures.

Pathology

In the field of phytopathology, the most obvious
of all applications of a new technique for the cul-
tivation of phanerogamic tissues is in the main-
tenance of such tissues as substrata for either
obligate or facultative pathogens. The method as

General Physiology of Plants

195

j

*' ■

X

m . |

#

Fig. 55. Optical longitudinal section of the region of matura-
tion of a growing excised tomato root, showing the well-differenti-
ated stele. x90. (From White, P. R. 1938. Am. J. Bot. 25:
223, fig. 1, 267.)

196

I'huit Tissue Culture

Fig. ."><;. A manometer attached to a single excised root growing
in its culture ll;isk. Water is secreted into the manometer, forcing
the colored index drop upward iii the capillary. (From White, P.
K. L938. Am. J. Bot. 25: 224, fig. 2, ?67.)

(lateral Physiology of Plants

197

Fig. 57. Details of equipment used in studying the basipetal
secretion of water by excised tomato roots, showing the glass mano-
meters, rubber hose, and metal clamps at various stages in their
assembly. (From White, P. B. 1938. Am. J. Bot. 25 : 225, fig. 5,
267.)

IDS

Plant Tissue Culture

Fig. 58. Assembly of four manometers with their attached roots
:iik1 culture Husks, coupled by a manifold to a tank of compressed
nitrogen in stn-li a manner thai the force with which water is driven
into the capillaries by the Becretory activity of the roots can be
balanced against a known gas pressure and thus measured. ( From
White, P. B. 1942. Sigma Xi Quarterly 30: L32, fig. 13, SS4.)

General Physiology of Plants 199

it stands today is hardly usable for the latter, for
the obvious reason that the basic nutrient for tis-
sues is also an excellent nutrient for many of these
organisms. It may, of course, be possible to
modify the nutrient in such a way as to make it
suitable for the host tissue but not for the infect-
ing organism. Lewis and McCoy (1933, 301) used
a primitive sort of organ-culture technique in the
study of bacterial nodule formation on the roots
of legumes. Although their results were not en-
tirely satisfactory, they suffice to indicate one
direction in which the more highly perfected tech-
niques may prove useful. Kiker and Berge (1935,
317) foresaw some such possibility in connection
with their studies of crown gall organisms, as did
Nobecourt and Dusseau (1938, 311) and Berthelot
and Amoureux (1937, 276). Nothing has been
done with fungi except Gioelli's observations on
the effects of chance contaminations on his cam-
bium cultures (1938, 292). If the roots of cereals
can be grown in the same manner as those of dico-
tyledonous plants, they should serve as interesting
substrata for the study of rust infections.

Viruses in tissue culture have been studied to a
somewhat greater extent than have bacteria and
fungi, though still barely enough to give a sugges-
tion of the possibilities. Viruses of a consider-
able number of diseases of the mosaic group have

200 Plant Tissue Culture

been cultivated in excised roots of tomato (White,
1934, 438, 1936, 439). (See Chapter VI.) Dis-
eases which cause only moderate local injury and
which move rapidly into all parts of the host can
be maintained indefinitely in tissue cultures with-
out difficulty and without essential change in the
methods. Here they produce no macroscopically
visible symptoms. By aliquot titration, it is pos-
sible to follow the rate of multiplication of the
virus as a function of host-tissue multiplication
(Fig. 59) and to show that the two rates are inde-
pendent of one another. Stanley (1938, 437) has
used such cultures as a source of virus in order to
demonstrate the essential identity of the chemis-
try of virus protein produced in these chlorophyll-
free tissues and of that derived from the usual
leaf tissues with their high chlorophyll content and
activity. Viruses which cause a high degree of
local injury and which have a slow migration rate
in the tissue are somewhat more difficult to main-
tain in culture. A considerable amount of older
tissue must be included in every explant along
with the usual meristem, since the meristems
themselves are often virus-free. Otherwise, the
virus may be left behind in the discarded tissue
and a strain of healthy tissue segregated for culti-
vation. Such healthy strains have been segre-
gated from cultures carrying "cucumber mosaic"

General Physiology of Plants

201

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202 Plant Tissue Culture

virus, Jensen's No. 16 strain of tobacco mosaic
virus, and some other virulent types (White, un-
published). Interestingly enough, no method has
yet been found for inoculating such cultures (they
must be isolated from systemically infected
plants), so that there is no danger of a virus-bear-
ing culture, once established, ever becoming con-
taminated with another virus (White, 1934, 438).

Tissue cultures have to date not been tested as
hosts for phanerogamic parasites such as Cuscuta,
Viscum, etc., but should prove useful in the study
of phanerogamic host-parasite relations.

One field of investigation in which tissue cul-
tures promise to be extremely useful, which repre-
sents essentially an extension of the nutrition
studies mentioned above, is that covering the
effects of toxic or injurious substances, either of
organic or inorganic origin, on the behavior of the
tissues of such cultures. The reaction to indole
acetic acid mentioned above is an excellent exam-
ple and serves to show the sorts of observations
which are possible in this field using even very
simple methods (Figs. 48, 51, 52). The reaction of
excised carrot tissue to colchicine has also been
studied, by Duhamet (1939, 210).

Histology and Cytology

Tissue and organ cultures furnish unusually
clean, uniform, easily handled material for histo-

General Physiology of Plants 203

logical study. Gautheret (1935, 15, 1937, 282,
1938, 283, 284, 1940, 286-289), Dauphine (1930,
279), Bobbins and bis colleagues (Bobbins and V.
B. White, 1936, 62), and Addicott (1941, 196) have
utilized root cultures for some anatomical and
histological studies. White (1939, 78) Gautheret
(1938, 283, 284) and Gioelli (1937, 290) have made
a few similar studies with callus cultures. There
remains, however, much to be done in this direc-
tion.

Summary

The tissue culture technique has been applied to
the study of the reactions of tissues, organs, and
cells to variations in concentrations of nutrient
materials, growth stimulants, toxins, and other
environmental variables. These reactions can be
measured in terms of mass increase, differentia-
tion, level of respiratory activity, enzyme or
water secretion, tropistic bending, and many other
sorts of behavior. Bacteria, molds, and viruses
have been cultivated on growing isolated plant
organs as hosts and the host behavior studied.
These represent some of the fields to which the
technique has already been applied. Many more
obviously remain as yet untouched.

General Physiology of Plants

205

Fig. 60. Optical longitudinal section of an excised tomato root
grown for one week in a complete nutrient to which was added 15
rag. of tyrosine per liter of solution, showing the typical blackening
of the plastids indicating the presence of tyrosinase, x 150.

206

Plant Tissue Culture

i

*

\

L4

\

Pig. 61. Above, successive stages (left to right) in the hyper-
hydric enlargemenl of the integuments of immature seeds of Antir-
rhinum upon cultivation in hanging drops of nutrient. These large,
thin-walled callus cells furnish excellent material in which to ob-
serve the details of cellular behavior. ■ '.W. ( From White, P. E.
1932. Arch. exp. Zellforsch. LI: 615, figs. 1 I. is. 19,89.) Below,
single cells from the margins of hanging drop cultures of immature
seeds of Antirrhinum showing the nucleus, protoplasmic strands,
|. last. .is, oil globules, etc. < 250. (Prom White, P. R. 1932.
Arch. exp. Zellforsch. 11 : 617 and 619, figs. 2] and 23, 89.)

General Physiology of Plants

207

Fig. (52. Detail of a mass of meristematic cells from the outer
region of a culture of undifferentiated callus from a plant of the
hybrid, Nicotiana langsdorffi x N. glauca. Delafield 's haematoxylin-
safranin. x 1000. (From White, P. R. 1939. Am. J. Bot. 26:
63, fig. 11, 78.)

•jus

Plant Tissue Culture

Fir,. <;:;. A shorl series of scalariforno <-clls forming an incom-
plete vascular strand in the interior of ;i Nicotiana callus culture.
L000. (From White, P. R. IJKW. Am. .1. Hot. I2t> : (53, fig. 8,

Chapter X
MORPHOGENESIS

It was suggested at the beginning of this volume
that the technique of cultivating excised tissues
had its chief raison d'etre and gave its greatest
promise in the study of the origins of form and
function, that is, in problems of morphogenesis.
Harrison 's original problem which led to the first
in vitro cultivation of animal cells — whether the
nerve fibrils originate in situ from the innervated
cells or their products or whether they originate
ab extra, by growth from the distant ganglia — was
a morphogenetic problem. So was Robbins ' orig-
inal problem — whether the root was dependent on
the leafy portions of the plant for anything more
than its basic carbohydrate nutrition. So like-
wise was the problem — what was the nature of the
stimulus by which crown gall organisms or their
products induce abnormal proliferation of the
host cells — which led the author of this volume to
undertake the development of a plant tissue cul-
ture technique (White, 1931, 32). The number of
problems which have been attacked to date by
means of this technique is not large. But the

210 Plant Tissue Culture

number which it should be possible to attack by
this means is legion. It seems desirable therefore
to outline some of the possibilities. (See Harri-
son, 1912, 415, 1933, 416 ; Fischer, 1922, 405, 1930,
409, 1931, 410; Fischer u. Parker, 1929, 411;
Parker, 1934, 427, 1936, 428, 429).

That the developmental and behavior pattern
in higher animals is greatly influenced by meta-
bolic products known as "hormones" is today
generally recognized. Of recent years, there has
grown up a considerable literature on "plant
hormones" as well. The first plant hormone,
"auxin," was originally studied as a factor in
geotropic and phototropic, hence purely orienta-
tion, responses (Paal, 1914, 367, 1919, 368; Boysen-
Jensen, 1910, 344). It has subsequently come to
be looked upon as a true morphogenetic hormone,
since it seems to induce the differentiation of roots
from tissues that ordinarily rarely, if ever, pro-
duce roots (Went, 1929, 380 ; Bouillenne et Went,
1933, 277 ; Thimann and Went, 1934, 229 ; Laibach,

1935, 218; Thimann and Koepfli, 1935, 376;
Cooper, 1935, 345, 1936, 346; Zimmerman et al.,
1933, 384, 1935, 385, 386), to control the formation
and development of buds (Thimann and Skoog,
1933, 377, 1934, 378; Snow, 1925, 372; LeFanu,

1936, 359; van Overbeek, 1938, 366), to influence
the abscission of leaves and of fruits (Gustafson,

Morphogenesis 211

1936, 350, 1938, 351, 352, 1939, 353 ; Gardner and
Marth, 1937, 348), to initiate the activity of cam-
bium (Snow, 1933, 373, 1935, 374-, Avery, Burk-
holder, and Creighton, 1937, 341), to regulate the
specific branching habit of plants (Lehmann, 1936,
360; Delisle, 1937, 347; Goodwin, 1937, 349), to
mediate between genes and growing tissues in con-
trolling the procumbent vs. erect habit (van Over-
beek, 1936, 363, 1938, 365), in producing dwarf ness
(van Overbeek, 1935, 362, 1938, 364), to take part
in the production of bacterial root nodules (Thi-
mann, 1936, 375), and many other responses.
(Compare Appleman, 1918, 340; Went, 1928, 379,
1932, 381). All these responses are attributed to
the same substance. If this view is correct, then
the differences in response must depend on corre-
lative factors resident in, or in the neighborhood
of, the responding cells. A growing, undifferen-
tiated mass of cells, such as a callus culture, or a
mass characterized by a definite but limited degree
of differentiation, such as an excised root, should
not only possess fewer of these internal variables
but should permit a more rigorous control of the
external variables as well. If auxin is truly a
specific root-forming hormone (Went, 1929, 380) f
it should be possible to set up conditions under
which its local application to such a callus culture
would regularly induce the local formation of

212 Plant Tissue Culture

roots. If auxin is truly a hormone of tropic re-
sponse (Boy sen- Jensen, 1910, 344), its unilateral
application to growing excised roots should regu-
larly induce unilateral curvature. Moreover,
there have recently been described a number of
other specific "hormones," a flower-forming hor-
mone (Melchers, 1937, 361; Hamner and Bonner,
1938, 356), a stem-forming hormone, "cauloca-
line" (Went, 1938, 382), and a leaf -forming hor-
mone, "phyllocaline" (Went, 1938, 382), in addi-
tion to the root-forming hormone, "rhizocaline,"
which Bouillenne (1938, 206) and Cooper (1935,
345, 1936, 346) believe to be different from the
auxin of Went (1929, 380) and Kogl, Haagen-
Smit, and Erxleben (1933, 357). If these sub-
stances are truly specific for the processes from
which they take their names, and if, as we have
suggested, the cells of tissue cultures are essen-
tially totipotent, it should be possible to set up
cultural conditions for such tissues such that ap-
plication of a flower-forming hormone would regu-
larly induce the formation of flowers, application
of rhizocaline would regularly induce the produc-
tion of roots, etc. The tissue culture technique
seems to offer possibilities for the analysis of
these relations, under conditions relatively free of
uncontrollable variables.

On the other hand, there is already available

Morphogenesis

•21:5

e

\ /- , . :

" 1 \ 1

. w

3 s^^BPfJ?

* <

Fig. 64. Above, two cultures of cambium of Sambucus JHtfra
grown side by side on a semi-solid nutrient in such a manner that
a graft union formed at the center. The exact line of fusion can-
not be distinguished, x 100. Below, cultures of cambium of Acer
pseudoplatanus grown as in the picture above. The graft union
was much less perfect here, x 150. (From Gautheret, R. J. 1935.
Recherches sur la culture des tissus vegetans. (These) 250, plate
XVIII, 1, 2, 15.)

214

Plant Tissue Culture

Fig. (>."). Transverse section of an 8-weeks-old culture of
Vicotiana callus, showing the compact, relatively organized central
portion surrounded by a broad region of younger, louse, unorgan-
ized, and mostly rapidly growing tissue. 20. (From White, P.
R. 1939. Am. J. Hot. 26: 61, fig. •"".. 78.)

I'm. (i(i. Detail from Lower margin of Pig, !>."">, showing meriste-

matic areas scattered through the upper half of the picture while

the lower portion, particularly at the left, is made up largely of
gianl cells of a mature type, x 138. (From White, 1'. R. L939.
An,. .1. Bot. 26: til', fig. 7, 78.)

Morphogenesis

215

•:• \

t

}. i L— -" J 9> *•

'/r?

P"»«s*

■■Ji r-

Fig. 66.

•J Hi

Plant Tissue Culture

m<~- -

a;

*

^ 4

xiv

>.-*-

V;

I

Vr 1 1

* J t

.'■\ •

**

•^ .

Pig. r>7. A typical whorl of disorganized vascular tissue from
the interior of a culture of bacteria free crown-gall tissue of sun-
flower. ■ \'l'i.

Morphogenesis 217

some evidence that the differentiation of specific
organs, while it may be influenced by specific sub-
stances, may also be controlled to some extent at
least by concentration gradients of non-specific
substances, hence by polar (physical) as well as
chemical relationships. The phenomena of polar-
ity mentioned at the very beginning of this book in
connection with the work of Vochting, Goebel, and
others thus offer another field in which the tissue
culture technique provides a means of approach
not heretofore available. Priestley (1928, 314,
1930, 315, Pearsall and Priestley, 1923, 313)
attributes the formation of cambium in radial
plant bodies to a radially distributed hydrogen-ion
gradient. Lund (1931, 247) and Rosene and Lund
(1935, 319) have shown that there is a correspond-
ing electropotential gradient which can be modi-
fied by imposing artificial oxygen-hydrogen gradi-
ents. White (1939, 333) has shown that an oxy-
gen gradient may be correlated with the response
by which undifferentiated callus cultures (Figs.
17, 18, 61, 65) are induced to form stems and
leaves without the intercession of a caulocaline
(Figs. 68, 71). White (1938, 267) and Rosene
(1937, 259, 1941, 260) have shown that there is a
hydrostatic flow correlated with the electrostatic
gradient along excised and intact roots. But it is
as yet quite unknown which of all these gradients

218 Plant Tissue Culture

— of pH, of redox-potential, of electrostatic poten-
tial, of hydrostatic potential, and of tendency to
differentiate — is primary and which secondary.
It should be possible in tissue culture to artificially
impose or modify any or all of these gradients,
except the last, along controlled spatial axes and
thus to determine with which other gradients they
bear a causal relationship. Brachet has applied
this method in animal embryology to the study of
effects of imposed oxygen gradients (Brachet,
1937, 387; Brachet and Shapiro, 1938, 388), and
Huxley (1926, 417, 1927, 418), Gilchrist (1928,
412), and Vogt (1932, 434) have applied it to the
study of temperature gradients. Fife and Framp-
ton (1936, 241) have applied it in plant pathology
to the study of the causal factors involved in the
orientation of a parasitic insect's proboscis in the
tissues of the host, with brilliant results. With
plant tissue cultures it is a virgin field except for
White's preliminary observations on oxygen gra-
dients and differentiation (Figs. 68, 71) (1939, 333,
1942, 334) and, in a sense, those of Nobecourt (1939,
448) and of Gautheret (1940, 286-288) on bud
formation. Yet these methods of approach
should prove effective with excised organs (roots)
and undifferentiated masses (callus) and perhaps
even more with young developing embryos, espe-
cially if these can be grown from the fertilized egg.

Morphogenesis 219

The forces which, in the egg cell of a vascular
Cryptogam, for example, result in the first three
mitoses segregating cells which normally develop
into stem growing point, cotyledon, root, and sus-
pensor, respectively, are sufficiently clean-cut to
offer material capable of clear and definite analy-
sis, once a technique for such studies is available.
The tissue culture technique should make possible
the development of a true science of plant em-
bryology.

One method of approach to problems of organi-
zation which has proved especially effective in
animal embryology is, as we have seen, that of
transplantation, developed to a high degree by
Harrison and by Spemann (1936, 431, 432). With
plant material, the Boysen-Jensen-Paal-Went
technique of transplanting Avena coleoptile tips,
or agar blocks containing extracts from such tips,
is essentially a comparable technique, the coleop-
tile or agar block (Went, 1928, 379) acting in the
same way as an organizer in the sense of Spemann.
If plant members such as coleoptile tips (Avery
and La Rue), root tips (White et al), stem tips
and buds (White), leaf primordia, etc., can be
grown in culture, can be subjected to controlled
environmental influences, and can then be trans-
planted into otherwise intact host plants, the ques-
tion of organ influences at a distance, of correla-

220 Plant Tissue Culture

tions of the type suggested by Sachs (1880, 370,
1882, 370, 1893, 320, 371), could be brought within
the scope of experimental analysis (see Went,
1941,555).

Another field in which this technique might pre-
sumably find valuable applications is the study of
the physiological aspects of heterosis and incom-
patibility. Bobbins has already studied the
growth behavior of excised roots from certain
hybrid tomatoes, compared with that of roots
from the two inbred parents (1941, 318). In cases
where the hybrid is ' ' physiologically sterile, ' ' due
to unsatisfactory endosperm development, as in
certain peaches (Tukey, 1933, 327; White, 1928,
332), the tissue culture method has proved its
value in permitting the propagation of embryos
which would otherwise perish. Where adventi-
tious embryos arise aposporously from the nucel-
lar wall, as in many Citrus fruits, Taraxacum,
certain Daturas, etc. (Blakeslee and van Over-
beek), the technique should prove valuable, not
only in propagating these embryos in refractory
cases, but also in determining the causes of poly-
embryony and aposporous embryogony. More-
over, where "incompatibility" is suspected of
being physiological rather than anatomical, the
cultivation of tissues of the two incompatible mem-
bers together in vitro might well throw light on

Morphogenesis 221

the nature of this incompatibility. Gautheret
(1935, 15) has carried out experiments of this type,
though for quite a different purpose, in his pre-
liminary study of heterologous and homologous in
vitro grafts, which indicate the feasibility of this
sort of approach (Fig. 64). (See also Figs. 69,
70.)

The tissue culture technique has recently per-
mitted the transfer of one problem group already
mentioned, the "crown gall problem," from the
field of pathology to that of experimental morpho-
genesis with extremely important implications.
Tissues isolated from secondary or metastatic
crown gall tumors have been grown in tissue cul-
ture (Fig. 19). Here they maintained a rapid,
disorganized type of growth (Fig. 67) which ex-
pressed itself, upon reimplantation into healthy
hosts, in the production of new tumors. The tis-
sues were shown to be free of bacteria. The
tissue culture technique has thus permitted the
experimental manipulation of crown gall tissues
free of the original pathogen and the study of the
factors involved in the origin of the tumefacient
property of the cells themselves. It thus offers a
new approach to problems of the origin of malig-
nancy (White and Braun, 1941, 335, 1942, 336, 337;
Braun and White, 1942, 436).

The physiological aspects of cellular morpho-

222 Plant Tissue Culture

genesis, the causal factors involved in the differen-
tiation of bast-fibers, stone-cells, medullary ray
cells, phloem tube elements, and of true meristems,
all from the same parenchymatous elements, are
also problems open to attack by tissue culture
methods.

These suggestions merely serve to point out
some of the as yet essentially untouched fields
opened up by the development of this technique.
The history of the progress of science is a history
of such tools.

SUMMAKY

In the study of the origin of form and function,
that is, of morphogenesis, tissue culture methods
provide a means of isolating cells, organs, and tis-
sues from the morphogenetic influences of neigh-
boring cells, organs, and tissues and placing them
in controlled environments. Questions of the
effects of growth substances or ''hormones," of
chemical and physical gradients, and of cytoge-
netic factors can be approached by this means with
considerable hope of success.

Morphogenesis

223

Fig. 68. A culture of Nicotiana callus maintained for 8 weeks
in an undifferentiated state by repeated transfer on a serni-solid
nutrient and then placed for 2 weeks in a liquid nutrient. Reduc-
tion in available oxygen supply under the latter conditions has
resulted in the formation of differentiated growing points and
leaves, x 7.5. (From White, P. R. 1939. Bull. Torrey Bot. Club
66: 509, fig. 2, 333.)

224

I'hi ul Tissue Culture

in,. 69. Transverse section of a Nicotiana callus culture main
tamed for two weeks iii the anterior chamber of a rabbit's eve.
(Courtesy of Dr. Harry s. X. Greene, Vale University Medical
School.) The deep-staining areas are animal cells, largely invasive
leucocytes, which have in many cases pushed their way between the
less deeply staining plant cells. The very dark, broken, lenticular

mass at the left is the lens of the eye. The plant tissue contains
many gianl cells at the margin and some vascular strands luit no

meristematic areas, indicating that, while cell division had stopped,

Cell enlargement and maturation had proceeded for some time after
insertion of the culture in the eve. ■ 41'.

Morphogenesis

225

t>

Fig. 70. Detail from the lower right-hand portion of Tig. 69,
showing plant tissue with a central vascular strand flanked both
above and below by animal cells which have in many cases pene-
trated into the intercellular spaces, x 400.

2'2i\

Plant Tissue Culture

PIG. 71. Two sister cultures of Nicotiana Callus tissue. That

above was grown for _<> weeks on :i semi-solid medium providing
adequate aeration. Thai below was grown for !<• weeks on the

semi solid medium followed by 10 weeks in ;i nutrient of the same

constitution except for the omission of the agar. Here the culture
was submerged and thus subject to reduced aeration, stem grow
ing points and Leaves of n high order of differentiation have, under
the latter conditions, developed from n tissue mass which, on the
semisolid nutrient, appeared to have lost the capacity to differ-
entiate. <1.5. (From White, P. R. L939. Bull. Torrey Bot. Club
66: 509, fig. 1, 888. i

BIBLIOGRAPHY

Keference has been made in the foregoing text to publications
in a variety of fields, some directly bearing on the cultivation of
excised plant organs and tissues, some having only an indirect
bearing on this subject. The following list of publications cited
must obviously be incomplete. It is believed to contain all the
major contributions on plant tissue cultures. The references on
collateral subjects, such as plant growth substance studies, animal
tissue cultures, etc., are intended to be merely suggestive. The
citations have been roughly classified according to subjects to
facilitate the reader's search for a given type of material. This
classification must obviously appear arbitrary in many cases, but
it is hoped may prove useful and suggestive.

Nos.

I. General treatises 1-39

II. Cultivation

1. Roots 40-66

2. Callus and cambium 67-78

3. Embryos 79-89

4. Tissues other than roots, calluses, and embryos 90-116

5. Animal tissues 117-132

III. Nutrition

1. Inorganic

a. Plant 133-144

b. Animal 145-165

2. Carbohydrate

a. Plant 166-173

b. Animal 174-175

3. Nitrogenous

a. Plant 176-182

b. Animal 183-194

4. Fat

a. Plant (None)

b. Animal 195

5. Vitamins and hormones

a. Plant 196-232

b. Animal 233-236

228 Plant Tissue Culture

IV. Physiology other than nutrition

1. Plant 237-270

2. Animal 271-274

V. Morphology and morphogenesis

1. Plant

a. General 275-339

b. Growth substances 340-386

2. Animal 387-435

VI. Pathology

1. Plant 436-439

2. Animal 440-442

VII. Miscellaneous

1. Plant 443-455

2. Animal 456-457

General Treatises

1. Bloom, W. 1937. Cellular differentiation and tissue cul-

ture. Physiol. Rev. 17: 589-617.

2. Borger, H. 1926. Verfahren pflanzlicher Gewebeziichtung.

(Nachtrag zur: Erdmann, Rh. Gewebeziichtung.) Handb.
norm. path. Physiol., Band 14, l.Halfte: 1000-1002.
Springer, Berlin.

3. Burlet, E. 1940. Ueber die pflanzliche Organkultur und

ihre Anwendung bei physiologischen Untersuchungen.
Ber. schweiz. bot. Ges. 50: 519-544.

4. Carrel, A. 1924. The method of tissue culture and its bear-

ing on pathological problems. Brit. Med. Jour. 2: 140-
145.

5. . 1924. Tissue culture and cell physiology. Physiol.

Rev. 4 : 1-20.

6. . 1928. Modern techniques of tissue culture and

results. Arch. exp. Zellforsch. 6: 70-81.

7. . 1931. The new cytology. Science 73: 297-303.

8. . 1937. Le present et l'avenir de la cytologic ex-

perimentale. Arch. exp. Zellforsch. 19: 121-128.

9. Ephrussi, B. 1932. La culture des tissus. pp. 233. Gauthier-

Villars, Paris.
10. Erdmann, Rh. 1920. Einige grundlegende Ergebnisse der
Gewebeziichtung aus den Jahren 1912-1920. Ergeb.
Anat. Entw.-geschichte 23: 420-500.

Bibliography 229

11. . 1930. Praktikum der Gewebepflege oder Explan-

tation besonders der Gewebeziiehtung. Ed. 2. Springer,
Berlin.

12. Fiedler, H. 1938. Die pflanzliche Gewebe- und Organkul-

tur. Ztschr. Bot. 33: 369-416.

13. Fischer, A. 1930. Gewebeziiehtung. Handbuch der Biolo-

gie der Gewebezellen in vitro. 3. Ausgabe. Miller u.
Steinicke, Munich.

14. . 1933. Gewebeziiehtung und ihre Beziehung zur

allgemeinen Biologic Zool. Anzeig., Suppl. 6: 83-99.

15. Gautheret, R. 1935. Kecherches sur la culture des tissus

vegetaux: Essais de culture de quelques tissus me>i-
stSmatiques. These, Univ. Paris, pp. 279.

16. . 1937. La culture des tissus vegetaux. Son 6tat

actuel, comparaison avec la culture des tissus animaux.
Actuality sci. industrielles. pp. 66. Hermann, Paris.

17. . 1938. La culture des tissus vegetaux. Sciences

(rev. franc) 20: 57-71.

18. Harrison, R. G. 1928. On the status and significance of

tissue culture. Arch. exp. Zellforsch. 6: 4-27.

19. Krontowski, A. 1928. Explantation und deren Ergebnisse

fur die normale und pathologische Physiologic Ergeb.
Physiol. 26: 370-500.

20. Kiister, E. 1909. Ueber die experimentelle Erforschung

des Zellenlebens. Naturw. Wochenschr. 24: 433-438.

21. . 1928. Das Verhalten pflanzlicher Zellen in vitro

und in vivo. Arch. exp. Zellforsch. 6: 28-42.

22. Lewis, W. H., and Lewis, M. R. 1924. Behavior of cells in

tissue cultures. (In: Cowdry, General cytology, Univ.
Chicago Press, pp. 385-447.)

23. Loeb, J. 1916. The organism as a whole, pp. 379. G. P.

Putnam's Son, New York.

24. . 1924. Regeneration from a physicochemical view

point, pp. 143. McGraw-Hill Book Co., Inc., New York.

25. Nobecourt, P. 1939. titat actuel de la question des cultures

de tissus vegetaux. Soc. Dauphinoise d 'etudes biol. 322:
54-55.

26. Parker, R. C. 1938. Methods of tissue culture. Paul B.

Hoeber, Inc., New York.

230 Plant Tissue Culture

27. Schleiden, M. J. 1838. Beitrage zur Phytogenesis. Arch.

Anat., Physiol., wiss. Med. (J. Muller) 1938: 137-176.

28. Schneider. 1926. Gewebekulturen bei Pflanzen. (In: E.

Krause, Enzyklopadie der mikroskopischen Technik. 3.
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29. Schwann, Th. 1839. Mikroskopische Untersuchungen iiber

die Ubereinstimmung in der Struktur und dem Wachstume
der Tiere und Pflanzen. Nr. 176, Oswalds Klassiker der
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30. Strangeways, T. S. P. 1924. Tissue culture in relation to

growth and differentiation. Heffer, Cambridge, England.

31. Vogt, W. 1934. Entwicklungsmechanik und Gewebeziicht-

ung. Arch. exp. Zellf orsch. 15 : 269-280.

32. White, P. E. 1931. Plant tissue cultures. The history and

present status of the problem. Arch. exp. Zellf orsch. 10:
501-518.

33. . 1936. Plant tissue cultures. Bot. Eev. 2: 419-

437.

34. . 1939. Eecent contributions from excised plant

tissue and organ culture studies to the science of plant
physiology. Chron. Bot. 5: 166-167.

35. . 1939. Plant organ and tissue physiology, as ex-
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before Am. Sci. Teachers Assoc, A.A.A.S., December,

. 1938. pp. 15-22.

36. . 1941. Plant tissue cultures. Biol. Eev. 16 : 34-48.

37. . 1942. "Vegetable Dynamicks" and plant tissue

cultures. Plant Physiol. 17: 153-164.

38. . 1942. "Vegetable Dynamicks" and plant tissue

cultures. Science 95: 520-522.

39. . 1942. Plant tissue culture. Ann. Eev. Biochem.

11: 615-628.

40. Willmer, E. N. 1928. Tissue culture from the standpoint

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41. . 1935. Tissue culture, pp. 126. Methuen, Lon-
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Cultivation — Eoots

42. Bonner, J., and Addicott, F. 1937. Cultivation in vitro of

excised pea roots. Bot. Gaz. 99: 144-170.

Bibliography 231

43. Chambers, W. H. 1923. Cultures of plant cells. Proc. Soc.

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44. . 1924. Tissue culture of plants. Jour. Mo. State

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45. Fiedler, H. 1936. Entwicklungs- und reizphysiologische

Untersuehungen an Kulturen isolierter Wurzelspitzen.
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46. Galligar, G. C. 1934. Growth studies on excised root tips.

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47. . 1939. Growth behavior of one-millimeter excised

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48. Gautheret, E. 1932. Sur la culture d 'extremites de racines.

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49. . 1933. Cultures de meristemes de racines de Zea

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50. Heidt, K. 1931. Ueber das Verhalten von Explantaten der

Wurzelspitze in nahrstofffreien Kulturen. Arch. exp.
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51. Kotte, W. 1922. Wurzelmeristem in Gewebekultur. Ber.

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52. . 1922. Kulturversuche mit isolierten Wurzel-
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53. Malyschev, N. 1932. Das Wachstum des isolierten Wurzel-

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54. . 1932. The growth of isolated meristems of roots.

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55. Marotto, L. 1935. I resultati di alcune culture ' ' in vitro ' '

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56. . 1936. Esperienze esequite con apici radicali iso-

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57. Bobbins, W. J. 1922. Cultivation of excised root tips and

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58. , Bartley, M. A., and White, V. B. 1936. Growth

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232 Plant Tissue Culture

59. , and Maneval, W. E. 1923. Further experiments

on growth of excised root tips under sterile conditions.
Bot. Gaz. 76: 274-287.

60. , and Schmidt, M. B. 1938. Growth of excised

roots of the tomato. Bot. Gaz. 99 : 671-728.

61. , and . 1939. Further experiments on ex-
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62. , and White, V. B. 1936. Limited growth and

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63. White, P. E. 1932. Influence of some environmental con-

ditions on the growth of excised root tips of wheat seed-
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64. . 1933. Liquid media as substrata for the cultur-

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65. . 1934. Potentially unlimited growth of excised

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66. . 1938. Cultivation of excised roots of dicoty-
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Cultivation — Callus and Cambium

67. Gautheret, K. 1934. Culture du tissu cambial. C. r. acad.

sci., Paris, 198: 2195-2196.

68. . 1937. Nouvelles recherches sur la culture du

tissu cambial. C. r. acad. sci., Paris, 205: 572-574.

69. . 1938. Sur le repiquage des cultures de tissu

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125-127.

70. . 1938. Eecherches sur la culture de fragments de

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71. . 1939. Sur le developpement de fragments de

tubercules de chou-rave. C. r. soc. biol., Paris, 130: 244-
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72. . 1939. Sur la possibility de rSaliser la culture

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73. . 1940. Sur la culture du tissu cambial de Carotte.

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75. . 1938. Sur les proliferations spontanees de frag-
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76. . 1938. Sur la proliferation in vitro du paren-

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77. . 1939. Sur la perennite et 1 'augmentation de vol-
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78. White, P. E. 1939. Potentially unlimited growth of excised

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Cultivation — Embryos

79. Dietrich, K. 1924. Ueber die Kultur von Embryonen

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80. Essenbeck, E., u. Suessenguth, K. 1925. Uber die aseptische

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81. Hannig, E. 1904. Zur Physiologie pflanzlicher Embryonen.

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82. La Bue, C. D. 1936. The growth of plant embryos in cul-

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83. , and Avery, G. S., Jr. 1938. The development of

the embryo of Zizania aquatica in the seed and in arti-
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84. Razdorskii, V. 1938. The culture of isolated plant embryos.

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85. Stingl, G. 1907. Experimentelle Studie iiber die Ernahrung

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86. Tukey, H. B. 1933. Artificial culture of sweet cherry

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87. . 1934. Artificial culture methods for isolated

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234 Plant Tissue Culture

88. Werckmeister, P. 1934. Tiber die kiinstliche Aufzucht von

Embryonen au3 Zns-Bastardsamen. Gartenbauwiss. 8 :
607-608.

89. White, P. R. 1932. Plant tissue cultures. A preliminary

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Cultivation — Tissues Other Than Boots, Calluses
and Embryos

90. Bobilioff-Preisser, W. 1917. Beobachtungen an isolierten

Palisaden- und Schwammparenchymzellen. Beih. bot.
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91. Bonner, J. 1936. Plant tissue cultures from a hormone

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93. Czech, H. 1926. Kultur von pflanzlichen Gewebszellen.

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94. Dauphine, A. 1929. Sur le developpement d'organes em-

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95. Gautheret, R. 1933. Cultures de cellules detachees de la

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96. . 1933. Nouvelles recherches sur la culture des

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97. . 1939. Nouvelles experiences sur la croissance

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236 Plant Tissue Culture

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303. . 1916. Further experiments on correlation of

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fcklBRAR
YE/

302.

250 Plant Tissue Culture

304. . 1917. Influence of the leaf upon root formation

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305. . 1917. The chemical basis of axial polarity in

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258 Plant Tissue Culture

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Pathology — Plant

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Pathology — Animal

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260 Plant Tissue Culture

Miscellaneous — Plant

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445. MacDougal, D. T., and Shreve, F. 1924. Growth in trees

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446. Mason, T. G., and Phillis, E. 1939. Experiments on the

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447. Mueller, J. H. 1935. Methionine as an impurity in natural

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INDEX

Author names are in black face type, plant and animal names in
italics. Page references in black face type indicate bibliography,
■while those in italics indicate illustrations.

Abies lasiocarpa, 55

Abies pectinata, cambium, cul-
tures of, 127

abscission, auxin a factor in,
210

Acer pseudoplatanus, grafts in
cambium cultures, 213

achenes, sterilization of, 118

acid, ascorbic, 99

acid, boric, 103, 104, 105

acid, chromic (see potassium
dichromate)

acid, humic, 56

acid, indole-acetic, effects on
growth, 98, 105, 179, 182,
186-188, 194, 202

acid, nicotinic, 97, 98, 100, 103,
104, 105

acid, sulfuric, as nutrient, 105
(see also cleaning solution,
71)

acids, amino (see amino-acids)

Addicott, 203, 241; A. and Bon-
ner, J., 97, 241; Bonner, J.,
and A., 34, 57, 93, 100, 131,
185, 230

adventitious embryos, origin and
cultivation of, 220

adventitious roots, 50, 88, 120

aeration, effect on differenti-
ation, 194

aeration, effect on geotropic re-
sponse, 192

aeration, requirements of roots,
56

Aesculvs, 252

agar, 102, 104, 105

air-conditioning, of culture and
transfer rooms, 74, 77

alanine, 31

alcohol, for cleaning glassware,
71

alcohol lamps for flaming in-
struments, 83

alcohol sterilization of tissues,
119

algae, growth in stock salt solu-
tions, 104

Allium, 28

amino-acids, 93, 99, 100

amino-nitrogen, as measure of
growth, 156

Amoureux; Berthelot and A.,
199, 247

amphibian embryos, 14, 15

animal tissue cultures, 21-30

Antirrhinum, cultures of seed
primordia, 206

apical dominance, in roots, 55

aposporous embryos, 220

apple seeds, aseptic removal of,
118

Appleman, 211, 252

area of cultures, measurement
of, 158

Arnold sterilizer, 69, 107

Arnon, 70, 93, 236; A. and
Stout, 70, 93, 237; Stout and
A., 70, 93, 237; Hoagland
and A., 70, 237

ascorbic acid, 99

asepsis, 117 et seq.

asparagin, 31

Aspidium, 28

atmospheric humidity, 134

264

Plant Tissue Culture

aucuba mosaic virus, 201

autoclave, 68

autonomy of cells, 4

auxin, effects of, 210-212

Avena, 252

Avena coleoptiles, 219

Avery and La Rue, 50, 245; A.,

Burkholder, and Creighton,

211, 252; La Rue and A., 45,

233
Axtman; Bonner, J., and A.,

98, 242

bacterial digestion of fibrin,
101

bacteriological contaminations,
74, 83, 121

bacteriologist's hood, for trans-
fers, 74

Baker, 22, 90, 101, 240, 244;
B. and Carrel, 22, 90, 240,
241; Carrel and B., 22, 90,
241

baker's yeast, 101

balances, 71

bark, removal for cultivation,
115, 122

Barker; Bennet-Clark, Green-
wood, and B., 94, 245

Bartley; Bobbins and B., 35, 95,
96, 97, 133, 157, 185, 239,
243; Robbins, B., and White,
V. B., 35, 231; Robbins,
White, V. B., McClary, and
B., 93, 237 (see also Robbins
and Schmidt [Schmidt=B art-
ley] 35, 95, 133, 157, 158, 185,
232, 243, 244)

bean pods, aseptic seeds from,
118

bean pods, use in testing wound
substances, 57, 114

beech, cambium cultures from,
123

Begonia, 28, 125

Behre, 47, 50, 247

belly epithelium of toad, 15

Bennet-Clark, Greenwood, and
Barker, 94, 245

Berge; Riker and B., 199, 250

Berkefeld filters, 107

Berry; Steward, B., and Broyer,
56, 246

Berthelot, 93, 104, 237; B. and
Amoureux, 199, 247

Berthelot 's solution, 104

Berzin; Thielman and B., 94,
247

Beta vulgaris, 19

biotin, 99

Blakeslee; van Overbeek, Conk-
lin, and B., 27, 220

Bloch; Sinnott and B., 58, 126,
251

blood as a nutrient, 10

blood plasma, 21, 22, 90

Bloom, 228

Bobilioff-Preisser, 28, 234

Bocconia, 27

Bonner, D.; Bonner, J., and B.,
99, 242

Bonner, J., 34, 96, 97, 105, 125,
132, 185, 234, 242; B. and Ad-
dicott, 34, 57, 93, 131, 185,
230; B. and Axtman, 98, 242;
B. and Bonner, D., 99, 242; B.
and Buckman, 97, 245; B. and
Devirian, 97, 242; B. and En-
glish, 57, 114, 126, 252; B.
and Koepfli, 187, 188, 252;
Addicott and B., 97, 98, 241;
Hamner and B., 212, 253; van
Overbeek and B., 99, 246

Borger, 28, 58, 228, 234

boric acid, 103, 104, 105

boron, 70, 92

Bouillenne, M. ; Bouillenne, R.,
and B., 212, 242

Bouillenne, R., and Bouillenne,
M., 212, 242; B. and Went,
210, 248

Bcysen-Jensen, 210, 212, 219,
252

Brachet, 218, 255; B. and Sha-
piro, 218, 256

branches, effect on measure-
ments of roots, 160

branching habit, auxin a factor
in, 211

Index

265

brass rings, as culture chambers,

81, 149
Brassica, 28
Brassica nigra, apical dominance

in roots, 55
Brassica rapa, 19
Braun and White, P. R., 221,

259; White, P. R., and B.,

52, 124, 221, 252
brewer 's yeast, 101
' ' Brewer 's yeast — Harris, ' ' 101
bromine, as sterilizing agent,

118, 119
brown sugar, 95
Broyer; Hoagland and B., 56,

245; Steward, Berry, and B.,

56, 246
Bryophyllum, 28, 125
Bryophyllum calycinum, 249,

250
Buckman; Bonner, J., and B.,

97,245
buckwheat roots, 63, 64, 105
bud cultures, 151
buffering effect of complex nu-
trients, 186
Bufo vulgaris, 15, 257
Bnrkholder; Avery, B., and

Creighton, 211, 252
Burlet, 118, 228, 242; Geiger-

Huber and B., 98, 187, 243
Burrows, 21, 22, 35, 131, 235,

236; Carrel and B„ 22, 236

C
calcium, 92, 96, 185
calcium hypochlorite, 119
calcium nitrate, 103, 104, 105
calcium sulfate, 105
callus "blasen," 194, 206
callus cultures, 52, 53, 99, 105
"calots," 66
cambial activity, initiation and

control by auxin, 211
cambium cultures, 33, 36, 116
cambium cultures, isolation of,

115, 121, 122
cambiums, 48-50 (see also,

meristems)
Camptosorus, 48, 125

cancer, 2
Cannon, 56, 245
Canti; Fell and C, 256
carbohydrate, 57, 93, 94-96
carbon, 92
carboys, Pyrex, 69
Carrel, 21, 22, 26, 35, 71, 75, 90,
131, 183, 228, 236, 241; C. and
Baker, 22, 90, 241; C. and Bur-
rows, 22, 236; Baker and C,
22, 90, 240, 241
Carrel flasks, 80
Carriere, 13, 248
carrot, 49, 99

carrot, cultures from 105, 124
caulocaline, 212, 217
cell theory, 4, 35
cellular reactions, 193-194
cellular totipotency, 4-8
celluloid rule, for measuring

roots, 128, 161
cereals, roots, cultivation of, 199
Chambers, 33, 131, 148, 151, 231
chemotropic response, 192
chlorides, 112
chlorine, 92
chlorine, as sterilizing agent,

119
Chlorophytum, 125
Chlorophytum datum, 250
chromel wire loops, 82, 85
chromic acid (see potassium di-

chromate)
' ' chymical implements, ' ' 66
Citrus, fruits, aposporous em-
bryos in, 220
"clarite," 151
Clark-Kennedy, 260
cleaning procedures, for glass-
ware, 71, 72
cleaning solutions, 69, 71
clones, importance of, 132
clover roots, cultures of, 40, 105
clover roots, dominance in, 55
clover roots, root cap, 61
coagulants, 101, 102
cobalt chloride, 105
coconut milk, as a nutrient, 27
colchicine, effects on carrot cul-
tures, 202

266

Plant Tissue Culture

eoleoptile, Avena, transplanta-
tion of, 219
Coleus, 28
Coleus arifolia, 19
colony shape, 133, 158
commercial sugar (see sucrose)
companion cells, phloem, meri-

stematic character of, 58
Compo sitae, bud formation on

roots, 47, 51
concentration gradients, 217
Conklin; van Overbeek, C, and

Blakeslee, 27
contaminations, 74, 83, 121
convection currents, 74
Cooper, 210, 212, 252, 253
copper, 96
copper sulfate, 105
cortical tissues, retention of

meristematic function by, 43,

58
Corydalis solida, 6
cotton plugs, 73
cotyledons, regeneration in, 47
cover glasses, 150, 151
Crassula, 28
Creighton; Avery, Burkholder,

and C, 211, 252
criteria of response to external

influences, 184-194
Crocker; Zimmerman, C, and

Hitchcock, 255
crocks for cleaning solution, 71
crown gall, 199, 209, 221
crown-gall tissue, cultivation of,

53, 124, 216
cucumber mosaic virus, 200
Cucurbita, 28

culture conditions, 144 et seq.
culture period, length of, 142,

143
culture rooms, 76
culture techniques, 131 et seq.
curvature, tropic, effect of auxin

on, 212
Cuscuta, 202
cuttings, development of roots

on, 88, 121
cyclosis, 194
cystei'n-HCl, 105

cystoscopic scissors, 83
cytology, 202
Czech, 28, 234

D
Dakin solution, 119
dandelion root, polarity and bud

formation, 51
Datura, 27, 220
Daucus, 28

Dauphine, 47, 203, 234, 248
Day, 98, 242
Delarge, 133, 248
Delisle, 211, 253
Demuth; Fischer and D., 22, 90,

101, 241
developmental patterns, 210-219
deviation from norm, growth

rates of cultures, 163-165
Devirian; Bonner, J., and D.,

97, 98, 242
dextrin, 95
dextrose, 32, 105, 107
diameter of roots, 159
diastase, digestion products as

nutrients, 31
Dicranum, 28
Dietrich, 45, 233
differentiation, as affected by

oxygen supply, 194, 223, 226
digests, peptic, of fibrin, 90
diphtheria bacilli, 156
disaccharides, 95
distilled water, 69, 70, 92
diurnal rhythms, of water secre-
tion and of respiration, 190
dominance, 55, 136, 159
Drew, 22, 90, 237, 238
Drosera, 247

drupes, sterilization of, 118
dry weights, as measure of

growth, 143, 157
Duhamet, 99, 187, 202, 242
Dusseau; Nobecourt and D., 34,

199, 250
dwarfness, auxin a factor in, 211

E
Ebeling, 22, 29, 90, 96, 133, 158,
240, 247, 256, 261; E. and
Fischer, 29, 30, 256

Index

267

egg, fertilized, as meristem, 44
egg cells, cultivation of, 45, 219
EichJwrnia crassipes, 20
electropotential gradient, 217
electrostatic potential, 218
elementary organism, 4, 29
' 'elixir proprietatis, ' ' 66
embryo juice as nutrient, 22, 27,

90
embryogeny, 218, 219
embryological watch glasses, 81,

ISO, 132
embryology, experimental, 219
embryos, adventitious, 220
embryos, amphibian, 14, 15
embryos, plant, 45-47
embryos, undifferentiated, 46, 47
endodermis, meristematic char-
acter of, 43
endosperm, abortive, 45, 220
endosperm, liquid, as nutrient,

27
energy sources, 94—96
English; Bonner, J., and E., 57,

114, 125, 126, 252
Ephrussi, 228
epicotyl, proliferative capacity

of, 48
epidermal tissue, meristematic

character of, 44
epidermis, cultivation of, 28
Epilobium, 254

epithelial cells, animal, in cul-
ture, 29
epithelium, 2, 30
epithelium, belly of toad, 15
epithelium, glandular, of kidney,

2
epithelium, of iris, 2
Erdmann, 228
erect habit, auxin as a factor in,

211
Erlichman, 90, 238; Vogelaar
and E., 22, 71, 90, 91, 239, 244
errors of measurement, 162 et

seq.
Erxleben; Kogl, Haagen-Smit,

and E., 212, 253
Essenbeck and Suessenguth, 45,
233

ether-alcohol, for cleaning cover

glasses, 150
ethyl-methacrylate, 151
Eutettix tenellus, 245
excised roots, early cultivation

of, 31-36
excision procedures, 87, 88
exudate, phloem, toxicity of, 27
eye-cup, of frog, 15

F
Fell, 131, 132, 184, 256; F. and
Canti, 132, 256; F. and Lan-
dauer, 132, 256; F. and Robi-
son, 132, 256, 257
ferric chloride, 109
ferric nitrate, 109
ferric sulfate, 103, 105, 106, 108,

109, 180, 181
ferric tartrate, 105
fertilized egg cells, as material
for morphogenetie study, 218-
219
fertilized egg cells, meristematic

character of, 44
fibrin digests, as nutrients, 90,

101
fibroblasts, apparent loss of toti-

potency in, 29, 30
Fiedler, 34, 95, 99, 100, 102, 131,

159, 187, 192, 229, 231
Fife and Frampton, 218, 245
Filatow, 15, 30, 257
fir, white, 55

Fischer, 29, 30, 210, 229, 257; F.
and Demuth, 22, 90, 101, 241;
F. and Farker, 143, 210, 257;
Ebeling and F., 29, 30, 256
flaming of tubes, practice, 76, 83
flasks, Carrel, 80, 81
flasks, culture, 131
flax roots, 105
floating of roots, importance of,

140
flower-forming hormones, 212
foliar leaf, differentiation of, 2
form, origin of, 1, 2, 11, 12
Frampton; Fife and F., 218, 245
Fraxinus Ornus, 19
frog tissues, 14, 15, 21

2(>8

Plant Tissue Culture

fructose, 94

fruits, fleshy, membranaceous
and woody, aseptic seeds from,
118

function, origin of, 1, 2, 11, 12

fungi, as parasites on tissue cul-
tures, 199

fungoid growth of cambium cul-
tures, 123

G

galactose, 95

Galligar, 34, 231, 239

ganglia, nerve fibers from, 209

Gardner and Marth, 211, 253

gas, toxicity of, 75, 76

gas interchange as a morpho-

genetic factor, 192, 194
Gautheret, 33, 34, 36, 38, 49, 50,

96, 99, 102, 104, 105, 116, 122,

123, 127, 144, 148, 158, 184,

203, 213, 221, 229, 231, 232,

234, 242, 245, 248; Plantefol

and G., 189, 246
Geiger-Huber, 98, 187, 243; G.

and Burlet, 98, 187, 243
Geinitz; Spemann and G., 259
gelatine, 102
' ' gelose, ' ' 104
gemmae, stipular, of Marattia,

125
gene mechanism, 30
genes, auxin in relation to, 211
genetic constitution and stabil-

ity, 132
geotropic response, 192, 210
Gey, G. O., 131, 146, 236; G. and

Gey, M. C, 146, 236
Gey, M. C; Gey, G. O., and G.,

146, 236
Gilchrist, 218, 257
Gioelli, 122, 199, 203, 249
glandular function of roots, 189,

190
glandular hairs, cultivation of,

20
glassware, 79 et seq.
glucenium sulfate, 105
glucose, 31, 94

glycine, 31, 99, 103, 104, 105, 106

glycocoll (see glycine)

Goebel, 6, 7, 217, 249

Goodwin, 211, 253

gourds, 13

grafting, 7, 13, 14, 15

grafts, in vitro, 33, 213

granulation of wounds, 2

grasses, meristems of, 48

Greene, 224, 225

Greenwood; Bennet-Clark, G.,

and Barker, 94, 245
Grossenbacher, 190, 245
growth, distribution of, in the

body, 10, 11
growth, restrictions on, 10
growth habit, 57, 142
growth measurements, 154 et

seq.
growth rates, 57, 141
Gruenewald scissors, 82
guard cells, loss of proliferative

capacity, 44
Guilliermond, 33
Gustafson, 210, 253
Gymnosperms, sterilization of

seeds, 118

H
Haagen; Elvers, H., and

Muckenfuss, 184, 259
Haagen-Smit; Kogl and H., 99,

253; KogL H., and Erxleben,

212, 253
Haberlandt, 8, 9, 10, 16, 18, 20,

21, 27, 28, 30, 31, 32, 33, 35,

44, 100, 126, 184, 234, 245,

249, 253
hairs, glandular, stamen and

stinging, cultivation of, 20
hairs, root, 62, 63
Hales, 66
Hamner and Bonner, J., 212,

253
hanging-drop cultures, 148, 149,

151 et seq.
hanging-drop cultures, equip-
ment, ISO, 149 et seq.
Hannig, 45, 233

Index

269

Harris yeast, 101

Harrison, 9, 14, 21, 23, 30, 35,

90, 131, 183, 209, 210, 219,

229, 236, 257
Heidt, 33, 231
Eelianthus annuus, 251
Helianthus tuberosus, 251
heterologous grafts, 221
heterosis, 220
Heyman scissors, 82
histograms, use of, 175
histology, 202, 203
Hitchcock; Zimmerman and H.,

255; Zimmerman, Crocker,

and H., 255
Hoagland and Arnon, 70, 237;

H. and Broyer, 56, 245
hollow-ground slides, 130, 149
homologous grafts, 221
hood, bacteriological, 74
hood, chemical, 77
hormones, 93, 96, 210-212
Hort, 42
humic acid, 56
Huxley, 218, 258
hybrid peaches, 45
hybrid tomatoes, 220
hydrogen-ion gradients, 217
hydrostatic gradients, 217
hydrostatic potential, 218
hydrostatic pressure, 190
Hyoscyamus niger, 254
hyperhydrie enlargement, 206
hypocotyl, prolif erative capacity

of, 47
hypotheses, working, 3

immature ovules, cultivation of,

58
immersion heater, 83
implements, 82 et seq., 85
incompatibility, 220
increment rates, 155 et seq.
indole-acetic acid, 98, 105, 179,

182, 186-188, 194, 202
innervated cells, 209
inoculation procedures, 138 et

seq.
insect galls, 126

insolation, danger from, 144
intercalary meristems, 48, 49,

124, 151
intumescences, 114
iodine, 92

iodine, effects of deficiency, 188

iridectomy scissors, 82

iris, epithelium, 2

iron, 92

iron, effects on growth, 180, 181

iron, optimal concentrations of,

185
iron chloride, 109
iron nitrate, 109
iron sulfate, 103, 105, 106, 109,

180, 181
iron tartrate, 105
isopleth diagrams, use of, 176
isotonicity, 94

J
Jaeger, 43, 48, 249
Jensen 's No. 16 virus, 202
Johnson, B.; Naylor and J., 44

125, 250

Johnson, D. S., 34, 46, 47, 260
juvenile cells, 48

K

Kalanchoe, 28

Kemmer, 28, 234

Kisser; Klein and K., 118, 249

Klein and Kisser, 118, 249

Knop's solution, 31, 104

Knudson, 32, 234, 239; K. and

Smith, R. S., 32, 239
Koepfli; Bonner, J., and K., 188,

252; Thimann and K., 210,

255
Kbgl and Haagen-Smit, 99, 253;

K., Haagen-Smit, and Erxle-

ben, 212, 253
kohl-rabi, cultivation of tissues

of, 124
Kotte, 31, 32, 33, 34, 35, 36, 37,

56, 94, 100, 101, 131, 192, 231
Krontowski, 229
Kubowitz; Warburg and K.,

184, 247
Kunkel, 28, 234
Kuster, 28, 126, 229, 249

270

Plant Tissue Culture

laboratory, 67 et seq.
Laibach/210, 243
Lamium, 20
Lamprecht, 28, 234
Landau er; Fell and L., 256
La Rue, 45, 47, 233, 235; L. and
Avery, 45, 233; Avery and L.,
50, 219, 245
lateral meristems, 48
lead, toxicity of, 92
leaf-mesophyll, cultivation of,

27, 43
leaf-prirnordia, cultivation of,

219
Le Farm, 210, 253
legume nodules, 199
Lehmann, 211, 254
lens, origin of in amphibian eye,

15
Leonian and Lilly, 188, 243
leucine, impurities in, 100
leucocytes, 224, 225
Lewis, K. H., and McCoy, 199,

249
Lewis, M. R., 22, 25, 34, 90, 96,
238, 240; L. and Lewis, W. H.,
22, 34, 90, 131, 149, 238
Lewis, W. H., 22, 24, 74, 81, 90,
147, 238, 258; L. and Lewis,
M. R., 22, 229
banes, 48
Liebig 's meat extract, use in

nutrients, 31, 32, 100
Lilly; Leonian and L., 188, 243
limb-buds, vertebrate, 2
line graphs, use of, 174
bnear increments, as measure of

growth, 159
Locke, 90, 238
Locke 's solution, 90
Loeb, 32, 79, 229, 249, 250
Loo, S. W.; Loo, T. L., and L.,

34, 100, 243
Loo, T. L., and Loo, S. W., 34,

100, 243
loss of function, 4
Lund, 217, 245; Rosene and L.,

217, 251
Lupinus alb us, 234, 242, 245

lymph, use as nutrient fluid, 10,
21

M

MacDougal, 44, 250; M. and
Shreve, 44, 260

macrophage, 30

magnesium, 92, 96, 185

magnesium sulfate, 103, 104,
105

maize roots, 98

malignancy, origin of, 221

maltose, 95

Malus, 252

Malyschev, 33, 95, 131, 144, 158,
231

Maneval; Robbins and M., 32,
95, 144, 157, 232, 246

manganese, 92

manganese sulfate, 103, 104, 105

mannose, 95

manometer for measuring root
pressure, 196, 197, 198

Marattia, 125

Marotto, 34, 231

Marth; Gardner and M., 211,
253

Mason and Phillis, 94, 260

Maximow, 81, 131, 258

Maximow embryological watch
glass, 81

Mayer, 33, 237

McClary, 97, 245; Robbins,
White, V. B., M., and Bartley,
93, 237

McCoy; Lewis, K. H., and M.,
199, 249

measurement, methods, 128, 154
et seq.

meat extract, Liebig 's, as nutri-
ent, 31, 32, 100

media, 90 et seq.

media room, 68 et seq.

medullary ray parenchyma, pro-
liferative capacity of, 43, 48

medullary rays, 58, 125

megaspore-mother-cell, 2
melanotic epithelium, of am-
phibia, 30

Melchers, 212, 254

Index

271

mercuric chloride, as disinfec-
tant, 119

meristematic function, retention
of, 58

meristematic regions, 57

meristematic tissues, 2 07, 215

meristems, 44 et seq., 59

meristems, intercalary, 48

meristems, lateral, 48

meristems, terminal, 50

metastatic tumors, 221

methionine, as impurity in leu-
cine, 100

mica cover glasses, 150

migration of cells, 148, 158

migration rate of viruses, 200

Moebius, 58, 125, 235

mobility of cell surfaces, 11

Molliard, 33, 47, 125, 235

monocotyledonous plants, 94

monosaccharides, 95

morphogenesis, 3-12, 13, 14, 15,
209 et seq.

mosaic diseases, 199-202

motility of cells, 11

Muckenfuss; Rivers, Haagen,
and M., 184, 259

Mueller, 100, 156, 240, 260

Musa seminifera, 45

mustard, 55

mustard roots, 105

N
Nagao, 99, 245, 246

nasal scissors, 82

Naylor, 44, 125, 250; N. and

Johnson, B., 44, 125, 250; N.

and Sperry, 44, 125, 250
necrotic lesions, 194, 201
nerve fibrils, origin of, 183, 209
neuroblast, 21
niacin (see nicotinic acid)
nickel chloride, 105
Nicotiana, callus cultures, 53,

207, 208, 214, 215, 223-226
Nicotiana glutinosa, 201
nicotinic acid, 97, 98, 100, 103,

104, 105
nitrates, 112
nitrogen, 92

nitrogenous materials, 93

Nobecourt, 33, 34, 36, 50, 96, 97,
99, 218, 229, 233, 246, 260; N.
and Dusseau, 34, 199, 250

nodules, bacterial, formation on
legumes, 199

notebooks, 129, 168

nucleated cells, cultivation of,
44

nutrient concentrations, sensitiv-
ity to, 57, 185

nutrient salts, 92, 93

nutrients, 90 et seq.

O

oak, cultivation of cambium, 123

oak, white, 55

obligate parasites, cultivation
of, 194, 199

Okado, 58, 250

oligodynamic organic sub-
stances, 96

omission of nutrient constitu-
ents, 110, 111

optimum concentrations, 185

optimum temperatures, 76

orchids, undifferentiated em-
bryos of, 46

organic complexes, in nutrients,
22, 90, 91, 100, 101

organic materials, 93 et seq.

organic nitrogen, 99

organism as a whole, 7, 29

Ornithogalum, 20

osmotic agents, 94

osmotic value, 57

ovaries, cultivation of tissues
from, 57, 125

Overbeek, van, 99, 210, 211,
246, 254; O. and Bonner, J.,
99, 246; O., Conklin, and
Blake slee, 27; Blakeslee and
O., 220

ovules, immature, cultivation of,
58

oxygen, 92

oxygen gradients, effect on dif-
ferentiation, 194, 218

oxygen-hydrogen gradients, 217

272

Plant Tissue Culture

Faah 210, 219, 254

palisade cells, cultivation of, 20

Palladin, 104, 260

parasites, facultative and obli-
gate, cultivation of, 194, 199-
202

parenchyma, cortical, medullary
ray, xylem, retention of meri-
stematic function, 43, 44, 58

Parker, 29, 67, 71, 75, 119, 131,
133, 143, 148, 149, 151, 152,
158, 210, 229, 258; Fischer
and P., 143, 210, 257

pathogens, facultative and obli-
gate, cultivation of, 194, 199-
202

pathology, 194 et seq.

patterns, developmental (mor-
phogenetic), 7

pea root, 105

pea seeds, digest of, as nutrient,
31

peaches, hybrid, 45

Pearsall and Priestley, 217, 250

Pelece; Thielman and P., 99,
244

pellicle, of animal cell, mobility
of, 11

pellicle, of plant cell, rigidity of,
9

Teperomia, 28

Peperomia hispidula, 46, 47, 260

Feperomias, undifferentiated
embryos in, 46, 47

pepsin, 101

peptic digests, 90, 101

peptone, in nutrients, 31, 101

percentages, use of 172, 173

pericycle, retention of meriste-
matic function by, 43, 125

Petri-dish cultures, 132

petroleum oil, toxicity of, 92

Pfeiffer, 28, 148, 235, 260

pH, 57, 218

phagocytosis, 11

phanerogamic parasites, 202

rhaseolus vulgaris, 242

phelloderm, as material for cul-
tures, 125
phellogens, 48

Phillis; Mason and P., 94, 260
phloem exudate, as nutrient, 27
phloem parenchyma, meriste-

matic character of, 43
phloem sap, 9
phosphatase, 184
phosphates, 112
phosphorus, 92
photosynthesis, 20, 189
phototropic response, role of

auxin in, 210
phyllocalines, 212
physiologically sterile plants, 45,

220
pierced slide, 80, 81, 82, 149
pigmentation of amphibian em-
bryos, 14, 15
pine, cultivation of cambium of,

123
Tisum, 28
pith, meristematie capacity of,

43, 58
pith cells, cultivation of, 20
placental tissue, cultivation of,

58
planimeter, use in measuring

growth, 158
plant extracts, toxicity of, 27
PlantefoL 189, 246; P. and

Gautheret, 189, 246
plasma, use as nutrient, 21, 90
plasmolytic value, 94
plasmoptysis, 182 , 194
plastids, 193

plumule, rooting of, 48, 50
polarity, 5-7, 217, 218
polyembryony, causes of, 220
Folypodium, 28
poplar, cultivation of cambium,

123
Populus nigra, 19
Fortulaca oleracea, cultivation

of embryos, 54
potassium, 92
potassium, effect of deficiency

of, 188

Index

273

potassium chloride, 103, 104, 105

potassium dichromate, 71

potassium iodide, 103, 104, 105

potassium nitrate, 103, 104, 105

potassium phosphate, acid, 105

potato tuber cultures, 124

Pothos celatocaulis, 19

Prat, 27, 28, 260

" Precision" still, 70

preparation of nutrients, 102 et
seq.

Priestley, 48, 217, 250; P. and
Swingle, 48, 250; Pearsall and
P., 217, 250

proboscis, insect, 218

procambial strands, technique
of excision, 123

procambial tissues, 48, 49

procambium, cultivation of, 49

procumbent habit, auxin a fac-
tor in, 211

protein-N, measurement of, 156,
157

proteoses as nutrients, 101

Prunus avium, 251

Pulmonaria, 20

"pulvis fulminans, " 66

Putnam, G. P., and Son, 42

pyridines, 97

pyridoxine, 98, 100, 103, 104

pyrimidine, 97

Q

qualitative reactions, 186 et seq.
quartz cover glasses, 151
quartz still, 70
Quercus alba, 55

K
rabbit 's eye, plant callus in, 224,

racks, for cover glasses, ISO, 152
radish roots, 105
Bana esculenta, 15, 257
Sana palustris, 14
Bana sylvatica, 14
Razdorskii, 45, 233
Rechinger, 19, 20, 235
records, 129, 168 et seq.
redox-potential, 218

regeneration, 6, 19, 30

Reiche, 58, 126, 254

reimplantation of tumors, 221

replications, 165 et seq.

respiration, 189

re-utilization of nutrient con-
stituents, 144

rhizocaline, 212

Bhoeo, 28

Richards, 71, 72, 260

Riker and Berge, 199, 250

Ringer, 90, 239

Ringer's solution, 90

rings, brass, as culture cham-
bers, 81, 149

Rivers, Haagen, and Mucken-
fuss, 184, 259; R. and Ward,
184, 259

Robbins, 27, 32, 33, 34, 36, 50,
56, 74, 82, 94, 98, 100, 101,
105, 131, 143, 157, 185, 209,
220, 231, 240, 243, 250; R. and
Bartley, 35, 95, 96, 97, 133,
157, 185, 239, 243; R. and
Maneval, 32, 95, 144, 157,
232, 246; R. and Schmidt, 35,
95, 98, 105, 133, 157, 158, 185,
232, 243, 244; R. and White,
V. B., 27, 35, 95, 133, 203, 232,
240; R., Bartley, and White,
V. B., 35, 231; R., White,
V. B., McClary, and Bartley,
93, 237

Robison; Fell and R., 256, 257

roller tube cultures, 146, 147

root caps, 61

root cultures, 117-121

root hairs, 62, 63

root nodules, auxin a factor in
formation of, 211

root pressure, 172, 190, 191, 196,
197, 198

root tips, cultivation of, 31, 50,
56

rooting response, 188

roots, adventitious, 50, 88, 120

roots, localization of initiation,
211, 212

Rosene, 190, 217, 246; R. and
Lund, 217, 251

274

Plant Tissue Culture

rust infections, 199

Rytz; Schopfer and R., 76, 244

S

Sachs, 220, 251, 254

Saeger, 76, 246

Saintpaulia, 28

Saintpaulia ionantha, 250

' ' sal volatile oleosum, ' ' 66

Salix eapraea, cultures of cam-
bium, 116, 123

Salix eapraea, respiration of
cambium cultures, 189

"salt-cellars" (culture dishes),
132

salt mixtures in nutrient solu-
tions, 90

saltations, 133

salts, 71, 92, 93

Sambucus nigra, grafts in cam-
bium cultures, 213

sand furnaces, 66

sap, xylern, as nutrient, 27

scalariform cells, differentiation
of, 194, 208

scalpels, 82, 83, 85

scar tissue, 2

Scheitterer, 28, 48, 148, 151, 235

Schilling, 43, 251

Schleiden, 5, 8, 230

Schmidt; Robbins and S., 35,
95, 98, 105, 133, 157, 158, 185,
232, 243, 244

Schmucker, 27, 43, 91, 235

Schneider, 230

Schopfer and Rytz, 76, 244

Schotte, 30, 258

Schwanitz, 7, 251

Schwann, 4, 5, 8, 230

scions, 7

scissors, cystoscopic, 83

scissors, Gruenewald, 82

scissors, Heyman, 82

scissors, iridectomy, 82

scissors, nasal, 82

seasonal variations in growth,
77,75

secondary thickening, 159

secondary tumors, 221

seed primordia, cultivation of,

125, 151, 206
seedling roots, as culture ma-
terial, 117
segregation of functions, 4
segregation of tissues and cells

for study, 5
Seifriz, 62
Seitz filters, 107
semi-solid culture media, 101,

102
Sempervivum, 28
serum, as nutrient, 90
serum bottles, 73
Shapiro; Brachet and S., 218,

256
shock of excision, 48
Shreve; MacDougal and S., 44,

260
silica still, 70
silver still, 70
sink, 69
Sinnott and Bloch, 44, 58, 126,

251
Skoog; Thimann and S., 99, 210,

255, 260
slides, depression (hollow-

ground), 130, 149
slides, pierced, 80, 81, 130, 149
Smith, L. H., 28, 47, 251
Smith, R. S.; Knudson and S.,

32, 239
Snow, 210, 211, 254
soap, 71
sodium, 92
sodium acid phosphate, 103, 104,

105
sodium chloride, 176
sodium pyrophosphate, 71
sodium sulfate, 103, 104
softener, water, 70
"soft" glass cover glasses, 150
soil solution, 56
Solarium, 28

Solatium tuberosum, 233
Solidago, 253

somatic cells, totipotency of, 30
Spemann, 30, 219, 258; S. and

Geinitz, 219, 259

Index

275

Sperry; Naylor and S., 44, 125,

250
Spirodela, 246
squash fruit, aseptic seeds from,

118
squash stem, cultivation of pro-
cambium, 49
stamen hairs, cultivation of, 20
Stanley, 200, 259
starch, resorption of, 193
statistical analysis, 165-168
statistical significance of re-
sults, 166
stele, 195

Stellaria media, 193, 235
stem tips, 50
sterilization of nutrients, 106,

107
sterilizer, Arnold, 69, 107
Steward, Berry, and Broyer, 56,

246
stills, 69

stinging hairs, cultivation of, 20
Stingl, 45, 58, 233, 251
stipular gemmae of Marattia, as

culture material, 125
stock cultures, 113, 132
stomatal guard cells, cultivation

of, 20
stone cells, 222
stone fruits, 45
Stout and Arnon, 70, 93, 237;

Arnon and S., 70, 93, 237
Strangeways, 230
Stukeley, 66

substrata, nutritive (see nutri-
ents)
sucrose, 95, 96, 103, 105, 106,

185
Suessenguth; Essenbeck and S.,

45, 233
sugar, 71
sulfur, 70, 92
sulfuric acid, 71, 105
sunflower, cultivation of callus

from, 52
sunflower roots, 105
suspensor, 219

Swingle; Priestley and S., 48,

250
Symphoricarpus, 28

T
tap water, 70, 92
Taraxacum, 220
temperature, optimal, 76, 144,

145
temperature effects, 57, 76, 144,

145
temperature gradients, 218
terminal growing points, 50
terminal meristems, 50 et seq.
test-tube cultures, 131, 146, 147
Theophrastus, 42, 260
thiamin, 97, 100, 103, 104, 106,

108, 185
thiazole, 97

Thielman (Tauja-Thielman),
28, 95, 235, 239; T. and
Berzin, 94, 247; T. and Pelece,
98 99 244
Thimann, 99, 211, 244, 255; T.
and Koepfli, 210, 255; T. and
Skoog, 99, 210, 255, 260; T.
and Went, 210, 244
Thunbergia, 28
tile, for walls, 75
' ' tinctura metallorum, ' ' 66
tissue broth, as nutrient, 90
tissue culture, 9, 12, 35
tissue juices, toxicity of, 27
titanium sulfate, 105
titration of virus concentration,

200, 201
tobacco, cultivation of procam-

bium, 49
tobacco callus, 105
tobacco fruits, aseptic seeds

from, 118
tomato roots, 40, 62, 64, 98, 105,

113, 179, 180, 181, 182
tomato seeds, 118
totipotency, 4, 8, 12, 30, 35, 44,

212
toxicity of gas, 75, 76
toxins, inorganic, 202
toxins, organic, 202

276

Plant Tissue Culture

Tradescantia, 20, 28

transfer room, 74—76

transplantation, 7, 8, 15, 30, 219

traumatin, 114

trays, enamel and galvanized, 72

trees, cambium, cultivation of,

49
trees, cambium, cultures of, 127,

213
trees, cambium, excision of, 121-

123
trichomes, loss of meristematic

function in, 44
Trifolium repens, 55
Triticum, 28
Triturus torosus, 257
tropic response, auxin as factor

in, 212
tropisms, 192
Tukey, 45, 58, 73, 125, 220, 233,

235, 251
tumefacient property of cells,

221
tumor metabolism, 184
tumors, 221
Tyrode, 90, 239
Tyrode's solution, 90
tyrosinase, 193, 205
tyrosine, 194, 205

U

tlehla, 28, 44, 101, 235

Vlmus campestris, 248

ultraviolet light, use as steriliz-
ing agent, 118

undifferentiated tissues, cultiva-
tion of, 34, 133

ungcrminated embryos as cul-
ture material, 46-48

uniformity of cultures, 133

Urtica, 20

"Uspulin," as disinfectant, 119

vaccines, cultivation of, 184
van Tiegham cell, 81, 149
variability, 133 et seq.
vascular strands, disorganized,
216

vascular strands, organization
of in culture, 190

vascular tissue, importance for
regeneration, 19

vaseline for sealing slide cul-
tures, 151, 152

Vicia faba, 250, 252, 255

Viola, 28

Virchow, 5, 259

virus molecules, determination
of shape, 79

viruses, cultivation of, 120, 199,
200

Viscum, 202

vitamin Bly 96, 105 (see thia-
min)

vitamin Ba, 98, 105, 108, 172 (see
pyridoxine)

vitamins, 93, 96, 99

vitreous sink, 69

Vbchting, 5, 7, 8, 30, 217, 251

Vogelaar and Erlichman, 22, 71,
90, 239, 244, 261

Vogt, 218, 230, 259

W
wall color, 75
Warburg, 184, 189, 247; W. and

Kubowitz, 184, 247
Ward; Rivers and W., 184, 259
washing of air, 75
washing glassware, 71, 72
watch glasses, Maximow, 81
water, distillation of, 70, 92
water, filtration, 92
water, purity, 70, 91, 92
water softener, 70, 92
Wehnelt, 57, 125, 126, 244
weight vs. length (as measure of

growth), 159
Weiss, 259
Went, 210, 211, 212, 219, 220,

255; Bouillenne, R., and W.,

210, 248; Thimann and W.,

210, 244
Werckmeister, 45, 234
Westinghouse immersion heater,

83
wheat roots, 105
white clover, root cap, 61

Index

277

white oak, 55

White, P. B., 31, 34, 36, 39, 45,
49, 50, 53, 54, 55, 57, 58, 61,
63, 64, 76, 77, 92, 93, 95, 96,
97, 98, 99, 100, 101, 105, 118,
119, 120, 122, 123, 125, 131,
133, 139, 144, 145, 146, 151,
159, 175, 176, 180, 184, 185,
188, 190, 191, 192, 193, 194,
195, 196, 197, 198, 200, 202,
203, 206, 207, 208, 209, 214,
217, 218, 220, 223, 226, 230,
232, 233, 234, 235, 237, 239,
240, 244, 247, 251, 252, 259;
W. and Braun, 52, 124, 221,
252; Braun and W., 221, 259

White, V. B.; Bobbins and W.,
27, 35, 95, 133, 203, 232, 240;
Bobbins, Bartley, and W., 35,
231; Bobbins, W., McClary,
and Bartley, 93, 237

Wilcoxon; Zimmerman and W.,
255

Wilhelm, 58, 252

Willmer, 230

willow, cambium cultures, 105,
123

willow, polarity in, 7

Wilson, 119, 260
Winkler, 58, 252
wire racks for cover glasses, 130,

152
wound healing, 2, 21, 43
wound hormones, 114

xylem fibers, loss of meriste-

matic function by, 44
xylem parenchyma, proliferative

capacity, 58
xylem sap, 9, 27

yeast, brewer's vs. baker's, 101
yeast, extract as nutrient, 32,
100, 105

Zimmerman, 56, 247; Z. and
Hitchcock, 210, 255; Z. and
Wilcoxon, 210, 255; Z.,
Crocker, and Hitchcock, 210,
255

zinc, 70, 92, 96

zinc sulfate, 103, 104, 105

Zizania aquatica, 233