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Plant Growth Substances 

This Volume is Published in Celebration of the 

of the Founding of the University of Wisconsin 


A. J. Haagen-Smit 
Kenneth V. Thimann 
Thomas Kerr 
Hans Burstrom 
Albert F. Blakeslee 
P. W. Zimmerman 
R. H. Burris 
W. D. Bonner, Jr. 
G. S. Christiansen 
George S. Avery, Jr. 
Frederick G. Smith 

A. R. Schrank 
John W. Mitchell 
J. M. Beal 

B. Esther Struckmeyer 
K. C. Barrons 

R. S. Dunham 
Frank E. Gardner 
J. van Overbeek 

Philip R. White 
Nancy Kent Ziebur 
Folke Skoog 
Cheng Tsui 
John R. Raper 
Gilbert M. Smith 
A. E, Murneek 
R. H. Roberts 
Felix G. Gustafson 
Robert M. Muir 
S. H. Wittwer 
R. S. de Ropp 
Albert C. Hildebrandt 
A. J. Riker 
J. E. Thomas 
T. C. Allen 
E. H. Newcomb 
Esmond E. Snell 
E. L. Tatum 
William J. Robbins 

"^ - .■> 


Edited by FOLKE SKOOG 








Front row: R. H. Roberts, R. H. Burris, and f. Lederberg. 

Back Row: K. P. Buchholtz, J. T. Curtis, T. C. Allen, F. Skoog, and A. J. Riker 

(xMissing, O. N. Allen) 

Symposium meetmgs were held in the Wisconsin Memorial Union Theater 

Publisher's Note 


OR bibliographical reasons, Professor Folke Skoog, chairman of the 
committee responsible for this book, has been designated editor. 
Listing volumes of essays written under separate authorship presents 
problems to bibliographers which do not easily lend themselves to prac- 
tical solution. The Press, therefore, feels that scholars will be grateful for 
a simple entry under which this book may appear in files, catalogues, 
and bibliographies. 


THE concept of hormonal regulation of growth dates back nearly a 
century to Julius Sachs, who deduced from his experiments on 
plants that special substances are responsible for the formation and 
growth of different organs. However, definite proof of hormonal action 
in plants was not obtained until 1926-28 when Went demonstrated a 
growth substance, auxin, in the tip of the oat seedling. This discovery 
marked the culmination of a long period of quantitative experimentation 
on the nature of plant movements and the beginning of a new approach 
to the study of plant growth. 

Twenty years ago the work on plant growth substances was the 
preoccupation of a few botanists in European laboratories, who dealt 
with fractions of a milligram of an active material, not yet chemically 
defined and present in plants in concentrations too small to be detected 
except through its physiological activity. In this country the first small 
laboratory for plant growth substance research was being constructed 
in Pasadena by Herman Dolk, as a result of T. H, Morgan's vision and 
interest in the subject. From this slow beginning, the work has developed 
with increasing rapidity, so that it now influences all branches of botany 
and has had far-reaching agricultural applications. 

Today several thousand persons are engaged in investigations, manu- 
facture, and applied work on growth-regulatory compounds with physio- 
logical properties resembling those of the material originally obtained 
from the oat seedling. In this country synthetic plant growth regulators 
have become one of two main groups of organic chemicals for agricultural 
use. For the purpose of weed control alone nearly twenty million acres 
of crops were treated with these compounds during the past year. 

A rapid stream of new information pertaining to the physiology, 
biochemistry, chemistry, and agricultural uses of growth substances is 
now pouring out from laboratories all over the world. To gain a per- 
spective of the present status and progress in the field as a whole, a 


committee representing different departments at the University of Wis- 
consin engaged in one phase or another of plant growth substance work 
arranged this symposium. The committee felt that, particularly since 
many of the persons who are responsible for the early development of 
the subject are still active and would be available for participation, 
much could be gained not only from a formal program of lectures 
covering principles, main lines of investigations, and recent develop- 
ments, but even more from group discussions and individual contacts 
between persons engaged in the fundamental and appHed aspects of the 

The present volume contains the papers presented In general meetings 
and round table discussions, September 5-7, 1949. 

The generosity of the Wisconsin Alumni Research Foundation in 
providing a grant through the University Research Committee of the 
Graduate School and of the Knapp Fund in providing a grant through 
the Wisconsin Centennial Committee has made this symposium possible. 

On behalf of the Growth Substances Research Committee grateful 
acknowledgement is made to the sponsors and to all members of the 
University Administration and Faculty who helped to plan and conduct 
the meetings. 

Folke Skoog 
Madison, Wisconsin, April 6, igso. 


Table of Contents 


The History and Nature of Plant Growth Hormones 3 

A. J. Haagen-Smit, California Institute of Technology 

The Synthetic Auxins: Relation Between Structure and Ac- 
tivity 21 
Kenneth V. Thimann, Harvard University 

Growth and Structure of the Primary Wall 37 

Thomas Kerr, United States Department of Agriculture Experi- 
ment Station, Beltsville, Maryland 

Mechanisms of Cell Elongation 43 

Hans Burstrom, University of Lund, Sweden 

Control of Evolution and Life Processes in Plants 57 

Albert F. Blakeslee, Smith College 

Twenty Years of Plant Hormone Research 67 

F. W. Went, California Institute of Technology 

Plant Hormones in Practice 81 

P. W, Zimmerman, Boyce Thompson Institute for Plant Re- 


The Study of Growth Substances in Plant Metabolism 93 

R. H. Burris, University of Wisconsin 
Changes in Metabolism During Growth and Its Inhibition 97 

K. V. Thimann, W. D. Bonner, Jr., and G. S. Christiansen, 

Harvard University 
Stimulation of Respiration in Relation to Growth 105 

George S. Avery, Jr., Brooklyn Botanic Garden 



Respiratory Changes in Relation to Toxicity hi 

Frederick G. Smith, loiva State College 


Electrical Polarity and Auxins 123 

A. R. Schrank, University of Texas 

Translocation of Growth-Regulating Substances and Their 
Effect on Tissue Composition 141 

John W. Mitchell, United States Department of Agriculture 

Experiment Station, Beltsville, Maryland 

Histological Responses to Growth-Regulating Substances 155 

J. M. Beal, University of Chicago 

Comparative Effects of Growth Substances on Stem Anatomy 167 

B. Esther Struckmeyer, University of Wisconsin 

Formative Effects of Hormone-Like Growth Regulators 175 

P. W. Zimmerman, Boyce Thompson Institute for Plant Re- 


Vegetation Control on Non agricultural Land 187 

K. C. Barrons, Dow Chemical Company 

Differential Responses in Crop Plants 195 

R. S. Dunham, University of Minnesota 

Growth Substances in Relation to the Production of Tree 
Fruits 207 

Frank E. Gardner, United States Department of Agrictdture 

Experiment Station, Orlando, Florida 

Use of Growth Substances in Tropical Agriculture 225 

J. van Overbeek, Agricultural Laboratory, Shell Oil Company, 
Modesto, California 


The Role of Growth Substances in Vegetative Development 
as Exemplified in Tissue Cultures 247 

Philip R. White, Institute for Cancer Research, Philadelphia 


Factors Influencing the Growth of Plant Embryos 253 

Nancy Kent Ziebur, University of Wisconsin 

Growth Substances and the Formation of Buds in Plant Tis- 
sues 263 
Folke Skoog and Cheng Tsui, University of Wisconsin 

The Development of Stems and Leaves 287 

F. W. Went, California Institute of Technology 


Chemical Regulation of Sexual Processes in Fungi 301 

John R. Raper, University of Chicago 

The Sexual Substances of Algae 315 

Gilbert M. Smith, Stanford University 

Growth-Regulating Substances in Relation to Reproduction 
OF Some Horticultural Plants 329 

A. E. Murneek, University of Missouri 

The Induction of Flowering with a Plant Extract 347 

R. H. Roberts, University of Wisconsin 

Fruit Development as Influenced by Growth Hormones 351 

Felix G. Gustafson, University of Michigan 

The Growth Hormone Mechanism in Fruit Development 357 

Robert M. Muir, University of Iowa 
Growth Substances in Fruit Setting 365 

S. H. Wittwer, Michigan State College 


Experimental Induction and Inhibition of Overgrowths in 
Plants 3°^ 

R. S. de Ropp, New Yor{ Botanical Garden 

In Vitro Experiments on Tissues of Pathological Origin 391 

Albert C. Hildebrandt, University of Wisconsin 
The Interaction Between Causative Agents in Diseased 
Growth 4^5 

A. J. Riker and J. E. Thomas, University of Wisconsin 


Deformities Caused by Insects 411 

T, C. Allen, University of Wisconsin 

Comparative Studies of Metabolism in Insect Galls and Nor- 
mal Tissues 417 
E. H. Newcomb, University of Wisconsin 


Growth Factors in Bacterial Nutrition 431 

Esmond E. Snell, University of Wisconsin 

Genetic Aspects of Growth Responses in Fungi 447 

E. L. Tatum, Stanford University 

Vitamin and Amino Acid Requirements for the Growth of 
Higher Plants 463 

William J. Robbins, New Yor\ Botanical Garden 

Plant Growth Substances 

The History and Nature of Plant Growth Hormones 


A LOOK at the index of the Biological Abstracts provides some 
indication of the general interest in the field of plant growth 
substances. In 1930 only fifty papers appeared, but now this number has 
increased to a few hundred a year. 

With the growing number of papers on this subject, there has arisen 
a confusion in the use of the terms growth substance, growth hormone, 
regulator, phytohormone, formative substance, and auxin. To avoid 
further confusion I shall adopt Thimann's (50) recent suggestion on the 
nomenclature of the auxins and phytohormones, which is as follows: 
"An auxin is an organic substance which promotes growth (i.e. irre- 
versible increase in volume) along the longitudinal axis, when applied 
in low concentrations to shoots of plants freed as far as practical from 
their own inherent growth promoting substances. Auxins may, and 
generally do have other properties, but this one is critical." 

This definition excludes sugars which promote longitudinal growth 
when the term "low concentration" is interpreted as meaning below 
0.00 1 molar. It also excludes nutrient salts and is intended to exclude 
substances such as mafic and other organic acids which promote growth 
of the Avena coleoptile in a 0.00 1 molar concentration and less, but 
only in the presence of auxin. In this concept, van Overbeek's (46) 
criterion that an auxin should be active in the Avena test is replaced by 
a more general statement on the longitudinal growth of shoots. This 
change made it possible to include also substances active in other tests 
such as the pea test. 

A phytohormone has been defined as: "An organic substance produced 
naturaUy in higher plants, controUing growth or other physiological 
functions at a site remote from its place of production, and active in 


minute amounts" (50). This definition includes those auxins which are 
of natural occurrence, certain of the vitamins, and other hormones such 
as those stimulating wound growth and the postulated hormones of 
flowering. It brings in certain restrictions regarding the place of forma- 
tion relative to the place of action not present in the original hormone 
concept of Fitting (13, 14). Since industry has recognized the sales value 
of the term hormone, many synthetic preparations appear under this 
name. It has, therefore, been suggested that the requirement for natural 
occurrence be omitted, thereby including the synthetic substances found 
to have growth effects on plants. This meeting of many plant-hormone 
workers might effect some agreement as to the proper use of the terms. 

The first efforts to determine the chemical nature of plant growth 
stimulators were made by Fitting (13, 14) forty years ago. While visiting 
the Dutch East Indies he made a study of the effects of poUination on the 
orchid flower and noticed that pollen initiated swelUng of the ovaries 
and other phenomena of post-flo ration. These effects could be reproduced 
by the application of dead pollen as well as by water extracts of poflen. 
A test method was devised whereby pieces of cotton wool soaked in the 
active extracts were brought in contact with the stigma, after which 
the growth effects could be observed. Fitting made several attempts 
to fractionate the pollen extract and found that the hormone was 
soluble in alcohol but insoluble in petroleum ether and ether. He even 
looked for sources other than orchid pollen and found that saliva caused 
similar responses. 

Although Fitting promised the continuation of his work, no further 
data were available until Laibach (34) showed that the active pollen 
substance was probably identical with one of the auxins. If Fitting had 
continued his studies the history of the plant hormones might have 
been quite different. After discovering the activity of saliva other excre- 
tions such as urine might have been tried, and using Fitting's test 
method, the auxins might have been isolated. Their parthenocarpic 
effect might then have been discovered directly. Other actions such as 
cell elongation in Avena, root formation, and the like, would have been 
recognized as additional properties of the pollen hormone. There were 
several reasons why history did not take this course. The microchemical 
techniques were in their infancy, although Pregl at just about the same 
time developed his organic microanalytical methods. The available physi- 
ological tests would not have been adequate for the accuracy and the 


number of routine analyses necessary for isolation of the actiye material. 
Moreoyer, most of these problems require research budgets, which, in 
the time of Fitting and long afterward, were extremely small. Fitting 
makes special mention of the fact that he bought some of the orchids 
himself. Finally, the borderline nature of the problem required the 
cooperation of seyeral workers, and team work in science belongs to a 
much later date. 

At about the time of Fitting's work, another field of plant physiology 
dealing with phototropic and geotropic phenomena was rapidly develop- 
ing. As early as 1880 it had been shown by Darwin (9) that some influence 
is transmitted from the upper to the lower part in seedhngs when they 
are exposed to one-sided illumination. Boysen-Jensen (7,8) showed in 
19 13 that this stimulus can be transmitted through a gelatin layer by 
cutting off the tips of Avena coleoptiles and pasting them on again with 
gelatin. After one-sided illumination the curvature appeared not only 
in the tip but also in the lower part. Boysen-Jensen was very cautious in 
the explanation of this phenomenon and assumed that a concentration 
change in the tip was responsible for this effect. 

Paal (48) in 19 19 demonstrated that the stimulus did not cross a 
layer of cocoa butter, mica, or platinum foil, and that a tip placed on one 
side of the coleoptile caused curvatures similar to those seen in photo- 
tropic experiments. He postulated the existence of a diffusible correlation 
carrier, a substance which is produced in the tip and moves downward. 
Phototropic effects were explained by an interruption of the normal flow 
of the substance through interference in its action due to some change 
in the protoplasm. This clear formulation of a substance as the active 
agent was an important step forward. 

Several unsuccessful attempts were made to collect the active material 
from crushed coleoptiles until Went (63) showed that it diffused into 
gelatin from living coleoptile tips. For the measurement of the growth 
effect Went improved the technique introduced by Stark and Drechsel 
(54). This made it possible to conduct the physiological experiments on 
a quantitative basis, a prerequisite for the chemical isolation of the 
growth substance. Because of the importance for all the subsequent work 
let us briefly review this measuring method and its later modifications. 

For this test etiolated oat seedlings are used, from which, three hours 
before testing, the tip is removed. After the primary leaf has been 
detached from its base and a second decapitation has been performed, 


an agar block containing the active material is placed on one side of the 
cut surface of the coleoptile, the primary leaf thereby serving as a 
support. After no minutes the curvature caused by the growth dif- 
ference between the two sides is measured. The curvatures are, up to 
a certain concentration, proportional to the amount of growth substances 
applied. While Went used this test primarily to measure the growth 
substance diffusing from tips into gelatin or agar, this method has been 
extended to the quantitative assay of extracts obtained by chemical 
procedures. This introduces complications which are often underesti- 
mated. Salt concentration, pH, and the preparation of the agar blocks 
have been shown to affect the curvatures. A more thorough investiga- 
tion of these factors will undoubtedly increase the reliability of the 
results obtained. The Avena test has a high degree of sensitivity; 25 
gammas of indoleacetic acid per Hter gives a curvature of about 10°. 
This means that each plant receives 1/20,000,000 milligram of growth 
substance. A three- to fivefold increase in the sensitivity of this test 
was obtained by removing the seed without damaging the embryo, 
twelve to eighteen hours before the test (53). Straight growth measure- 
ments have also been used for the determination of the auxins and 
resulted in the development of the coleoptile cylinder method, whereby 
short pieces of the coleoptiles are immersed in the test solution. The in- 
crease in length is proportional to the logarithm of the concentration (4). 

A test which has found wide application was devised by F. W. Went 
(66) who observed that the internodes of pea stems, when spUt 
lengthwise, curved inward when in contact with auxin solutions. The 
inward curvature is due to the difference in growth between the two 
sides of the organ, the outer side being more sensitive to auxin stimulation 
than the inner side. The pea test has been of great value in the testing 
of a number of synthetic substances, which, because of their lack of 
transportability, do not react in the Avena test. In comparing the 
activity of these substances it would be beneficial to apply the same 
rigid standardization to the pea test as Went introduced for the Avena 

Many plants, among them the Avena, show this behavior, and it 
might be of advantage to use this more uniform material. Using Avena 
a greater sensitivity was obtained by splitting the coleoptile into quarters 
instead of halves (60). A further refinement of this method consists 
of removing the quarters containing the vascular bundles, which have 


a somewhat lower sensitivity than the other two quarters (52). With 
this method indoleacetic acid can be measured in concentrations of one 
gamma per hter. The curvature observed is proportional to the logarithm 
of the concentration. 

For special purposes other test methods have been developed in which 
the auxins are taken up in lanoHn or other water-insoluble material and 
applied to different regions in the plant. These methods are especially 
useful for work outside the laboratory where the conditions cannot be 
as strictly controlled as is necessary for the Avena test. 

In principle, any reaction of plants to the auxins can be used for 
measuring auxin activity. For example, the sweUing of decapitated 
stems of Viciafaba has been utiHzed for an assay method (33). Hitch- 
cock and Zimmerman (22) have made use of the fact that application 
of auxins changes the angle between the stem and the petiole in a 
number of plants. 

In carrying out measurements of auxin activity all experimenters are 
aware of fluctuations in the sensitivity of the test object, although the 
temperature, humidity, and Hght conditions are kept constant. Measure- 
ments of the same amount of indoleacetic acid at different hours of the 
day gave values which may differ considerably from the average value. 
No explanation of this interesting phenomenon has yet been given, and 
attempts to prevent these variations have not been successful. In de- 
terminations where results have to be compared with those previously 
obtained, it is therefore general practice to test known concentrations 
of indoleacetic acid at the same time and express the results as indole- 
acetic acid equivalents. With proper technique the measurement of 
the over-all growth activity of active material has reached a satisfactory 
degree of accuracy, and the difficulties at the present time are found in 
the interpretation of these results. 

Quantitative determination of the growth-hormone content of ex- 
tracts can be seriously influenced by the presence of growth inhibitors. 
While the study of these inhibiting substances is of importance for 
understanding the mechanism of plant growth, it also has a direct 
bearing on the evaluation of the auxin analysis. In low concentrations 
of the growth substances these interfering agents may cause a positive 
curvature. In higher concentrations the activity curve may be con- 
siderably depressed and consequently give low values for the amounts 
actually present. Lipoid-soluble inhibitors have been found in extracts 


of corn, tomato, radish, and other plants. In testing for the inhibiting 
action on Avena coleoptiles it is necessary to supply the plant with 
sufficient growth hormone so that depression of the activity can be 
measured. Larsen (38) measured in this way the inhibitory effects of the 
seed-germination inhibitors, anemonin andparasorbic acid. Effects were 
noticed, respectively, in concentrations of o.i gram per liter and of 
i.o gram per liter. The biological activity of a given amount of indole- 
acetic acid is neutralized in the Avena test by 2x10^ to 2x10^ times the 
amount of parasorbic acid. The number of known, naturally occurring, 
inhibiting substances is rapidly increasing, and future determination 
of the auxin content of plant and animal extracts must take these facts 
into account. It seems necessary, therefore, that in these determinations 
activity curves be compared with those of known growth substances. 
If a significant difference occurs it will then be necessary to purify the 
extracts before measuring their activity. Recovery experiments, whereby 
a certain amount of synthetic growth hormone is added to the extract 
to be analyzed, will also be of value. Such a procedure is to be recom- 
mended in any auxin determination where extractions have been carried 
out. The large amounts of solvents used to extract the exceedingly small 
quantities of hormone may easily introduce considerable uncertainty 
about the result. A routine treatment of the ether with peroxide- 
destroying agents is not a guarantee for the reliability of the results. 
A good example of the effect of inhibitors on the curvature in the 
Avena test is found in a study made by Larsen (38). The addition of a 
neutral extract of tomatoes depresses the curve of both auxins consider- 
ably. The acid growth substance can be easily separated from such a 
neutral inhibitor. However, this is not possible with the neutral auxins. 
To compUcate matters further, inhibitors have been found which are 
acidic in nature. In such cases a comparison of growth curve of the pure 
auxin and that of the crude extract must be made in order to interpret 
the results correctly. 

The Avena test made possible important discoveries in the field of 
geotropism and phototropism. It was shown that these phenomena 
depended on the presence of the growth hormone and its lateral dis- 
placement. As a direct result of the availability of a testing procedure 
we may also list attempts to isolate the growth substances. The first 
was made by Nielsen (40,41) who found that two pathogenic fungi 
produced in the nutrient medium substances which were strongly active 


in the Avena test. This material proved to be thermostable, and soluble 
in water, ether, and alcohol. It was soon evident that auxin production 
was shown not only by the two fungi used by Nielsen, but also by other 
fungi, bacteria, and yeasts. In the isolation in Kogl's (25) laboratory in 
Holland several of these organisms were used as producers of starting 
materials, but attempts to isolate the growth substance from their media 
were abandoned when it was found that urine causes strong bending of 
the Avena coleoptile. After several purification steps the active agent 
was obtained in chemically pure form and proved to be a nitrogen-free 
substance (C18H32O5). 

Diet experiments showed that auxin secretion was directly connected 
with food intake, and when the secretion was found to be increased 
considerably by corn oil, this was used for isolation purposes (24). The 
auxin found was identical with the one from urine, but in addition a 
second nitrogen-free substance with auxin activity was obtained. These 
were distinguished by the names auxin-a and auxin-b. Using a different 
extraction procedure with urine, a third substance active in the Avena 
test was isolated (28). This substance proved to be 3-indoleacetic acid, 
a compound discovered by Salkowski (51) about 75 years before, during 
his investigations of protein decomposition. Indoleacetic acid was then 
isolated from yeast (29), and in this country Thimann (56) proved by 
isolation that the organism with which Nielsen had begun his studies, 
Rhizopus suinus, also produced indoleacetic acid. 

During the years immediately following the discovery of the action 
of indoleacetic acid as a growth hormone, it was generally assumed that 
this substance was formed only in lower organisms. This belief was 
supported by its isolation from Rhizopus and yeast and from the evidence 
obtained using indirect means of identification. However, evidence 
gradually accumulated that indoleacetic acid could be present in higher 
plants too. The final proof was supplied by its isolation from wheat and 
corn by extraction under mild alkaUne conditions (2,19), and from 
immature corn (18). 

As a result of Larsen's (37) investigations, indoleacetaldehyde must 
be added to our list of naturally occurring auxins, although it is recog- 
nized that its activity is due to its conversion into indoleacetic acid in 

the plant. 

The auxins-a and -b were shown to have the following structures 
(23,24). (See Fig. i). Auxin-a is a trihydroxy acid, whereas auxin-b 


CH3 -CHg CH3 




CH3 ^CHj CH3 

Ig Wl 12 


Figure i. Formulae for auxins-a and -b. 

contains only one hydroxyl and a ketone group. The relation between 
the two compounds is well established since it is possible to convert 
auxin-a to auxin-b by the use of dehydrating agents. It has been observed 
that the auxins gradually lose their activity, and this change is explained 
by an allyl rearrangement. (See Fig. 2). The positions of the hydroxyl 
and carbonyl groups are such that lactone formation takes place easily. 
This lactone in equilibrium with the free acid undergoes, upon ultra- 
violet radiation, rapid conversion to an inactive product named lumi- 
auxone. (See Fig. 3). This conversion also takes place by irradiation in 
the visible range of the spectrum when carotenoid pigments are present 
(30). While it was not yet possible to synthesize the entire molecule, 
a major part of the proposed structure was confirmed by synthesis of 
one of the degradation products, auxin-a glutaric acid. (See Fig. 4). 
The synthesis is compHcated by the presence of four asymmetric carbon 
atoms. Auxins-a and -b, with 7 and 5 asymmetry centers respectively, 
will present an even greater problem. 

The substances isolated fulfill all the requirements necessary for ac- 
tivity in the Avena test. They are transported from the top of the 
coleoptile to the base in a polar fashion. It has been shown that the 



* Oris uHj (-'Ha 

PSEUDOAUXIN-CIi CH3-CH2-CH-CH ):h-chch2-ch3 


Figure 2. Comparison of auxin-a with pseudo auxin-a. 



A — 



6 COH 





Figure 3. Conversion of auxin-a to lumiauxone. 

coleoptile cylinder test, and especially the pea test, does not require 
the transportability characteristic of substances which are active in the 
Avena test (57). When these tests are used in the isolation, substances 
will be found which do not possess all the qualities necessary for a true 
auxin, but which show the primary growth activity. Using the pea test 
as a guide, indican could have been isolated from urine. In plants 
substances like phenylacetic acid, occurring free and esterified in pepper- 
mint oil and in oil of neroli, would have been recognized as growth 
substances. The same is true for m-cinnamic acid, which occurs free 
and esterified in numerous plants, the most abundant sources being 
Peru and Tolu balsam. These substances could well be included in the 
list of naturally occurring growth substances since they show the primary 
growth activity. Their presence in the plant could hardly fail to cause 
some response from the neighboring cells. In some cases where they occur 
in oil glands and resin ducts, we could speculate that they played a role 
in the cell proliferation during the formation of the glands. 

As a result of the chemical investigations several substances occurring 
in plants and fulfilling the requirements for activity in the Avena test 
have been identified. The next question, therefore, is: which auxin 


9 A 9 

c-c c-c 

I I I I 

AUXIN-a ' 




■C— C 


CH3 CH2 
\ / \ 


Figure 4. Formulae for auxin-a and auxin-a glutaric acid. 


occurs in the Avena coleoptile tip and in other plants? For the identifica- 
tion of the auxins in plant tissues it would be preferable to isolate the 
active components in a pure condition. This is, of course, generally not 
feasible. In the case of the Avena tip which contains 1/50,000,000 of a 
milligram, it would take years to collect enough material. An additional 
difficulty in the case of auxins-a and -b is that both substances are con- 
verted into inactive products within a few months. 

Indirect methods of determination are therefore usually the only 
means of answering the question of which growth substances are present 
in plant tissues. Went used the molecular weight determination by 
diffusion as a means of determining the size of the active material 
diffusing from the Avena tip. Since that time this method has served in 
a number of cases to decide between the occurrence of auxins-a and -b 
or indoleacetic acid. Went found for the tip auxin a molecular weight of 
378, which was close to the molecular weight of auxin-a; but recent work 
has shown that when a diffusate purified by ether extraction is used, a 
much lower figure is found (32,71). We must accept this revision of 
the molecular weight with caution, since acid and alkali destruction 
tests were in agreement with the presence of auxin-a. It has been found 
in the case of the pure auxins that heating with hydrochloric acid 
destroyed indoleacetic acid, whereas auxin-a was resistant. On the other 
hand, treatment with sodium hydroxide showed the opposite effect. 
Auxin-b and indoleacetaldehyde were destroyed by both treatments. 
In the inactivation of indoleacetic acid by acid, oxygen plays an im- 
portant role, and it has been shown that in a nitrogen atmosphere no 
destruction takes place. Since the destruction method is used extensively 
it should be more carefully standardized. 

A third possibility of detecting the difference between the action of 
indoleacetic acid and the tip growth hormone is based on the phototropic 
behavior of plants. In the Avena coleoptile Went showed that with low 
light intensity of 20 to 100 meter candle seconds a lateral movement of 
the auxins takes place, causing an increased concentration at the dark 
side. At the same time there is a lowering of the total auxin content, 
this destruction being of the order of 25 per cent. If one or the other 
auxin is responsible for the phototropic curvature, artificial application 
of one of these substances to growth-hormone-free coleoptiles should 
give similar response to that of the intact coleoptile. This experiment has 
been carried out by Koningsberger and Verkaaik (31) and by Op- 


penoorth (42), who found that a coleoptile cylinder suppHed with auxin-a 
showed phototropic behavior, whereas those with indoleacetic acid did 
not. The destruction under the influence of Hght has been explained by 
the conversion of auxin-a-lactone to inactive lumiauxone. These changes 
take place rapidly by irradiation with ultraviolet light, and in the 
presence of both a- and /3-carotene the inactivation occurs in the visible 
spectrum. The proposed mechanism of phototropic action finds support 
in the presence of carotenes in the coleoptile and in the agreement 
between the spectral sensitivity of the coleoptile to light and the ab- 
sorption spectra of the carotenes. Recently, however, Galston (15) has 
shown that indoleacetic acid is easily destroyed by light when a suitable 
activator is present. Lactoflavin was found to have such an action, and 
rapid decarboxylation and oxidation is observed in vitro in sunlight, 
and the action spectra also agree with that of the coleoptile. Van 
Overbeek (44) found that the growth inhibition of Avena coleoptiles 
after exposure to light does not seem to occur when the growth hormone 
applied to the top of the coleoptile is indoleacetic acid instead of auxin-a. 

There is a possibility that an additional method of distinguishing 
indoleacetic acid and auxin-a may be found in the experiments of 
Guttenberg (17) on deseeded, derooted coleoptiles of Avena and hypo- 
cotyls of Helianthus annuiis. After one-sided application of tip growth 
substances, the curvatures appear during the first two hours, whereas 
those from application of indoleacetic acid take from 3 to 10 hours to 
develop. This slow reaction of indoleacetic acid is explained as being due 
to the production or release of auxin-a under the influence of indoleacetic 
acid. Further support for this theory is found in experiments whereby 
auxin-free coleoptiles are treated on the outside with indoleacetic acid. 
After extraction, the extracted growth substance behaves like auxin-a in 
acid and alkali stabihty tests. 

Another method which holds promise in distinguishing between the 
auxins could be based on enzymatic destruction. Thimann (58) observed 
a considerable loss in activity when auxin was incubated with leaf 
extracts of Vicia faba and Helianthus and attributed this effect to the 
enzymatic destruction of the auxin. The importance of this phenomenon 
for the regulation of plant growth was shown by van Overbeek (43), who 
found that dwarf corn contained greater than normal amounts of this 
destructive agent. Larsen (36) made some steps toward purification of 
the auxin inactivating substance and found it to be of enzyme nature. 


Tang and Bonner (55) showed that the optimum range of activity fell 
between pH 6.2 and 6.7. It is reported that the enzyme does not attack 
indoleacetamide, indolebutyric acid, indolepropionic acid, indolecar- 
boxylic acid, or tryptophan, and therefore seems to possess a considerable 
degree of substrate specificity. 

It would be desirable to have chemical methods to determine quanti- 
tatively the different auxins. Up till now such methods have been de- 
veloped only for indoleacetic acid. Salkowski (51), analyzing the products 
of putrefaction of proteins, found that one of the products formed gave 
a red color with nitrite or ferric chloride and a mineral acid. Both 
reactions have been carefully studied (39) and the optimum conditions 
for the reactions determined. Methods were developed whereby indole- 
acetic acid can be determined over a range of 5 to 200 gammas per 
milliliter. Especially recommended is the nitrite method, where the 
coefficient of variability is less than three per cent. The ferric chloride 
reaction has often been used for the identification of indoleacetic acid 
in plant material, as, for example, in the media of fungi. Wildman and 
Bonner (70) found that a considerable portion of the Avefia tip growth 
activity could be explained by the presence of indoleacetic acid. 

After discussing the question as to which auxins occur in plant tissues, 
another problem connected with their production, storage, transport, 
and action presents itself. It had been observed that a part of the auxins 
is readily extracted from the tissue, but that considerable additional 
amounts of auxins could be obtained by continued ether extraction 
or by treatment with hydrolytic agents. Thimann and Skoog (61) have 
shown that many ether extractions spaced over several months are 
required to extract all the ether-soluble auxins present in Lemna. They 
concluded from their experiments that the continued production of 
auxin in ether was due to an enzymatic liberation from an inactive form, 
probably a protein. These observations have since been confirmed by a 
number of investigators. Determination of free auxins prevents this 
enzymatic production by boiling previously frozen and ground material, 
or by carrying out the ether extraction at zero degrees. 

Wildman and Bonner (69) obtained from spinach leaves a fraction 
which is homogeneous in ultracentrifugation and electrophoresis experi- 
ments and has phosphatase action. This fraction gave, on treatment with 
alkali or proteolytic enzyme, a growth hormone which is probably 
identical with indoleacetic acid. The bound auxin seems a part of the 


broken molecule since repeated fractional precipitation of the protein 
with ammonium sulfate did not remove the auxins. 

In seeds free and bound auxins have also been shown to occur. In corn 
oil some of the auxin can be readily extracted with water, and consists 
mainly of auxins-a and -b. Hydrolysis of the oil gives additional quantities 
of auxins which are probably present in the ester form. When the entire 
seed of cereal grains is treated with dilute alkaU, amounts of auxins are 
found which are several times greater than those available from seeds 
after extraction with water. This liberated auxin was isolated and shown 
to consist mainly of indoleacetic acid (2,19). 

As Gordon and Wildman (16) have shown, some of this liberated 
auxin has its origin in tryptophan, which releases under mild treatment 
small quantities of indoleacetic acid measurable in the Avena test. It is, 
for example, unstable at 37°C. at pW 10.5, or in contact with cold 
phosphate buffer at pW 4.6. Even melting with agar releases some growth 
hormone. Similar degradations of tryptophan take place within the 
plant, and it has been shown that an enzyme is responsible for this 

These findings do not invalidate all conclusions based on previous 
auxin extraction methods. However, they must be considered in future 
work. When, for example, wheat or corn is treated at a pW of 10.5 for 
two days, an increase of 5 milligrams per kilogram in the auxin content 
is noted over the readily extractable auxin. As Avery and Berger (i) 
have shown, it is safe to conclude that this amount represents auxin in 
bound form and is not due to the conversion of proteins containing 
tryptophan. Avery and Berger purified the auxin complex and found 
it to be protein-like. Skoog (53) has shown in the Avena that inactive 
precursors originating in the seed travel upwards and are able to induce 
growth of the coleoptile after 2 to 6 hours. It is possible that the auxin 
complexes mentioned serve as the auxin reserve for the plant. Since 
Wildman et al. (71) discovered an enzyme which converts tryptophan 
to indoleacetic acid we have to include this substance and tryptophan- 
containing proteins as potential sources of auxins in the plant. 

The proportion of free to bound auxin varies greatly in different plants 
and plant parts. In hemna only two per cent is free according to Thimann 
(59), whereas the free auxins in Avena coleoptile tips account for nearly 
all of the total auxin. The study of the relative proportions of free and 
bound auxin is of importance for the understanding of several growth 


phenomena. For example, van Overbeek et al. (47) have shown that 
the geotropic response of horizontally placed sugar cane is due to the 
formation of free auxin at the expense of the bound form in the lower 
side of the node. 

In a search for growth factors other than auxins the effect on the 
seedling of removal of the endosperm was studied. Even after supplying 
sugars and mineral salts the growth of young seedlings is then greatly 
retarded. In experiments on pea seedlings it was shown that when a 
cotyledon was placed in the nutrient medium together with the embryo 
considerably better growth is obtained. Since it was known that several 
vitamins were present in the endosperm these were tried, and it was found 
that biotin, vitamin Bi, ascorbic acid, and even the animal hormone, 
estrone, caused increased growth of the seedlings over the controls. The 
addition of thiamin was characterized by a greatly increased root system 
(26). This effect is also observed in isolated roots, and Bonner (5) has 
shown that vitamin Bi is a major factor in the development of the root, 
and that its function is supplemented by pyridoxine and probably 
nicotinic and pantothenic acids (6). The material diffusing from the 
cotyledons is apparently rich in growth substances, a conclusion sub- 
stantiated when pea diffusate was found to be active in promoting leaf 
growth. Using the growth increase of circular discs of tobacco and radish 
leaves as a test method, adenine was isolated and recognized as one of the 
active agents. Embryonic pea leaves also showed increased growth under 
the influence of adenine. For this tissue, therefore, adenine acts as a 
typical plant hormone (3). Some investigators have found that the 
auxins also affect leaf growth. Went (65) has given the name rhizocaline 
to the complex of chemical factors other than auxin which are involved 
in root formation. They are found in buds and cotyledons and are formed 
by leaves in the presence of light. The nature of these substances is 
unknown. The same is true of caulocalines, factors which influence stem 
growth and are produced in roots, and the postulated flower hormones. 

We are better informed regarding the nature of a different type of 
growth activator: the wound hormones. Upon injury of a plant the intact 
cells surrounding the wound show a greatly increased rate of division. 
Wiesner (68) suggested in 1892 that special substances are probably 
produced by wounded cells. Haberlandt (20,21) found that the surface 
of potato tuber responded to the application of phloem tissue and to 
crushed cells or their extracts. Similar phenomena can be studied on 

F. VV. Went, left, and A. F. Blakeslee 

C. A. Elvehjem, left, and A. J. Haagen-Smit and Kenneth V. Thimann 

R. S. Dunham, K. C, Barrens, and K. P. Buchholtz 

Kenneth V. Thimann, George S. Avery, Jr., Frederick G. Smith, and 

R. H. Burris 

R. H, Roberts, left, and Felix G. Gustafson 

J. van Overbeek, left, and P. W. Zimmerman 

Left, R. Alexander Brink and E. L. Tatuni; center, Hans Burstrom; and right, 

Esmond E. Snell 

O. N. Allen, R. S. de Ropp, Albert C. Hildebrandt, T. C. Allen, A. J. Riker, 

and E. H. Newcomb 


other plants. Wilhelm (72) introduced a test method using the response 
of the parenchyma tissue which hnes the hollow stem of the Windsor 
bean, and Wehnelt (62) developed a test using the lining of the kidney 
bean pod. A drop of crushed tissue applied to this layer causes rapid 
cell division, and a small intumescence about one to two millimeters 
high appears. The height of this new tissue measured after 48 hours is 
to a large extent proportional to the concentration of the wound hor- 
mone. The beginning of the curve represents reactions nonspecific in 
nature, such as the result from appUcation of water, strong sugar solu- 
tions, and toxic substances. After fractionation of the bean pod juice, 
the active material proved to be an a, /3-unsaturated dicarboxylic acid 
of twelve carbon atoms (11). This i-decene-i,io-dicarboxylic acid has 
been named traumatic acid, and is active in amounts as little as o.i 
gamma in the bean pod. Since in growth a considerable expenditure 
of building material as well as stimulatory substances are necessary, it is 
not surprising that several cofactors can increase the activity of traumatic 
acid. Most striking is the effect caused by glutamic acid, which enhances 
the activity of the wound hormone about ten times. 

English (12) has prepared several analogues of traumatic acid, and 
shown that the activity is confined to the dicarboxylic acids. Both 
saturated and unsaturated acids are active, but the double bond, while 
not essential, increases the hormone action. For example, decane-1,10- 
dicarboxylic acid has only half the activity of traumatic acid. The 
structure of traumatic acid seems to indicate that it is a result of the 
breakdown of fatty acids or their derivatives such as lecithins and fats. 
No efforts have been made to establish its origin, and there is no informa- 
tion on its mode of action. 

Since the time that the hormone concept was introduced in plant 
physiology considerable progress has been made. Some of the correlation 
carriers have been isolated and their actions have been studied. Still 
many of the hormones remain to be discovered, and in addition a great 
number of new problems have appeared. In striving for simpHfication 
deductions in this field are often based on the results of work on a variety 
of plants all through the plant kingdom. These extrapolations have had 
a stimulating effect on the development of the hormone field, but with 
the increase in our knowledge it is evident that there are a large number 
of individual problems which require special investigation. Consequently, 
the complete picture of hormone action on plant growth will un- 



doubtedly be a complicated one. If we consider tliat the important 
applications in agriculture are due to the exploitation of only one of the 
hormones we can expect a great deal from the future. 


1. Avery, G. S. and Berger, J., Science, 98:513 (1943). 

2. Berger, J. and Avery, G. S., Am. J. Botany, 31:199, 203 (1944). 

3. Bonner, D., Haagen-Smit, A. J., and Went, F. W., Botan. Gaz., loi :i28 


4. Bonner, J.,/. Gen. Physiol., 17:63 (1933). 

5. , Science, 85:183 (1937). 

6. Bonner, J. and Addicott, F., Botan. Gaz., 99:144 (1937). 

7. Boysen-Jensen, p., Ber. deut. botan. Ges., 28:118 (1910). 

8. , ibid., 31:559 (1913)- 

9. Darwin, C. and F., The Power of Movement in Plants (John Murray, 

London, 1880). 

10. DoLK, H. and Thimann, K. V., Proc. Nat. Acad. Sci. U. S., 18:30 (1932). 

11. English, J. Jr., Bonner, J., and Haagen-Smit, A. J.,/. Am. Chem. Soc, 

61:3434 (1939)- 

12. , Proc. Nat. Acad. Sci. U. S., 25:323 (1939). 

13. Fitting, H., Z. Botan., 1:1 (1909). 

14. , ibid., 2:225 (19 10). 

15. Galston, a. W., Proc. Nat. Acad. Sci. U. S., 35:10 (1949). 

16. Gordon, S. A. and Wildman, S. G., /. Biol. Chem., 147:389 (1943). 

17. Guttenberg, H. von and Buchsel, R., Planta, 34:49 (1944). 

18. Haagen-Smit, A. J., Dandliker, W. B., Witwer, S. H., and Murneek, 

A. E., Am. J. Botany, 33:118 (1946). 

19. Haagen-Smit, A. J., Leech, W. D., and Bergren, W. R., ibid., 29:500 


20. Haberlandt, G., Sitz. ber. \. preuss. A\ad. Wiss., 318 (1913). 

21. , ibid., 1096 (1914). 

22. Hitchcock, A. E. and Zimmerman, P. W., Contrib. Boyce Thompson 

Inst., 9:463 (1938). 

23. Kogl, F. and Erxleben, H., Z. physiol. Chem., 227: 51 (1934). 

24. , and Haagen-Smit, A. J., ibid., 225:215 (1934). 

25. Kogl, F. and Haagen-Smit, A. J., Proc. Kon. A^ad. Wetensch. Amsterdam, 

34:1411 (1931). 

26. , Z. physiol. Chem., 243:209 (1936). 

27. Kogl, F., Haagen-Smit, A. J., and Erxleben, H., ibid., 214:241 (1933). 

28. , ibid., 228:90 (1934), 

29. Kogl, F. and Kostermans, D., ibid., 228:113 (^934)- 

30. Kogl, F. and Schuringa, G. J., ibid., 280:148 (1944). 

31. Koningsberger, V. J. and Verkaaik, B., Rec. trav. botan. tieerland., 

^35:1 (1938). 

32. Kramer, M. and Went, F. W., Plant Physiol., 24:207 (1949). 

33. Laibach, F. and Fischnich, O., Ber. deut. botan. Ges., 53:469 (1935). 

34. Laibach, F. and Maschmann, E., Jahrb. wiss. Botan., 78:399 (1933). 


35. Larsen, p., Naturwissenschaften, 27:549 (1939). 

36. , Plania, 30:673 (1940). 

37. , ylndoleacetaldehyde as a Growth Hormone in Higher Plants. Diss., 

Copenhagen (1944). 

38. , Am. J. Botany, 34:349 (1947). 

39. Mitchell, J, W. and Brunstetter, B. C, Botan. Gaz., 100:802 (1939). 

40. Nielsen, N., Planta, 6:376 (1928). 

41. , Jahrb. wiss. Botan., 73:125 (1930). 

42. Oppenoorth, W. F. F., Rec. trav. botan. neerland., 38:287 (1941). 

43. Overbeek, J. VAN, Proc. Nat. Acad. Sci. U. S., 21:292 (1935). 

44. , ibid., 22:187 (1936). 

45. , Plant Physiol., 15:291 (1940). 

46. , Ann. Rev. Biochem., 13:631 (1944). 

47. , Olivo, G. D., and de Vasquez, E. M. S., Botan. Gaz., 106:440 


48. Paal, a., Ber. dent, botan. Ges., 32:499 (1914). 

49. , Jahrb. wiss. Botan., 58:406 (1919). 

50. PiNCUS, G. and Thimann, K. V., The Hormones, I (Academic Press, 1948). 

51. Salkowski, E., Z. physiol. Chem., 9:8, 23 (1885). 

52. Santen, a. M. a. van, Groei, Groeistof en Ph., Diss., Utrecht (1940). 

53. Skoog, F., /. Gen. Physiol., 20:311 (1937). 

54. Stark, P. and Drechsel, O., Jahrb. wiss. Botan., 61:339 (1922). 

55. Tang, Y. W. and Bonner, }., Arch. Biochem., 13:11 (1947). 

56. Thimann, K. V., /. Biol. Chem., 109:279 (1935). 

57. , Proc. Kon. Al^ad. Wetensch. Amsterdam, 38:896 (1935). 

58. , /. Gen. Physiol., 18:23 (1934). 

59. , The Chemistry and Technology of Grotvth (Princeton University 

Press, 1949), p. 61. 

60. •-, and Schneider, C. L., Am. J. Botany, 26:792 (1939). 

61. , and Skoog, F., ibid., 27:951 (1940). 

62. Wehnelt, ^., Jahrb. wiss. Botan., 66:773 (1927). 

63. Went, F. W., Proc. Kon. A/{ad. Wetensch. Amsterdam, 30:10 (1926). 

64. , Rec. trav. botan. neerland., 25:1 (1928). 

65. , Proc. Kon. Akfld. Wetensch. Amsterdam, 37:445 (1934)- 

66. , ibid., 37:547 (1934). 

67. , and Thimann, K. V., Phytohormones (Macmillan, New York, 


68. WiESNER, J., Die Elementarstructur and das Wachstwn der lebenden Substanz 

(Vienna, 1892), p. 102. 

69. WiLDMAN, S. G. and Bonner, }., Arch. Biochem., 14:381 (1947). 

70. , Am. J. Botany, 35:740 (1948). 

71. WiLDMAN, S. G., Ferri, AI. G., and Bonner, J., Arch. Biochem., 13:131 


72. WiLHELM, A., Jahrb. wiss. Botan., 72:203 (1930). 

The Synthetic Auxins: Relation Between 
Structure and Activity 


THE discovery of the auxins in the early 1930's was accompanied by 
a great increase in physiological experimentation. This broadened 
the basis of our knowledge of auxins, for it brought to light the fact 
that the auxins control many activities of the hfe of plants. In addition 
to promoting cell elongation of stems and coleoptiles, which was the 
first known function, the following processes (reviewed in 10) are all 
controlled or promoted by auxins: i) the initiation of roots on stems; 
2) the initiation of roots on roots (but only to a hmited extent) ; 3) cell 
division both in the cambium and other stem tissues; 4) growth of the 
ovary into a fruit (involving both cell division and enlargement); 5) 
growth of parenchymatous tissues into tumors; 6) streaming of proto- 
plasm (studied only to a hmited degree). However, the development 
of buds and the elongation of roots are inhibited by auxin in all but the 
very lowest concentrations. 

The first widely held view of auxin action was that it acted in some 
way to increase the plasticity of the cell wall, thus allowing elongation 
to take place under the cell's own osmotic force. However, the participa- 
tion of auxin in so many processes, involving also cell division and the 
inhibition of enlargement, makes it clear that this concept is insufficient 
and that some much more profound action or group of actions must 
take place. This leads at once to the idea that auxin probably acts in 
some process of metabolism common to nearly all plant cells and capable 
of leading to varied results depending on the influence of additional 
factors on the cell. Such a process of metaboHsm would of course be 
enzymatic. Since the amount of auxin needed is far too small for it to 


be a substrate it must act catalytically and thus in some way become a 
part of some enzyme system. Tliis of course raises the question as to 
what enzyme systems are involved in growth. In addition, however, 
the participation of a small molecule in an enzyme system presupposes 
special properties in that molecule. Hence this idea focuses attention 
on the nature of the auxin molecule itself; what are the properties of the 
substance — a relatively simple molecule — which might enable it to act 
in an enzyme system? We are in a position to attempt an answer to this 
question now because of the large number of synthetic auxins that have 
been prepared. 

In other comparable cases, like those of the coenzymes and prosthetic 
groups, there are strict limitations of structure, and quite small changes 
in the molecule such as, for example, the substitution of two ethyl for 
two methyl groups in pyridoxine, make the compound inactive. In 
other cases, such as the replacement of the thiazole ring by a pyridine 
ring in thiamin, a relatively small change may make the compound a 
competitive inhibitor. The case of the auxins is somewhat diflerent since 
even the first known compounds, the naturally occurring auxins, show 
wide differences in structure and appear virtually unrelated. However, 
if we consider auxin-a (I) and indoleacetic acid (II) more carefully it 
will be seen that in fact they have many properties in common. In the 





first place, both have an unsaturated 5-membered ring with an acid 
group situated at a distance from the ring. Second, the presence of the 
N atom in the ring of one compound but not in that of the other indicates 
that this atom is not essential for activity. A study was therefore made 
of the activity of compounds resembHng indoleacetic acid but without 
the nitrogen. It was found at once (9) that indeneacetic acid, (III) is 
highly active; the ring N atom is therefore not important for activity. 
However, III shows some curious properties. In the Avena test it gives 




good curvatures, though with only about i per cent of the activity of 
indoleacetic acid, but the curvatures are hmited to a very short apical 
zone. In growth tests in which the plant parts are immersed in the solu- 
tion, the activity is much higher relative to that of indoleacetic acid 
than in the Avena test, reaching 20 per cent in the curvature of slit 
pea stems. Further, in the formation of roots on pea stems III has low 
activity when applied to the apex of the stem but is highly active when 


I I 

CHjCOOH ^<^ ^0' 

applied to the base; the difference is of the order of 100 times. (In later 
studies we have found this compound excellent for the rooting of woody 
cuttings, when appHed to their bases.) These observations show that the 
activity of III is being hmited by the ability of the plant to transport it. 
A similar analysis of the activity of benzofurane-2-acetic acid (IV) gave 
comparable though more extreme results. It is completely inactive in 
the Avena curvature test but active on immersed sections; and in root 
formation it is inactive when appHed to the tip but active at the base. 
Benzofurane-3-acetic acid (V), the spatial analogue of II, is about twice 
as active as IV but is also limited by transport. 

Activity in inducing growth thus depends not only on activity of the 
compound per se, but on its abiUty to be transported in the plant. It is 
obvious that there may be many other such secondary characteristics 
which influence the apparent activity of a compound. The following may 
be suggested: i) ability to enter the cell (solubility in the plasma 
membrane); 2) resistance to inactivation by plant enzymes; 3) stability 
to hght (in certain tests), or to the combination of light and photo- 
dynamic substances hke eosin, riboflavin, or carotenoids; 4) dissociation 
constant of the acid group, since the anion is in general less active than 
the undissociated acid. 

The influence of many such factors can be minimized by using a short- 
time test with a single standard tissue immersed or floating in a solution 
of known pW in darkness. The curvature of slit halves of etiolated pea 
stems has been most widely used and most of the conclusions which 
follow are based on this test. To avoid curvatures due to release of 
residual auxin in the tissues, the sht halves are placed in water for two 
hours before exposure to the test solution (16). 


Structural Requirements for Primary Activity. — Interpreting the pea 
test as indicative of primary growth activity, a great many substances 
have been prepared and tested. In general it appears that only compounds 
with an unsaturated ring system are active. The saturated compounds 
cyclohexaneacetic and decahydronaphthaleneacetic acids are both in- 
active. In the Avena test, even saturation of the heterocyclic double bond 
of indoleacetic acid abolishes activity (5). Further, all the active com- 
pounds are acetic, propionic, or butyric acid derivatives. Examples are 
naphthalene- 1 -acetic acid (VI), anthraceneacetic and even phenylacetic 
acid, but not cyclohexaneacetic acid. The salts are in general less active 
than the acids, though at the/?H of the cell (5.5-6.0) they are partly con- 
verted to the free acid. Acids stronger than indoleacetic acid (pK 4.75), 
however, are more fiilly dissociated, and their apparent activity should 
be corrected for the extent of dissociation (i). Esters of the acids are in 
general active, and methyl esters have about the same activity as the 
acid; ethyl esters, generally less. In the Avena test Kogl and Kostermans 
(5) found that activity decreased with the increasing size of the alkyl 
esterifying group and concluded that the esters must be hydrolyzed to 
the free acids to produce growth. It is probable that such hydrolysis is 
only necessary for transport and that, for primary activity, the ester 
is active per se. Amides present a similar problem. In the case of naphtha- 
leneacetamide, although the activity is lower than that of the free acid, 
the curve relating activity and concentration is quite different from that 
of the acid (11), whereas if hydrolysis were involved one would expect 
the two curves to be parallel. Further, at the optimum concentration 
no trace of ammonia could be detected in the solution (i i). It is probable, 
therefore, that amides too are active per se for primary activity. In one 
or two cases nitriles show activity, which can safely be ascribed to 
hydrolysis to the acid. 

Spatial configuration evidently plays an important part in determin- 
ing activity. In phenylacetic acid (VIII), substitution of one of the side- 
chain hydrogen atoms by a methyl group (IX), or of both of them by a 
methylene group (X), does not appreciably change the activity,* but 
substitution of them by two methyl groups (XI), abolishes it completely 
(3). This suggests steric hindrance and indicates that the carboxyl must 
bear a certain spatial relation to the ring. Clear evidence for this is given 

*The ^-propyl and ally! derivatives are also active, both being more active 
than phenylacetic acid, though the isopropyl derivative is almost inactive (14). 




















by the cinnamic acids; the aV-form (XII) is active and the trans-iorm 
(XIII), inactive. The /^-methyl and the o-methoxy derivatives of these 
two acids behave similarly. 

Putting all these facts together, Koepfli, Thimann, and Went ten 
years ago (3) concluded that the minimum structural requirements for 
primary activity are: i) a ring system as nucleus; 2) a double bond in 
this ring; 3) a side chain; 4) a carboxyl group (or a structure readily 
convertible to a carboxyl) on this sidechain at least one carbon atom 
removed from the ring; and 5) a particular space relationship between 
the ring and the carboxyl group. 

The term carboxyl should be broadened, however, because of the 
activity of naphthalene- i-nitromethane, which in the ad-iorm (\ II) 
has an acid group isosteric with a carboxyl (13). Indican also has weak 
activity (13), though no sulfonic acid yet tested is active. It may thus 
be that the requirement is merely for an acid group which is not too 
highly dissociated. 

The Ring. — The nature of the ring is important. As a rule 5-membered 
rings do not confer activity. Pyrroleacetic acid, in spite of its resemblance 
to the indole structure, is inactive, as are the acetic derivatives of 
imidazole, furane, and thiazole. Neither A^-cyclopenteneacetic nor 
A--cyclopenteneacetic acid is active, which is of interest in comparison 
with auxin-a (I), (11). The fusion of a 6-membered ring with the 5- 



membered ring confers activity, however, as shown by the acetic deriva- 
tives of indole, benzofurane, and thionaphthene. The activity of auxin-a 
and -b seems exceptional, but if we consider formula I it might be 
suggested that the secondary butyl group has almost the configuration 
of a ring. This phenomenon, of course, is well known in the case of the 
estrogens, where the substituted derivatives of stilbene reach a maximum 
activity with the diethyl derivative (XIV), which most nearly resembles 
estradiol (XV). Further studies with alkyl-substituted 5-membered rings 
are desirable to clear up this point. 

The need of unsaturation in the ring is characteristic and cannot be 
replaced by unsaturation in the side chain. Thus A^-cyclohexeneacetic 










(XVI) is active, but cyclohexylideneacetic (XVII) is not (3) ; indene-3- 
acetic (III) is highly active, but benzofulvenecarboxylic (XVIII) is very 
weak (11), although, of course, it retains unsaturation in the benzene 
ring. It is not that unsaturation in the side chain destroys activity, for 
aV-cinnamic acid(XII) and atropic acid (X) are active. Thus, irrespective 
of the degree of saturation of the side chain, there must be unsaturation 
in the ring itself. Veldstra in 1944 (13) brought forward evidence that 
the unsaturation of the ring is needed to confer interfacial or surface 
activity upon it. This evidence depends on the behavior of a series of 
substances when their current-voltage curves are determined with the 
dropping mercury electrode in acid methanol solution. Under these 
conditions the control curve passes through a sharp maximum, ascribed 
to oxygen adsorbed on the cathode, while active auxins strongly depress 
this maximum. Seven active compounds behaved in this way while 
seven inactive ones had no such effect. Eight other inactive compounds 
did depress the maximum, however. Veldstra concludes that the ring 
must have strong surface activity, that is, it must be capable of ad- 















sorption to some reactive surface, a property essential for activity but 
of course not sufficient by itself. Such a view agrees with the concept of 
a coenzyme or similar function discussed above. It should, however, be 
pointed out that A--cyclohexeneacetic acid (XIX) Is inactive, (compare 
the activity of XVI) from which it seems that the double bond must 
be adjacent to the side chain (3,17), as indeed it is in all the other active 
substances. This would suggest that the adsorption to a surface may not 
be as unspecific as it sounds and may require very definite configuration. 

The Side chain. — The acid group must be separated from the ring. 
In the Avena test (with the indole ring) the one methylene group is 
sharply optimal, but for primary activity the butyric acid with 3 
methylene groups has about the same activity. The propionic and valeric 
derivatives have definitely less activity in all tests, and this holds both 
for indole and for naphthalene compounds. 

There are one or two cases of carboxyl groups attached directly to the 
ring which must be considered. It was claimed that 2-bromo-3-nitro- 
benzoic acid was active, but its action appears to rest on synergistic 
effects rather than true primary activity. Veldstra and van de Westering 
(.14) have kindly made available their studies on the naphthoic acids; 



COOH ^/^ /CH 




the a-acid (XX) apparently has real but sUght activity, while the /3-acid 

(XXI) is inactive. However, hydrogenation of the substituted ring 

(XXII) increases the activity 5-10 times. It may be pointed out that in 
these compounds the COOH is attached to one ring but may be con- 
sidered separated from the other, that is, the compounds may be thought 
of as derivatives of phenylacetic acid. This is particularly true when the 
ring is hydrogenated. It is significant that the 5,6,7,8-tetrahydro- 
derivative has very Httle if any activity, and this compound is of course 
analogous to cyclohexane-i -acetic acid. The activity of naphthoic acid 

will be referred to again below. 




A heteroatom may take the place of carbon in the side chain, as in 
naphthoxy-2-acetic acid (XXIII), with an oxygen atom, and 2-methyl- 
4-chIorophenylthioacetic acid (XXIV), (11) with a sulfur atom, which 
is highly active. (See Table i.) Phenoxyacetic acid and phenylthioacetic 
acid, however, are inactive (10), while phenylpropionic acid has a little 
activity, so that the oxygen and sulfur atoms are less effective than the 
carbon. Phenylglycine with a nitrogen atom is also inactive. 

A striking effect is caused by introduction of a hydroxyl into the 
side chain. The activities are as follows (compared to indoleacetic acid 
as 100): 

Parent Compound 
Phenylacetic 10 

a-phenylpropionic (XXV) 6 

Indeneacetic (III) 






Hydroxy Derivative 

Atrolactic (XXVI) 
Tropic (XXVII) 
ethyl ester (XXVIII) 












II ' 








Recently Went has reported (17) that mandelic and tropic acids are 
also virtually devoid of preparatory activity (see below). Thus the 
hydroxyl in the side chain essentially destroys all activity. An original 
explanation will be offered below for this clear-cut effect. It must be 
noted, however, that auxin-a with its 3 hydroxy Is is again an exception. 

The Problem of Spatial Configuration. — To the cases of the cinnamic 
acids and the chain-substituted phenylacetic acids many interesting 
observations have been added. Unfortunately their meaning is not yet 

There are now three cases in which optical isomerism influences ac- 
tivity. The optical isomers of indole-a-propionic acid (XXIX) were 
tested by Kogl and Verkaaik (6) in the standard Avena test. The (+) 


acid was found to be 30 times as active as the (— ). On straight growth 
of immersed sections, however, the two isomers have the same activity. 


H H 


0^ /^"s ^^ ^0. /COOH 

^CH C ^^1 CH 

CllL ^Cl iOOH CllL ^Cl CHj 

The difference is not one of primary activity therefore, a fact which was 
confirmed by direct demonstration of a difference in transport rate and 
in rate of inactivation by tissue brei. The isomers of 2,4-dichlorophenoxy- 
a-propionic acid (XXX) present a different picture. In this case the 
d- and racemic acids* show differences in the pea test, the d-iorm being 
almost exactly twice as active as the racemic, which means that the 
activity of the /-form is very low (11). This, then, is a difference in 
primary activity. The third case is that of the 1,2,3,4-tetrahydro- 
naphthoic acids (XXII) of which the (-) form has nearly the activity 
of indoleacetic acid, while the activity of the (+) form is very low; this 
again refers to activity in the pea test (14). Optical isomerism, therefore, 
does influence primary activity. 

The geometrical isomerism is perhaps more interesting because it has 
given rise to a consistent theory. Veldstra (13) has pointed out that in 
/ra;2^-cinnamic acid the dipole of the carboxyl is held in the plane of the 
ring, while in the aV-isomer it is held at an angle to it. He has thus 
generalized the five requirements of Koepfli, Thimann, and Went to 
two, namely, "a non-polar part (ring-system) active interfacially, carry- 
ing an acidic polar group (preferably a carboxyl group) in such a spatial 
position with respect to the nucleus that this group is situated out of the 
ring system as far as (or as frequently as) possible" (14). To the cmnamic 
acids, tetrahydronaphthyhdeneacetic (XXXI) and a-naphthaleneacrylic 
(XXXII) have been added. In each case one form is active and the other 

*Kindly supplied to me by Dr. Franklin Jones. 










not, and there is evidence from the ultraviolet absorption spectra that 
the active form of both has the m-configuration (2). The surface activity, 
determined by the polarographic procedure, is the same for the two 
isomers of XXXI. A detailed criticism of Veldstra's theory would be 
out of place here, but at least in the case of XXXI the argument is open 
to doubt. For, according to principles of organic chemistry, derived from 
study of tetramethylethylene and other simple cases, when a carbon 
atom forms a double bond the other two bonds He in the same plane. 
Hence in both forms of XXXI the carboxyl would be expected to lie in 
the plane of the ring. Because the right-hand ring is saturated, however, 
it can be somewhat buckled, raising carbon atom number 2 above the 
plane of the ring and hence making it possible for the COOH to he 
slightly below the plane, and thus to avoid interference with the hydro- 
gen atom on the aromatic ring. Judging from models, the effect is very 

The same theory has been appUed to the nitrophenoxyacetic acids. 
Of these only the ;;7-isomer shows appreciable activity. It is pointed out 
by Veldstra that the 0- and /7-isomers are capable of resonance with a 
quinoid structure, in which the oxygen atom would be held in the plane 
of the ring. This restricts freedom of rotation and favors positions in 
which the acid dipole is at an angle to the plane of the ring. But careful 
examination of scale models shows that this restriction would in fact 
hold the carboxyl at least as much out of the plane of the ring as in it, 
that is, the o- and /^-compounds should have at least as high activity. 

Another more general consideration opposes this view also. If the 
maintenance of the carboxyl out of the plane of the ring were the princi- 


pal requirement for activity then a compound like aV-cinnamic acid, in 
which this position is fixed, should be much more active than, say, indole- 
acetic acid, in which this is only one of many positions attainable by free 

The effect of ring substitution can be considered in this connection. 
Introduction of a methoxy group into the benzene ring of indole- 
propionic acid, in any one of 3 positions, 4, 5, or 6,* inactivates it com- 
pletely (3). On the other hand introduction of halogens into the ring of 
phenyl derivatives greatly increases activity. The phenoxyacctic acids 
show the following primary activities (10) (relative to that of indole- 
acetic as 100): 

Phenoxyacctic ca. 

o-chlorophenoxyacetic 4 

/7-chlorophenoxyacetic 200 

2,4-dichlorophenoxyacetic 1000 

2,4,5-trichlorophenoxyacetic 500 

Similarly, 2,4-dichlorophenylacetic acid has very high activity. A strik- 
ing case is that of phenylbutyric acid whose activity is virtually zero; 
f-bromination raises it to about 15 per cent of that of Indoleacetic acid 
(see Table i). Methyl groups In the o- and p- position apparently 
increase the activity in the phenoxyacctic series about as much as 
chlorine atoms (7).! Many similar examples can be drawn from the work 
of Norman and his colleagues at Camp Detrick, though they used 
different tests. 

It Is difficult to see how substitution In the remote para- position could 
twist the carboxyl out of the plane of the ring, or how in Indoleacetic 
acid substitution of a methoxyl In the remote 5-posItion could have 
the opposite effect. According to Veldstra the influence of the halogens 
may be exerted on the lipophilic properties of the molecule, which 
are increased thereby; however, if the molecule becomes too lipophilic 
the balance between this and its hydrophihc properties is disturbed 

*On the other hand, S. P. Findlay and G. Dougherty [/. Biol. Chem., 183:361 
(1950)] have recently reported, but without quantitative data, that 5- 6-, and 
7-methoxyindoleacedc acids are active. The activity is probably rather low. Their 
data also confirm the inactivity of A'-cyclopenteneaceric acid. (See page 25). 

fR. M. Muir, C. H. Hansch, and A. H. Gallup [Plant Physiol, 24:359-366 
(1949)] have recently reported in some detail on the halogenated phenoxy 



List of compounds whose activity is here quantitatively compared for the 

first time 

Name Activity* 

(Indoleacetic acid = I go) 

Benzofulvenecarboxylic acid 0.5 

Benzofulvenehydroxycarboxylic acid o 

Indene-3-gIycolic acid ethyl ester o 

Phenylthioacetic acid O 

2-methyl-4-chlorophenylthioacetic acid 200 

Methylbenzoquinonethioacetic acid 0.2 

Trimethylbenzoquinonethioacetic acid o 

Phenoxyacetic acid ca. o 

2-chIorophenoxyacetic acid 4 

4-chlorophenoxyacetic acid 200 

2,4-dichlorophenoxyacetic acid 800-1,200 

2,4,5-trichlorophenoxyacetic acid 500 

2,3,4,6-tetrachIorophenoxyacetic acid I 

Pentachlorophenoxyacetic acid ca. 'y 

Bis- (2, 4-dichlorophenoxy) -acetic acid 1.5 

2,4-difluorophenoxyacetic acid 12 

2,4-dichlorophenylacetic acid 15 

Racemic 2,4-dichlorophenoxy-Q:-propionic acid . . 600 

D-2,4-dichlorophenoxy-Q;-propionic acid .... i , 200 

i,4-naphthoquinone-2-butyric acid O 

4-bromophenylbutyric acid 15 

4-hydroxyphenyl-7-ketobutyric acid o 

Naphthalene-i-acetamide (approx.) 10 

*Based on slit pea stem curvatures. 

and activity is reduced (15). It seems to the present author that the 
evidence for this conception is far from complete though it is in principle 

Enhancement of Auxin Action. — To complete the factual picture one 
other phenomenon should be mentioned. Many compounds, inactive 
by themselves, increase the apparent activity of the auxins. Went, who 
discovered this phenomenon, studied it by pretreating the pea sections 
with the inactive substance and subsequently placing them in a low 
concentration of the auxins (16). He views the growth process as taking 


place in two steps, the first or preparatory reaction being pH insensitive 
and being capable of accomplishment by a wider variety of structures 
than the growth reaction can be. In general, substances which have all 
but one of the structural requirements of an auxin have preparatory 
activity (17), and these are called hemi-auxins. They include 2-bromo-3, 
5-dichlorobenzoic acid (carboxyl adjacent to ring), cyclohexancacetic 
acid (saturated ring), thiazoleacetic acid (5-membered ring) and vinyl- 
acetic acid (no ring). There are many other examples. In some ways 
2,3,5-triiodobenzoic acid, "TIBA" (XXXIII), behaves similarly both 
in the pea test and in other growth reactions, but when the concentration 
ratio between this substance and the auxin becomes large then growth 
inhibition takes place (12). This was interpreted by Thimann and Bonner 
In terms of competition between the TIBA and the auxin for the same 
active centers in the cell. Several workers have found that TIBA pro- 
motes flowering and have explained this in terms of Its Inhibition, at 
high concentrations, of the action of auxin in the plant. It is evident that 
the structure of TIBA comes within the grouping of hemi-auxins men- 

/1-C5H,,— CH-/7-CjH„ »z^ ^CHCOOH 

ioOH H^C^^CH, 

XXX III xxxivo xxxiy b 

tioned above. However, Veldstra, under the term "synergism," describes 
a similar phenomenon brought about by quite unrelated compounds, 
especially dl-w-amylacetlc acid (XXXIV), (14). Although it Is true that 
the related amine, di-7z-amylmethylamlne, has no synergistic activity, 
and hence that the carboxyl may be needed, still the relationship be- 
tween XXIV and the typical auxin structure is somewhat remote.* (If 
the formula is written as shown at XXXIVb it suggests perhaps a relation- 
ship with phenyl-a-propionic acid or auxin-a.) Even if such exceptions 
should show that Went's generahzatlon is only partly correct, it still 
seems fair to visualize the enhancement phenomenon as due in some way 
to the ability of related substances to combine with a given structure. 

The Mechanism of the Ac/ion of Auxin: A Theory.— h remains only to 
consider what light all this sheds on the mechanism of auxin action. The 
discussion of growth as a metabolic process will take place in other papers 

*A recent direct comparison, using a sample of XXXIV kindly supplied by Dr. 
Veldstra, shows that TIBA is the more active of the two. 


SO only one or two points need be mentioned here. The growth process 
involves the metabolism of the organic acids. One of the enzymes in 
this system, which has a sulfliydryl structure, is a critical link; sulfhydryl 
reagents, such as iodoacetate, inhibit growth strongly and specifically. 
Malate, pyruvate, succinate, and other acids promote growth; they are 
used up during growth, and malate or citrate may accumulate when 
growth is inhibited. Malate and other acids, including malonate, protect 
growth against the inhibiting influence of iodoacetate. Thus enzymes 
acting upon the 4-carbon acids play a major role in growth. \'iewed in 
this light the structural requirements for auxin activity take on a strik- 
ing significance. The protecting action of malonate against inhibition 
by iodoacetate is only part of a general phenomenon in which substrates 
and interfering substances or inhibitors compete for the active locations 
on catalyst molecules, as for example, the relation between O2 and CO 
with activated charcoal, or between succinic and malonic or maleic 
acids with succinic dehydrogenase. In this case malonic acid will protect 
the enzyme against inactivation by oxidized glutathione. The parallel 
with succinic dehydrogenase is indeed very close, for Quastel and 
Wooldridge (7) showed long ago that besides malonic acid and related 
substances, phenylpropionic acid inhibits this enzyme. Thus the phenyl 
group can substitute on the enzyme for one of the carboxyl groups of 
succinic acid. If now w^e view the auxins as protective substances, we see 
that the ring takes the place of a carboxyl. The requirement for one car- 
bon atom between the ring and carboxyl means that the auxin is really a 
malonic acid derivative. Suppose the tissue contains a natural inhibitor of 
succinic dehydrogenase. (The argument may apply equally to certain 
other enzymes of the 4-carbon acid series.) For the enzyme to have 
activity and thus to allow growth it must be protected against this 
inhibitor; the best protector is indoleacetic acid. 

This line of thought at once brings many observations together: 
i) Succinic dehydrogenase is inhibited not only by malonic acid but 
by maleic acid. If auxins are envisaged as malonic acid derivatives, the 
corresponding maleic acid derivatives should have the same action. 
These, of course, are the aromatic acids of aV-configuration like cis- 
cinnamic acid. If this is true it is the c/V-structure itself, rather than the 
angle of the carboxyl, which is important. We should note, too, that 
the active naphthalene- 1 -acetic acid and the almost inactive naphthalene- 
2-acetic acid can be considered as analogous to cis- and trans-ioxms,. 


2) The inhibitors of succinic and oi lactic dehydrogenase show marked 
specificity. Quastel and Wooldridge (7) showed that hydroxy acids, 
such as hydroxymalonic, which. inhibit lactic dehydrogenase, are quite 
inactive on succinic dehydrogenase. This parallels the marked effect of 
a side-chain hydroxyl in abolishing auxin activity. The auxin analogue of 
hydroxymalonic acid is, of course, mandelic acid. 

3) The action of malonic acid itself is most suggestive in this connec- 
tion. In low concentrations (lo'^'M) it protects against the inhibiting 
action of iodoacetate on growth. In high concentrations (lO'^M and 
above) it inhibits growth both of coleoptiles and of pea stems. To the 
plant tissue poisoned with iodoacetate, then, malonic acid acts like an 
auxin, promoting growth at low concentrations and inhibiting it in 

4) The 4-carbon acid enzymes, including succinic dehydrogenase, are 
of critical importance to the hfe of cells. To protect them against 
poisoning is essential not only for growth but for hfe. But the action of 
a protecting substance implies that it is adsorbed on the enzyme more 
readily than the inhibitor against which it protects. Hence when the 
concentration of a protecting substance becomes high its molecules begin 
to be adsorbed on the enzyme to a degree comparable with that of the 
substrate; in other words the protecting substance now begins to inhibit. 
As the concentration increases at first only growth is inhibited, but 
when inhibition becomes complete the cells are killed. In this way the 
fact that at high concentrations the auxins become toxic and herbicidal 
would be explained. 

This hypothesis has the advantage of bringing together two hitherto 
unrelated aspects of the auxin studies, namely the role of auxm in 
metabolism and the chemistry of the active substances. Perhaps also 
it may be suggestive to workers in other fields of biology which involve 
the fascinating relation between structure and activity. 


1. Bonner, D. M., Botan. Gaz., 100:200-14 (1938)- 

2. Havinga, H. and Nivard, R. J. P., Rec. trav. chim. Pays-Bas, 67:846-54 

3. KoE?FLi,* J. B., Thimann, K. v., and Went, F. W., /. Biol. Chem., 

122:763-80 (1938). 

4. KoGL, F., Ber. deut. Chem. Ges., 68:16-28 (1935). 

5. , and Kostermans, D. G. F. R., Zeii. Physiol. Chem., 235:201-16 



-, and Verkaaik, B., Zeit. Physiol. Chern., 280:167-76 (1944). 

7. QuASTEL, J. H. and Wooldridge, W. R., Biochem. ]., 22:689-702 (1928). 

8. Templeman, W. G. and Sexton, W. A., Proc. Roy. Soc. (London) 

133 6:300-13 (1946). 

9. Thimann, K. v., Proc. Kon. Al^ad. Wetcnsch. Amsterdam, 38:896-912 


10. , "Plant Growth Hormones" in The Hormones, Physiology, Chem- 
istry and Applications (Academic Press, 1948). 

11. , Unpublished data. 

12. , and Bonner, W. D., Jr., Plant Physiol., 23: 158-61 (1948). 

13. Veldstra, H., Enzymologia, 11:97-136, 137-63 (1944). 

14. • , Paper read at Ilnd Int. Congress of Crop Protection, London, 

July 21, 1949. 

15. , and Booij, H. L., Biochem. Biophys. Acta, 3:278-95 (1949). 

16. Went, F. W., Bull. Torrey Bot. Club, 66:391-410 (1939). 

17. , Arch. Biochem., 20:131-36 (1949). 

Growth and Structure of the Primary Wall 


THE primary wall is the membrane surrounding the growing cell 
showing reversible changes in surface area with variations in cell 
turgor and permanent changes in surface area as a result of growth. The 
structure of a membrane capable of undergoing such changes has not 
been satisfactorily explained. 

All cells of higher plants Irrespective of their size, shape, or cell-wall 
thickness have primary walls, and as long as they are in a rapid state 
of enlargement, the cells have only primary walls. Thus the thickened 
walls of collenchyma and the outer walls of epidermal cells which are 
known to be cutlnlzed must be classified as primary membranes. 
Throughout this discussion the structure of the primary wall will be 
based chiefly on the structure of a typical parenchyma cell such as 
one finds in the Avena coleoptlle. 

Chemically the primary walls of higher plants are known to be com- 
posed of cellulose and pectic substance. In the cotton hair where thorough 
investigations have been made, the cellulose of the primary wall gives 
a typical X-ray diffraction pattern (2), has an average chain length 
approximately 50 per cent that of the secondary wall (6), and shows 
standard solublUtles of cellulose. Considering the wide variations In 
chain length In different types of secondary walls it may be concluded 
that the cellulose molecules of the primary membrane are Identical with 
those of the secondary wall. 

For many years Investigators working on cell walls have realized that 
the structure of the primary wall cellulose Is fundamentally different 
from secondary wall cellulose without knowing the basis for the dif- 
ference. The recent work of Miihlethaler (9) on the structure of the 
primary wall under the electron microscope shows why these differences 


exist. (Fig. i) Miihlethaler's excellent micrographs demonstrate that the 
primary membrane is composed of a network of microfibrils in contrast 
to the more or less parallel arrangement of the fibrillar structure in the 
secondary wall. The sharply defined microfibrils of the primary mem- 
brane are fairly constant in diameter (200-250 A) and seem to be partly 
interwoven as in a textile fabric. 

Inasmuch as the fibrils of the secondary wall are arranged parallel to 
each other, fine details of structure can be studied under the ordinary 
microscope. On the other hand, since the microfibrils of the primary 
wall are in the form of a network and the fundamental units of the 
network are below the limits of microscopic visibility, details of primary 
wall structure under the ordinary microscope cannot be worked out in 
an entirely satisfactory manner. However, it has been known from studies 
using crossed nicols, that the microfibrils of the primary wall are not 
randomly arranged even though they are in the form of a network. It can 
be assumed that the majority of fibrils composing the network are 
oriented more or less in one direction. It has been known for years that 
the cellulose chains in parenchyma cells are oriented chiefly perpen- 
dicularly to the major axis of the cell and that this orientation does not 
change during growth. Striking preparations showing the orientation 
of the cellulose in the primary wall may be seen when cells are stained 
deeply in congo red and viewed under a polarizing microscope (Fig. 2). 
It is well known that congo red accentuates the weak birefringence of 
cellulose. The stain is prepared as a 0.2 per cent solution of congo red in 
I per cent solution of sodium hydroxide. Pieces of tissue (e.g. whole 
Avena coleoptiles) placed in the dye solution for 48 hours may be 
macerated easily on a slide by a tap on the coverslip. Thus it is possible 
to study the orientation of the cellulose in single cells, and even single 
walls are also easily obtained by allowing a slide prepared in the above 
manner to dry with a weight on the coverslip. The upper walls of many 
cells adhere to the coversfip while the lower walls adhere to the slide. 

Some years ago, a large number of various primary wall types were 
studied under the polarizing microscope and from these studies certain 
generalizations may be made. Whenever a cell possesses an elongated 
shape, the main orientation of the cellulose fibrils and the major orienta- 
tion of the primary pit fields are perpendicular to the major cell axis. 
The regions of the wall in contact with the intercellular spaces, show a 
second orientation, for here additional fibrils are oriented parallel to 

Figure I. Electron micrograph of the primary wall from a cell of a ger- 
minating corn root. Photographed by Miihlethaler. 

Figure 2. Primary wall of a parenchyma cell from an Athena coleoptile 
stained in congo red and photographed between crossed nicols. 


the major cell axis. The two opposite orientations within the same wall 
frequently show optical compensation and the parts of the wall adjoining 
the intercellular spaces may appear isotropic. When one examines a series 
of cells grading from parenchyma to coUenchyma, the second orientation 
parallel to the cell axis gradually becomes the dominant one. This 
parallel orientation is the arrangement of the cellulose in the thickened 
corners of coUenchyma. Thus two types of cellulose orientation, one in 
which the fibrils are perpendicular to the major cell axis, and a second 
in which the fibrils are parallel to the major cell axis, may be seen in cells 
that are undergoing rapid enlargement. In neither case is there any 
appreciable change in the orientation of the microfibrils during growth. 
Therefore it follows that growth in any direction is independent of the 
major orientation of the microfibrils within the wall. 

The changes that occur in the primary wall during growth have 
recently been considered by Frey-Wyssling (5). He postulates that the 
microfibrils of cellulose form a network which is held together by lateral 
forces, presumably by hydrogen bonding or some equivalent force. 
During growth the lateral forces holding the fibrils must be loosened, 
the empty spaces enlarged, and new strands must be interwoven. Frey- 
Wyssling's hypothesis will explain the plastic extension or growth of the 
wall. On the other hand, it is extremely difficult considering our present 
knowledge to see how a wall built in this manner could possess the elastic 
extensibility of primary membranes. Primary walls are known to expand 
and contract as much as 30 per cent with turgor changes in the protoplast. 
Such elastic properties could be explained if the wall were composed of an 
elastic or rubber-like substance or it could also be explained if the wall 
were composed of a highly hydrophyllic material that would change 
in volume and in surface area with the water relations of the protoplast. 
Cellulose is known to possess rigid molecules having only very limited 
elasticity and does not display anything of a rubbery nature. Further- 
more sheets of cellulose do not change appreciably either in volume or 
surface area in different neutral solutions with a range of four or five 
atmospheres. If the wall were composed of a skeletal framework of 
cellulose, it would be expected to be a nonelastic structure similar to a 
sheet of cellophane. 

The picture is unfortunately more complicated. Properties of cellulose 
have been evaluated from dried material, and it is now recognized that 
natural cellulose, that has never been dried, possesses somewhat different 


properties. This can be illustrated from studies made on the secondary 
wall of the cotton hair (3). Cotton fibers of commerce are twisted, ribbon- 
hke structures, whereas the same fibers before drying for the first time 
are hollow cylinders. During the initial dehydration twists or convolu- 
tions first appear and once formed are irreversible. Before the initial 
drying the cellulose does not give an X-ray diffraction pattern or at the 
most, only a faint indication of one. This must mean that crystallization 
or hydrogen bonding of cellulose does not occur at the time of deposition 
but takes place chiefly during the initial dehydration. The cellulose of 
the primary walls of cotton hairs likewise gives an X-ray diffraction 
pattern only after drying. Heyn (7) has reported a similar situation 
in the parenchyma of Avena coleoptiles. Undried cotton fibers show 
considerable plasticity but when stretched under water, crystallization 
of the cellulose occurs even without drying. The artificial stretching of 
primary cells reported by Bonner (4) unquestionably involves not only 
a reorientation of the cellulose but also crystallization. Dried primary 
walls do not show the plastic behavior of undried membranes. It is 
difficult to see how a primary wall composed of a network of microfibrils 
undergoing a rapid increase in surface area as a result of a sharp increase 
in turgor, could avoid crystallization of the cellulose. Nevertheless, it 
is known that crystallization does not take place. Therefore, it is still 
highly improbable that undried cellulose could form a skeletal network 
with the elastic extensibility of the primary membrane. 

In order to have a better understanding of the primary wall, it is 
necessary to consider the structure and properties of pectic substances. 
Our knowledge of the manner in which these substances occur in the 
wall is still somewhat vague. Preston (11) has recently considered that 
they are encrusting substances in the same category as lignin. Frey- 
Wyssling (5) does not mention the pectic substances in his recent dis- 
cussion of the growth of the primary membrane. Materials in the primary 
wall grouped under the term "pectic substances" are unquestionably 
mixtures, including arabans, galactans and possibly other hemicelluloses. 
However, true pectic substances are apparently restricted to the primary 
walls and intercellular substance of higher plants and do not seem to be 
present elsewhere. Pectic substances are now recognized to be composed 
of straight, long chained molecules of anhydrogalacturonic acid units 
(8). The carboxyl groups of the polygalacturonic acid may be partly 
esterified by methyl groups, in which case the substance is called "pectin," 


or partly or completely neutralized by one or more bases as in calcium 
pectate. The term "protopectin" is applied to the very long chained, 
parent substance which occurs in primary walls. Protopectin is insoluble 
in water, but upon restricted hydrolysis gives rise to pectic acid (free 
of methyl groups) or pectinic acid (partly methylated). The intercellular 
substance is commonly considered to be calcium pectate. 

It is possible to dissolve the cellulose of the primary wall and leave a 
structural residue of the protopectin. Conversely the pectic substances 
may be removed and there remains a structural framework of the 
cellulose. One might expect that long chained molecules of protopectin 
would be arranged in the primary membrane similar to the long chained 
molecules of cellulose, but this apparently is not the case. The pectic 
residue of the wall after the removal of cellulose is isotropic. At no time 
do the pectic substances within the wall give an X-ray diffraction pattern, 
and the removal of the pectic substances does not affect the diffraction 
pattern which is already present. On the other hand, pectins removed 
from the wall, formed into threads, stretched, and dried, will give an 
X-ray diffraction pattern (8,10). Furthermore, Owens and his co-workers 
have given excellent evidence to indicate that pectins possess rigid, 
rod-hke molecules. The length of the protopectin chain is unknown but 
pectinic acid derived from protopectin by hydrolysis has been reported 
to have molecular lengths varying from 530 to 1650 A. (10). Considering 
all these facts, the arrangement of the protopectin chains within the 
wall is probably at random or the molecules might possibly be oriented 
with respect to each other and yet incapable of crystallization. 

There are several other properties of pectic substances which are 
important from the standpoint of their presence in primary walls, i) 
Pectic substances are well known for the ease with which they form 
gels. 2) In calcium pectate, the calcium ion is shared between two 
carboxyl groups of adjacent chains of pectic acid, resulting in cross 
linkages. 3) Pectins undergo degradation or depolymerization with ex- 
treme ease when in aqueous solutions, and the degradation is markedly 
influenced by various cations, particularly hydrogen ions. 

At least one property of the wall, rigidity, can be associated with the 
properties of the pectic substances in the wall. Pickles of various kinds 
from cucumbers, watermelon rinds, or green tomatoes are essentially 
skeletons of the primary wall preserved in acid, usually after fermenta- 
tion. During fermentation, cucumber pickles sometimes become soft, 


causing considerable financial loss. The softening of pickles is associated 
with a decrease in the chain length of the pectic molecules (i). Con- 
versely, it is well known that calcium salts harden the texture of pickled 
vegetables presumably by forming calcium pectate with cross linkages. 

Considering all the properties of pectic substances, the property of 
the primary wall could be explained if it were assumed that the pectic 
substances were the continuous phase of the wall and the cellulose micro- 
fibrils were a discontinuous phase. The cellulose fibrils might be con- 
sidered as structural reinforcement. Changes that occur during growth 
would affect the easily hydrolyzable pectic substances and not the 
cellulose. Changes in elastic extension of the wall could be explained as 
changes in hydration of the pectic gel. This hypothesis has not been 
proved but it has been brought forward to get around the impasse that 
the properties of the primary membrane are not the properties of a 
sheet of cellulose. 

The primary wall has been pictured as a fibrillar network of cellulose. 
A wall built in this manner would not have the elastic extensibility 
characteristics of primary membranes. Considering the properties of 
cellulose and pectic substances, the properties of the primary wall can 
be explained if it is assumed that the protopectin forms a continuous 
phase and the cellulose microfibrils form a discontinuous phase. 


1. Bell, T. A. and Etchells, J. L., Food Technology (In Press). 

2. Berkley, E. E., Textile Res., 9:355-375 (1939)- 

3. , and Kerr, T., Ind. and Eng. Chem., 38:304-309 (1946). 

4. Bonner, ]., Jahrb.f. wiss. Bot., 82:377 (1935). 

5. Frey-Wyssling, a., Growth Symposium, 12:151-169 (1948). 

6. Hessler, L. E., Merola, G. V., and Berkley, E. E., Textile Res., 

18:628-634 (1948). 

7. Heyn, a. N. S., Botan. Rev., 6:515-574 (1940). 

8. JosLYN, M. A. and Phaff, H. J., Wallerstein Lab. Communication 

10:39-56 (1947). 

9. MuHLETHALER, K., Btochem et Biophys, 3:15-25 (1949). 

10. Owens, H. S., Lotzkar, H., Schultz, T. H., and Maclay, W. D., 

/. Am. Chem. Sac, 68:1628-1632 (1946). 

11. Preston, R. D., "The Organization of the Cell Wall in Plants," in 

Fiber Science (edited by J. M. Preston,The Textile Institute, Manchester, 
England, 1949). 

Mechanisms of Cell Elong-ation 


General Course of the Cell Elongation 

THE mechanism of cell elongation can be viewed from different 
angles: as a hormone problem, or as a question of morphologic 
and metabolic changes of the cell. Invited to read a paper on the 
mechanism of cell elongation, I should like as far as possible to avoid 
discussing hormones and hormone actions, and concentrate upon the 
course of the cell elongation. In its general features this problem recently 
has been awarded an excellent treatise by Frey-Wyssling (8) in a growth 
symposium paper, but when growth is considered in connection with 
hormones, the crucial point is not only to find out what happens during 
the elongation but to find the real inciting cause of the process, where 
the hormones are fikely to exert their action. 

Many opinions have been advanced for the cause of the cell elonga- 
tion, and there is scarcely any part of the metabolism that has not 
been assumed to form the point of action of auxins and connected with 
the elongation. Therefore the title, "Mechanisms of cell elongation," 
obviously covers such a wide subject that I must restrict myself to 
certain aspects of the problem only. 

The superficial cytological course of the elongation has proved to be 
rather similar in roots and shoots, although these are different with 
respect to their physiological behavior. Figure i shows some well-known 
main features of cell elongation. The example is taken from the elonga- 
tion of the epidermis of roots. Growth follows the usual S-shaped curve 
with a very slow start. The increase in length of the cell is in this case from 
about 1 8 to 300Ai, and, as is well known, the increase in volume involves 
largely an absorption of water. Of the osmotic properties the suction 
pressure of the cell can be determined easily on elongating cells, and this 












Figure i. The change in the elastic tension of root epidermis cells during 
the cell elongation. 

has been done both on roots and coleoptiles (2,11). Regarding roots 
growing in a solution the conditions are simple, the cells are constantly 
saturated with water during the whole elongation process. In coleoptiles, 
on the contrary, there is always a large water deficit. In any event it 
is obvious that the often advanced opinion that elongation must cease 
in a water-saturated cell is undoubtedly erroneous, and such an opinion 
cannot even be theoretically sound. 

The properties of the cell wall are indubitably of first-rate importance 


to the cell elongatfbn. Of the mechanical properties of the wall the 
reversible, elastic extensibility can be determined with a fair degree 
of accuracy. Experiences with both roots and coleoptiles have taught 
that the elasticity always increases at the start of the cell stretching 
but decreases again before the cells have attained their full size. Figure 
I shows how, in a root, the elastic tension as measured per cell increases 
rapidly at the start to a maximum value, which is maintained during 
the main part of the elongation. Relative to the increasing cell length 
this means a decreasing tension of the cell walls. This increasing elasticity 
cannot cause the elongation, but it signifies that there are changes in 
the cell wall at a very early stage of elongation, which undoubtedly 
are connected with the elongation process. 

These observations on the osmotic and wall properties of the cells 
have led to the assumption that cell elongation proceeds in two phases, 
which may be rather sharply distinguished from one another and during 
which different conditions prevail. The first phase involves an increasing 
elasticity of the wall, which has been explained on the basis of a loosening 
of the joints between the micellae (7). The second phase is characterized 
by a hardening of the wall, as evidenced by a decreasing elasticity. During 
the second phase there is, further, a very rapid supply of nutrients to 
the cell so that the osmotic concentration does not decrease in spite of 
the rapid increase in cell volume. 

Similar results had earlier been obtained by Ruge (i i) with coleoptiles, 
and he has likewise concluded that the growth proceeds in two steps, 
the first one involving stretching of the cell without synthesis of new 
wall materials but with an increasing extensibiUty of the walls. The 
consequence is that during this part of the elongation the wall becomes 
thinner in proportion to the increasing cell surface, and other observa- 
tions (6,12) have verified that such a spreading of the wall material 
takes place during the early part of the elongation. During the second 
phase there is a deposition of new wall material, undoubtedly through 
intussusception, probably also through apposition. This results in a 
concomitant hardening of the cell wall. It seems probable that Ruge's 
two phases for coleoptiles are identical with those shown to exist also in 
roots, and that consequently shoots and roots behave similarly. 

This distinction between the two phases has been further supported 
by the fact that auxins affect them in different ways (3). The first phase 
is accelerated so that elongation starts earlier, and the second phase is 


shortened, which results in a reduced cell length* and an over-all in- 
hibited growth in roots. These two actions can manifest themselves 
simultaneously in one root, which supports the idea that there are two 
different processes going on in the cell elongation. 

The first phase above all deserves attention in this connection. We 
may find there the primary cause of the cell stretching, whereas the 
ensuing growth by intussusception may be assumed to follow only as 
a consequence of the preparation made during the first phase. 

Cell Elongation and Proteins 

It is wrong to assume, however, that this is all that takes place during 
the elongation. Frey-Wyssling (7) has especially emphasized that there 
is a considerable increase in the amount of cytoplasm during the cell 
stretching. That this is so in elongating coleoptiles and anther filaments 
is less significant because they lack a meristem, but the same holds true 
in roots as shown by Miss Kopp (9). It is correct that the increase 
up to 90 per cent in volume is caused by water intake, but nevertheless 
as much as half of the protein synthesis takes place during the cell 

That there is some connection between proteins and elongation is to 
be seen from the fact that an increase in the supply of nitrogen increases 
the rate of cell elongation, whereas no such effect is found, for instance, 
with phosphorus (4). Table i shows what happens when excised wheat 
roots are supplied with increasing amounts of these two nutrients. High 
concentrations of nitrogen increase the root length. Analyses have veri- 
fied that an increasing supply of nitrogen leads to increased synthesis 
of proteins. See Figure 2 for the rate of cell-elongation increases. Note 
that the time of starting and the duration of the elongation as well as 

Growth of isolated wheat roots with different phosphate and nitrate supplies 

Increase in Increase in 

Solution root length Cell length cell number 

MM. fi longitudinally 

Low P, low N 13. 7 ±1.7 177 ±3 35 ± 4 

High P, low N 25.5 ±2.0 170 ±3 120 ±5 

Low P, high N 25 . 2 ± 1 . 3 269 ±4 5 1 ± 3 



40 h 



I > ^200 


/ 1 


^'n 1 


1 1 

/• / 

\ \ 



1 1 


1 1 

i , 
i 1 
1 1 





i\ 1 

/ / 

\\ \ 

/ '''■ 

» \ \ 


\ \ * 

^i^ f 

'• M 

— — , 

^'^ / 

\ ^ 


y , 

^ Vw 


y\ 1 

1 •■, 1 vs 




Figure 2. The grand period of cell elongation with dilTercnt supplies of 
nitrate and phosphate. 

the frequency of cell divisions are unaffected by the nitrate concentra- 
tions above the nitrogen starvation level. Only the rate of stretching is 
affected. Phosphorus, on the contrary, given in concentrations above 
deficiency levels does not interfere with the cell elongation but only 
with the cell divisions. It may be that a certain synthesis of proteins is 
an indispensable prerequisite for cell elongation, because we do not know 
any instance of cell elongation at present without a protein synthesis. 
On the contrary, however, there is httle possibility that a formation 
of cytoplasm evokes the elongation. If so, there ought to be a more rapid 
start or a longer duration of the elongation in response to nitrogen 
supply and resulting protein synthesis. Further, such a synthesis is a 
characteristic feature of the meristem, and even though it continues 
during the elongation it cannot be the cause of the incipient vacuoliza- 
tion and elongation of the cells. Their explanation must be sought in the 
osmotic properties of the cell or in the conditions of the cell wall. 

Elongation and Absorption of Water 

The increase in volume depends directly upon an absorption of water, 
and thus it is necessary to consider under what conditions an uptake of 


water may be brought about, with the natural reservation that not every 
kind of water uptake results in growth. This is also the usual starting 
point for a discussion of cell elongation, but it is regrettable that there is 
an obvious uncertainty as to the importance and nature of the osmotic 
conditions of the cell that regulate the absorption of water. I need hardly 
remind you of the fact that there is still no unanimously accepted 
physical explanation of osmotic pressures. 

The fundamental formula underlying the water absorption is the 
one given by Ursprung: S = O — W, where S denotes the suction 
pressure of the entire cell against the external medium, is the osmotic 
value or the diffusion pressure deficit of the vacuolar sap including 
sweUing pressures, and W is the elastic tension of the wall, directed 
towards the protoplast. Unfortunately the properties 5 and O are often 

As the driving force of the growth it is customary to refer to turgor 
pressure, or the pressure acting from within the cell as a force striving 
to expand it. What turgor signifies can be theoretically deduced and 
expressed by the formula T = O - E, where T denotes the turgor 
pressure, and E the osmotic value or diffusion pressure deficit of the 
solution surrounding the cell, that is, the external solution. The turgor 
pressure can thus be defined as the difference in diffusion pressure of the 
water outside and inside the cell, or the force with which water tries 
to enter into and expand the cell (5). 

These two formulae are, as a matter of fact, quite independent of each 
other. They contain three independent variables: O, E, and W, the 
last of which is in reahty not an osmotic property in the strict sense, 
but is, as already mentioned, the elastic wall tension. 

The equations further signify that there are two existing forces or 
pressures in the cell and not more, namely W, the elastic pressure of the 
wall, directed inwards, and T, the diffusion pressure of water, i.e., the 
net diffusion pressure, which normally as in turgid and expanding 
cells causes a pressure directed outward toward the wall but in shrinking 
cells is directed inward. These two pressures are in principle quite inde- 
pendent of each other. If they are equally large, that is, if T = W, the 
cell is in equilibrium with its external medium. Then also S = E, which 
is the usual way of expressing that the suctions of the cell and of the 
external medium are equal. We must assume that before elongation 
starts the cell is in such an equilibrium with its surroundings. The rela- 



tion between turgor and wall pressure is illustrated in the osmotic 
diagram in Figure 3. 

In order for absorption of water to occur it is necessary that T exceed 
W. We may therefore say that the pressure from the inside causes an 
expansion of the cell; but from the equations it follows that in such a case 
S must also exceed E to the same extent, so we may quite correctly 
also say that the suction of the cell is greater than that of the external 
medium. Hence water is absorbed and the cell expands. Expansion is 
caused by a difference, T — W, or in other words, S — E; it may be 
called a turgor-over-pressure or water deficit of the cell. It is thus im- 
material if we call the resulting increase in volume an absorption of water 
owing to a water deficit or an expansion owing to a turgor-over-pressure. 

Volumel ,4 

Figure 3. Diagram showing the osmotic conditions of one cell at equilibrium 

or nonequilibrium with one external medium. O O osmodc value 

of the cell sap, T T turgor pressure, W W wall pressure, 

E osmotic value of the external medium, S suction of the entire cell. I = 
incipient plasmolysis, II = the cell absorbing water. III = equilibrium with 
the medium E. 


Note, however, that when the cell is at equilibrium, the turgor pressure 
is balanced by the wall pressure and therefore cannot cause any expansion 
of the cell, because water cannot be absorbed without a suction difler- 

These relations are still under discussion, but for the given reasons I am 
inclined to believe that the computations recently made by Frey- 
Wyssling (7,8) of the osmotic work performed in the elongation of cells 
are not correct. He has assumed the cell to be expanded by a force = W, 
whereas in reality it should have been T, and these two values need 
not be equal, certainly not during water saturation, and perhaps not 
during the elongation. 

This connection between turgor and water absorption is not generally 
realized. Audus (i), in his monograph on auxin actions, brings out 
Heyn's opinion that the turgor pressure can call forth a stretching of the 
cell, which ultimately implies a reduced hydrostatic pressure of the cell 
contents, the consequences of which are an increased suction force and 
water uptake in active cell stretching. Such a proposed sequence of 
events has no purpose. The conditions are much simpler, for "stretching 
under a turgor pressure" is but another way of saying "water absorption 
owing to a suction difference." 

Looking for the cause of such a water uptake we must find out what 
can cause a change from the equilibrium conditions when T = W and 
S = E. This is possible by changing one of the three variables of the 
equations, E, O, or W. E can, of course, be omitted from the discussion 
leaving O and W to be considered. 

An increase in O impHes an increase in the amount of osmotic material 
in the cell. That an actual increase in nutrients occurs from the start 
of the elongation is easily demonstrated. The result should be an increased 
turgor pressure, — E — W, causing absorption of water, a rise in the 
wall pressure, and a decreasing turgor until a new equilibrium is attained, 
with O, W, and T all higher than before. In practice there is a decrease 
in O during the most rapid elongation, so that such a process alone 
cannot be responsible for the cell elongation, Nevertheless it is necessary 
to consider to what extent absorption of water in itself can contribute 
to the cell elongation. Especially so, because such mechanisms and even 
an auxin-induced water uptake without any change in the osmotic 
conditions proper play a certain role in the discussion of the cell- 
elongation process. 


The most elaborate theory to that effect has recently been advanced 
by Pohl (10). By studies on plasmolysis and deplasmolysis of coleoptiles 
he has verified in a highly convincing way the point that auxins increase 
the permeability of the cytoplasm to water. He has concluded that the 
cause of the elongation is an auxin-induced rise in the water permeability, 
causing a water uptake and an increase in volume of the cell. There is a 
difficulty in this interpretation, however. It presupposes that the increase 
in volume of the cell is limited by the rate of water absorption. Now it 
is generally assumed, for good reasons, that the water permeabihty of 
the cytoplasm is very high and that it can scarcely hamper the water 
uptake if the osmotic conditions permit an absorption of water. Of 
course the rate may be affected but not the end result. Pohl has therefore 
been compelled to assume that initially the tonoplasts are practically 
impermeable to water. This is a highly hypothetical assumption which, 
of course, ought to be verified cytologically. 

The immediate consequence of this or any water absorption is a 
reversible elastic extension of the wall along the curve W of Figure 3, 
and it need not be emphasized that this is different from the elongation 
which occurs in growth. The problem is, however, whether such an 
elastic extension may change into an irreversible, plastic deformation. 

This concept of plasticity in the physical meaning applied to living 
cell walls has been repeatedly criticized with regard to the special 
micellar structure of the wall and the extreme and unnatural experi- 
mental conditions necessary for a demonstration of such a passive, 
irreversible stretching of the cell wall. Not even Pohl was able to explain 
the elongation by means of a water absorption alone, but he had to round 
off his theory by assuming an additional change in the cell wall, without 
which cell stretching is precluded. 

The reason Pohl has found it impossible to explain elongation and 
auxin action by a change in the wall and the wall pressure only is his 
emphatic postulation that the suction pressure of the coleoptiles amounts 
to 6 to 7 atmospheres and the wall pressure to only i to 2 atmospheres. 
Thus any change in the wall pressure must be of relatively httle im- 
portance and cannot explain the rapid elongation following an auxin 
treatment. This deduction is wrong, however. Before growth in length 
starts the wall pressure is only some few atmospheres whereas the cell is 
probably in equilibrium and the turgor is as low as the wall pressure. 
There exists no turgor-over-pressure or suction deficit with respect to 


the surroundings. Every decrease in TiT below Tmay thus be of relatively 
great importance even if its absolute amount is small. 

In fact, when the water deficit of a cell is large and the wall pressure 
is low a decrease of the latter permits a relatively much greater absorp- 
tion of water and an increase in volume of the cell than when the cell is 
more nearly water saturated. That follows from the shape of the W^-curve 
in Figure 3. To obtain the same increase in volume only by a water ab- 
sorption without active changes of the wall, enormous forces of water 
absorption would be required, irrespective of whether they were of 
osmotic or so-called nonosmotic origin. 

There remain to be considered inevitable changes of the wall pres- 
sure as a starting point of the elongation. It is hardly necessary to 
emphasize the fact that if there is an active change in the wall structure, 
it is futile to speak of plastic extension as the cause of the elongation 
because this presupposes only a passive stretching under the influence of 
an external load. 

Elongation and Changes in the Cell Wall 

The next step is to find out what direct evidence there is of active 
changes within the cell wall. One fact seems to be firmly established — 
that the walls of growing cells always have a tubular structure (8). This 
means that the micellae are generally orientated in a transverse direction, 
neither the stretching during the first phase of the elongation nor the 
intussusception seems to alter this structure of the wall. 

As already mentioned it is probable that the growth during the first 
phase involves an increase in surface of the cell wall partly at the expense 
of its thickness. This means that wall material is translocated within 
the wall itself, and the increase in surface depends upon a kind of growth 
by intussusception, even though the material deposited in the wall is 
not delivered from without. If this were a mere plastic remodeling of 
the wall substances this fact would become apparent from changes in 
elastic properties of the wall which are most conspicuous signs of some- 
thing happening in the wall at the start of the elongation. 

To take some figures from one series of measurements on roots, the 
cell length increases from 20 to about 400/i. The elastic stretching under 
a constant turgor pressure increases, as already mentioned, from about 
3 to a maximum of about 40/i per cell when the cell reaches a length of 
about i20/i. From then on the elastic stretching remains constant. 



The specific tension of the wall showing its elastic properties can be 
computed in dilferent ways. The tension must of course be related to 
the cell length, but attention must also be paid to the thickness of the 
wall. The theoretical formula reads 

dl = 

1(1 - n)P 
2Ed ' 

where dl is the elasticity, / the cell length, d the wall thickness, and the 
rest are constants. In applying this formula to my material Frey-Wyssling 
(7) has assumed the wall thickness to be constant and has constructed a 
graph on this assumption (Fig. 4). The graph gives the modulus of 
elasticity which runs inversely to the tensibihty of the wall. During the 
first phase of the elongation this computation gives a tenfold increase 
in the elasticity of the wall. 














Figure 4. The elastic properties of the walls of elongating epidermis cells. 
Both graphs deduced from one series of measurements (2). E the modulus of 
elasticity according to Frey-Wyssling, f the rigidity of the wall according to 
the formulae of Tamiya. 


As a matter of fact we do not know whether the wall thickness 
remains constant. It is possible that it decreases during this first phase, 
which would cause an increasing tension in the wall even if its structure 
remains unchanged. On such an assumption the real increase in the 
elasticity should not amount to more than a doubling. These are mini- 
mum figures and those of Frey-Wyssling are maximum values for the 
loosening of the wall structure. 

There cannot be merely a loosening of the wall structure, however. 
Assuming a constant amount of wall material and taking into account 
the fact that the cell wall maintains its micellar structure during the 
whole elongation process, there must be some factor responsible for this 
organized translocation of material within the wall. Assuming with 
Frey-Wyssling that the wall maintains its thickness from the start of 
the elongation, it follows that there must be from the very onset a 
considerable formation of new wall material. In neither case is there 
reason for believing that the initial phase of the elongation is only a 
loosening of the wall structure. Although such a change occurs and plays 
a prominent role, we must always also consider some mechanism estab- 
lishing the fixed structure of the wall. 

All these computations have been carried out under the assumption 
of a proportionality between pressure and tension of the wall, but this 
is erroneous as is shown by Figure 3. Tamiya (13) has deduced formulae 
of wall pressure and suction in relation to turgor tension which include a 
factor for the rigidity of the cell wall. The formula reads: 

Tension = ^^ 

/C, - 1 + e-^^' 

where d denotes the osmotic value and f the rigidity of the wall. 
If his formulae are applied to elongating cells it is found that the rigidity 
factor/is remarkably constant in spite of the very large variations in the 
elasticity as computed according to the ideal physical formulae. This 
is shown by Figure 4. The cause of this disagreement is that in this case 
the cell walls are assumed to obey ideal laws of mechanics, whereas the 
rigidity is computed with attention paid to the empiric formulae of the 
osmotic conditions (13). The two graphs of Figure 4 are constructed 
from one series of measurements. 

Obviously there are several ways of computing the elastic properties 
of the wall, and difterent results can be obtained. We have certainly 


too incomplete a knowledge of the physical properties of the cell wall 
to be entitled to draw any definite conclusions, but it seems probable 
that even if some changes occur in the wall properties, nevertheless 
during the elongation these properties remain remarkably constant. 

Another complication in the evaluation of all data on the mechanical 
properties of the cell walls is the fact that in several instances the elonga- 
tion does not take place uniformly over the whole cell surface. Apical 
growth is a well-known feature of many cells, and the differential growth 
reported by Sinnott is probably a widespread phenomenon in ordinary 
tissues. The consequence is that the walls of growing cells are also 
heterogeneous with respect to their mechanical properties. In the case 
of root-hair cells the basal parts may be elongated on an average 9 per 
cent under a given turgor pressure whereas the apical parts will be 
changed as much as 25 per cent. This shows that the two ends of one 
growing cell must have different structure. Thus, average figures for 
whole cells do not show the properties of the restricted areas where 
growth takes place at any given moment. 

The conclusion to be drawn at present from available data is, however, 
that we cannot abandon the old idea that the fundamental principle 
of cell elongation is an organized, active growth of the wall. It is also 
obvious, however, that the experimental data concerning the cell-wall 
properties are too meager to permit more than a vague hypothesis of 
what takes place during the cell stretching. Nevertheless, any theory 
of the cell elongation and hormone action in growth must take into 
account the active formation of the wall. 


1. AuDUs, L. I., Biol. Rev., 24:51 (1949). 

2. BuRSTROM, H., Ann. Agric. Coll. Sweden, 10:1 (1942). 

3. , ibid., 209 (1942). 

4. , Fysiografiska Sdllskapets Vbrhandl. (Lund), 17:1 (1947). 

5. , Physiol. Flantarum, 1:57 (1948). 

6. DiEHL, I. M., GoRTER, C. I-, VAN Iterson, G. Ir., and Kleinhoonte, a., 

Rec. trav. bot. neerland., 36:709 (1939). 

7. Frey-Wyssling, a., Viertelj. Naturf. Ges. Zurich, 93:24 (1948). 

8. , Growth Symposium, 12:151 (1948). 

9. Kopp, M., Schweiz. Berichte Bot. Ges., 58:283 (1948). 

10. PoHL, R., Planta, 36:230 (1948). 

11. RuGE, U., ibid., 32:572 (1942). 

12. ScHOCH-BoDMER, H., and Huber, P., Verhandl. Naturforsch. Gesellsch. 

Basel, 56:343 (1945). 

13. Tamiya, H,, Cytologia, 8:542 (1937). 

Control of Evolution and Life Processes in Plants 


AS A college student I was often driven to employ a not uncommon 
technique of asking questions when I was unprepared to recite. 
This must have had a measure of success since one of my professors who 
was unable to say anything more favorable in my behalf, wrote as a 
recommendation that I asked intelligent questions. I feel impelled to 
use the same technique here since I feel woefully unprepared to con- 
tribute much in the way of explanation of the mechanisms of chemical 
control of cell division in plants — the topic originally assigned me for 
this symposium. I can, however, think of many questions I should like 
to have answered regarding these important processes. 

A question is the basis of every research. To be fruitful, however, a 
question need not be inteUigent, if by intelligent is meant approved by 
conventional judgments. Such conventional questions will often be found 
to have been already answered. It is the unusual that most easily arouses 
our curiosity. For this reason botanical gardens generally feature exotic 
plants and not the native flora. The commonplace— what is continuafly 
happening under our observation or continually not happening — al- 
though more fundamental than rare occurrences, is usually considered 
as an axiom and not as a matter for inquiry. Unconventional questions 
and those which would generally be called foolish may be the most 
rewarding. Laymen who are unhampered by too great familiarity with 
one's field of research and stereotyped ways of thinking often ask the 
most penetrating and suggestive questions though they generally apolo- 
gize for asking what they think may be a foolish question when they 
know so httle about the subject. It is one of these foolish questions 
which has been asked me on several independent occasions that I wish 
to pass on to you as an introduction to my discussion of the control of 


evolution and life processes. The question has each time been conceived 
as a jest and yet it touches at the very heart of our understanding, or 
rather lack of understanding, of the processes of sex and reproduction. 
If when we gather here again to celebrate the 1 50th anniversary of the 
natal day of the University of Wisconsin, we have a complete answer 
to this question we can feel that the intervening 50 years of research 
have been well spent. The challenging question which has been asked 
of me and which I am passing on to this symposium is— why can't 
you cross corn and beans and get succotash? I confess I don't know the 
answer. Does anyone in the audience.? 

We will return more specifically to the subject of crossability later on. 
It might be pointed out now that the question mentioned implies the 
desirability of translating knowledge through action toward a directed 
goal. For a long time the various aspects of biology tended to be studied 
as separate entities. The structure and behavior of chromosomes for 
example were of interest to cytologists without relation to the trans- 
mission of hereditary traits, and some leading geneticists seemed to 
resent the use of chromosomes in interpreting genetic behavior. This 
stage is passing and cytology and genetics have profitably joined, with 
evident hybrid vigor, to form cytogenetics. Structures and processes 
are being studied in order to interrelate and understand what the 
organism is and does. A still newer phase of research is under develop- 
ment — the utiUzation of this understanding of all the aspects of the 
plant in consciously controlling its Hfe processes and directing its evolu- 
tion. It is about this phase of research, which we may call that of the 
genetics engineer, that I wish to speak a bit. I shall use largely the genus 
Datura for examples to show how new knowledge can be put to work in 
interpreting biological behavior and in molding form and function. 
This is because I am personally more familiar with this plant than 
with most other plants, but especially because I am familiar with the 
important research of associates who have worked with Datura. Though 
I have had something to do with raising certain of the problems it is 
to these others that we are generally indebted for their solution. In 
acting as spokesman for the Datura workers, therefore, I feel as if I 
were shining by reflected light. 

Because of its ease of handling Datura has been much used by the 
early hybridizers. With it Naudin discovered segregation in the F2 
generation before Mendel's classical paper on inheritance in garden peas. 


He thought he had discovered visual evidence for the separation of 
maternal and paternal essences in the Fi generation since in a hybrid 
between a form with spiny capsules and one with smooth capsules he 
sometimes found capsules one half of which was smooth and the other 
half spiny. This is an example of a correct conclusion from faulty in- 
terpretation, since more than 50 years later we were able to show that 
Naudin's findings were due to a virus disease which can be propagated 
by grafting. This environmentally induced type we called "Quercina" 
from its lyrate leaves. Almost identical effects are produced by a recessive 
gene which arose from X-ray treatment. DeVries used the segregation 
of two pairs of characters in Datura in rediscovering the laws of Mendel. 

Of modern students of Datura I want first of all to speak of John 
Belling, a brilliant pioneer in cytogenetics. His finding that certain 
mutant types in Datura had single extra chromosomes, which in many 
cases he could distinguish by size and structure, not only furnished an 
interpretation of our trisomic ratios from Primary 2n + i types but 
showed that such types were due to the unbalancing effect of specific 
chromosomes. This was at the time when some of the leading students 
of Oenothera believed that extra chromosomes were the effect and not 
the cause of 2n + i types. It was nearly 20 years after Belling's findings 
before the successor of de Vries publicly acknowledged that extra 
chromosomes were the cause of mutant types in the Evening Primrose. 

In another group of 211 + i types which we now call Secondaries, 
Belling found that the extra chromosome was often associated with 
two others at metaphase to form a closed trivalent. This could only 
mean, he believed, that the extra chromosome of the trivalent must be a 
doubled half chromosome. He was the first to put effectively to work 
the idea that at metaphase similar chromosome ends are attached like 
dominos. This interpretation not only explained the morphological 
pecuUarities and breeding behavior of Secondaries but laid the way 
for the later discovery of segmental interchange. 

In 1 92 1 there appeared in our cultures a compensating chromosomal 
type "Nubbin" among the offspring of a plant treated by Gager with 
radium emanation. This is probably the first induced chromosomal 
mutation. A couple of gene mutations in the selfed offspring of only 
18 parents also were found from the same treatment but their number 
was too small to warrant being sure that their appearance was due to the 
radiation. Later Muller from his brilliant work with Drosophila gave 


ample evidence that genes may be caused to mutate by radiation treat- 
ment. Ratios of purple to white flower color in the offspring of our 
induced type Nubbin showed it to be a convenient form with which to 
separate the A and B chromosomal types in races from nature which 
we had earlier distinguished by tedious breeding procedure and by the 
fact that B races occasionally threw certain abnormal in + i types. 
It was in one such type that Belling found two large chromosomes 
attached to a small chromosome to form a trivalent at metaphase. The 
fact that like chromosomal ends become attached, which he had earlier 
used in interpretation of Secondary in + i types, he again put to work 
in explaining our B races. He concluded that two of their chromosomes 
could have been derived from the A race by interchange of segments of 
two nonhomologous chromosomes. As shown by Bergner and Satin, 
segmental interchange has been responsible for the formation of so- 
called Prime Types in all the species of Daiura of which an adequate 
number of races have been investigated. It has accompanied the forma- 
tion of species in this genus. The theory of segmental inrri^hange laid 
the basis for interpretation of the peculiar genetic end c ; ^- j'jrcal be- 
havior of the Oenotheras which had baffled students of hei cu 'y '^ince 
de Vries first published on their mutability. 

Through the utilization of Secondary chromosomes and TertuTy 
chromosomes which had been formed by segmental interchange, it 
was possible to synthesize pure-breeding types with predicted ch?rac- 
teristics. As examples the following types all with similar characters due 
to the extra 2 half chromosome may be mentioned: in — (11-12)2 + 
(2- II- 12)2: 2«- (1-2)2+ (-1)2+ (2-2)2:2^- (13-14)2- (23-24)2 + 
(2-14)2 + (13-23)2 + (-24)2. It has been pointed out that such types 
should not be called synthesized new species since they possess no 
isolating mechanism which would prevent them from crossing with the 
normal type. However, if their chromosome number is doubled (and 
this has been done through treatment with colchicine) such crossing 
is almost completely prevented. 

Since tetraploids are a source of triploids and thus of the full range 
of the Primary in + i types, methods have been sought for doubling the 
chromosome number. In 1937 we stumbled on the use of colchicine for 
this purpose. Colchicine apparently prevents the process of spindle 
formation without interfering with chromosome division. Hence the 
chromosomes may divide several times before the effect of the drug 


wears off and spindle fiber formation and cell division is resumed. It 
should be pointed out that a pure /\n branch is probably never produced 
as the immediate effect of treatment with colchicine or other poly- 
ploidizing agents. Dr. Bergner has shown that individual chromosomes 
are dropped out either in connection with the doubling or without any 
doubling of the chromosome number. Colchicine, therefore, is a method 
of securing 2;z — i plants. Furthermore it is only the cells which are 
in division which have their chromosome number doubled. The resting 
cells are not affected. The first effect of the treatment is a rough-leaved 
mixochimera branch in which some of the cells are /[n and others normal 
272. Out of such a mixochimera may grow a smooth-leaved branch which 
may have 2n or 4« cells. As Dr. Satin has shown, such a branch may be 
a perichnal chimera with different numbers of chromosomes in each of 
the three germ layers. She has put this fact to work by labehng the 
three germ layers with different polyploid chromosome numbers and 
in this way has been able to determine the contribution which each germ 
layer makes to the organs of the adult plant. She has thus been able to 
show that the classical interpretation of the stamen as a modified leaf 
is incorrect. From the fact which she discovered, that the transmitting 
tissue in the style through which the pollen tubes grow is of epidermal 
origin, we have been able by using a periclinal chimera with a 472 outer 
layer to get abundant crosses between a m female and a 4/2 male. Such 
a cross would not usually be possible between normal 272 X ^n parents 
since the pollen tubes from the 422 parent, as Buchholz has shown, burst 
in the 222 tissue of the female style. Dr. Satin's findings have been 
extended by others to spontaneous bud sports of fruit trees which 
investigation has shown are often periclinal chimeras. 

Autotetraploidy in which each kind of chromosome is represented 4 
times is of interest to floriculturalists since it usually causes an increase 
in flower size. It has also occurred in nature. The greatest interest in 
methods of doubling chromosome number lies in the ability which it 
affords of producing new fertile and pure-breeding forms with hybrid 
vigor from sterile "mule" plants. This is known to have been a method 
of evolution in nature and has given us some of our best economic 
varieties in wheat, oats, tobacco, cotton, and timothy grass. In the 
development of these forms we have had to wait for the chance hy- 
bridization between distantly related species and the rare doubling 
of the chromosomes of the sterile hybrid. Now by the techniques of 


embryo culture we should be able greatly to Increase the number of 
wide hybrids between species, and by the use of colchicine we should be 
able to transform many of them into pure-breeding double diploids or 
new species. 

Having found a method for doubling the chromosome number it 
seemed desirable to find a method for halving the number. Such a 
method would have considerable theoretical and economic importance. 
A plant breeder, for example, may wish to combine in a single variety 
of wheat the resistance to different rusts and smuts, high yield, good 
milling, and other desirable qualities by crossing two varieties together. 
The resulting hybrid will be highly heterozygous and it may take several 
generations and much labor before he is able to secure a pure-breeding 
type with the combination of characters he desires. If he could only 
induce the reduced egg cells to develop without fertilization he could get 
homozygous individuals in two jumps since the m plants from unfertil- 
ized eggs can be readily induced to double their chromosome number — 
at least such is the case in Datura. We confess we have not yet succeeded 
in our attempts to induce unfertilized eggs to develop but we beheve 
the problem is soluble since the plant does it as shown by the fact that 
we have found over 200 spontaneous m plants in our cultures of Datura 
stramonium. A related question is what are the factors which determine 
whether a cell shall divide mitotically retaining the 2« condition in the 
daughter cells or divide meiotically thus halving the chromosome num- 
ber. Perhaps work now under way in the University of Wisconsin will 
help us to the answer. Through the use of auxins one has a measure of 
control over the production of roots. It would be of convenience to the 
plant breeder if he were able similarly to induce shoot production. 
(This sentence was written before I attended the meeting of the Growth 
Societv in New London where I learned of the work of Skoog in con- 
trolling bud and root formation through interaction of adenine and 
auxin.) The plant breeder's next request would probably be for a 
method of inducing flower formation or usable reduction divisions 
wherever and whenever he wanted them. 

We now return to the question of our introduction: Why can't one 
cross corn and beans and get succotash? This naturally leads us to 
consideration of the barriers to crossability between species. We need 
not mention at this time the various ways in which pollination is hindered 
or prevented such as geographical separation, or flowering at different 


times. There are a series of problems the plants have to solve or barriers 
that have to be overcome before a hybrid between species is possible, 
assuming pollination has taken place. The first problem is that of ger- 
mination on the stigma of the female parent. Pollen germination may 
be induced by manipulation of the osmotic pressure and the chemical 
constituents of artificial media. The function of a pollen grain is by 
means of the pollen tube to carry the two sperm cells down the style 
and discharge them into the ovule. As Buchholz had shown, in many 
species' combinations the pollen tubes burst in the foreign style and are 
put out of commission before they reach the ovary. In one case we 
were able to overcome this bursting. One of our students, Miss Carmen 
Sanz, found that most of the tubes burst when pollen of tomato was 
used on diploid styles of Datura stramonium but that most of them grew 
without bursting when used on tetraploid styles. Some of our students 
have had a measure of success in getting slow-growing pollen tubes 
down to the ovary by shortening and splicing the styles. By these two 
methods, therefore, the barriers of slow-growing and bursting pollen 
tubes have been partially overcome. It is surprising how many species 
are able to overcome the earliest barriers to crossability. Miss Sanz 
tested the pollen of over 60 species on the pistil of Datura stramonium. 
She found that in nearly half the cases the pollen germinated. It was 
especially surprising that several of the monocots such as Freesia and 
Tulip showed relatively good germination though their pollen tubes 
grew only a short distance in the stramonium styles. What is it that slows 
up the growth of pollen tubes in such wide pollinations or causes their 
tubes to burst? Perhaps the difficulty is related to the cause of bursting 
of tubes from a ^n parent in the in style of a female, the cause of which 
is still unexplained. 

A special study has been made of crossability in the 90 combinations 
among the 10 species oi Datura. We had anticipated that failure of egg 
and sperm to unite might be an important barrier to crossability. There 
is no evidence that this is the case in Datura. Apparently whenever the 
egg and sperm are brought into contact they fuse. Barriers come later 
which may prevent their further development. In some hybrid com- 
binations no division of the fusion cell takes place. In other combinations 
cessation of growth occurs after 6 or 8 cells at most are formed. In slill 
other combinations young hybrid embryos of various stages of develop- 
ment are produced. In only 19 species' combinations were viable seeds 


formed, and two of these produced a single good seed only after several 
hundred pollinations. In some cases, especially when long-styled species 
are pollinated by short-styled species, the barrier to crossability may be 
due to inability of pollen tubes to reach the ovary before the style 
abscises, because of their slow growth or early bursting. The major 
barrier to crossability in our group, however, is the cessation of develop- 
ment of the hybrid embryos. What stops the growth of the embryo? 
For this we have some evidence. 

In selfs and compatible crosses. Dr. Satin has found that the cells of 
the integument are filled with starch grains at early stages of develop- 
ment. Apparently the endothelium, a single layer of cells immediately 
surrounding the embryo sac, functions as nurse cells and through its 
activities the cells of the endosperm become filled with fat and granules 
of aleurone which are ultimately digested and passed on to the normally 
developing embryo. The endothelium itself also becomes digested. In 
incompatible crosses, however, the picture is quite different. The number 
of starch cells of the integuments increases and the endothelium, instead 
of remaining a single layer of cells, proliferates and may invade the 
embryo sac and form a tumorous tissue. In consequence, apparently, 
the embryo aborts. This abortion is evidently not due to any lack of 
food but rather to inability of the embryo for some reason to utilize 
the abundant food present or to some factor responsible for the inhibi- 
tion or digestion of the embryo. 

Rappaport has given further information regarding the causes of 
embryo abortion. He finds that there is a water-soluble, thermostable 
inhibitor which can be extracted from the disintegrating material which 
surrounds the aborting or arrested hybrid embryos. This inhibitor stops 
the growth of normal selfed embryos both in vitro and in vivo. Normal 
ovules which have been arrested by injection of a solution of the Inhibitor 
furnish an extract which Inhibits embryo growth when Injected Into 
another young ovary which again develops an extractable Inhibitor. 

Why wide crosses between certain species cause the production of 
ovular tumors and embryo inhibitors is not clear. It cannot be due to 
Incompatibility of different kinds of genes from the two parents since 
the same type of ovular tumors with embryo abortion had been earlier 
found in ihc cross /\n X 2;; and 2/2 X ^n when both the cHploid and 
tetraplold parents were In the same highly inbred line. Moreover a 
similar production of ovular tumors is obtained, as shown by van Over- 


beek and Conklin, when an auxin such as naphthaleneacetic acid is 
injected into young castrated ovaries. It appears that the endothelial 
layer has a lower sensitivity threshold to certain chemicals than the 
other tissues of the ovule. We are attempting to induce the endothelial 
tumors to grow in tissue cultures but so far without success. Why certain 
tissues are easily cultivated and others are difficult is not clear. Also we 
do not know why a fertilized 7.n egg in a diploid ovary develops readily 
to a mature seed but an unfertilized i.n ^gg in a tetraploid ovary usually 
fails to develop. The obvious objective in our endothelium problem is 
to find an anti-inhibitor which will neutralize or prevent the activity 
of the embryo tumors. 

Now how can these facts be put to work? Drs. van Overbeek and 
Marie Conklin several years ago developed special methods for the 
culture of excised normal embryos of Datura stramonium on artificial 
media. It was only the larger embryos which could thus be induced to 
develop further under the conditions used. The fact that sometimes 
haploid (177) seedlings have come from twin embryos within the same 
seed coats suggested that there might be some stimulating substance 
in the normal embryo sac which induced the development of the twinned 
177 embryo. Following this suggestion, coconut milk, which is a natural 
endosperm, was tested and found to be an effective embryo stimulator 
which incited growth in excised embryos as small as o.i mm. in diameter. 
Later, on coming to Smith College, we used the embryo culture tech- 
nique to secure hybrids in wide species crosses. We have found malt 
extract more convenient than coconut milk as an embryo stimulator. 
Since when autoclaved it develops toxic substances, however, we have 
had to sterilize it with a Seitz filter. 

The embryo technique we have put to work in getting hybrids with 
which to determine the interchanges which have taken place in the 
evolution of our ten species of Datura. It also has given us information 
regarding important barriers to crossability. We have already said that 
of the 90 species' combinations only 19 gave viable seeds capable of 
germinating. In addition we have secured dissectablc embryos from 45 
species' combinations. Of these 31 have been grown to a stage of maturity 
at which it could be certain they were the result of hybridization. The 
others may have been hybrids, but it sometimes happens that the 
dissected embryo turns out to be a haploid (177) derived from the 
development of an unfertilized egg, and sometimes an embryo is due to 


a slip In technique and not to the cross intended. In 5 cases sectioned 
material showed that fertiUzation had occurred but growth had not 
proceeded far enough to be detected with a dissecting microscope. There 
were 21 cases which have been recorded as negative, presumably because 
the pollen tubes were not able to reach the ovary. 

Embryo culture of hybrids of our 10 species of Datura raises many 
problems. It has taught us to think of growth and development as 
determined by the internal microenvironment through the interaction 
and balance of chemical stimulators and inhibitors. Even now with our 
limited knowledge and inadequate techniques the possibilities of greatly 
increasing the number of wide species' hybrids, some of which may 
be transformed into pure-breeding new species, is certainly alluring. 
With further knowledge and improved techniques more of the barriers 
to crossability may be found to be removable, and plant breeders may 
inaugurate an age of massive miscegenation. It may then be possible 
to answer the question of our introduction and to say that we can cross 
corn and beans. It will be interesting to see what the offspring will 
be from all these crossings. 

In closing I wish again to apologize for speaking only of work on 
Datura. Other investigators might make similar statements about the 
plants with which they have been working. With examples from studies 
of the plant which I know best I have tried to emphasize the desirability 
of learning all we can regarding the intimate structure and behavior of 
the ultimate elements of the plant, but also of interrelating this knowl- 
edge toward a better understanding of the whole life of the plant and 
especially toward the utilization of this knowledge in the conscious 
control of its life processes and evolution. 

Knowledge is indeed power — potential power — but knowledge which 
is not put to work is sterile. 

Twenty Years of Plant Hormone Research 


IN TALKING on the history of plant hormones, I want to trespass first 
on the era before definite information on the subject was available. 
It is most illuminating to see how many excellent starts were made 
experimentally and theoretically before the overwhelming pressure of 
evidence broke through the inertia of plant science as a whole, and plant 
hormones became generally accepted as important factors in plant de- 
velopment. I still remember an afternoon in 1933 when I was discussing 
plant growth with some students and assistants of one of our big uni- 
versities. Inadvertently I used the words "plant hormone" which im- 
mediately illicited the condescending question, "Do you really believe in 
plant hormones?" 

In and before the eighteenth century the biological sciences were still 
predominantly descriptive, with only here and there some remarkable 
inroads by the experimental method, such as were made by Stephen 
Hale and Jan Ingenhousz. When we find here the first beginnings of the 
hormone concept, it is merely descriptive without any vestige of experi- 
mental evidence. Thus Duhamel Du Monceau makes descending sap 
responsible for root formation, and Agricola is even slightly more specific 
in that he talks about a "materia," or substance which causes root 

Then in the nineteenth century, after a period of contemplation or 
Natur philosophic, botany unfolded into an experimental science. It was 
largely the genius and untiring work of Julius Sachs which led to the 
creation of a picture of the fife processes in the plant. 

These first years of plant physiology were largely given to analysis, 
in which many processes inside the plant were studied separately without 
trying to find links between them. There was so much to be done before 



a synthesis could be attempted. Yet a number of investigators, among 
whom Sachs, Beijerinck, and Darwin should be mentioned, tried to view 
the plant not only as an agglomeration of diverse reactions but as an 
organism. Thus they came to an appreciation of interrelationships be- 
tween different parts. Darwin (7), for example, found that in grass 
seedlings and in roots the response to light and gravity is produced by a 
bending of the zones several millimeters below the tip of the organ, yet 
that the tip itself is essential to the execution of the curvature. His 
clear mind saw immediately the implications of this behavior, namely, 
that some sort of connection between tip and responding region of the 
organ existed. To make this behavior clear to his readers, he drew some 
parallels with animal behavior, which caused a storm of protests. Thus the 
essence of the phenomenon itself was overlooked by fighting about its 
wording. Certainly his tentative suggestion that a substance brought 
about this correlation was never taken seriously. 

Whereas Darwin came upon the idea of links between different parts 
of a plant through a study of tropisms, Sachs (18,19,20,21) hit upon the 
existence of chemical messengers through his studies of the flowering 
behavior of begonias and squashes, and the rooting responses of cuttings. 
He clearly showed that in addition to ordinary nutrition, which makes 
nongreen parts dependent upon leaves, more specific responses were 
evoked by leaves, and through clear reasoning he came to the conclusion 
that minute amounts of chemicals, moving polarly through the plant, 
were responsible for differentiation of roots and flower parts. 

Beijerinck (2) came to a similar conclusion based on entirely different 
facts. He had observed that galls were produced by extremely minute 
quantities of substances given off by the developing larvae of gall insects, 
or in one specific instance by the mother insect while it laid its egg. He 
coined the name "growth enzymes" for such substances, because their 
effect did not seem to depend on their quantity but rather their quality. 
Both Sachs and Beijerinck made some abortive experiments to extract 
their correlation carriers or growth enzymes. But these arguments were 
sufficient by themselves to indicate their existence, according to our 
present-day views. In those days, more than half a century ago, their 
arguments were not considered vaUd, and even in the present century 
much experimental effort was expended in the disproval of Darwin's, 
Sachs', and Beijerinck's views. 

F. W. WENT 69 

It should be stressed that all these considerations, showing how essential 
the idea of chemical messengers was for the interpretation of phenomena 
observed in plants, were published 10-25 years before the hormone 
concept was introduced in animal physiology. If the other botanists 
of that time had been more constructive in their thinking and less of 
the flaw-picking variety, plant hormones might have been a reality 
long before the more tedious proofs for the existence of hormones in 
animals had been produced. However, there should be no crying over 
spilt milk; we should try to learn by the achievements and the faults of 
our predecessors. 

After this first brilliant period in which from so many different angles 
evidence for the existence of correlation carriers in plants was adduced, 
and after the first reaction to this in the form of negations, a period of 
consolidation started. Many investigators (Haberlandt, Ricca, Errera), 
among whom I should like to single out two, J. Loeb and H. Fitting, 
accumulated facts which demonstrated the action of correlation carriers. 
But all remained isolated instances which only during the last 20 years 
could be integrated. 

Loeb (14), the zoologist, became fascinated with the regeneration of 
buds on severed leaves of Bryophylhtm and always had his laboratory 
window sills full of those plants. For years he experimented with them, 
came to the conclusion that this regeneration, the geotropic response of 
the stems, and the outgrowth of axillary buds were all regulated by hor- 
mones, and in several papers he presented evidence that these responses 
all might be due to a single agent. It was impossible, however, to get 
direct evidence concerning the nature of this agent, and thus Loeb (15) 
rejected his earlier idea that a plant hormone was involved, and he 
assumed that all his observations were an expression of a mass action 
of some food constituent. 

In the work of Hans Fitting (10) we see the opposite development of 
ideas. In an extensive investigation of the transmission of tropistic stimuli 
in the Avena coleoptile he rejected any suggestion that this might be 
accomplished by chemical or even physical means. He believed that his 
experiments showed that light induced a polarization in cells, which 
could be transmitted from cell to cell, even when by complicated 
incisions there was no continuous linear connection between the stimu- 
lated and reacting cells. This was different from the results of Darwin, 


Rothert, and others, and it seemed to disprove the existence of a correla- 
tion carrier, or of electrical transmission of a stimulus. This hopelessly 
confused the issue, so that for years its solution was impossible. 

But a few years later when Fitting (i i) worked in Buitenzorg, Java, on 
the flowering of orchids, he found that the swelling of the ovary and the 
fading of the flowers after pollination was due to a water-soluble, heat- 
stable substance, which occurred in the pollinia, and which he compared 
with a hormone. It is unfortunate that this paper of Fitting, which so 
clearly showed the existence of a hormone-like compound in the plant 
and proved that it could be handled outside the plant body like any other 
chemical, had so little influence on botanists, whereas his paper on the 
transmission of the light stimulus, which we might designate as almost 
contrary to the facts as we know them now, had such a profound 

It was only natural that the work of Fitting stimulated PfefFer, and 
he assigned Boysen Jensen, who was just then visiting his laboratory, 
to repeat Fitting's work. Whereas Fitting had made deep incisions in 
grass coleoptiles in many different ways without preventing transmission 
of the phototropic stimulus, Boysen Jensen went one step further. In 
a few experiments he completely severed the tip of an Avena coleoptile 
and then replaced it. This drastic treatment did not prevent the trans- 
mission of the phototropic stimulus either. This experiment invalidated 
Fitting's hypothesis of transmission of a polarity of the coleoptile cells 
induced by light. 

Four years later Boysen Jensen (3,4) published a full account of his 
experiments in Pfeffer's laboratory and of additional work in Copenhagen 
and discussed their impHcations. Within the framework of current ideas, 
especially of Pfeffer and his school, the experiments did not fit very well. 
According to them transmission of a stimulus was a complicated process, 
comparable to the transmission of stimuH in animals, with the tacit 
implication that some electrical phenomenon was involved. This seemed 
to be contradicted by Boysen Jensen's experiment, but in its discussion 
it was pointed out that potential differences could arise by concentration 

Thus in explaining transmission of the phototropic stimulus Boysen 
Jensen arrived at the mental picture of the transmission of a concentra- 
tion gradient of substances, which could pass a cut and which could 
produce the necessary electrical gradient in the base of the coleoptile. 

F. W. WENT 71 

This means that Boysen Jensen definitely thought in terms of diflfusible 
materials, either substances or ions, hut that the complexity of the 
prevailing views on stimuli in plants prevented him from assuming any 
connection between such substances or ions and simple growth. Not 
only Boysen Jensen failed to see this connection, but so did all his 
contemporaries, including Pfeffer. It seems that on the whole more 
significance was attributed to Fitting's experiments, which were pub- 
lished in much greater detail, and the decapitation experiment was 
considered a curiosity. Many investigators tried to explain it away, 
pointing out that since decapitation involved very serious wounding, 
the transmission might, therefore, be simulated by wound reactions. 

Pfeffer was also much worried by Boysen Jensen's experiment, and 
he induced another visitor to his laboratory, A. Paal from Budapest, to 
repeat it. This Paal (16,17) ^^^ i^ great detail, varying the experiment 
in every conceivable way. And even a modern statistician would have 
been satisfied with the total numbers of plants used. The most important 
thing, however, was that Paal probed deeply into the nature of the 
transmission and found that it was just a case of unequal distribution of 
a growth-promoting substance, which is formed in stem tips all the time. 
This liberated plant physiology from some of the mysticism connected 
with tropisms and the stimulus concept. It opened the way to many new 
and interesting experiments, and it was the first generally accepted 
demonstration of the existence of a correlation carrier In plants. 

We come now to the second stage In the development of our knowledge 
about plant hormones. This really started with the work of Paal, who 
clearly stated that in the normal coleoptile tip a growth-promoting 
substance was formed continuously, which regulated the growth of the 
cells below the tip. This, and not Boysen Jensen's work, broke the 
spell cast by Fitting and so many others on the problem of the trans- 
mission of the phototropic stimulus. Many investigators, such as Stark, 
Drechsel, Seubert, Soding, Brauner, and Nielsen, started to work on 
growth-regulating substances In stems and coleoptlles, all within a few 
years from the publishing of Paal's paper. Everything pointed towards a 
speedy solution of this problem. 

Since so much of the basic knowledge about tropisms in plants had 
come from my father's laboratory in Utrecht, and since at the time the 
growth-regulating substance seemed to be particularly important in the 
understanding of tropisms, it is obvious that this substance was much 


discussed among staff and students. It was partially to settle an argument 
that I did my first experiment with auxin. Paal had pointed out that 
there were three possible explanations for a positive phototropic curva- 
ture in grass seedlings: i) decreased production of the growth regulator 
in the front side of the coleoptile tip; 2) decreased translocation along 
the front side; or 3) destruction by light along the front side of the 
coleoptile. Some of my fellow students were in favor of this third possi- 
bility, which seemed to be incompatible with many facts as I saw them. 
If the growth regulator could be handled outside the plant its light 
stability could be investigated. Therefore, I devoted most of the free 
evenings and nights which my miHtary training left me to experiment 
with Avena seedHngs in the laboratory darkroom. Those were exciting 
nights when the effects of decapitation and regrafting of the tip were 
studied and it was gradually revealed that the wounding as such did not 
have such a severe effect as had been supposed. And then on the night 
of April 16, 1926, the first coleoptiles made their bow to the tip diffusate 
which had been collected in gelatin. By 3:00 a.m. the negative auxin 
curvatures were clearly visible, and it was hard for me to realize that this 
momentous experiment could not move my father to get up in the 
middle of the night and accompany me to the laboratory! This work 
was reported for the first time by my father at the International Botan- 
ical Congress in Ithaca (26). I then worked out a quantitative method for 
the assay of this growth-promoting substance or auxin (28), as it was 
later named by Kogl and Haagen-Smit. In this way a good basis was 
laid for a further physiological and chemical analysis of the auxin and its 

In a short time some important principles were established: without 
auxin there is no growth; and as a corollary of this, the Cholodny-Went 
theory of tropisms, saying that tropistic curvatures are due to differential 
distribution of auxin within the responding stem or organ. 

It is typical of the timeHness of the subject, that first in 1926 for 
geotropism, and then in 1927 for phototropism, Cholodny (5,6) pub- 
lished two papers assuming, on theoretical grounds, that gravity or 
unilateral light deflects the normal symmetrical downward stream of 
auxin. And each time a paper of mine (27,28) was in press giving experi- 
mental proof of this assumption. 

Concerning the role of auxin in the plant, Dolk found that after de- 
capitation of a coleoptile the reduction in growth rate was due to 

F. W. WENT 73 

removal of the auxin-production center, and that auxin was produced 
again at the cut surface two and one half hours after decapitation, 
simultaneously with a rise in growth rate of the stump. Avery et al. (i), 
and W. Zimmermann (31) found a close parallelism between auxin 
production by the terminal bud of woody branches and their growth 
rate. Went and Thimann (29) found the same parallelism in the Avena 
coleoptile. It should be stressed that all this work was carried out by 
measuring the auxin which diffused from the tissues into agar. 

It turned out that the responses to gravity of all plants which were 
investigated fitted excellently into the general auxin theory of tropisms. 
The work of Dolk (9) should be mentioned here specifically. 

Thus the morphological polarity existing in the plant turned out to 
be based on the polar transport of auxin, and a typical morphological 
problem was brought into the realm of physiology. It seems amazing 
that morphologists and physiologists have not made more use of this 
common meeting ground. 

It would take too long to follow in detail all the work which was 
carried out in the thirties. First a period of consolidation set in, when the 
knowledge gained about auxin was extended in breadth. In numerous 
talks and speeches my father disseminated the new knowledge, especially 
in Europe, so that soon botanists there were auxin-conscious. In America 
it took longer, and it was really my predecessor in Pasadena, Herman 
Dolk, who brought the experimental attack on auxin to this country. 
With Thimann he did pioneer work, and their first two students, James 
Bonner and Folke Skoog, of course followed an auxin career. Yet 
in those early days the existence of auxin was still questioned by many 
otherwise well-informed scientists. Then it was almost an adventure 
to give a talk on auxin before a botanical audience; the reactions 
ran the whole gamut from enthusiastic acceptance to disdainful rejection. 
It is remr.rkable to observe here, how this situation has changed in the 
last fifteen years. 

Gradually a differentiation in the research on auxin set in. Originally 
the main emphasis had been laid on a study of its biological and physio- 
logical role, its function as correlation carrier, as chemical messenger. 
This research had opened remarkable vistas of the whole regulation of 
plant growth; it had shown that a single agent, auxin, tied together a 
large number of activities inside the plant. It had become clear that the 
effects of stem tip and young developing leaves on stem elongation were 


wholly exerted by auxin; that this same auxin was the agent which caused 
apical dominance in stems, preventing the lower lateral buds from grow- 
ing out. Auxins were intimately tied up with root initiation, fruit devel- 
opment, leaf and fruit abscission, and many other phenomena. Morpho- 
logical polarity in organ formation was apparently largely due to polar 
auxin transport. On the cellular level auxin influenced a number of 
protoplasmic properties and activities, such as permeability, viscosity, 
water uptake, cyclosis, etc. Although none of these phenomena were 
completely and exhaustively studied, the evidence was so strong that the 
participation of auxin hardly seemed a problem any more. Yet if we 
consider in detail the physiological role of auxin in particular cases, we 
find that there are more discrepancies than we suspect when we view 
the problem as a whole. As a particular case I might mention the role of 
auxin in phototropism. After the first main objections against the 
Cholodny-Went theory were allayed through the work of van Overbeek, 
who bridged the chasm between this theory and Blaauw's by showing 
that both principles did coexist in a single plant, the problem seemed 
almost solved, especially when the carotene activation of auxin-a destruc- 
tion by light was found. But now in view of the stability of indoleacetic 
acid towards small amounts of light and many other conflicting facts 
it seems necessary to reconsider the problem of phototropism from an 
auxin angle. 

The work on the chemical nature of auxin in the plant, so brilliantly 
Initiated by Kogl and Haagen-Smit (12) in Holland, and Thimann (23) 
in this country, led to developments along two difl^erent lines. In the 
first place the discovery of the activity of indoleacetic acid and many 
related substances as auxins, by Kogl and Haagen-Smit (13), and its 
extension in this country by Zimmerman and Hitchcock (30) and many 
others, notably Norman and collaborators (25), made it possible to 
apply growth-promoting substances, and later, related growth-inhibiting 
and herbicidal substances, in concentrations far beyond what the plant 
tissues are normally subjected to. This led to what has often been 
referred to as plant pharmacology and made practical applications 

The other development which grew out of the chemical work is the 
biochemical study of indoleacetic acid inside the plant. Since Haagen- 
Smit and collaborators, and Avery and Berger, isolated and chemically 
identified indoleacetic acid in extracts from plants it has become bio- 

F. W. WENT 75 

chemically very attractive to study its production, source, and fate. 
This is now being carried out by many groups of investigators, notably 
Bonner and Wildman, and Thimann and co-workers. It is now evident 
that tryptophan can act as a precursor for indoleacetic acid in the 
plant and that enzyme systems exist which carry out this transformation. 
Thus we can see how in particular instances (for example, the corn 
endosperm) large amounts of indoleacetic acid can be produced. Another 
enzyme system widely occurring in plants inactivates indoleacetic acid. 
Thus we have the possibility of a complicated interplay between these 
enzyme systems which gives us a fine opportunity to explain physio- 
logical phenomena. This has not been done as yet, so we do not know to 
what extent the new biochemical intelligence is able to explain the 
physiological phenomena of growth and correlation. 

There is a basic objection against these biochemical studies of indole- 
acetic acid inside the plant. It is usually tacitly assumed that indoleacetic 
acid is the one auxin in plants, ignoring all the evidence for the role of 
substances hke auxins-a and -b. Thus a necessarily one-sided and incom- 
plete picture is obtained of the biochemistry of auxin. 

Another development has had a decisive influence on the auxin field. 
That is the use of organic solvents for extraction of auxin. In the earlier 
work auxin was obtained only by diffusion from the producing or 
transporting tissues. This had the advantage that only the auxin on its 
way as correlation carrier was caught and measured. But it was impossible 
to make a balance of the source and fate of auxin. Thimann (22) suc- 
ceeded in extracting auxin from Avena coleoptiles with chloroform, 
which later was replaced with peroxide-free ether. First this method gave 
most interesting results, showing that as a coleoptile grew the extractable 
auxin disappeared proportionately with the amount of growth. It also 
could be shown that less auxin could be extracted at any one time than 
could be obtained by exhaustive diffusion, indicating continuous produc- 
tion and utilization of auxin. It could also be shown that the diffusible 
and extractable auxin were correlated with completely different phe- 
nomena inside the coleoptile. The diffusible auxin bore a direct relation- 
ship to tropisms, whereas the correlation between extractable auxm 
and growth was as direct as Thimann and Bonner (24) had found m 
their earlier studies. 

The extraction method, however, soon degenerated into a race for 
the most auxin that could be extracted from any one object. The 


exhaustive extraction became a fad and was in no way correlated any 
more with the physiological role of auxin. From all we know about 
indoleacetic acid and its formation from, for instance, tryptophan, we 
can easily see that under certain circumstances large amounts of indole- 
acetic acid could be released from tryptophan by way of protein break- 
down, which during the life of the tissue never would have been available. 
I would like to urge not the abandoning of different extraction methods, 
but their simultaneous use coupled with a physiological analysis. Thus 
it may be possible to find for each process a form of auxin or a fraction 
with which it is correlated. In this way it may be possible to make some 
sense of the enormous amount of experimental data which are amassing. 

Let me make clear what I wanted to say with a simile. Suppose that 
we want to find the role of water in a steamship. By judicious experi- 
ments we can find that a certain amount of fresh water is necessary for 
steam generation, and sea water is needed for cooling purposes. Such 
experiments would be physiological (for example, plugging the supply 
line for cooling water, excision and regrafting of fresh water tanks and 
so on) . A biochemical study of the steamship would presumably start with 
sectioning of the ship into segments and squeezing each. Some sections 
would yield large amounts of water (those containing the fresh water 
tanks and ballast tanks), others would have intermediate amounts, such 
as the boiler room, and again others would yield almost no water, such 
as the turbine room. Only by judicious separation of water from boilers, 
steam from turbines, and storage tank water could such a biochemical 
analysis of the steamship yield intelligible results. Total extraction of 
water would not give any correlations with the functions of the steam- 

The development of the auxin field is a typical example of how science 
works. Originally growth of a plant was considered almost as a category, 
in the way the Greeks considered water, fire, or earth. It was taken as a 
property of the living organism. When it turned out that in the absence 
of auxin there was no growth, and that stem growth could be controlled 
at will by the application of measured amounts of auxin, it seemed to 
many that we had an explanation of growth. This was partly because 
some of the mystic quality of growth was possessed by a relatively 
simple substance which could be stored in crystalline form on the 
chemical shelf of the laboratory. Some of my more mystically inchned 
friends actually disapproved of the idea of an unromantic chemical 

F. W. WENT 77 

being involved in the growth process. To convince them of the physical 
reality of the growth-promoting substance, I had to do the diffusion rate 
experiments proving that the coleoptile tip extract was not an imponder- 
able emanation, but something possessing the common attributes of 
chemical substances. 

After the first enthusiasm for the growth hormone had worn off, 
it became generally recognized that auxin was not a panacea or the 
stone of wisdom, but simply that we had pushed back the frontiers of 
science slightly, creating a much longer and more complex line demarcat- 
ing fields of knowledge and the unknown. But at the same time it should 
not be forgotten that even though the discovery of auxin complicated 
knowledge about growth, it also produced links between many different 
botanical fields of endeavor, and it brought chemists, horticulturists, 
anatomists, physiologists, and many others together into a strong unity of 
common interests. 

It is sometimes said that botanists are not sufficiently aware of the 
practical implications of their work, or are staying too much aloof from 
practical appUcations, or choose impractical problems to work on. All 
these criticisms are either not based upon fact or are not germane. I can 
assure you that at present only a small percentage of all the practical 
auxin applications we were talking about twenty-two years ago have 
materialized as yet. But a botanical laboratory is usually not rich enough 
nor equipped with large enough experimental fields or greenhouses to 
carry out the semipractical experiments necessary to apply theoretical 

The criticism of the impractical subject matter of botanical research 
can easily be disposed of by pointing towards the auxin research. In the 
early years all auxin work sprang from previous work on phototropism 
in seedlings. This was considered so unimportant by more practically 
incUned botanists, that the plant physiology textbook used most ex- 
tensively in this country fifteen years ago did not even mention the word 
tropism. And not only in this country, but also in Holland and elsewhere, 
auxinolosists were criticized for their use of anemic etiolated seedlings. 
We should be aware of the fact, however, that not until the advent of the 
completely air-conditioned greenhouse or artificially lighted culture 
room, were really conclusive experiments with fully mature plants pos- 
sible. The basic principles of growth are not changed by the growing 
conditions, but quantitative research requires reproducible plant ma- 


terial, and that was available inexpensively, conveniently, with minimal 
space requirements as seedlings. Such seedlings will continue to be an 
ideal source of experimental material, and only for processes typical of 
the mature plant, such as flowering and fruiting, will we be restricted in 
our experimental material to mature plants. 

History is not something which abruptly ends. If I had written my 
speech an hour ago, I would have had to include the advances made in 
the auxin field this morning and afternoon. I think all of us this morning 
were aware that history was being made. Something which had been 
coming slowly, almost imperceptibly, was suddenly spotlighted. I refer 
here to the demotion of auxin by Dr. Thimann from an executive 
position to a simple policing job. This actually creates a number of new 
positions to be filled, and I hope that soon not only nominations for 
the position of Director of Root Formation, or Coordinator of Cambial 
Activity will be made, but that in competitive experimental examina- 
tions these positions will be filled by purified and recrystallized chemicals, 
which will take a place of honor on the shelf of any self-respecting 
chemist, and which will become known by some cryptic combination 
of letters and numbers. 


1. Avery, G. S., Burkholder, P. R., and Creighton, H. B., Am. J. 

Botany, 24:51 (1937). 

2. Beijerinck, M. W., Botan. Zeit., 46:1, 17 (1888). 

3. Boysen Jensen, P., Ber. deut. botan. Ges., 28:118 (1910). 

4. , Bull. acad. roy. sci. et lettres Danemar\, No. i, 3 (191 1). 

5. Cholodny, N., Jahrb. wiss. Botan., 65:447 (1926). 

6. , Biol. Zentr., 47:604 (1927). 

7. Darwin, C, The Power of Movement in Plants (John Murray, London, 


8. DoLK, H. E., Proc. Koninkl. Nederland Akad. Wetenschap, 29:1113 (1926). 

9. , Geotropie en ^rom/o/" (Kemink & Zoon N. V., 1930). 

10 Fitting, W., Jahrb. wiss. Botan., 44:177 (1907). 

11. , Z. Botan., 1:1 (1909). 

12. KoGL, F. and Haagen-Smit, A. J., Proc. Koninkl. Nederland AJ^ad. IVeten- 

schap, 34:1411 (1931). 

13. , and Erxleben, H., Z. physiol. Chem., 228:90 (1934). 

14. LoEB, J., Botan. Gaz., 63:25 (1917). 

15. , /. Gen. Physiol., 5:831 (1923). 

16. Paal, a., Ber. deut. botan. Ges., 32:499 (1914). 

17. , Jahrb. wiss. Botan., 58:406 (1918). 

18. Sachs, J., Botan. Zeit., 2y.iiy (1865). 

19. , Arb. Botan. Inst. Wiirzburg, 2:452, 689 (1880, 1882). 

F. W. WENT 79 

20. , ibid., 3:372 (1887). 

21. , Pliysiologische Notizen VI Uehcr Wachstluansperiodeti itnd Bildungs- 

reizen, Marburg (i8g8). 

22. Thimann, K. v., /. Gen. Physiol., 18:23 (1934). 

23. -, /. Biol. Chem., 109:279 (1935). 

24. , and Bonner, J., Proc. Roy. Soc. (London), 113:126 (1933). 

25. Thompson, H. E., Svvanson, C. P., and Norman, A. G., Botan. Gaz., 

107:476 (1946). 

26. Went, F. A. F. C, Plant Movements, Proc. Intern. Congr. Plant. Sci. 

(George Banta Publishing Co., Menasha, Wisconsin, 1929). 

27. Went, F. W., Proc. KonikJ. Nederland. A\ad. Wetenschap, 30:10 (1926). 

28. , Wuchsstoff tmd Wachstum, Utrecht (1927) and Rec. trav. botan. 

neerland., 25:81 (1928). 

29. , and Thimann, K. V., Phytohormones (Macmillan, 1937). 

30. Zimmerman, P. W. and Hitchcock, A. E., Contrib. Boyce Thomps. Inst., 

7:439 (1935)- 

31. Zimmermann, W. a., Z. Botan., 30: 209 (1936). 

Plant Hormones in Practice 


AS A concept lying along the frontiers of science, plant hormones 
appeared on the horizon with the pubhcation in 1880 by Charles 
Darwin of a book called The Power of Movement in Plants. Startling 
advances have been made and in a way each new phase of the subject 
has constituted a new horizon. 

My assignment today concerns a chemical revolution in science and 
horticultural practice; that is chemical regulation of the pattern of 
growth and development of plants. The subject is new but the progress 
has been rapid. The chemicals involved are called hormones, auxins, 
or growth-regulating substances. The early experiments and results were 
looked upon as laboratory curiosities, but they finally led to botanical, 
agricultural, and horticultural apphcations which are sweeping the world 
today. Owing to the varied types of responses induced by plant hor- 
mones, the subject drew the attention of botanists, horticulturists, and 
chemists. Professors in universities found the field a fertile one for 
graduate students and assigned projects which resulted in published 
theses for advanced degrees. Publications are now pouring out at a 

rapid rate. 

No one person or small group of persons could ever have discovered 
all the facts that are now known about plant hormones. It is hardly 
conceivable that extensive horticultural applications could have arisen 
from the simple but profound researches involving the bending of the 
oat coleoptile exposed to unilateral illumination and the realization 
that this was due to an influence of a chemical nature. It was by no means 
easy for scientists to agree on the meaning of such growth responses, 
but through fear, envy, suspicion, interest, and curiosity one worker 
picks up where another leaves off and advances our sphere of knowledge. 


However, no man's word is law; all claims must be tested and proven 
before they are acceptable in science. 

Interest in the subject of plant hormones from the standpoint of 
fundamental science is still uppermost in the minds of many workers. 
It is increasingly evident, however, that an attack on practical problems 
in the field of plant hormones also contributes to fundamental science. 
It may be said that when practical problems are properly studied, the 
results of the investigation contribute to both science and practice. 
Laboratory curiosities pointed the way to horticultural applications. 
To show the wide interest in the field of plant hormones, the following 
titles of publications which recently came to my desk are taken at 

Inducing fruit set and seedless tomatoes 
Hormones retard bud development 

Hormone sprays and their effect upon the shipping and keep- 
ing quahty of Bartlett pears 

Effects of certain growth-regulating compounds on Irish 

Inhibition of bacterial growth by auxins 
Apple-bloom thinning with chemicals 
2,4-D hits cotton again 
New weed killer plant for Australia 

Direct introduction of chemical substances into herbaceous 

2,4-D injury to trees 

Spraying is a new method of applying root-promoting sub- 

The use of 2,4-D in rice fields for the control of weeds 
Quack grass conquered by new chemical powder 
Method of defoliating 

The enzymatic inactivation of indoleacetic acid 
Synthetic plant hormones and the pineapple industry 
Foresee new uses for 2,4-D 

The groups of chemicals best known as growth regulators are as 
follows: Indole compounds involving ,S-indoleacetic and /3-indolebutyric 
acids; naphthalene compounds involving a-naphthaleneacetic acid and 
its derivatives, /3-naphthoxyacetic and j8-naphthoxypropionic acids and 


their derivatives; substituted phenoxyaliphatic acids especially 2,4-di- 
chlorophenoxyacetic acid (2,4-D) and higher homologs, a-(2-chloro- 
phenoxy) propionic acid, 2-methyl-4-chlorophenoxyacetic acid, 2,4,5- 
trichlorophenoxyacetic acid and higher homologs; and unsaturated hy- 
drocarbons, especially ethylene and acetylene. While there is some varia- 
tion in uses and responses induced with acids, salts, esters, and amides, 
for our purpose they may be thought of as similar in bringing about 
desired results. There are many natural hormones which have not been 
isolated and definitely identified but nevertheless are known to exist. 
Indoleacetic acid and ethylene are two compounds manufactured by 
plants and definitely identified by chemical methods. 

Today there are many lines of research as indicated by the titles of 
publications just read. From the fundamental research angle workers 
are trying to find how hormones work and how they are synthesized. 
Much emphasis is being placed on enzymatic activities, precursors, and 
reactions ultimately leading to active substances. Another group is trying 
to harness plant hormones and put them to work. Physiological activity 
is found with simple structures like ethylene and with the more complex 
structures hke naphthaleneacetic acid and substituted phenoxyaUphatic 
acids. New compounds are being discovered almost daily, and it is 
likely that the most effective ones are yet to come. 

Methods are adapted to meet the needs for each particular kind of 
work to be accomplished. New and better methods are constantly being 
developed. Among others the following apphcations resulting from plant 
hormone research have been established : 

Propagation of plants through hormone treatment of cuttings 
and scions. 

Prevention of pre-harvest drop of fruit, especially apples and 
oranges, with naphthaleneacetic acid or 2,4-D. 
Increasing fruit set and induction of seedless tomatoes with 
numerous hormone-like substances. 

Inhibition of buds to prevent potatoes from sprouting by 
the use of vapors from esters of naphthaleneacetic acid. 
Inhibition of fruit tree buds or prolonging dormancy to pre- 
vent loss through late frost though the methods are as yet not 
well perfected. 

Inhibition of buds of nursery stock to prevent growth until 
the young plants are established. 


Regulation of flowering of pineapples by the use of ethylene, 

acetylene, naphthaleneacetic acid, naphthoxyacetic acid, and 

substituted phenoxy compounds. In this way a crop can be 

staggered to facilitate harvest and canning operations and 

also increase size of fruit of certain varieties. 

Defoliation of plants with ethylene, acetylene, propylene 

gases, and other chemical means. 

Prevention of leaf fall in contrast with defoliation. 

Thinning of fruit, especially apples, with hormone sprays 

bringing about better size and quality of fruit and causing 

biennially bearing trees to bear annually. 

The use of hormone-like chemicals as selective weed killers. 

Defoliation and inhibition of growth were given great prominence 
during the war. It is conceivable that chemicals may be developed which 
can be sprayed on the forest to defoliate the plants and thereby expose 
the enemy. Crops may be killed over large areas by the use of chemicals 
dusted or sprayed by means of an airplane or allowed to float over the 
fields through artificial fogs or smudges. 

Extensive use of plant-hormone-like substances became evident during 
the past year when the sale and use for 2,4-D practically equaled that 
of DDT which has been leading the list of all other organic agricultural 
chemicals. a-Naphthaleneacetic acid was also high on the list since large 
orchards were sprayed to prevent preharvest fruit drop. 

One of the best illustrations of growth regulation of plants is the 
modification of the pattern of new organs growing under the influence 
of applied hormone-like chemicals. Substituted phenoxy, benzoic, and 
naphthoxy acids are particularly effective for this purpose. Mature 
organs do not change their shape, but all new organs which grow under 
the new chemical influence are modified. This leads to the assumption 
that normal leaf patterns are determined by natural chemical influences 
within the plant. Under new and stronger chemical influences the leaves 
and other organs assume a new pattern. So far no practical applications 
for this odd response have been found. However, we should not be 
surprised if one crops up since many other curiosities in the plant- 
hormone field have eventually led to practical applications. 

To date no hormone-like chemicals or any chemical alone have been 
found to stimulate growth of the entire plant in the same sense as is 


recognized for complete fertilizers. The scant claims for such stimulation 
have not stood the test of time. 

Flowers of solanaceous plants last for an abnormally long time after 
being treated with hormone-like substances which induce parthenocarpic 
development. This fact is very suggestive and gives the idea that the 
Hfe of cut flowers can be prolonged. To date, unfortunately, these 
phenomena seem to apply only to intact plants. No effective means for 
substantially increasing the life of cut flowers have been discovered. 
Attempts, however, to find methods or chemicals for this purpose should 
not be discouraged. 

From our experience with substances which induce roots, modify 
leaves, and otherwise regulate growth, it appears evident that aU organs 
of the plant are under some regulating influences probably of a chemical 
nature. It would seem, therefore, that we should find flower-inducing 
substances and shoot-inducing substances. While there have been various 
claims for shoot-inducing substances, this is not a reality in the same sense 
that we have root-inducing substances. There is a special case reported 
where leaves of the pineapple plant produce many adventitious buds 
after treatment with 2,4-D. Other species have not responded in like 
manner. It is logical, however, to assume that such chemicals do exist in 
nature and eventually may be found. Once located, if they ever should 
be, progress should be rapid and results spectacular. 

One of the greatest single problems in agriculture and horticulture 
is the control of weeds. For many years one of the farmer's greatest 
problems has been cultivation largely to rid the field of weeds. However, 
that situation is changing rapidly since the discovery that hormones can 
be used to regulate and actually kill troublesome weeds. Lawns can be 
sprayed to rid them of such weeds as dandelion, plantain, and hawkweed 
without kilhng the grass. Interestingly enough, 2,4-D can be applied 
along with fertilizer to kill the weeds while the grass is being stimulated. 

Weed killing is by far the most important practical use of hormone-hke 
chemicals. The second in importance is the use of naphthaleneacetic 
acid to prevent preharvest drop of apples. 2,4-D alone has a potential 
annual market of more than 100,000,000 pounds and may soon top the 
list of all organic agricultural chemicals. 

There are also other achievements with plant hormones, and these 
illustrate the varied lines of attack from the practical point of view. 
In Hawaii several kinds of plant hormones are being used to force 



pineapples into flower or to increase fruit size. Still other hormones 
prevent premature flowering of pineapples, and fmally the vegetative 
propagation of pineapples is facilitated with plant hormones. Thev are 
rapidly becoming a part of our everyday life, and, as with the telephone 
and the X-ray, we shall soon wonder how we ever got along without 
plant hormones. 

Fortunately monocotyledonous species are more resistant than many 
of the dicotyledonous species. Since corn covers such a vast area of 
land, it is encouraging to find that at least some types of weeds can be 
controlled in the field without harming the corn. The results reported 
are somewhat variable but sufficiently promising to warrant further 
testing. It may develop that certain varieties are resistant enough to 
permit sufficient amounts of 2,4-D per acre to kill noxious weeds without 
affecting corn. Treatment of the soil after planting and before weed 
seeds have started may be the answer to some of the problems. This 
pre-emergence treatment applied after the corn is planted appears to 
control both broad-leaved annuals and weedy grasses. One to five pounds 
per acre can be appfied as a spray on the ground without preventing 
corn from germinating and growing. If applied after corn starts growing, 
care must be taken to hit at the base of the stalk rather than at the 
tops. When tops are sprayed the corn leaves fail to separate and remain 
rolled up so that the tassel must push out at the side. Also abnormal 
flowering and prop roots appear, and the crop may be decreased. How- 
ever, the results are sufficiently promising to indicate that with selection 
of proper varieties of corn and improvement of methods controlling 
weeds in a corn field will be a reality. 

Weeds in sugar cane fields, particularly alligator weed, have been con- 
trolled by applying 0.2 per cent of 2,4-D at the rate of 100 gallons per 
acre. Both dusting and spraying were done without noticeable decrease 
in the sugar production. Weed control in the tropics was demonstrated 
by van Overbeek and shown to be of tremendous economic importance. 

There are some 10,000 miles of waterwavs in the southern district 
in and around the state of Louisiana which have become infested with 
water hyacinth and alligator weed. This constitutes a major problem 
assigned to the army engineers of the southern district. Navigation, 
drainage, health of people and wild life are all affected by this problem. 
There are a number of contact weed killers which are effective on 
aquatic plants, but they are poisonous to fish and other animals and also 


hazardous to humans. Consequently the use of hormone-Uke herbicides 
appears to be the most feasible method for controUing water weeds on a 
large-scale basis. Working with the army engineers during the last two 
years, Hitchcock, Kirkpatrick, Earle, and I have shown that waterways 
can be kept free of aquatic weeds by means of 2,4-D sprays and proper 
maintenance control. The pounds per acre, methods of applying the 
spray, and weather conditions are all important when the first application 
is made. Complete eradication may not be possible, but practical patrol 
maintenance is feasible and keeps weeds within bounds. By means of a 
helicopter the equivalent of 600 acres per day can be sprayed at a cost of 
fifty cents an acre for the use of the plane. The plane was used on the 
marsh lands as well as waterways. The results are striking, and large- 
scale applications of hormone-like herbicides on forest areas, cereal crops, 
and swamp land can be predicted with certainty. 

Let us try to look beyond the present horizons for plant hormones 
and predict some of the things to come. We shall have hormone-Uke 
selective weed killers for every conceivable use — for corn fields, wheat 
fields, orchards, gardens, forests, waterways, swamp lands, hedge rows, 
and yards. Any hormone which has specificity in its effects is likely to 
find special uses. Perhaps the forest can be sprayed to kill all but pines 
or other desired species. With a little stretching of the imagination we 
can picture a lawn without crab grass, a bayou in Louisiana without 
water hyacinth and aUigator weeds, pastures without thistles, onion fields 
not weeded by hand, and helicopters available for spraying at twenty- 
five cents an acre. 

Bud-inducing chemicals are seriously needed. They would facilitate 
propagation of plant parts where buds have not appeared or where they 
have been lost. Day liUes, Gloriosa lilies, and dahlias are often lost because 
the storage organ does not have a natural bud. Internodes which do not 
normally produce adventitious buds could be used for propagation 
purposes. A shoot-inducing substance should cause new shoots to arise 
where desired on the plant to make possible propagation of budless 
parts or improve the shape and appearance of intact plants. There is no 
end to the conceivable uses of bud- or shoot-inducing substances. 

Flower-inducing substances which are thought to exist in nature may 
be isolated, identified, and used as a common tool. If this becomes a 
reality it should be possible to force long-day types to flower during 
short days or short-day types to flower during the long days. In short, 


it should be possible to induce flowering of plants at will and to force 
flowers to grow at unusual places. We can imagine plants with flowers 
on internodes, on leaves, and even on roots. 

At the present time there appears to be considerable variation in the 
time of ripening of fruit. Under the influence of chemicals the time of 
ripening should fit into our needs. During the past season it has been 
shown that apples treated with certain hormone-like chemicals ripened 
prematurely. To a lesser degree this has been noticed for tomatoes. 
Tomato flavors are not affected under the influence of the chemical. 
Apples, however, change flavor and consistency, but the fact that modi- 
fications in time of ripening have been demonstrated offers encourage- 
ment for practical methods applicable to all or many fruits. 

Since it has been demonstrated that fruit buds can be delayed through 
treatment with growth substances, one is led to the assumption that 
fruiting of tropical species and flowering of plants in general can be 
staggered to extend throughout the entire season. Mangoes, for example, 
flower and ripen fruit at definite periods of the year. During the rest of 
the year they are not available as food. Since this is an important tropical 
food, it would be desirable to extend it throughout the season. This 
should become a reality by the proper hormone applications to growing 
buds. The idea is particularly applicable to tropical plants because the 
temperature and other growing conditions would not limit the time of 
fruiting. We should, however, be able also to stagger flowering of spring 
shrubs in the north so that we may enjoy them over longer periods. 

Without making further predictions, it appears that the field of plant 
hormones presents a challenge for scientists with varied attacks. From 
the fundamental research angle, it might be said that the work is just 
beginning. We have found only a few natural hormones, and we still 
know little about how hormones work. From the standpoint of horti- 
cultural applications progress has been more rapid than in fundamental 
research, and the results so spectacular that interest in the subject is 
now sweeping the world. Research holds much for the future and more 
important applications are sure to be made. 

There is much competition in the fields covered by this paper. Perhaps 
that is why progress has been rapid. Research in some phases of the 
subject can be done by amateurs and practical horlicullurists as well 
as by trained scientists. It is well to recognize the fact that several 
techniques have played a part in solving difficult problems. We take 



new courage when we remember that this is the age of research. We 
must follow where research leads. Thousands of researchers are looking 
into the unknown or fields that have no other limits than man's imagina- 
tion. Research is not confined to the laboratory. Its origin is in the 
individual. All of us are researchers no matter what our jobs, and we 
must surpass what has already been accomplished to keep ahead. We 
must search for new and better methods, for even that which we now 
do well must be done better tomorrow. 

Growth Substances 
in Plant Metabolism 

The Study of Growth Substances in Plant Metabolism 


TO A group such as this the title Usted on the program, statement of 
the problem, must appear superfluous. I think this is particularly 
true in view of the fact that Dr. Thimann's paper constitutes a superb 
statement of the problem in addition to an answer to certain phases of it. 
Perhaps it would be more accurate to say that we would now present a 
restatement of the problem. 

Our concern today is to describe in biochemical terms the mode of 
action of plant growth substances. It is immediately apparent that this 
is a big order and one which, in our present state of knowledge, we can 
complete in a sketchy manner at best. However, an integration of 
current information and a projection of this into working hypotheses 
may be useful in directing subsequent efforts. 

In dealing with plant growth substances we must first conscientiously 
define the particular effect in which we are interested and then employ 
caution when we translate results obtained there to explain effects of 
a different nature. This shifting is analogous to shifting from lane to lane 
in traffic, unless you signal your intentions to others you are likely to 
be bumped ignominiously in the rear. 

The need for caution in definition of effects and transfer of information 
from one effect to another arises from the multiplicity of actions of plant 
growth substances. Not only will a given substance cause different actions 
on various tissues but even more intriguing is the fact that a variation 
in the concentration of a growth substance not only will cause a quanti- 
tative variation in the intensity of a plant response but also may cause a 
quahtative change in the response. This property is not confined to 
plant growth substances, for many inhibitors, for example cyanide, may 
exert a stimulation at very low concentrations. However, the variety 


and range of responses elicited by plant growth substances appear to be 

With due caution to the definition of effects studied and experimental 
conditions employed, we now return to the question of how can we 
describe in biochemical terms the mode of action of growth substances 
in eUciting particular responses in plants. As Dr. Thimann has pointed 
out, the minute quantities in which growth substances bring about their 
most interesting effects immediately suggests that they are not acting 
as substrates but in all likelihood are serving as cofactors, constituents, 
or antagonists of enzyme systems. If we accept this point of view we 
can rephrase our basic query as, how can we describe in biochemical 
terms the effect of growth substances on the enzymatically catalyzed 
processes of the cell. This rephrasing does not simpHfy our problem 
particularly, for it does not define which of the multiplicity of cellular 
enzymes we must examine for growth substance effects. 

Our reasoning to this point forms a rather logical approach to our 
problem. But now we are confronted with the necessity of choosing 
one particular enzyme or group of enzymes and determining whether it 
can be related to any particular effect of growth substances. Let us, for 
example, choose herbicidal action as the growth substance effect. What 
enzymes shall we study? Data in the hterature suggest that herbicides 
cause the mobilization of carbohydrate in the plant, so we might logically 
test the influence of various concentrations of herbicides in vitro on 
amylases. Other reports show an increased percentage of nitrogen in 
seeds from plants treated with sublethal doses of herbicides. The herbi- 
cides may influence nitrogen anabolism or catabolism, or the observed 
effect could merely reflect an enhanced carbohydrate depletion. Enzymes 
concerned with transamination or reductive amination might logically 
be examined. A considerable number of reports indicate that herbicidal 
action is accompanied by increased respiratory activity and carbohydrate 
depletion. In contrast we find that herbicides in high concentrations may 
inhibit respiratory enzymes. Hence, we can adopt either of two working 
hypotheses; first, that herbicides kill a plant by stimulating its carbo- 
hydrate oxidation to a point where it burns itself out, and second, that a 
herbicide acts by directly inhibiting respiratory processes. 

These two opposite possibilities reveal another problem which besets 
the investigator — namely, the type of test material to employ. In general, 
although there are exceptions to this statement, the concept of herbicidal 

R. H. BURRIS 95 

action by enhanced respiration has been derived from observations of 
intact plants, whereas the idea of herbicidal action by respiratory inhibi- 
tion has arisen from studies of tissue sHces or cell-free enzyme systems. 
We must accept Dr. Went's warning that the percentage of water in 
any given segment of a steamship does not necessarily define the role of 
water therein. Whenever possible we must devise experimental checks 
with intact plants to test our observations made with isolated enzyme 

Of the enzyme systems investigated for their relation to herbicidal 
action the respiratory enzymes have received most attention. This is 
not surprising, for they are perhaps the most intriguing and widely 
investigated group of enzymes in other regards, and we all admit their 
vital importance in supplying the driving force for cellular processes. 
In addition, work with growth substances has often turned up effects 
which strongly implicate respiratory processes. 

In this statement of the problem we have chosen to discuss the mode 
of action of herbicides, but this has merely served as an example. It is 
open to question whether herbicidal action and other effects of growth 
substances stem from an influence on a common enzyme system. We are 
still some distance from a complete explanation in biochemical terms of 
the mode of action of plant growth substance. It is our hope that the 
accumulation of additional data and the synthesis of information in 
common meetings, such as we have here today, may lead to an elucidation 
of the basic mechanisms of the many faceted activities of plant growth 

Changes in Metabolism During Growth and 

Its Inhibition 


IN THE growth of isolated sections of Avena coleoptiles it was observed 
some years ago (5) that iodoacetate acts as a powerful inhibitor. A 
more careful study of this inhibition and the circumstances surrounding 
it has revealed some interesting facts. The procedure is simple; seedlings 
are grown on moist filter paper and the coleoptiles decapitated; three 
sections each 3 mm. long are mounted on combs floating in indoleacetic 
acid solution containing i per cent sucrose. After growth in darkness 
at ifC they are measured, usually at 7, 24, and 48 hours. All conditions 
are rigidly controlled, for there are numerous possible sources of vari- 
ability. One of these is the age of the plants from which the sections are 
cut (10). The younger the plants, the more vigorously the sections grow. 
However, younger plants are also less sensitive than the older plants 
to inhibition by iodoacetate. In Figure i (from 10) the growth of the 
uninhibited controls is in each case set at 100 per cent. It will be seen 
that coleoptiles 54 hours old yield sections which are only incompletely 
inhibited even at concentrations as high as iq-^M. At the other extreme 
the sections from coleoptiles 120 hours old show threshold inhibition 
even at lo-^M, and at 5 X io~%4 are 100 per cent inhibited. The curves 
for the intermediate ages show the complication of growth promotion 
at low concentrations of iodoacetate, but in other respects the sensitivity 
is intermediate between the two extremes. Thus in general the resistance 
of the plants to iodoacetate decreases steadily with age. 

A characteristic feature of the iodoacetate inhibition is that it is 
completely prevented by the presence of certain organic acids (5,10). 



Malate, fumarate, succinate, isocitrate, and pyruvate are all effective 
in this way (Fig. 2). Concentrations of about M/iooo are needed. With 
these acids added to the auxin and sucrose solution the coleoptile sections 
grow at least as much as though the iodoacetate were not present, and in 
general somewhat more. Even malonate, which at higher concentrations 
is itself a growth inhibitor, protects against iodoacetate. 

2 3 4 5 6 7 



Figure i. Effect of iodoacetate on the growth of A vena coleoptile sections 
after 48 hours at 25°C. in darkness in indoleacetic acid i mg. per liter plus 
sucrose i per cent. Sections cut from plants aged: curve A, 74 hours; B, 64-66 
hours; C, 54-56 hours; D, 96 hours. The growth of the uninhibited controls 
is placed at 100 per cent for each age. (10). 

It follows from these observations that the amount of the dicarboxylic 
acids normally present in the coleoptile must exert considerable influence 
upon the response of the plant to iodoacetate. If the content of these 
acids were high enough, presumably iodoacetate would not inhibit 
growth at all, and if it were to decrease with increasing age, the decrease 
in resistance to iodoacetate would be explained. Accordingly analyses 
were made for total organic acids, malic and citric acids, in coleoptiles 
of different ages. The methods of Pucher and Vickery (8) were used, 
the same 9 mm. zone of the coleoptiles being analyzed as was used for 










Figure 2. Protection against iodoacetate inhibition. Sections cut from 64 
hour coleoptiles, in solutions containing indoleacetic acid i mg. per liter 
plus sucrose i per cent. Iodoacetate was added 7 hours after placing the 
sections in solutions; this accounts for the incompleteness of the inhibition 
at 7 X io~'^M. (10), 

growth studies. The results (Table i) show that the above expectation is 
reahzed. It is of interest to recall that Sweeney and Thimann (9) some 
years ago deduced from the study of protoplasmic streaming that the 
content of organic acids in the coleoptile must decrease with increasing 

Pea stem sections, at the age used for our growth tests, have a content 
of malate and citrate equal to that of very young coleoptiles. This cor- 
responds well with their relative insensitivity to iodoacetate, of which a 


Organic acid content oi Avena coleoptiles as a function of age, and oi Pisum 
stems. Plants grown in darkness at 25°. All figures are micro-equivalents per 

gram dry weight 

Age of tissue Total Acids Malic Acid Citric Acid 

(hours) (/X-EQUIV.) ()Li-EQUIV.) (jU'EQUIV.) 












Pisum fy days 
Stems \ 




concentration as high as 6 X io~^ M is needed for 50 per cent growth 

The question now arises as to whether this disappearance of organic 
acids is a result simply of time or whether they are actually used up in 
growth. To answer this question sections were cut and allowed to grow 
in the usual way in auxin; after measurement they were analyzed for 
organic acids as before, with an additional test for pyruvic. This work is 
tedious, involving large numbers of stem sections. A number of experi- 
ments were also carried out in which growth was inhibited by iodoacetate 
or arsenite. Arsenite differs from iodoacetate in that its action is not 
prevented by organic acids, but it probably reacts with the same 
sulfhydryl enzyme (i i). Some of the results are given in Table 2. Malate, 
but not citrate or pyruvate, is evidently used up when the sections are 


Organic acid content of pea stems before and after growth. 

All values expressed as micro-equivalents per grain dry weight 

Condition of 

Per cent 
















After 24 hours in: 



Auxin + Arsenite 









^Inclole-3-acetic acid, I mg. per liter. 


merely kept in water, that is, when little elongation takes place, but all 
three acids are used up rapidly when the sections grow. As we shall 
see below, these acids are indeed not the only metabolites which are con- 
sumed in the process of growth. Furthermore it is clear that when growth 
is inhibited by arsenite, the consumption, especially of malate and 
pyruvate, is prevented. 

Now the metabolism of the organic acids is interrelated with many 
aspects of the oxidative metabolism of tissues so that the consumption of 
organic acids in growth, and their preservation when growth is inhibited, 
would indicate that growth and inhibition involve deep-seated changes 
in metabolism. Yet it is a curious fact that when growth is promoted by 
auxin the total oxygen consumption is not increased detectably in the 
coleoptile,* and only 15 per cent in the pea; correspondingly also, 
growth can be inhibited without detectable decrease in oxygen consump- 
tion. In the case of inhibition by fluoride there is even a small increase in 
oxygen consumption. These considerations led us to make a more careful 
study of the changes that go on during inhibition. For this purpose we 
have used a growth inhibition of 50 per cent as a reference point because, 

(a) this is achieved with relatively low concentrations of inhibitors, and 

(b) it takes place with only minor changes in respiration rate. Pea stem 
sections were used rather than coleoptiles because they do not require 
added sucrose for growth. The three inhibitors, iodoacetate, arsenite, 
and fluoride were all used. 

We have previously reported (4) that when growth is inhibited re- 
ducing sugars disappear. They disappear steadily with time, whether the 
sections are growing or not, but in the presence of inhibitor (at a 
concentration sufficient to reduce growth by 50 per cent) their disap- 
pearance is accelerated. Arsenite, iodoacetate, and fluoride all had the 
same effect. Since the oxygen consumption of the sections is not in- 
creased, the polysaccharides were determined. However, there was no 
significant change in any of the cell-wall constituents, and the sections 
do not contain starch. The sugars, therefore, were neither being oxidized 
nor polymerized. An examination of the neutral ether-soluble material 

*There is some difference of opinion on this. Van Hiilssen (1936), J. Bonner 
(1936), and Commoner and Thimann (1941) found no increase, while Berger, 
Smidi, and Avery (1946) and J. Bonner (1949) record increases of 20—25 per cent 
when physiological concentradons of indoleacetic acid are added. Kelly and 
Avery (1949) found that 2,4-D increased respiradon in concentrations below the 
toxic level. 



(fats) was then made, and this revealed the fate of at least part of the 
reducing sugars. As shown in Figures 3 and 4, the inhibitor causes a 
lar<^e increase in the content of fats in the sections. The extent of the 
reaction is proportional to the concentration of the inhibitor. The action 
of iodoacetate is in the same direction but less marked. In the case of 
arsenite the final fat level is equal to the initial value, but in the case of 
fluoride there is an actual formation of additional fat. These phenomena 









Figure 3. Changes in the fat content (neutral ether soluble fraction) of 
pea stem sections after 24 hours in indoleacetic acid i mg. per liter with and 
without sodium arsenite. The As concentration giving 50 per cent growth 
inhibition was i x io~^M. 

indicate far-reaching metabolic changes, the occurrence of which is 
concealed by the constancy of the oxygen consumption. 

Still other far-reaching changes take place in the nitrogen compounds. 
Analyses for proteins, amino acids, amides, and ammonia, of which a few 
selected data are given in Table 3, show the following facts: i) as the 
sections are maintained in water the amino acids are consumed; 2) this 
consumption is more complete if the sections are actually growing in 



Figure 4. As Figure 3 but with sodium fluoride. The NaF concentration 
giving 50 per cent growth inhibition was 5 x io~^M. 


Changes in nitrogen compounds of pea stem sections during growth and 

All analyses as per cent of initial dry weight 






Per cent 


(of plasma) 






Water control 





Auxin I mg./Uter 



20. 1 

II. 5 

Auxin plus: — 

lodoacetate 6 x io~^M 





Arsenite i x io~^M 





Fluoride 5 x io~^M 






auxin; 3) the nitrogen of the amino acids is converted partly to asparagine 
and partly to the protein of the cytoplasm; 4) these changes are in large 
part prevented by all the three inhibitors. 

It is of interest that the process of cell elongation does involve con- 
siderable synthesis of protein. It is also of interest that, while there are 
definite differences, all three inhibitors act, at least qualitatively, in the 
same way. This might well be expected for iodoacetate and arsenite, 
since both are sulfhydryl reagents (11), but fluoride almost certainly 
acts on another enzyme system and hence similarity between its action 
and that of the others would hardly have been expected. 

Lastly it must be pointed out that these results, incomplete as they 
are in themselves, are consistent with the idea that it is organic acid 
metabolism which is primarily responsible for growth. For it is only by 
way of the organic acids that the oxidation of sugars, the formation of 
fats, and the conversion of amino acids to asparagine are linked. This 
interpretation therefore agrees well with the concept put forward in an 
earlier paper, that auxin acts on an enzyme concerned in four-carbon 
acid metabolism. 


1. Berger, J., Smith, P., and Avery, G. S. Jr., Af}7. J. Botany, 33:601-04 


2. Bonner, J.,/. Gen. Physiol., 20:1-11 (1936). 

3. , Atn. J. Botany, 36:323-32 (1949). 

4. Christiansen, G. S., Kunz, L. J., Thimann, K. V., and Bonner, 

W. D. Jr., Plant Physiol., 24:178-81 (1949). 

5. Commoner, B. and Thimann, K. V., /. Gen. Physiol., 24:279-96 (1941). 

6. HuLssEN, C J. van, Ademhaling, Gisting and Groei. Dissertation (Utrecht, 


7. Kelly, S. and Avery, G. S. Jr., Am.]. Botany, 36:421-25 (1949), 

8. Pucher, G. W., Wakeman, A. J., and Vickery, H. B., Ind. Eng. Chem. 

Anal. Ed., iy.2/^^-^6 (1941). 

9. Sweeney, B. M. and Thimann, K. V.,/. Gen. Physiol., 25:841-54 (1942). 
10. Thimann, K. V. and Bonner, W. D. Jr., Am. J. Botany, 35:271-81 (1948). 
ri. , il^id., 36:214-21 (1949). 

Stimulation of Respiration in Relation to Growth 


THE widespread natural occurrence of plant growth substances is 
one of the striking physiological attributes of the plant kingdom. 
From higher plants to lower plants hardly an organism, or any of its 
parts, has ever been tested and found to be without hormones. This 
wide distribution of such substances in plants, in the light of their 
high physiological activity, argues for their having some sort of universal 
role in metabolism. 

There is ample circumstantial evidence to connect these substances 
with the stimulation of respiration. For example, seeds of certain plants 
which have been carefully investigated are known to possess a hormone 
precursor which is hydrolyzed in the course of germination, giving a 
continuous high level of hormone supply during early growth. This 
high hormone supply goes hand in hand with the accelerated respiration 
and growth which is typical of germinating seeds. The growing root 
and stem tips of plants, as well as the cambium, constitute another 
example: meristems are regions characterized by the rapid production of 
new cells, the basis of which is the synthesis of new protoplasm. Wherever 
new protoplasm is produced hormone concentration is high as are the 
respiration and growth rates. In spite of these commonly known facts, 
to date there is surprisingly little experimental evidence hnking hormone 
concentration with the stimulation of respiration, and growth. 

Now briefly as to how phytohormones exercise their growth-regulating 
roles. We have the simple picture that growth responses, in higher plants 
at least, depend on differential distribution of phytohormones in their 
tissues and on the relationship of these hormones to other substances 
which are important in growth. If it is finally shown that hormones 
exert their growth-controlling influence through respiration, it seems to 


become a matter of different rates of metabolism being responsible for 
different growth responses, such rate differences being due to dissimilar 
hormone concentrations in various tissues. It has never been demon- 
strated, however, that hormones occur in tissues in concentrations that 
would inhibit metabolism. But let us look at the literature on the subject, 
to see what evidence there may be for linking hormone-accelerated 
respiration with growth. 

The first study of this sort is that of Bonner (6). He suspended small 
segments oiAvena coleoptiles in solutions of an impure hormone prepara- 
tion. At suitable concentrations the segments elongated considerably 
(over the controls), and this speeded growth was accompanied by an 
increase in the rate of respiration as measured by oxygen uptake in a 
Warburg respirometer. The author concluded, "it seems possible that 
the increase in respiration caused by growth substance may be an essential 
part of its action in growth." Three years later Bonner (7) again reported 
on the same general experiments, but this time carried out with improved 
Warburg vessels and crystalline auxenolonic acid (auxin b) from the 
Utrecht laboratories. From this work he concluded, in agreement with 
similar results obtained at Utrecht, that auxenolonic acid failed to 
stimulate respiration; indeed, a threefold purification of the impure 
hormone he had used in 1933 removed most of its stimulatory power. So, 
although growth was stimulated in the presence of the purified hormone 
preparations, Bonner reported no increase in respiration. 

In 1940 duBuy and Olson (11) studied the respiration of single 
infiltrated Avena coleoptiles and found that indoleacetic acid alone or 
in the presence of fructose had no accelerating effect on respiration. 
Sweeney (18) pointed out later that the failure to obtain accelerated 
respiration was caused by the use of water-infiltrated coleoptile segments, 
which were low in oxygen tension. 

In 1941 Commoner and Thimann (10), in an experiment of somewhat 
similar design, reported a small but significant stimulation of respiration 
in Avena coleoptile segments in the presence of certain four-carbon 
acids, mahc and fumaric in particular. They found that the acids ac- 
celerated growth as well as respiration if the hormone indoleacetic acid 
was present in a concentration of i to 10 mg. per liter. When the data 
were plotted the curves for growth and respiration were found to 
parallel one another roughly. The authors concluded that "the four- 
carbon acids provide a respiratory system which is part of the chain of 


growth processes, and which is in some way catalyzed by auxin. It 
represents a small but variable fraction of the total respiration." Here 
for the first time was promising evidence of hormone stimulation of 
respiration and stimulation of growth. At this point growth studies seem 
to have fallen by the wayside for a number of years. Respiration studies, 
as influenced by hormones, were no longer carried out with parallel 
fact-finding on growth. 

Evidence for the stimulation of respiration with the hormone indole- 
acetic acid comes from the work of Berger and Avery (1,2,3,4). They 
found both malic and alcohol dehydrogenase enzyme systems isolated 
from coleoptiles to be stimulated by pretreatment of the tissue with 
indoleacetic acid, but not by the addition of indoleacetic acid to the 
in vitro preparations. The activity of alcohol and malic dehydrogenases 
in Avena coleoptile tissue was increased respectively 200 and 150 per 
cent when segments of the coleoptile were treated with relatively high 
concentrations of indoleacetic acid (10 mg. per hter — the same concen- 
tration found most effective by Commoner and Thimann). Although 
not linked experimentally with studies on growth this evidence supports 
the view that both these dehydrogenases are closely concerned with 
growth, and that the hormone indoleacetic acid controls growth, at 
least in part, by activating them. 

Employing the same high concentration of indoleacetic acid, Berger, 
Smith, and Avery (5) reported increases of 35 per cent or more in 
oxygen uptake in Avena coleoptile segments in sucrose solutions. But 
they found malate, and therefore presumably fumarate, to function as a 
substrate rather than as a catalyst in this reaction. Skoog (15) points out 
that the role of substrate rather than catalyst agrees with Lundegardh's 
conclusion that fumaric and malic acids may be intermediaries but not 
catalysts in respiration of wheat roots. 

The most extensive recent study on Avena coleoptile tissue (8) includes 
a single experiment that should be mentioned here. It concerns hormone 
stimulation of respiration and growth. Bonner now reports growth and 
respiration, as determined at the end of a 24-hour growth period, to be 
stimulated 40 and 38 per cent respectively in the presence of indoleacetic 
acid at a concentration of 10 mg. per liter. 

Coleoptile tissue has also been investigated for its respiratory response 
to the synthetic hormone, 2,4-dichlorophenoxyacetic acid (2,4-D), and 
increases of 20 per cent or more in oxygen uptake in coleoptile segments 


are reported by Kelly and Avery (13) for 2,4-D in concentrations ranging 
from somewhat less than i to approximately 100 mg. per liter. In the 
presence of malic acid the stimulation of 2,4-D is much enhanced. In 
similar investigations on young pea stem tissue it was found that the 
concentration of 2,4-D required to give a 20 per cent stimulation was of 
the order of i/iooo that needed for Avena. Growth measurements, 
unfortunately, were not included in the study. 

At least brief mention ought to be made of several other types of 
plant tissue which have also been investigated for their respiratory 
response to 2,4-D. For example, the carbon dioxide output of ripening 
pears was shown by Hansen (12) to increase as a result of dipping in 
2,4-D at concentrations of from 50 to 100 mg. per liter of solution. Bean 
and morning-glory plants sprayed with 2,4-D (1,000 mg. per liter) 
showed up to 80 per cent greater carbon dioxide output than did un- 
sprayed plants (9), Rhizomes and roots of bindweed showed an average 
of 70 per cent increase in the uptake of oxygen as a result of spraying 
intact plants with 2,4-D at 1,000 mg. per liter (16); and Southwick (17), 
employing various 2,4-D treatments for several varieties of peaches and 
one apple variety, hastened ripening and increased carbon dioxide pro- 
duction by 10 to 30 per cent. 

One of the most recent studies (in press) involving stimulation of 
respiration in relation to growth on tissue other than Avena coleoptiles 
is that of Louis Nickell (14). He has kindly given me his. permission 
to tell you about it. The tissue used was the Rumex virus tumor of L. M. 
Black, grown in culture on a fully known substrate containing no 
hormones. For the respiration experiments various naturally occurring 
and synthetic hormones were added to the Warburg vessels, among them, 
indoleacetic acid and 2,4-D. 

Nickell observed that oxygen uptake was stimulated 40 to 60 per cent 
when indoleacetic acid (concentration range 0.00 1 to i.o mg. per liter) 
was added to the solution in the Warburg vessels. When this same 
hormone was added to the tissue culture substrate greatest growth 
(about 20 per cent over the control) was obtained at 0.0 1 mg. per liter. 
With 2,4-D the oxygen uptake was stimulated approximately 20 per 
cent over the control in a wide concentration range, and greatest growth 
in culture (about 20 per cent over the control) occurred when the syn- 
thetic hormone was added to the substrate at a concentration of o.i mg. 
per liter. It might be pointed out that although the tissue culture experi- 


ments ran for three weeks, and the Warburg determination for only, 
three hours, the stimulation range was common to both the growth and 
the respiration experiments. 

Thus in the past decade it has been found that indoleacetic acid 
stimulates both respiration and growth in two widely different tissues, 
one of them carrying a virus disease. In one study where 2,4-D was 
employed, the general pattern of stimulation closely followed that 
for indoleacetic acid. Other studies on respiration in response to added 
hormone (that do not include growth) show that in living tissues certain 
concentrations of hormone generally stimulate respiration. There is as 
yet no evidence that hormones occur in tissues in concentrations that 
would inhibit respiration, and therefore presumably inhibit growth. 

If we are to understand how hormones control metabolism, and 
ultimately, growth, it is important that we have further studies on the 
stimulation (and inhibition) of respiration in relation to growth. It 
is Important, too, that such studies be carried out on tissues capable 
of giving a growth as well as respiratory response. 


1. Berger, J. and Avery, G. S., Jr., Science, 98:454 (1943). 

2. , Am. J. Botany, 30:290 (1943). 

3. , ibid., 30:297 (1943). 

4. ,/^/fl'., 31:11 (1944). 

5. Berger, J., Smith, P., and Avery, G. S., Jr., ibid., 33:601 (1946). 

6. Bonner, ].,]. Gen. Physiol., 17:63 (1933). 

7. , ibid., 20:1 (1936). 

8. , Am. J. Botany, 36:323 (1949). 

9. Brown, J. W., Botan. Gaz., 107:332 (1946). 

10. Commoner, B. and Thimann, K. V., /. Gen. Physiol., 24:279 (1941). 

11. DuBuY, H. G. and Olson, R. A., Am. J. Botany, 27:401 (1940). 

12. Hansen, E., Plant Physiol., 21:588 (1946). 

13. Kelly, S. and Avery, G. S., Jr., Am. J. Botany, 36:421 (1949). 

14. Nickell, L. G., Am. J. Botany (in press). 

15. Skoog, F., Ann. Ret'. Biochem., 16:529 (1947). 

16. Smith, F. G., Hamner, C. L., and Carlson, R. F., Plant Physiol., 

22:58 (1947). 

17. SouTHWicK, F. W., Proc. Am. Soc. Hort. Sci., 47:84 (1946). 

18. Sweeney, B. M., Am. J. Botany, 28:700 (1941). 

Respiratory Changes in Relation to Toxicity 


GROWTH substances cause a variety of toxic effects in plants which 
often involve inhibition or alteration of respiratory metabolism. 
The nature of these effects is important not only as a clue to the role of 
growth substances in plant metaboHsm but also as a basis for their more 
successful application as herbicides. The distinction between the normal 
or physiological effects of growth substances and the abnormal or non- 
physiological effects is not a sharp one, and for the present purpose it is 
useful to consider nonphysiological or toxic action in broad terms as any 
alteration in metabolic processes which is deleterious to plant function. 
We must include, then, the response to intermediate levels of growth 
substances ranging from those characteristic of physiological hormone 
action to those used in herbicide work where toxic action may become 
less specific and too complex for present interpretation in metabolic 

The object of the present discussion is to summarize the evidence 
relating toxicity and respiratory changes and to consider its interpreta- 
tion in terms of present views of growth substance action and plant 
metabolism. The data are necessarily drawn from many types of experi- 
ments often not primarily designed to study toxic action and the results 
can be described only in broad terms. Data are confined to the effects 
of indoleacetic acid (lAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), 
the chief natural and artificial growth substances used in this kind of 
experimental work. 

Respiratory Changes in 
Elongation, Streaming, and Water Uptake 

Much of the work on the nature of hormone action has shown a 
close relation between cell elongation, protoplasmic streaming, and water 


uptake and respiratory activity measured by oxygen uptake or dry 
weiglit loss. Higher than optimum levels of growth substances usually, 
though not always, have caused parallel depression in over-all respiration 
and the associated physiological responses. Table i summarizes some of 
the data comparing these responses at optimum and inhibitive or toxic 
levels. Both lAA and 2,4-D begin to inhibit coleoptile or stem elongation 
and oxygen uptake in the range of 10 to 100 ppm. (13,15,18). Root 
elongation is much more sensitive but so far there is little if any evidence 
of an accompanying change in respiration (12). Protoplasmic streaming 
in Avena coleoptile has a range of sensitivity similar to elongation 
(33,34). In roots, however, while the optimum level for streaming is 
similar to that for elongation the former is strongly inhibited only at 
10 ppm. or more. Active or nonosmotic water uptake and respiration 
in potato tuber slices (14,28) and Avena coleoptiles (17) are both 
inhibited in the 10 to 100 ppm. range. In wheat roots (25), however, 
2,4-D inhibited nitrate uptake down to o.i ppm. with an actual increase 
in oxygen uptake at the upper range of 5 ppm. It appears that the corre- 
lation in stem and coleoptile between the inhibition of respiration and 
of other physiological effects does not hold in roots. 

Respiratory Changes in Germination 

Respiratory changes have also been observed in the toxic action of 
growth substances in the germination and growth of whole seedlings. 
Here in addition to elongation, water uptake and streaming, cell division, 
and other physiological processes must be considered. Table 2 summarizes 
this work. Pratt (26) first showed that wheat-seedling growth by dry 
weight was inhibited beginning as low as o.oi ppm. of lAA while oxygen 
uptake increased to about 50 ppm. and was strongly inhibited only at 
150 ppm. This suggests that root tissue accounted for the major part of 
the seedling response at this stage. Hsueh and Lou (16) have reported 
some interesting though brief data comparing the nature of the res- 
piratory changes in barley and rice after 2,4-D treatment. Rice, charac- 
teristically an anaerobic seed, was relatively resistant even to 1,000 
ppm. of 2,4-D while barley, an aerobic seed, was completely inhibited 
between 140 and 700 ppm. Furthermore, there was a clear indication 
that carbon dioxide evolution was less inhibited than oxygen uptake. 
This was especially true of rice and was most marked at the two and 
three-day stage where control samples of barley and rice, respectively, 



Growth substance effects in isolated organs and tissues 












:. IN PPM.) 


Avena coleoptile 



O2 uptake 
+ 50 (max.) 

+ 25 (max.) 

Commoner and 
Thimann (13) 



D 100 

+ 20 

+ 25 (max.) 

— 10 

Kelly and 
Avery (18) 

Pea stem 



D 0. i-io 



+ 10 

Avena root 




+ 30 

Bonner and 

Wheat root 




No change in 

— 2 



Koepfli (8) 
Thimann (37) 
Burstrom (12) 

Avena coleoptile 


Potato tuber 

lAA 10 +50 Sweeney (33) 

ca. 70 —30 

Avena root lAA o.oooi- +25 to + 30 Sweeney (34) 

0.0000 I 

I +10 to o 

10 -13 

Dry weight Water uptaJ^ 
1+34 +34 Reinders (28) 

10 +71 +46 

100 (became flaccid and died) 
10 +25 Commoner, 

Fogel and 
100 —12 Muller (14) 

O2 uptake 
20 max. Kelly (17) 

40 max. 

o . I Nitrate uptake 

— 13 Nance (25) 

I —42 

5 +20 to +40 ~6t, 



Avena coleoptile lAA 
Wheat root 2,4-D 

♦Percentage change from control; many figures estimated from graphs. 





























,,— ^ 




c ,-> 



rt VO 



3 3 
t« O 




E J 



n 2 ^ 


O * 


w w 


















•<*- ir\ iTN "<> 





















LTi ^ 

■s» 0\ 



lA M 

(N -T3 

-«; fs 




1 + + ^ 

f 1 









OO - 





" r) 


+ 1 


+ 1 







ON ON oo ir\ 



« -. t^ O 














o o o 
^ o o 
'-' r^ o 


« n ro 


<U C 

« s 

TD 4-' 





ir\ m 


I I 

It It 

"D CJ 

CJ V3 

1« ^ 


_C 3 























had reached complete germination. The suggestion was made that it is 
the aerobic phase of respiration which is most sensitive to 2,4-D inhibi- 
tion. Taylor (36) has made a similar study of the respiratory changes 
following treatment of wheat and mustard seedlings but at lower levels 
of 2,4-D. His data also show some evidence that CO2 evolution is less 
affected than oxygen uptake expecially in wheat and in the earlier 
stages of development. It is interesting to note further that aerobic 
bacteria seem to be much more sensitive to 2,4-D inhibition than 
anaerobic types (39). The aerobic species were inhibited from 2,000 to 
as low 0.2 ppm. in some cases while facultative and obligate anaerobes 
were unaffected or even slightly stimulated. Further comparisons of this 
type would be valuable, especially if organisms were selected which were 
more similar metabolically except for oxygen requirement. 

Respiratory Changes in Larger, Intact Plants 

Most work specifically on toxic or herbicidal effects of growth sub- 
stances has been on larger intact plants with a single dose treatment of 
the aerial parts. Morphological effects vary widely with the conditions 
from a stunted or suppressed development to accelerated, abnormal 
growth. Metabolic changes may be equally profound. In general altera- 
tions in gross carbohydrate and nitrogen metabolism are found with 
both lAA and 2,4-D treatment at levels as low as 25 to 50 ppm. in the 
applied solutions. It is obviously difficult to compare such dosage levels 
with those in the work on isolated tissues or seedlings since the effect 
may be at some distance from the point of application and the amount 
absorbed is uncertain. However, there is characteristically a marked 
acceleration in hydrolysis, translocation, and utilization of the carbo- 
hydrate reserves (2,22,23,24,27,31,32) accompanied by increased protein 
synthesis (29,30), water content (10), and mineral content (11), and 
by varying degrees of cell division and enlargement. Further evidence 
of accelerated metabolism was shown in Luecke, Hamner, and Sell's 
recent report (19) of large increases in treated bean stems of the B 
vitamins which are coenzymes in intermediary carbohydrate metabolism. 
In some of these cases there was also a marked increase in respiratory 
activity which may have accounted for most if not all of the carbohydrate 
utilized (27), while in others the over-all respiratory level seemed to be 
unchanged (23). 

A more detailed examination has been reported by the author (30) 


of the changes in respiratory capacity, chemical composition, and gross 
histology in bean stems after treating the plant with i,ooo ppm. of 
2,4-D. Within 24 hours there was a significant difference between treated 
and control tissues in oxygen uptake on a dry weight basis, though not 
on total nitrogen basis, and before any signs of abnormal meristematic 
activity. Maximum differences in oxygen uptake and anaerobic carbon 
dioxide evolution occurred by the seventh day again with the treated 
tissue higher on a dry weight basis and lower on a nitrogen or protein 
basis. Anaerobic glycolysis on a nitrogen basis showed the largest dif- 
ference, with the treated slices less than one-third the control value. 
This difference was also reflected in the characteristically lower res- 
piratory quotient of treated tissue. While this type of analysis gives a 
more complete picture of the respiratory changes following treatment 
it is not sufficient. For example, it does not distinguish between direct 
action of the growth substances on the respiratory mechanism per se and 
indirect effects due to changes in the available substrate. The best we can 
conclude at present from the toxic effects on larger intact plants is that 
there is a pronounced alteration in metabohsm that seems to be closely 
associated with respiratory processes. 

Respiratory Changes in Tissue Slices and Enzyme Effects 

Another type of approach is found in the in vitro treatment of tissue 
slices which has the advantage of limiting the variability in the plant 
material and affording better control of treatment. Bean stem sHces 
similar to those described above (30) were treated with o.i to 100 ppm. 
of 2,4-D in aerated aqueous media and the changes in oxygen uptake 
measured after 24 to 48 hours. No significant effects were found at o.i 
ppm.; at i and 10 ppm. results ranged from slight inhibition to marked 
acceleration but still without clear evidence of stimulated meristematic 
activity; and at 100 ppm. the treated slices were inhibited about 80 
per cent. Mitchell, Burris, and Riker (21) have made a more extensive 
study of this kind in which the inhibition of oxygen uptake by 0.002 
M. 2,4-D and lAA (350 and 410 ppm. respectively) in root and stem 
slices of several species was measured over shorter intervals. The per- 
centage inhibition was greater in roots than in stems, except for lAA 
on tomato, and greater with 2,4-D than lAA, except for tomato roots. 
They also found that lAA of about 100 ppm. caused no change in res- 
piratory quotient although there was 47 per cent inhibition of oxygen 


uptake; and they found that the extent of inhibition was directly 
proportional to the oxygen tension indicating that the same systems were 
hmited by oxygen as inhibited by lAA. They found further that the 
inhibition by lAA was largely reversed by washing stem slices which is 
similar to Audus' observation (3) that root growth inhibition by 2,4-D 
was similarly reversible. Further in vitro studies with these mature tissue 
slices should provide a valuable link between the classical seedling tissue 
studies and the herbicidal investigations with intact plants. 

Finally, there have been a few investigations of the in vivo and in vitro 
effects of growth substances on respiratory enzymes themselves. Berger 
and Avery (4,5) working with Avena coleoptile homogenates found a 
marked rise in alcohol dehydrogenase activity after 15 to 24 hours 
treatment with 10 ppm. lAA but no effect of />2 vitro treatment until 
inhibitive concentrations were reached between 100 and 1,000 ppm. 
The Wisconsin workers (21) have failed to find any in vitro effect on the 
cytochrome, ascorbic acid, catechol, or glycolic acid oxidases by either 
lAA or 2,4-D in the range of about 2 to 200 ppm. The direct inhibition 
of oxidases or dehydrogenases seems, at present, an unUkely basis for 
the toxic action of growth substances. 

Discussion and Conclusions 

The evidence relating toxic action of growth substances with respira- 
tory metabolism is still rather scattered and fragmentary but some 
interesting possibilities are apparent. In the relatively simple cases of 
inhibition of elongation, streaming, and water uptake in Avena coleoptile 
there seems to be a close parallel. The major evidence, in fact, for a 
respiratory role of plant growth substances, physiological, or nonphysio- 
logical is from studies on this classical tissue. With roots the evidence 
is limited, though it looks as if the inhibition of cell elongation and water 
uptake may occur at concentrations of growth substances which do not 
affect over-all respiration. This, of course, should not be considered 
evidence against any connection with respiratory metabolism since the 
fraction of total respiration involved in these processes may be small 
(13) or may not be expressed in terms of gross oxygen uptake or dry 
weight change. Our greatest need is clearly for a closer examination of the 
metabolic changes which accompany growth substance treatment. 

In the case of intact plants, seedlings or larger, toxic effects are more 
varied and complex, and there is no close parallel between respiratory 



inhibition and the other physiological effects. Toxic action in more 
mature plants may actually result in increased total respiration, with or 
without accelerated growth and development. Probably the fundamental 
effect, however, is the tendency to a change in type of respiration. 

What evidence do we have from these toxic effects of the way growth 
substances affect respiratory metabolism.? One of the most interesting 
possibilities is the greater sensitivity of the aerobic than of the anaerobic 
phase of respiration in seedlings to 2,4-D inhibition. This may also be 
involved in the differing sensitivities of roots and shoots if Taylor's 
observations on rice and wheat (35) on the relative abilities of these 
organs to grow anaerobically are of general occurrence. Mitchell, 
Burris, and Riker's (21) failure to find any change in respiratory 
quotient in bean stem tissue after short term in vitro treatment may 
indicate only that this aerobic-anaerobic difference is confined to less 
mature tissues. There is, in fact, considerable evidence for a change in 
respiratory mechanism from seedling to mature leaf tissue (1,20). It 
would be interesting to know as well whether the difference in monocot 
and dicot sensitivity to 2,4-D at emergence might be explainable in 
these terms. The special sensitivity of aerobic phases of respiration to 
growth substance inhibition is at least consistent with the fact that plant 
growth in general is aerobic, and with the present views of Thimann, 
Bonner, and others (7,38) that lAA affects growth through a sulfhydryl 
enzyme system and is involved in aerobic phosphate transfer. 

In the light of recent work of Thimann and W. D. Bonner (38) and 
J. Bonner (7) it is interesting to compare the action of known respiratory 
inhibitors with that of the growth substances, themselves. At appropriate 
concentrations most of the respiratory inhibitors like the growth sub- 
stances will either accelerate or inhibit growth. In some cases the concen- 
trations for growth inhibition and respiratory inhibition are similar, for 
example, the action of cyanide and lAA on Avena coleoptile (6). In 
other cases growth may be more sensitive than respiration, as in iodo- 
acetate (13) and fluoride action (9) and the effects of growth substances 
on elongation and water uptake in roots. Furthermore, at a given 
concentration growth may be inhibited and respiration stimulated as in 
the case of 2,4-dinitrophenol (7) and in some types of 2,4-D treatment. 
There are also some parallel changes in the inhibitive effects of respiratory 
inhibitors and growth substances with the age of tissues (38). These com- 
parisons are not meant to imply that lAA or 2,4-D act necessarily by the 


same mechanism as any of the respiratory inhibitors but only to indicate 
that the various types of growth and respiratory effects shown by the 
growth substances have parallels in the action of metabolic inhibitors 
whose action is more or less well known. This is merely a further justi- 
fication for the useful working principle that growth substances act, 
either in a physiological or in a toxic way, by some effect on respiratory 

In conclusion, we are likely to make more progress toward understand- 
ing the mechanism of growth substance action when we have a more 
complete knowledge of the respiratory machinery of the tissues in which 
the action of growth substances is studied, or possibly when we are able 
to study their action in tissues whose respiratory systems are already 
better known. 


1. Albaum, H. G. and Eichel, B., Am. J. Botany, 30:18 (1943). 

2. Alexander, T. R., Plant Physiol, 13:845 (1938). 

3. AuDus, L. }., New Phytologist, 47:196 (1948). 

4. Berger, J. and Avery, G. S., Jr., Science, 98:454 (1943). 

5. , Am. J. Botany, 30:297 (1943). 

6. Bonner, J.,/. Gen. Physiol, 20:1 (1936). 

7. , Am. J. Botany, 36:323, 429 (1949). 

8. , and KoEPFLi, J. B., ibid., 26:551 (1939). 

9. , and WiLDMAN, S., Sixth Growth Symposium, 51 (1946). 

10. Brown, J. W., Botan. Gaz., 107:332 (1946). 

11. Brunstetter, B. C., Myers, A. T., Mitchell, J. W., Stewart, W. S., 

and Kaufman, M. W., ibid., 109:268 (1948). 

12. BuRSTROM, H., Ann. Agr. Coll Sweden, 10:209 (1942). 

13. Commoner, B. and Thimann, K. V., /. Gen. Physiol, 24:279 (1940). 

14. Commoner, B., Fogel, S., and Muller, W. H., Am. J. Botany, 30:23 


15. DuBuY, H. G. and Olsen, R. A., ibtd., 27:401 (1940). 

16. HsuEH, Y. L. and Lou, C. H., Science, 105:283 (1947). 

17. Kelly, S., Am. J. Botany, 34:521 (1947)- 

18. , and Avery, G. S., Jr., ibid., 36:421 (1949). 

19. LuECKE, R. W., Hamner, C. L., and Sell, H. M., Plant Physiol, 24:546 


20. Marsh, P. B., and Goddard, D. R., Am. J. Botany, 26:724 (1939)- 

21. Mitchell, J. E., Burris, R. H., and Riker, A. J., ibid., 36:368 (1949). 

22. Mitchell, J. W. and Brown, J. W., Botan. Gaz., 107:120 (1945)- 

23. Mitchell, J. W. and Martin, W. E., ibid., 99:171 (i937)- 

24. Mitchell, J. W., Kraus, E. J., and Whitehead, M. R., ibid., 102:97 


25. Nance, J. F., Science, 109:174 (1949). 


26. Pratt, R., Am. J. Botany, 25:389 (1938). 

27. Rasmussen, L. W., Plant Physiol., 22:377 (1947). 

28. Reinders, D. E., Rec. trav. botan. neerland., 39:1 (1942). 

29. Sell, H. M., Luecke, R. W., Taylor, B. M., and Hamner, C. L., 

Plant Physiol., 24:295 (1949). 

30. Smith, F. G., ibid., 23:70 (1948). 

31. Smith, F. G., Hamner, C. L., and Carlson, R. F., ibid., 22:58 (1947). 

32. Stuart, N. W., Botan. Gaz., 100:298 (1938). 

33. Sweeney, B. M., Am. J. Botany, 28:700 (1941). 

34. , ibid., 31:78 (1944). 

35. Taylor, D. L., ibid., 29:721 (1942). 

36. , Botan. Gaz., 109:162 (1947). 

37. Thimann, K, v.. The Hormones, (Academic Press, 1948), vol. i, p. 5. 

38. , and Bonner, W. D., Jr., Am. J. Botany, 35:271 (1948); 36:214 


39. Worth, W. A., Jr., and McCabe, A. M., Science, 108:16 (1948). 

Tissue Responses 
to Growth Substances 

Electrical Polarity and Auxins 


GROWTH curvature responses of plants are dependent on an integrat- 
ing mechanism which permits the individual cells of a system to 
operate collectively as a coordinated unit. In the absence of a highly 
specialized nervous system in plants some other more primitive device 
must be functioning. Evidence has been presented to show that living 
organisms are surrounded and permeated by electrical fields which are 
generated by the polar components of the system (17). These fields 
have been looked upon as an electrical correlation mechanism, which 
operates by transferring electrical energy within the field. In the simplest 
aspect the flow of current could be accomplished by the movement of 
ions or other electrically charged particles. This, however, should not 
be taken to imply that the transfer of electrically charged particles is 
necessarily accomplished by electrophoresis. 

A prodigious mass of experimental data (3,37), which has been 
accumulated over a period of years, serves as the basis for the far- 
reaching Cholodny-Went (9) theory of plant growth responses. More 
recent evidence also continues to support the postulation that growth 
curvatures are due to an unequal distribution of auxin in the opposite 
sides of the curving organ (21,22,38). Although the detailed explanation 
of precisely how the uneven distribution is accomplished is not always 
apparent, it is generally accepted that the lateral transport of auxin is 
a rather significant intermediate link in many of the curvature responses. 
It is obvious that auxins cannot be transported of themselves, and that 
some additional mechanism in the form of an oriented force is essential. 
A challenging hypothesis can be formulated by assuming that the in- 
herent electrical fields function as the required directing forces, and 
that auxin is thereby transported in the form of electrically charged 


In many studies of auxin-controlled curvatures the Avena coleoptile 
has been used as the standard experimental material. Therefore, this 
discussion is to analyze the inherent electrical pattern, as a possible 
correlation mechanism, of the same plant. For convenience of organiza- 
tion this paper will be divided into the three following parts: examination 
of the bioelectrical pattern of the nonstimulated coleoptile; changes of 
certain components of the electrical field that result from stimulation 
by different types of energy; effects of altering the inherent electrical 
field by superimposing an electrical polarity of external origin. 

The Electrical Field of the Nonstimulated Coleoptile 

By performing a large number of experiments in which every possible 
precaution was taken to keep all of the environmental factors constant, 
Wilks was able to work out the distribution of electrical potentials of 
the Avena coleoptile in considerable detail (39). His observations were 
completely confirmed and extended by Schrank during the course of a 
later investigation (27). The results of these many experiments can be 
briefly summarized by the use of a diagram, which shows the dimensions 
of the electrical pattern quite distinctly. In presenting the diagram, 
shown in Figure i, a few explanatory remarks should be included. First, 
it is important to remember that one of the general characteristics of 
electrical polarities of living systems is that they vary quite appreciably 
from one instant to the next, even though a source of stimulation is not 
apparent (39). It is not uncommon for the longitudinal electrical polarity 
of an intact Avena coleoptile to change from 10 to 20 millivolts during 
the course of ten minutes. Second, the magnitude of a given polarity is 
not necessarily the same in all plants which are otherwise similar. These 
circumstances naturally demand that the magnitudes of the voltages 
expressed in the figure are only approximate averages. 

Examination of Figure i reveals several relevant points to which 
attention should be directed. In so far as the external longitudinal 
electrical polarity is concerned (circuits labeled X, Y, and Z), it becomes 
apparent that the apical region is electronegative to the base; the magni- 
tude of the polarity is of the order of about 50 millivolts. The most 
negative region, with respect to the base, is the section about 5 to 8 
millimeters below the apex. This also appears to be the region of the 
greatest radial polarity (circuits L, M, N, O, and P). It is noted that the 
orientation of the radial polarities of the apical 17 millimeters is such 










Figure I. Diagram showing some of the components of the electrical field 
of the Avena coleoptile under constant experimental conditions. Arrows m 
the external circuits indicate the direction of current flow. 


that the Inside Is electropositive to the outside. In the more basal regions 
the radial polarity is reversed. This would Indicate that for the Internal 
longitudinal polarity the basal region Is electronegative to the apex 
(27,39). ^^^ ^1^ of the data in the literature are in agreement on this 
point (12). Finally, Figure i shows that no transverse polarity (circuits 
A, B, C, D, and E) Is manifested by the nonstimulated coleoptile when 
it Is kept In the vertical position. 

The possibUity of a definite system of electrical currents within the 
coleoptile becomes apparent from the figure. The implication is that the 
electromotive forces of the cells would supply the energy required for 
continuous cell correlation. This concept of electrical correlation natu- 
rally assumes that some cells of the system are capable of absorbing the 
electrical energy that is generated by other cells some distance away. 
Whether or not coleoptile cells can absorb electrical energy will be 
Indicated by experiments included later In this paper. 

One additional statement about the direction of auxin movement 
must be made at this time. It Is known that the transport is from the 
apex toward the base In the coleoptile in the vertical position. In terms 
of the external longitudinal electrical polarity this means that the 
transport Is toward the electropositive region of the system. 

Transverse Electrical Responses to 
Various Types of Stimulation 

Stimulation by gravity. — The effect of stimulation by gravity on the 
electrical field of the Avena coleoptile has been investigated extensively 
by Schrank (23,27). His data disclose a number of changes In the various 
electrical polarities when a plant is shifted from the vertical to the 
horizontal position. Of these several changes only the transverse com- 
ponent will be included in the present treatment. Figure 2 was selected 
to show the typical results. Figure 2A shows that a transverse electrical 
polarity in the apical portion of the coleoptile (contacts 2 millimeters 
below the apex) Is nonexistent as long as the seedling remains In the 
upright position. When it Is placed in the horizontal position the under 
side becomes positive to the upper side; even the first reading, which 
was taken one minute after the change of position, Indicates the beginning 
of the polarity. It is worth while to note that this transverse electrical 
change occurs all along the longitudinal axis of the coleoptile, but 
smaller potential differences are established In the more basal regions 



(27). Similar electrical changes have also been observed by other workers 
for stems of other plants (2,4,5). However, Brauner and others reported 
comparable results from plants that had been killed in boiling water and 
from models constructed out of nonliving membranes (6,7,8). Because 
of the results obtained from the models these workers were led to 
maintain that the electrical responses were nonliving phenomena, and 
Brauner and Amlong ascribed the geoelectrical changes to streaming 
potentials (7). The data shown in Figure 2 were obtained from non- 


+ 10 - 

Figure 2. A. Transverse electrical changes in Avena coleoptiles that are 
rotated from vertical to horizontal position. Contacts 2 millimeters below 
apex. Average of 6 experiments. B. Upward curvature of coleoptiles in hori- 
zontal position measured in ocular scale divisions of 18 per millimeter. Average 
of 10 experiments. 


injured living tissues exhibiting normal growth and bending responses 
(27). Coleoptiles, which were immersed in boiling water for 15 seconds, 
did not show any electrical changes when they were placed in the hori- 
zontal position. Further control experiments also disclosed that the 
electrical responses were not due to electrode phenomena, which might 
have been the case in earlier work. 

The curvature induced by gravity, represented by movement of the 
coleoptile tip measured in ocular scale divisions (18 per millimeter), is 
shown in Figure 2B. This curve indicates that upward bending starts 
only after the plant has been in the horizontal position for 22 minutes. 
When the relationship between the known velocity of transport (19, 
36,37) and the minimum distance that the auxin has to be displaced in 
lateral redistribution is evaluated, it is at once apparent that the trans- 
verse electrical polarity in Avena is established before an unequal distri- 
bution of auxin is considered possible (23). This sequence of events also 
indicates that the electrical polarity in the coleoptile is not dependent 
on the metabolic process for which the auxin is directly responsible. 
These results and inferences permit the arrangement of coleoptile re- 
sponses to gravity in the following order: establishment of a transverse 
electrical polarity in which the underside becomes electropositive; un- 
equal distribution of auxins (in this instance the auxin is again trans- 
ported toward the electropositive portion of the plant); and upward 

Stimulation by light. — Only a limited number of investigators have 
been concerned with the various phases of the effects of incandescent 
light on the electrical potentials of etiolated seedlings (11,26,35). When 
the field is narrowed to the effect of unilateral illumination on the trans- 
verse electrical polarity of the Avena coleoptile the only information 
that is available comes from the preliminary experiments of Schrank 
(25) and Oppenoorth (20). Figure 3 A shows the typical electrical 
response of the most apical cells of an isolated Avena coleoptile (contacts 
0.5 millimeter below the apex), which was illuminated continuously by 
a light intensity of 16 foot-candles at the coleoptile position. As indicated 
by the curve in Figure 3A, after ten minutes of illumination the shaded 
side becomes electronegative to the lighted side. This first negative 
variation was observed in about 65 per cent of the experiments. Later 
the shaded side always becomes electropositive to the lighted side, 
reaching an average maximum of 8.2 millivolts. The corresponding bend- 



ing toward the light expressed as horizontal tip movement is shown in 
Figure 3B. As in the previous experiments the final transverse electrical 
polarity is again established before the curvature starts. The orientation 
of the electrical polarity with respect to the subsequent bending is 
the same as it was when the plants were stimulated by gravity. 

These observations are beset by several limitations. The indicated 
electrical changes have been observed only for the most apical cells of 
the isolated sheath. Whether or not unilateral illumination induces a 
transverse electrical polarity along the entire longitudinal axis is not 
known. Furthermore, the quantity of light that was used in these 


+ 10 
+ 5 





100 WATT 


■ ' ' ' 


- Curvature 

I I I 

10 20 MINUTES 60 70 

Figure 3. A. Effect of continuous unilateral illumination of an isolated 
Avena sheath on its transverse electrical polarity 0.5 millimeter below the 
apex. B. Phototropic bending of the same coleoptile. 


experiments was much greater than the amount required to give the 
first maximum positive curvature. In this instance the imphcation is 
that photoinactivation of auxin may have been an unusually prominent 
factor in the induction of the curvature. Finally, the data in Figure 3A 
are not in apparent agreement with the results obtained by Oppenoorth 
(20), who found that unilateral illumination of the coleoptile by 500 
meter-candle-seconds of unfiltered mercury light caused the lighted side 
to become electropositive to the shaded side. He obtained this polarity 
by subtracting the longitudinal electrical changes of the shaded side 
from the simultaneous changes of the lighted side. A common basal 
contact was used in measuring these electrical responses. 

Mechanical stimulation. — Growth curvature responses to mechanical 
stimulation, which were first studied in tendrils, are also prevalent in 
etiolated seedlings. Years ago Stark (33) demonstrated that he could 
induce bending of the coleoptile by stroking it on one side with a cork 
rod. These observations, along with the frequently reported fact that 
mechanical stimulation of a segment of living tissue causes it to become 
electronegative to the unstimulated portion (16,18), led Schrank (24) 
to investigate the relationship between the electrical and curvature 
responses of the Avena coleoptile to mechanical stimulation. Preliminary 
experiments confirmed Stark's observation that the coleoptile would 
bend toward the side that was lightly tapped and demonstrated that a 
transverse electrical polarity was established with the stimulated side 
becoming electronegative to the opposite side. In subsequent experi- 
ments mechanical stimuli were applied by the use of an electrically 
operated vibrator, which was mounted on a micromanipulator in order 
that the position of the stimulating device could be accurately duplicated. 

Curves in Figure 4A show the average magnitude of the transverse 
electrical polarity that is established in the apical region oi Avena when 
the apical 10 millimeters of one side are mechanically stimulated by the 
vibrator for the duration indicated. The magnitude of the polarity and 
the rate of its decrease are dependent on the duration of the stimulation. 
In Figure 4B the corresponding curvatures are shown. The magnitude 
and rate of bending are also dependent on the duration of the stimulation 
with the direction of bending being toward the negative side of the plant. 
Since mechanical stimuli cannot be applied to the coleoptile without 
bending or displacing it from its original position (the reason for starting 
the curves below the zero line), there is no way to determine exactly 



when the curvature starts toward the stimulated side. (A good guess 
would be that curvature due to growth starts as the coleoptile tip passes 
its prestimulation position.) Thus it is impossible to state in this situation 
whether or not the electrical polarity is established before the curvature 
starts. Logically it would seem that it is, because the rate and magnitude 
of the curvature can be approximately predicted from the magnitude of 
the electrical response and its rate of change. 



' I 
1 / 

ns.- / 



€ 4 seconds 
• 6 seconds 
o 8 seconds 

J L_J L_J L— L 



Figure 4. A. Transverse electrical polarities established by Avena coleoptiles 
1.67 millimeters below the apex in response to mechanical stimulation of one 
side of the apical 10 millimeters. Each curve is an average of 10 experiments. 
B. Corresponding average curvatures in scale divisions of 18 per millimeter. 


Without arrogating the space to review the specific data, two other 
impHcations of these experiments should be contemplated. It was pre- 
viously noted that when the coleoptile is placed in the horizontal 
position the under side becomes positive to the upper. Simultaneous 
mechanical stimulation of the apical 10 millimeters of the under side 
establishes a reversed electrical polarity (opposite to the polarity induced 
by gravity alone), inhibits upward curvature for 25 minutes, and de- 
creases the subsequent rate of bending. Geotropic bending can be 
entirely prevented by repeated appHcation of mechanical stimuli to the 
under side at five-minute intervals. This inhibition apparently is not 
due to an injury phenomenon because similar stimulation of the upper 
side does not inhibit upward curvature. Stimulation of the upper side 
results in an electrical polarity larger than that obtained from gravity 

Indications are that the curvature observed in these experiments 
is due to growth. Coleoptiles that have had the apical 3 millimeters 
removed 2 hours and 10 minutes before stimulation cannot be induced 
to bend by a combination of mechanical and gravitational stimuli. This 
is taken to verify the necessity of the presence of auxin for the curvature 
responses that were previously observed. The fact that the electrical 
polarity established by these plants is similar to that in nondecapitated 
coleoptiles has been interpreted to indicate further that the auxin- 
controlled mechanism is not required for the generation of electrical 
polarities of this nature. 

In the series of experiments that has just been reviewed it was demon- 
strated that the Avena coleoptile responds to stimulation by gravity, 
light, or mechanical means by establishing a transverse electrical polarity. 
These stimuli also induce bending. In all of these instances the polarity 
is established before the bending starts, and the direction of bending is 
such that the electropositive side always becomes the convex side. If it 
is assumed that the lateral transport of auxin is the fundamental inter- 
mediate link in these curvature responses, then it follows that the auxin 
is invariably transported toward the electropositive side of the plant. 
This implies that the auxin is transported as a negatively charged particle. 
These data are considered to be compatible with the Went-Kogl 
elect rophoretic transport theory. However, extreme caution should be 
exercised in drawing categorical and far-reaching conclusions about the 
causal relationship between the electrical polarity and the auxin transport. 

A. R. SCHRANK 133 

Effects of Applied Current on 
Electrical Pattern and Curvature 

Perhaps the most direct way to alter the electrical field of a living 
organism is to superimpose a field or polarity of external origin. This 
approach has been taken by several investigators. Wilks (39), Clark 
(12,13), duBuy and Olson (14), Cholodny and Sankewitsch (10), and 
Kogl (15) have all studied the various aspects of applied direct current 
on growth and auxin transport in the Avena coleoptile. Some of their 
reports (10,15,39) indicate that the effects of applied current on growth 
responses are dependent on the polarity of the current, but one main- 
tains that the curvature obtained is not dependent on the polarity of 
stimulation (14). Since the publication of these observations an additional 
series of papers has appeared in which the effects of applied current on 
the electrical polarity and curvature of the coleoptile were observed 
simultaneously. In the experiments to be examined at present the current 
was applied either transversely or longitudinally. 

Current applied transversely. — When 5 to 20 microamperes of direct 
current are applied transversely for 2 to 10 minutes the coleoptile always 
responds by establishing a transverse electrical polarity (28). (Control 
experiments show that such electrical responses are obtained only from 
living tissue.) In Figure 5 a group of average curves are shown, which 
represent the transverse electrical polarities induced by applying a given 
current (10 microamperes for 10 minutes) at various distances below 
the apex. These curves show that a given quantity of current induces 
the maximum electrical polarity when it is appHed to levels more than 
5 millimeters below the apex. The fact that these electrical polarities 
are established is taken to indicate that the applied current did flow 
through the Hving tissue, a point not always clear in previous papers. 
Also current applied transversely in the manner just described always 
causes the coleoptile to bend. Figure 6 presents a diagrammatic account 
of this bending process when 10 microamperes were applied for 10 
minutes at a level 10 millimeters basal to the apex. The initial curvature, 
in and above the contact region, is toward the positive pole of the 
current-applying circuit; however, there is a subsequent bending, basal 
to the contact region, in the opposite direction. The maximum angular 
curvatures that were observed in the initial direction, resulting from 
current (10 microamperes for 10 minutes) applied transversely to various 
levels below the apex, are shown in Table i. 




+ 60- 

+ 50- 

+ 40- 

+ 20- 

+ 10 

+ 30- 

Figure 5. Electrical polarities induced by applying 10 microamperes for 
10 minutes transversely to the Avena coleoptile at 2, 5, 10, 15, 20, and 25 
millimeters below the apex. Each curve is an average of 10 or more experiments 
and numbered beginning with the most apical level. 





U W X Y Z 


wwy^wwmv^ \ A\\\v\\\\\v\\\\\\\\ 




Figure 6. Successive stages of Avena coleoptile curvature after transverse 
application of 10 microamperes for 10 minutes. Current applied 10 millimeters 
below the apex. 

These curvature data permit several interesting observations. First, 
it would appear that the applied current is inducing the curvature by 
affecting the auxin-controlled mechanisms. This tentative conclusion 
is drawn from the fact that the zone of curvature moves toward the base, 
which is clearly shown in Figure 6. More specifically, this could be 
interpreted to mean that the current has actually affected the lateral 
transport of the auxin. Secondly, from Thimann's work on the distri- 
bution of auxin along the longitudinal axis of the coleoptile (34) and the 

Degrees of curvature of the Avena coleoptile 


10 15 20 











12 6 3 
6 3 2 
9 7 4 







II. 8 

8.2 5.2 3.2 


data shown in Table i, it appears even more likely that the hormone 
mechanism is involved in the bending induced by current. Finally, 
it has been demonstrated that phototropic bending can be inhibited by 
transversely applied current, and that curvature responses to electrical 
and light stimulation are apparently limited by a common factor (29). 
The premise that the responses to both types of stimulation are con- 
trolled by auxin would explain this observation. 

Perhaps the most striking fact is that the initial curvature is toward 
the electropositive side of the plant and not toward the negative side 
as it was in all of the previous instances. Several implications must be 
considered in the evaluation of this fact. What is needed most are 
additional experiments which would expose the details of the pathways 
of current flow through the tissue. This might help to visualize how 
bending is induced apically to the contact region, as well as to help 
account for the direction of the initial bending. Another point to be 
kept in mind is that the electrical polarity measured in these experiments, 
in contrast to the previous ones, very likely is due to polarization phe- 
nomena rather than changes in the inherent electrical pattern. This 
inference is made only on an indirect basis from the work of Berry et al. 
(i) on the onion root. 

Current applied longitudinally. — DuBuy and Olson (14) were the first 
to report that the Avena coleoptile could be made to bend by applying 
direct current longitudinally to one side of the apical 5 millimeters. 
The curvature, which they observed, was always toward the side on 
which the contacts were placed and, according to them, not dependent 
on the direction of the current flow. When these experiments were 
duplicated and extended, several additional facts were disclosed (30). 
Figure 7 presents some of these observations. Ten microamperes applied 
longitudinally to one side of the apical 5 millimeters (polarity indicated 
in the insets) cause marked changes in the longitudinal polarity of the 
coleoptile. Current flowing from the apex toward the base reverses the 
inherent electrical polarity and gives the response shown by the average 
curve I in Figure 7A. Effects of the same current flowing in the opposite 
direction are shown by curve II, which is quite obviously difl^erent from 
curve I. Current flowing from the apex toward the base enhances rather 
than reverses the inherent electrical polarity. The magnitude of the 
initial response is not as large as in curve I, and the sequence of events 




+ 40 

+ 30 
+ 20 

+ 10 






-0.5 ■ 




.•-•-• .-.'^* ••-•■•'• •../^ 

^ ON 



' ■ ' ' 

' ' ' ■ 

' ■ ' ' ' ■ ' I ■ ' ■ ■ I ■ ' ' 


'' I I I *''''*' I 

50 MINUTES 100 

Figure 7. A. Electrical responses oi Avena coleoptiles to 10 microamperes 
of direct current applied longitudinally for 2 minutes to a 5 millimeter apical 
segment. Each curve is an average of 5 or more experiments. B. Correspondmg 
average curvatures plotted as millimeters of horizontal tip movement. 
Numerals at the right of the curves show the average angular curvatures at 
the end of the experiments. 


is distinctly different. These facts definitely show that the polarity of the 
applied current has an effect on the electrical response. 

In Figure 7B the corresponding curvatures are represented by graphs 
of the movement of the apex and by numerals indicating the angular 
curvature at the end of the experiments. The direction of bending is 
always toward the side on which the contacts were placed, thus confirm- 
ing, in this part, the observations of duBuy and Olson (14). However, 
curves I and II in Figure 7B demonstrate that a given current flowing 
from the base toward the apex through the coleoptile induces curvature 
more effectively than the same current flowing in the opposite direction. 
On the basis of known effects of current on elongation (10,39) '^his can 
be interpreted to indicate that current flowing toward the apex inhibits 
elongation more effectively than the same current flowing in the reversed 
direction. These observations, in contradiction to the results previously 
published by duBuy and Olson (14), show that the magnitude and the 
temporal sequence of the curvature are dependent on the polarity of 
the applied current. This point very hkely will assume considerable 
importance in the final and complete explanation of how current affects 

Finally, one additional group of experiments will be introduced. 
It has recently been shown that direct current, (10 microamperes for 
2 minutes) apphed in the manner just described, always inhibits bending 
toward 200 meter-candle-seconds of unilateral illumination (31). The 
extent of the curvature inhibition and the temporal sequence of events 
are again dependent on the polarity of the apphed current and on the 
position of the contacts with respect to the light source. Results of these 
experiments show that current applied longitudinally on the hghted 
side inhibits rather than accelerates curvature toward the light, while 
current applied to the shaded side inhibits bending toward the light 
somewhat more effectively. The polarity of the applied current which is 
most effective on the lighted side is least effective on the shaded side. 

The results of these last experiments seem to indicate that longi- 
tudinally applied current brings about the growth curvature by in- 
fluencing the auxin-controlled mechanism, but it is rather difficult to 
explain, on the basis of the available evidence, precisely how applied 
current affects growth. It has been observed that current applied in this 
manner reversibly inhibits protoplasmic streaming (14). If the transport 
of auxin is dependent on the streaming, it follows that current could have 

A. R. SCHRANK 139 

some of its effects by inhibiting the movement of the active auxin (32). 
This explanation, however, is inadequate to account for the inhibition 
of the phototropic bending by current appHed longitudinally to the 
illuminated side. Furthermore, the data that are available (14) indicate 
that the inhibition of streaming is independent of the polarity of the 
applied current, while many of the experiments which have been re- 
viewed in this paper show that the magnitude and temporal sequence of 
the curvature of the coleoptile in response to current are dependent on 
both the polarity and strength of the electrical stimulation. It is obvious 
that protoplasmic streaming, at best, can play only a limited role in 
these electrically induced growth phenomena. 

A number of experimental observations that have been presented 
would permit the possibiHty of accepting the inherent electrical field 
as the primary integrating mechanism of the Avena coleoptile. 1) In 
stimulation by light, gravity, or mechanical means the electrical polarity 
changes apparently precede the hormone redistribution and curvature. 

2) The orientation of the transverse polarity with reference to the 
direction of the subsequent bending is consistent in all of these instances. 

3) Experimental evidence has been presented to show that the inherent 
electrical field of the coleoptile is not dependent on the same auxin- 
controlled process that is required for elongation. 4) Cells of the 
coleoptile are capable of absorbing electrical energy; this is demonstrated 
by the fact that curvature can be induced by either transversely or 
longitudinally appHed current. These growth responses in every instance 
are dependent on the polarity of the applied current. 5) Additional 
evidence indicates that applied current induces its effect on growth via 
the same mechanism that unilateral illumination does; at least both are 
limited by a common factor. 

Obviously the summarized facts are still inadequate to prove con- 
clusively that the inherent electrical field functions as the primary 
correlating mechanism in the Avena coleoptile. It Is now certain that an 
externally imposed electrical field does affect the growth of the coleoptile 
in a polar fashion, but much more Information is needed to clarify the 
details of this relationship. Additional data are also necessary to account 
for the fact that the coleoptile bends toward the electropositive pole of 
the current-applying circuit, while in the remainder of the troplsms the 
curvature is away from the positive side of the plant. 



1. Berry, L. Joe and Hoyt, R. C, Plant Physiol., 18:372 (1943). 

2. BosE, J. C, Comparative Electro- physiology (Longmans, Green, and Com- 

pany, 1907). 

3. BoYSEN Jensen, P., Growth Hormones in Plants (McGraw-Hill, 1936). 

4. Brauner, h., Jb. tviss. Botan., 66:381 (1927). 

5. , ibid., 68:711 (1928). 

6. , Revue de la Faculte des Sciences de FUniversite d" Istanbul, 7:46 


7. , and Amlong, H. U., Protoplasma, 20:279 (1933). 

8. , and Bunning, E., Ber. dent, botan. Ges., 48:470 (1930). 

9. Cholodny, N. G., Planta, 7:461 (1929). 

10. , and Sankewitsch, E. Ch., Plant Physiol., 12:385 (1937). 

11. Clark, W. G., Proc. Nat. Acad. Sci. U. S., 21:681 (1935). 

12. , Plant Physiol., 12:409 (1937). 

13. , ibid., 12:737 (1937). 

14. DuBuy, H. G. and Olson, R. A., Biodynamica, 2:1 (1938). 

15. KoGL, F,, Angeiv. Chem., 46:469 (1933). 

16. Lund, E. J., Plant Physiol., 6:507 (1931). 

17. , Bioelectric Fields and Grotvth (University of Texas Press, 1947). 

18. Marsh, G., Protoplasma, 11:497 (^93o)' 

19. Nuernbergk, E. and DuBuy, H. G., Recueil des Travaux Botaniques 

Neerlandais, 27:417 (1930). 

20. Oppenoorth, W. F. F., Jr., ibid., 38:287 (1941). 

21. Overbeek, J. VAN, Botan. Rev., 5:655 (1939). 

22. , Ann. Rev. Biochem., 13:631 (1944). 

23. Schrank, a. R., Plant Physiol., 20:133 (1945). 

24. , ibid., 20:344 (1945). 

25. , ibid., 21:362 (1946). 

26. , ibid., 21:467 (1946). 

27. , Bioelectric Fields and Grotvth (University of Texas Press, 1947), 


28. , Plant Physiol, 23:188 (1948). 

29. , /. Cellular Comp. Physiol., 32:143 (1948). 

30. , ibid., 33:1 (1949). 

31. , ibid., 35:353 (1950). 

32. .Showacre, J. B. and DuBuy, H. G., Am. J. Botany, 34:175 (1947). 

33. Stark, P., Jb. wiss. Botan., 57:198 (1916). 

34. Thimann, K. v.,/. Gen. Physiol., 18:23 (1934). 

35. Waller, J. C, Ann. Botany, 39:515 (1925). 

36. Wei J, H. G. van der, Recueil des Travaux Botaniques neerlandais, 29:379 


37. Went, F. W. and Thimann, K. V., Phytohormones (Macmillan, 1937)- 

38. , Ann. Rev. Biochem., 8:521 (1939). 

39. WiLKS, S. S., Bioelectric Fields and Growth (University of Texas Press, 

1947), p. 24. 

Translocation of Growth- Regulating Substances and 
Their Effect on Tissue Composition 


PLANT growth regulating substances are readily absorbed by plants. 
These chemicals penetrate the living surface cells of most plant parts 
and seem to move as readily through intact epidermal cells as through 
injured ones. Synthetic growth regulators readily gain entrance to the 
plant when applied to roots, stems, leaves, flowers, or fruits. They are 
absorbed by these organs even though the surface cells of some may be 
protected by thick walls and a layer of cutin. 

Three methods have been used in studying the rate of absorption 
of growth-regulating substances: i) the detection of morphological or 
histological responses that occur some distance from the treated area 
has been used to indicate that the plant has absorbed the growth- 
regulating substance; 2) measurement of the amount of residual growth 
regulator left on the surface of a treated area has been used to indicate 
the amount of the chemical absorbed; 3) radioactive tracers have been 
used to some extent in studying the absorption of these chemicals. As 
yet, however, we do not have a direct and completely reliable method of 
measuring the absorption of growth regulators by plants. All three 
methods have drawbacks. If we attempt to measure absorption by 
means of radioactivity, or by evaluating a morphological or histological 
response that occurs some distance from the treated area, then transloca- 
tion of the chemical is involved as well as its absorption. If we attempt 
to measure absorption by detecting the amount of the chemical remain- 
ing on the treated surface of the plant, then we are confronted with the 
problem of how to remove this surface residue quantitatively so that it 
can be measured accurately. In spite of these difficulties some reliable 


results have been obtained regarding the absorption of plant growth 

Such substances as indoleacetic acid and the phenoxy compounds are 
apparently absorbed by most leaf-surface cells of plants. Their absorption 
by leaves does not seem to be related to the presence of stomatal openings 


Because of the Inadequacy of methods at hand we have not yet 

been able to measure directly the effect of age or stage of development 

on the rate of absorption of growth regulators by plants. It Is known that 

when older, more mature leaves or stems are treated the plants do not 

respond as readily to growth-regulating substances as when the chemicals 

are applied to young vigorously growing parts, but this may be due to 

factors other than absorption of the chemical (59). 

The evidence, so far, Indicates that leaves of certain dicotyledonous 
plants may absorb certain growth-regulating substances somewhat more 
readily than do leaves of monocots such as oats and barley (62). Thus 
when 10 micrograms of radioactive 2-iodo-3-nitrobenzoIc acid was ap- 
plied the young leaves of bean plants absorbed roughly twice the amount 
absorbed by the leaves of barley plants. Thus far, most of the work 
concerning the absorption of growth regulators by plants has not dealt 
with the direct measurement of the rate of absorption of the chemical. 
However, Rice (48), using a direct method of measurement, reports that 
a large part of the 2,4-dIchlorophenoxyacetic acid that he placed on 
bean leaves was absorbed during the first 4 hours following treatment. 
He measured the amount of the chemical that was left on the surface 
of the leaf then subtracted this amount from that which had been 
Initially added thus obtaining the rate of absorption. 

Bean plants have absorbed a sufficient amount of 2,4-D within 4 
to 6 hours to bring about a maximum response (60). Absorption of 
2,4-D apparently begins as soon as the chemical comes in contact with 
the plant. In order for activated charcoal to absorb effectively 2,4-D 
that had been added to the surface of a plant, Weaver (57) had to apply 
the charcoal to the surface within 15 minutes after the chemical came 
into contact with the plant. It Is a matter of common observation that 
young and succulent plants often show marked growth responses within 
30 to 60 minutes following the application of a growth regulator of 
the phenoxy type, and it has long been known that indoleacetic acid is 
quickly absorbed by succulent tissues of such plants as tomato, bean, 


and oat colcoptlles (22,35,61). The rate of absorption of growth-regulat- 
ing substances is rapid, on the basis of these data, but the absorption 
of these chemicals is influenced to some extent by environmental factors, 
such as light, temperature, and the presence of a wetting agent or of 
surface-active substances. That light influences the rate of absorption 
of 2,4-D was shown by Rice (48). Absorption of the ammonium salt of 
2,4-D by bean leaves was greatest immediately after the chemical was 
applied. More of the salt was absorbed by illuminated leaves than by 
others kept in darkness. Leaves do, however, absorb 2,4-D at a slower 
rate during periods of darkness. Rice reported that increasing the light 
intensity from 100 to 900 foot-candles had no appreciable effect on the 
rate of absorption of the ammonium salt of 2,4-D. Temperature, on 
the other hand, affected absorption of the salt since the amount taken 
up by a plant at 46° to 58°F, was less than at a temperature of 78° to 
8o°F. Injurious effects of 2,4-D are more pronounced in plants grown 
at relatively high temperatures (7o°-85°F.) than in plants grown at 
relatively low temperatures (5°-i5°F.) (23,27). 

Wetting agents and some hygroscopic substances that dissolve growth- 
regulating chemicals have a marked effect upon the activity of the 
growth regulator. This is apparently so because they make it possible 
for the plant to absorb the growth-regulating substances more readily 
than when the wetting agents are not added to the aqueous mixture. 
Polyethylene glycols are among these activating substances (64). Growth- 
regulating chemicals are generally soluble in polyethylene glycols. Some 
of the glycols are hygroscopic. They serve as solvents and wetting agents 
and tend to keep the growth-modifying substance in close contact 
with the surface of the plant (12,37). I^ ^^ reasoned that growth regulators 
applied as aqueous mixtures of the salts, such as the sodium or ammonium 
salt of 2,4-D, tend to crystallize out as the water carrier evaporates. 
Absorption of the growth regulator is thus reduced since upon crystal- 
lization the chemical is no longer in close contact with the surface of 
the plant. By actual measurement the rate of absorption of ammonium 
salt of 2,4-D in water by bean leaves decreased after the first 4 hours. 
When Carbowax was added to the mixture of the salt and water, a 
relatively rapid rate of absorption prevailed for an extended length 
of time (11). 

In a similar way 2,4-D in a dust carrier was relatively ineffective as an 
herbicide, but when 3 per cent of Carbowax 1500 was added, a 0.05 


per cent concentration was sufficient to kill morning glory plants (28). 
With respect to the mode of action of Car bo wax 1500, its hygroscopic 
properties are probably not of prime importance, since glycerine and 
Carbowax both increased the effectiveness of 2,4-D in a dust carrier, 
while calcium chloride had no effect in this respect. Dispersing agents, 
such as Tween 20 and Emulfors, and surface-active substances, such as 
soapless washing powders which contain lauryl sulphates, are effective 
in increasing the activity of the acid and salt forms of 2,4-D (30,54). 

The nature of the action of surface-active substances is not understood 
fully. Their effectiveness may be due in part to the fact that they serve 
to spread the growth regulating chemical and make it adhere closely 
to the surface of the plant. 

Staniforth and Loomis (54) concluded from experiments with corn, 
flax, and soybeans that the power of these detergents to reduce surface 
tension is not directly responsible for their effect on the activity of 
aqueous 2,4-D mixtures. Five hundredths of one per cent of a detergent 
gave a maximum reduction in surface tension, but the detergent in- 
creased the effectiveness of 2,4-D up to a concentration of 2 per cent of 
the surface-active substance. It is of further interest that these detergents 
may increase the effectiveness of water mixtures containing salt forms of 
2,4-D by five or more times, but they had little effect on a water mixture 
of the ester form of 2,4-D. 

Staniforth and Loomis believe that a surface-active agent fike lauryl 
sulphate merely accelerates the initial responses to 2,4-D, such as 
epinasty and stem curvature, and that in the end these detergents do not 
really increase the herbicidal properties of 2,4-D. This effect may be due 
to more rapid absorption and translocation of 2,4-D when surface active 
agents are used than when the growth regulator is applied alone. 

Crafts (11) has proposed that herbicides might be divided into 2 
general classes, those that are polar and those that are nonpolar. This is 
of interest here because the growth regulator 2,4-D is now an important 
herbicide. Crafts concludes on the basis of general results with a variety 
of herbicides, that polar compounds are most effective when applied to 
the roots of plants and that nonpolar ones are most effective when 
applied to the above-ground parts of plants. He suggests that this 
difference may be due to a difference in the rate of absorption. Since 
radioactive polar and nonpolar forms of the 2,4-dichlorophenoxy com- 


pounds are now available, a means of testing this proposition is at hand. 
Regarding the absorption of growth regulators, we can conclude 
that the uptake of these substances by plants is greatly influenced by 
such factors as the age of the tissues to which the chemical is applied, 
light, temperature, and the presence of surface-active substances which 
tend to increase the rate and extend the period of absorption. 


Two methods have been used to measure translocation of synthetic 
growth-regulating substances in intact plants: first, evidence of a growth 
response some distance from the treated area has been used as an indica- 
tion that the growth regulator was translocated within the plant; second, 
some growth-regulating substances have been tagged with radioactive 
isotopes and the course of their movement through the plant followed 
by means of usual tracer techniques. The path of translocation of growth 
regulators depends to some extent upon the way the chemical is ab- 
sorbed by the plant. Taken in through the roots the growth regulator 
in most instances is moved rapidly upward through the water-conducting 
tissues (22,35). Absorbed by leaves, however, the chemical is trans- 
located mainly in living cells of the phloem. If, for instance, a few 
micrograms of 2,4-D are placed on a leaf, the plant can, under some 
conditions, rapidly absorb and translocate the chemical to the stem 
where it is moved in both an upward and a downward direction (12). 

The amount of growth-regulating substance translocated from a leaf 
depends upon several factors. Young rapidly growing leaves may absorb 
2,4-D but fail to translocate it to other parts of the plant (35). Phenoxy 
compounds are translocated more readily from leaves of medium age 
than from either younger or older ones (22,39). Similarly, in stem tissues 
most marked over-all responses have been observed when the chemical 
was applied to the young, succulent portion of the stem, least when the 
chemical was applied to the older, hgnified portion near the soil level 
(24). Movement of 2,4-D from leaves apparently involves the same 
mechanism as that used by the plant to translocate the products of 
photosynthesis or a similar one. Growth regulators of the phenoxy type 
are not translocated from leaves under conditions unfavorable for the 
production and translocation of photosynthate (25,35,48,58). Thus 2,4-D 
was not translocated from leaves kept in C02-free air or in darkness. 


Rice (48) has reported, however, that absorption of 2,4-D by leaves of 
bean plants was not afTected by increasing the light intensity from 100 
to 900 foot-candles. 

The chemical nature of the growth regulator may in itself influence 
the rate of its absorption and translocation. The morphoHne salt, the 
butyl ester, and the acid forms of a phenoxy compound (parent com- 
pound 2,4-dichloro-5-iodophenoxyacetic acid) have been applied in 
equal molecular amounts to bean leaves and their absorption and trans- 
location followed by means of radioactive tracers (39). Of the three 
forms, the acid was translocated in the greatest amount, the salt in the 
least amount. 

Under constant conditions bean leaves apparently absorb and trans- 
locate 2,4-D at a relatively uniform rate for a period immediately 
following treatment. In recent experiments (39) 2,4-D acid was apphed 
in an aqueous mixture containing Tween 20 to bean leaves, 10 micro- 
grams per leaf. On the basis of tracer measurements the amount ab- 
sorbed and translocated to the stems was essentially linear with time 
during a period of four days following treatment. 

Not all growth-regulating substances are translocated from leaves even 
under the most favorable conditions. Certain nicotinium compounds, 
such as 2,4-dichlorobenzylnicotinium chloride, have a systemic regulat- 
ing effect when apphed to succulent stem tissues of bean plants (44). 
These same compounds were not effective when applied in amounts of 
even a milligram or more to leaves or to the cotyledons of the plants. 
There is reason to believe that the method by which plants absorb and 
translocate different growth-regulating substances may vary widely in 
some respects, but so far these differences are not understood. 

Summarizing the data so far, it is evident that the rate of translocation 
of growth-regulating substances from a leaf is not related to the rate at 
which the chemical may be absorbed by the leaf. Translocation of such 
regulators as the phenoxy compounds is associated in some way with 
the translocation system involved in the movement of the products of 
photosynthesis. If activated diffusion plays a part in the translocation 
of growth regulators, as has been suggested by Clark (10), then such a 
phenomenon must be governed by external factors, including light 
and carbon dioxide supply; for without adequate illumination and 
carbon dioxide, growth-regulating substances are apparently not trans- 
located from leaves of plants. 


Tissue Composition 

After growth-regulating substances have been absorbed and moved 
to the different parts of the plant they incite specific physiological 
responses, and these are sharply reflected in or evidenced by the amount 
and kind of chemical constituents in the plants. 

Tissues of stems that respond most easily to such substances as 2,4-D 
are those that possess a high level of oxidation-reduction activity (18). 
In bean plants these tissues are phloem, endodermis, cambium, and 
xylem parenchyma. When 2,4-D, indoleacetic acid, or naphthaleneacetic 
acid come in contact with these tissues a series of chemical changes is 
generally set in motion. The end result of these responses depends in 
part upon the amount and the kind of growth-regulating substances 
used. If for example, a minute amount of 2,4-D is apphed to a sensitive 
plant, then the chain of responses does not extend far and only those 
reactions involved in cell elongation may be affected. If more of the 
compound is applied the chain of responses may be carried on through 
the process of cell division, the organization of these new cells into tissues, 
and finally their orientation into organs such as roots. 

If we consider this series of responses from the chemical standpoint, 
the first obvious effect is an increase in the water content of the cell 
(17,32,7,45), which is paralleled by an increase in the size of the cell. 
Brown (7) in testing bean plants found that after several days of treat- 
ment the water content of leaf tissues was depressed by 2,4-D while that 
of stem tissues was increased. Thus, the response by some leaf tissues may 
differ from those of stem tissues with respect to the effect of 2,4-D on 
their water relations. 

Two theories have been proposed to explain why stem tissues take 
up water when treated with certain types of growth regulators. First, 
the chemical may bring about the degradation of certain cell constituents 
so that the osmotic pressure of the sap is increased (17). There is also 
evidence that cells under the influence of indoleacetic acid, for example, 
absorbtions, such as potassium, more readily than do untreated cells. 
Both the absorption of ions and the degradation of cellular constituents 
would tend to increase osmotic pressure and favor water uptake. 

The second theory deals with the effect of growth regulators on the 
cell wall. The resistance of the wall to extension is thought to be lowered 
by the chemical so that the cell enlarges, thus allowing absorption of 
water until a new equihbrium is reached (45). 


Argument has been leveled against the theory based on a rapid 
increase in osmotic pressure. So far it has not been possible to demon- 
strate experimentally such an increase in osmotic pressure following 
application of a growth regulator. On the other hand, the theory based 
on wall extensibility may not entirely explain the phenomenon since 
it has been repeatedly demonstrated during the last three years that once 
a cell has expanded and taken up water under the influence of the 
regulating chemical, the cell has then changed in a way that makes it 
better able to retain the water it holds against forces of evaporation, than 
is the case with an untreated cell (32). Wall extensibility would hardly 
account for this increased water-retaining capacity of treated tissues. 
Increased osmotic pressure, on the other hand, might in part account 
for this effect. 

There is an abundance of evidence indicating that growth regulators 
accelerate either directly or indirectly the activity of some enzymes 
in plants. However, direct proof of their effect on enzyme systems is 
meager. Berger and Avery (3,4,5) obtained more highly active de- 
hydrogenase from oat plants treated with indoleacetic acid than from 
untreated ones. Eyster (14,15,16) believes that growth stimulation re- 
sults from the release of diastase from an inactive to an active form. Gall 
(18) demonstrated that starch in agar media was more readily digested 
by enzymes that diffused from sections of bean plants treated with 2,4-D 
than by enzymes from comparable untreated sections. His results are 
inconclusive for he states that the growth regulator may either have 
increased production of enzymes or increased their activity, or the 
chemical may have affected the tissues so that, although they contained 
the same amount of enzyme, more of it diffused from the treated than 
from the untreated section. His work shows clearly, however, that 
enzyme digestion of starch outside of the treated stems was greater than 
it was outside the untreated ones. 

Within the plant the activity of the enzyme system involved in the 
conversion of starch to sugar is also affected by some growth-regulating 
chemicals. In leaves of bean and morning-glory plants, for instance, 
starch hydrolysis has been accelerated by the application of relatively 
large amounts of such substances as indoleacetic acid, indolebutyric acid, 
naphthaleneacetic acid, naphthoxyacetic acid, and 2,4-D (34,38,43). In 
treated leaves a marked increase in sugar content at first paralleled 
starch degradation, but as the starch was depleted their sugar content 


fell below that of untreated ones (38,50,51). The net result was a 
depletion of the readily available carbohydrates in leaf tissues. Phenyl- 
acetic acid did not accelerate starch degradation in leaves nor did 
naphthaleneacetamide. The effect of such growth-regulating substances 
as indoleacetic acid and 2,4-D on reserve carbohydrates in other parts 
of plants is similar to that described for leaves (1,2,6,34,46,50,56). 

Bausor (2) obtained evidence that tomato cuttings retained their 
starch reserves when kept in darkness and supplied with sugar. Treated 
plants, on the other hand, utilized their starch reserves irrespective of 
the external carbohydrate supply. In these same experiments 0.02 per 
cent indoleacetic acid inhibited starch digestion in thin stem sections 
but hastened starch digestion in intact stems. This behavior may be 
explained, however, on the basis of the magnitude of treatment, there 
being much more growth regulator applied per cell in the case of the 
sections than in the case of the entire stems. 

Hydrolysis of complex carbohydrates, such as hemicelluloses, may 
also be accelerated by the apphcation of 2,4-D to plants (9). Thus, the 
action of some hydrolytic enzymes in plants is either directly or in- 
directly accelerated by some kinds of growth regulators. 

Hagen et al. (19), on the other hand, have recently shown that the 
activity of castor bean lipase in hydrolyzing olive oil was less in the 
presence of small amounts of 2,4-D acid than when used alone. They 
believe that only the acid form of 2,4-D was directly effective in reducing 
the lipase activity under their test conditions. Some hydrolytic enzymes, 
therefore, may under some circumstances be inhibited by the presence 
of a chemical such as 2,4-D. 

Nitrogenous compounds in plants are also affected by the application 
of growth substances. In general most growth-modifying chemicals bring 
about an increase in protein and amino acid content of stems when the 
regulator is appHed in relatively large amounts (31,41,42,49). In succulent 
plants regulating chemicals generally cause a mobilization of nitrogenous 
constituents towards the basal parts of the stems, both in cuttings and 
intact plants (2,42,52,55). This is not true, however, of all regulating 
chemicals. Applied to bean plants, for instance, naphthaleneacetic acid 
brought about a twelvefold increase in water-soluble nitrogenous com- 
pounds in the stems. Naphthaleneacetamide, on the other hand, brought 
about a decrease in amount of these compounds in the stems (31). 
Similar differences were also observed in carbohydrate fractions when 


the effects of the two compounds were compared. Naphthaleneacetic 
acid increased starch hydrolysis and the accumulation of sugars. Naph- 
thaleneacetamide caused the simple forms of carbohydrates to be built 
into more complex forms, such as lignin and cellulose, as was indicated 
by microscopic examinations of stem sections (31). Thus two regulators 
that are closely related chemically brought apparently opposing responses 
when applied to stem tissues. 

The mineral content of the stem tissues of bean plants has been 
increased through the apphcation of growth regulators to the stems (8) 
and to the roots (8,20). The movement of potassium, magnesium, 
manganese, and boron into treated regions of stems was increased a 
measurable amount within 30 hours after indoleacetic acid was applied. 
Phosphorus and copper were mobilized in treated stem tissues within 
48 hours after treatment. Iron and aluminum were also mobilized by 
treatment but more slowly than were other elements. The calcium 
content of stems was not affected by treatment with indoleacetic acid. 

In these experiments a sufficient amount of the regulator was used 
to bring about cell proliferation. It is obvious that certain elements were 
used for the structure of these new cells, and some elements, such as 
phosphorus, were found to increase in the treated stems at a rate about 
equal to the increase in total solid substance. On the other hand, there 
is evidence that some metals, such as copper, magnesium, and iron, 
are essential to enzyme systems, and it is suggested that these elements 
accumulate in tissues where meristematic activity has been induced, 
and play a part in the relatively large amount of enzyme activity in- 
volved (8). It is of interest that calcium, which is not abundant in 
meristematic tissues, was not mobilized as the result of treatment of 
stems with indoleacetic acid. 

Recently Rhodes and Templeton (47) reported that 2-methyl-4- 
chlorophenoxyacetic acid interfered with the potassium metabolism of 
rape but not of oats. They suggested that this adverse effect on potassium 
metabolism may account for the herbicidal effects of 2,4-D and for the 
difference in sensitivity of these 2 kinds of plants to this type of herbicide. 

The effect of 2,4-D and other growth regulators on the composition 
of fruits has recently received attention. In general, growth regulators 
tend to hasten starch hydrolysis in the tissues of relatively mature 
detached fruits like the banana, pear, and apple. With respect to starch 


this response is similar to that which occurs in stems of plants treated 
with these substances (21,40,53). 

It was recently found that the accelerating effect of 2,4-D on the 
ripening of bananas is greatly reduced by the lack of adequate aeration 
following treatment (29). Although the reason for this is not understood, 
the results indicate that in the absence or the presence of certain gases, 
tissues of fruits such as the banana may be relatively insensitive to 2,4-D. 

Recently attention has been directed toward the effect of growth- 
regulating substances on the vitamin content of plants. In recent experi- 
ments with beans the initial vitamin C content of the pods was not 
affected when the plants were sprayed with 4-chlorophenoxyacetic acid. 
The rate at which the vitamin was broken down during storage of the 
treated fruits was, however, much slower than for untreated ones. This 
effect was thought to be indirect since the growth regulator increased 
the storage hfe of the pods (36). 

According to recent tests with bean plants, application of 2,4-D 
brought about reduction of thiamin, riboflavin, and nicotinic acid in 
the leaf tissues (26). Stems of treated plants, on the other hand, con- 
tained higher concentrations of thiamin, riboflavin, nicotinic acid, and 
pantothenic acid than did comparable parts of untreated ones. The 
concentration of carotene in both leaves and stems was depressed by 
2,4-D, while the concentration of pantothenic acid was increased in 
stem and leaf tissues by its appHcation. These data indicate clearly that 
2,4-D brought about definite shifts in the concentrations of certain 
vitamins between stem and leaf tissues, but information on its effect 
on total vitamin production per plant is not at present available. 

2,4-D has increased the protein content of wheat seedlings when 
appHed in amounts ordinarily used for weed control (13). Increases of 
approximately 5 per cent or less in the protein content of seeds have 
been reported on the basis of field tests. Smaller increases were readily 
observed in greenhouse experiments, but in most instances these were 
accompanied by a depression in the yield of wheat seeds (33). The effect 
of growth-regulating substances on the chemical composition of fruits 
and seeds offers a relatively new and fertile field of study, which is just 
now beginning to receive the attention it deserves. 



1. Alexander, Taylor, R., Plant PhysoL, 13:845 (1938). 

2. Bausor, S. C, Botan. Gaz., 104:115 (1942). 

3. Berger, J., and Avery, G. S., Jr., Am. J. Botany, 30:297 (1943). 

4. , ibid., 31:11 (1944). 

5. , Science, 98:454 (1943). 

6. BoRTHwicK, H. A., Hamner, K. C, and Parker, M. W., Botan. Gaz., 

98:491 (1937). 

7. Brown, J. W., ibid., 107:332 (1946). 

8. Brunstetter, B. C, Myers, A. T., Mitchell, }. W., Stewart, W. S., 

and Kaufman, M. W., ibid., 109:268 (1948). 

9. Christiansen, G. S., Kunz, L. J., Bonner, W. D., and Thimann, K. V., 

Plant Physol., 24:178 (1949). 
ID. Clark, W. G., ibid., 12:409 (1937). 

11. Crafts, A. S., Science, 108:85 (1948). 

12. Ennis, W. B., Jr., and Boyd, F. T., Botan. Gaz., 107:552 (1946). 

13. Erickson, L. C, Seeley, C. I., and Klages, K. H., /. Am. Soc. Agron., 

40:659 (1948). 

14. Eyster, H. C, Plant Physol., 21:68 (1946). 

15. , ibid., 21:366 (1946). 

16. , Science, 97:358 (1943). 

17. Fogel, S., and Muller, W. H., Am. J. Botany, 30:23 (1943). 

18. Gall, Harold, J. F., Botan. Gaz., 110:219 (1948). 

19. Hagen, C. E., Clagett, C. O., and Helgeson, E. A., Science, 110:116 


20. Hamner, C. L., Botan. Gaz., 103:374 (1942). 

21. Hanson, E., Plant Physol., 21:588 (1946). 

22. Hitchcock, A. E., and Zimmerman, P. W., Contrib. Boyce Thompson 

Inst., 7:447 (1935). 

23. Kelly, Sally, Plant Physol., 24:534 (1949). 

24. LiNDER, P. J., and Mitchell, J, W., Unpublished data. 

25. LiNDER, P. J., Brown, J. W., and Mitchell, J. W., Botan. Gaz., 110:628 


26. Luecke, R. W., Hamner, C. L., and Sell, H. M., Plant Physol., 24:546 


27. Marth, p. C, and Davis, F. F., Botan. Gaz., 106:463 (1945). 

28. , and Mitchell, J. W., ibid., 107:129 (1945). 

29. Marth, P. C, and Mitchell, J. W., ibid., 110:514 (1949). 

30. , Unpublished data. 

31. Mitchell, J. W., Botan. Gaz., 101:688 (1940). 

32. , In press. Proceedings 2nd. International Congress for Crop Pro- 
tection, London (1949). 

33. , Unpublished data. 

34. , and Brown, J. W., Botan. Gaz., 107:120 (1945). 

35. , and Brown, J. W., ibid., 107:393 (1946). 

36. , EzELL, Boyce, D., and Wilcox, Marguerite S., Science, 109:202 


37. , and Hamner, C. L., Bot. Gaz., 105:474 (1944)- 

38. , Kraus, E. J., and Whitehead, M. R., ibid., 102:97 (1940). 


39. , and LiNDER, P. J., Unpublished data. 

40. , and Marth, P. C, Botan. Gaz., 106:199 (1944). 

41. , and Martin, W. E., ibid., 99:171 (1937). 

42. , and Stuart, N. W., ibid., 100:627 (1939). 

43. , and Whitehead, Muriel R., ibid., 102:393 (1940). 

44. , WiRWiLLE, J. W., and Weil, L., Science, 110:252 (1949). 

45. OvERBEEK, J. VAN, Am. J. Botany, 31:256 (1944). 

46. Rasmussen, L. W., Plant PhysoL, 22:377 (^947)- 

47. Rhodes, A., and Templeman, W. G., Nature, 160:825 (1947). 

48. Rice, E. L., Botan. Gaz., 109:301 (1948). 

49. Sell, H. M., Lucke, R. W., Taylor, B. M., and Hamner, C. L., Plant 

PhysoL, 24:295 (1949). 

50. Smith, F. G., ibid., 23:70 (1948). 

51. , Hamner, C. L,, and Carlson, R. P., ibid., 22:58 (1947). 

52. Smith, O., Nash, L. B., and Davis, G. E., Botan. Gaz., 102:206 (1940). 

53. SouTHWicK, F. W., Proc. Amer. Soc. Hort. Sci., 47:84 (1946). 

54. Staniforth, D. W., and Loomis, W. E., Science, 109:628 (1949). 

55. Stuart, N. W., Botan. Gaz., 100:298 (1938). 

56. TuKEY, H. B., Hamner, C. L., and Imhofe, Barbara, /<5'/^., 107:62 (1945). 

57. Weaver, R. J., ibid., 110:300 (1948). 

58. , and De Rose, R, H., ibid., 107:509 (1946). 

59. , ibid., 107:509 (1946). 

60. Weaver, R. J., Minark, C. E., and Boyd, F, T., ibid., 107:540 (1946). 

61. Went, F. W., and Thimann, K. V., Phytohormones (Macmillan, 1937). 

62. Wood, J. W., Mitchell, J. W., and Irving, G. W., Jr., Science, 105:33 


63. , ibtd., 105:337 (1947). 

64. Zimmerman, P. W., and Hitchcock, A. E., Contrib. Boyce Thompson 

Inst., 12:321 (1942). 

Histological Responses to 
Growth-Regulating Substances 


BOTH gross and histological responses of plants to different growth- 
regulating substances show a rather wide variety of patterns. Not 
only may plants of different species, genera, and families behave dif- 
ferently, but the age and succulence of individual plants within a 
variety at the time of treatment and the environmental conditions 
under which they are grown play important roles in the responses which 
they make. It is the purpose of the present paper to review the various 
types of responses to a number of these substances that have been re- 
ported in recent years. 

Early attempts to analyze the responses of plant tissues to specific 
chemical substances were made by Erwin F. Smith as far back as 19 17 
(34). He performed certain experiments in an effort to determine the 
mechanism of tumor formation resulting from infection with Phytomonas 
tumefaciens, using simple inorganic compounds, mainly ammonium salts 
dissolved in water. These were injected into the hollow pith of Ricinus 
and similar stems where they induced cellular proliferations of a callus- 
like character, with the differentiation of some vascular elements. 

Interest in plant hormones developed in the late 1920's and informa- 
tion on the gross morphological responses of plants to such substances 
accumulated rapidly. Boysen Jensen, Avery, and Burkholder (8) have 
presented the observations recorded up to 1936. Zimmerman and 
Wilcoxon (40), Q)oper (12), and Brown and Gardner (9) had reported 
the production of tumors following the application of indoleacetic acid 
to plants. Laibach and Fischnich (25) reported the effects of an indole- 
acetic acid-lanoHn mixture on the stems of Coleus, Viciafaba, and tomato. 


In the treated stems of Coleus Fischnich (14) found that the cells of the 
parenchyma enlarged, the cells in the vicinity of the cambium pro- 
hferated, the cambium became active, new vascular bundles appeared 
between the old ones, and adventitious roots arose principally in the 
borders of the vascular bundles. Czaja (13) studied the effects of indole- 
acetic acid-lanolin paste on Helianthus hypocotyls and described some 
of the tissue responses. Snow (35) showed that auxin-a, indoleacetic 
acid, and extracts of urine stimulated cambial activity in Helianthus 

Relatively little detailed description of the histological responses of 
plants to any growth-regulating substance had appeared, however, prior 
to the publication of the paper by Kraus, Brown, and Hamner in 
1936 (23), dealing with the gross and histological reactions of the red 
kidney bean to applications of indoleacetic acid. This paper stimulated 
tremendous interest and gave direction for numerous papers which 
followed, deahng not only with the responses induced by indoleacetic 
acid but with a number of other chemical substances. Because of the 
accurate and detailed observations presented, as well as of the influence 
exerted on subsequent histological studies following the application of 
indoleacetic acid and other growth-regulating substances, it is deemed 
advisable to point out some of the most significant of their findings. 

Responses of Young Decapitated Stems to Indoleacetic Acid 

The indoleacetic acid was apphed as a 3 per cent mixture in lanohn 
to the cut surface of the second internode of young bean plants which 
had been cut off squarely about i mm. below the base of the petiole 
of the first compound leaf. In some instances axillary shoots were used. 
Essentially the same responses were obtained in both cases. 

Following decapitation and application of the lanohn mixture, little 
gross response of the stem was noticeable within 1 8 hours. Swelling of the 
stem for i to 2 mm. down from the cut surface then began, and by the 
end of 48 hours the topmost portion of the stem had become distinctly 
enlarged. At 72 hours the end of the stem attained a diameter nearly 
twice that at 5 mm. below the cut surface, and by the end of 1 10 to 120 
hours glistening tips of root primordia were visible about the periphery 
of the tumor. At 144 to 168 hours the roots were more evident and some 
of them emerged from the surface of the tumor. Such tumors may con- 
tinue to grow for periods longer than six months and frequently attained 

J, M. BEAL 157 

diameters of 2 cm. or more, and a height above the original cut surfaces 
of 2-2.5 *^rn- Similar appearing tumors were induced on bean pods, 
with roots developing from them also. 

Histological Details. — The cells of the epidermis and pericycle re- 
sponded less actively than those of the other tissues. The cells of the 
cortical parenchyma enlarged somewhat and those near the endodermis 
became meristematic. The cells of the endodermis were highly responsive; 
they were the first to show meristematic activity with nuclear divisions 
being greatly accelerated shortly after treatment, and extending as far 
as 5 cm. below the point of apphcation after a week. The derivatives 
differentiated as phloem and xylem elements and large, apparently 
multinucleate, parenchymatous cells. Many derivatives remained meri- 
stematic while others gave rise to root histogens and eventually to 
adventitious roots. Especially over the vascular bundles long proliferat- 
ing strands of vascular tissues developed from endodermal derivatives. 
These frequently enlarged sufficiently to rupture the tissues exterior 
to them. The parenchyma of the primary phloem showed the same 
general type of response and subsequent differentiation of tissues. Some 
of the cells derived from it formed a part of the cortical tissues of the 
apical crown of adventitious roots, A part of the tissues composing the 
cortical portion of these adventitious roots, however, was derived from 
the parenchyma of the secondary phloem. Other cells matured into 
parenchymatous tissue, tracheids with simple pits, sieve tubes, and 
companion cells. The cambium divided actively. Its derivatives often 
remained active over long periods of time, later maturing as various 
phloem or xylem elements, or continued as meristematic zones from 
which such elements continued to form. 

Near the surface of application the cells of the rays adjacent to the 
xylem proliferated greatly. Many of their derivatives matured as 
tracheids, with meristematic areas intermingled with them. This con- 
fused mass of tissue often persisted and continued development for 
weeks. The ray cells adjacent to the phloem and just within the pericycle 
also proliferated greatly and, in conjunction with the phloem cells 
flanking them, gave rise to root histogens and also directly formed large 
portions of the adventitious roots. 

The pith cells also proliferated greatly, with the first marked activity 
adjacent to the surface of apphcation, but later activity progressed 
down the stem next to the elements of the primary xylem. Large pitted 


tracheids intermingled with large parenchymatous cells and meristematic 
zones continued to develop for a long period of time and accounted for 
much of the overgrowths which continue development for periods longer 
than six months (20). 

Decapitated stems treated only with lanolin generally enlarged but 
little. When decapitated and otherwise untreated, calluses derived mainly 
from cells of the primary and secondary phloem parenchyma might 
be formed. 

The main responses shown by various tissues of the tomato (7,27), 
cabbage (17), pea (33), and broad bean (32) to indoleacetic acid were 
similar to those in the bean, differing from it and from one another 
chiefly in degree and a few details. 

Decapitated and treated stems of etiolated peas (32) developed a 
meristem cylinder from division of the cells of the inner cortex, endo- 
dermis, pericycle, and phloem parenchyma. The root primordia varied 
in origin in younger and older stems. In younger seedlings the identity 
of the endodermis became lost in the meristem cylinder complex. The 
root primordia arose from a group of ray cells within this complex. In 
older stems the endodermal cells did not divide and the root primordia 
were pericyclic and intrapericyclic in origin. Iresine (21) and Mirabilis 
(19) differed from most other species investigated in that the pericycle 
was more responsive than the endodermis. The phloem of Iresine was 
strongly reactive while that of Mirabilis was not. Adventitious roots 
developed mostly from pericycle and ray tissues. 

In Vicia faba (32) adventitious roots were rarely formed, although 
large, rather unorganized masses of meristematic tissue occasionally 
developed. These appeared to be potential roots, which had not organ- 
ized merlstems or orderly systems of derivatives. The cabbage plant (17) 
was interesting because it developed adventitious shoots from the tumors; 
apical ones from callus, and lateral ones from inner cortex and ray tissues. 

All of the preceding plants are dicotyledons. The only detailed histo- 
logical investigation of the responses of monocotyledonous stems to 
indoleacetic acid was that on three species of L///ww (3). In L.philippinense 
formosanum and L. longiflorum the first detectable changes in decapitated 
stems occurred in the cells of the fundamental parenchyma, lateral and 
centlfugal to the vascular bundles, usually only the outer bundles. These 
cells became meristematic, and by repeated divisions gave rise to cells 
which later differentiated as adventitious roots. In L. harrisi (L. longi- 

J, M. BEAL 159 

florum var. eximium) the region of response was limited largely to the 
cells of the epidermis and outer cortex in the immediate vicinity of the 
leaf axils adjacent to the surface of application. Adventitious roots were 
rarely formed. The epidermal cells of the stem immediately above the 
axil elongated radially, while the outer cortical cells centripetal to as 
well as slightly below them enlarged and in the course of about five 
days began dividing. This marked the initiation of the region at which 
buds later developed. Neither buds nor bud primordia were present in 
these leaf axils at the time of treatment. The groups of meristematic 
cells in the leaf axils divided repeatedly, accompanied by radial divisions 
of the cells of the epidermis over them. The subsequent growth of their 
derivatives resulted in a hump of cells which through further develop- 
ment and differentiation became a bud. As many as three buds may 
develop in one axil. If permitted to grow on the plant for 8 to 10 weeks, 
the induced buds became bulbils with roots at their bases. Bulbils were 
removed from the parent plant and grown separately. No visible ana- 
tomical differences were observed which could be used in explaining 
the differences in the behavior of the three species. 

Responses to Other Growth Substances 

A number of other growth substances have been applied to the red 
kidney bean. Indolebutyric acid (20) produced apical tumors similar to 
those resulting from indoleacetic acid, but with fewer, thicker roots in 
the upper zone, more roots i cm. or so below the treated surface, and 
several tiers of roots between these two regions. Naphthaleneacetic acid 
(20) resulted in the formation of apical tumors with greater and more 
uniform diameter for a distance of about i cm. below the cut surface, 
where a circle of large root primordia was formed, each primordium over 
one of the main vascular bundles. The responses to tryptophan (22) 
are less vigorous but similar insofar as the responsive tissues are concerned. 
No adventitious roots developed but abundant anastomosing vascular 
strands were differentiated, mostly from derivatives of the endodermis. 
Whether these effects are due to indoleacetic acid derived from trypto- 
phan is not wholly certain. Histological studies indicate they may not 
be; biochemical determination of material in situ must be made. An 
extract of maize pollen (28) applied to bean stems produced responses 
similar to those from tryptophan. Tetrahydrofurfurylbutyrate (29) pro- 
duced Uttle or no effect on epidermis, cortex, pericycle, or cambium, 


and only a slight effect on the endodermis. Xylem, phloem, and rays 
proliferated, but no roots developed. Naphthoxyacetic acid (2) appUed 
to tomato stems produced responses similar to those from indoleacetic 
acid. Root primordia resulted from the activity of the pericycle and 
phloem parenchyma, the root cap arising from the endodermis. 

The next important group of compounds to be investigated was the 
substituted phenoxy group. These compounds attracted great interest 
because of their marked formative and telomorphic effects, as well as 
the fact that many plants treated with them are killed. Numerous 
investigations on the reactions of plants to them led to their use as 
selective herbicides. Recently some of them have been employed for 
several other important purposes. 

The first report dealing with the histological responses induced by 
any of these substances was a rather brief description of the histological 
changes in bindweed and sow thistle following applications of 2,4- 
dichlorophenoxyacetic acid (2,4-D) in herbicidal concentrations (37). 
Following this there were three papers (5,6,36) deahng with the reactions 
of the bean to these compounds, especially 2,4-D and 2,4,5-trichloro- 
phenoxyacetic acid (2,4,5-T). In two of these reports (5,6) both lanolin 
and Carbowax 1500 had been used as carriers of the growth substances, 
and it was found that the carrier played a role in the effectiveness of 
some of the substances. In general the tissue responses were similar 
in kind but greater in degree than those induced by indoleacetic acid 
and several of the other substances. Endodermis, cambium, phloem, and 
ray parenchyma were strongly activated, and in most instances roots 
developed abundantly, mainly from the phloem and ray parenchyma. 

Somewhat similar experiments were done on decapitated bean plants, 
using 2,4-D and four of its salts, namely, ammonium, copper, calcium, 
and magnesium 2,4-dichlorophenoxyacetates (30). Histological responses 
in the early stages showed considerable similarity. Later certain dif- 
ferences became evident, but no response induced by one substance was 
entirely absent in the reaction to another substance, since all responses 
appeared to fall within the range of effects distinctive for 2,4-D. 

A 2 per cent phenylacetic acid and lanolin mixture (38) applied to 
the cut surface of decapitated young bean plants produced flat-topped, 
somewhat tuberculate tumors. The bulk of the tumor arose from a 
marked proliferation of the inner cortex, endodermis, and primary 
phloem parenchyma. The derivatives matured as tracheids or paren- 



chyma, continued as patches of meristematic tissue, or infrequently 
differentiated as small vascular bundles. There was increased activity 
of the cambium, its derivatives maturing entirely as tracheids. Root 
primordia were rare. 

Alpha-naphthylmethylacetate applied to the cut surfaces of decapi- 
tated second internodes of the bean (i) produced in general the same 
tissue responses as had occurred following the application of most other 
growth-regulating substances. The production of considerable wood was 
one of the notable responses. In this respect it resembled the response 
to a-naphthylacetamide (24). 

Responses of Roots 

Studies of the effects of growth-regulating substances on roots were 
for a time limited largely to observations on the stimulation or inhibi- 
tion of root elongation, the development of laterals, and the accompany- 
ing physiological activities. Some histological and cytological responses 
of Allium roots to several chemicals were described by Levan (26). 
Noirfalise (31) reported the cessation of elongation of the primary root 
of young seedlings of Viciafaba when placed in certain aqueous solutions 
of heteroauxin. Relatively high concentrations inhibited elongation but 
resulted in marked increase in diameter and the production of numerous 
lateral roots. Microscopic preparations showed that the increase in 
diameter resulted chiefly from the activity of the cells of the pericycle. 
Morphological changes in induced roots of wheat following treatment 
with heteroauxin were reported by Burstrom (10). A study of the histo- 
logical and cytological changes induced in the roots of Allium cepa, 
Narcissus (var. Paper White), and Tulipa (vars. John Ruskin and Louis 
XIV) by several growth-regulating chemicals was presented by Carlton 
(11). Six substances were used: a-naphthaleneacetic acid, indoleacetic 
acid, indolebutyric acid, /3-naphthoxyacetic acid, a-naphthylacetamide, 
and tryptophan. These were used at concentrations of 10 or 20 parts 
per million in a three-salt solution. The bulbs were rooted by placing 
their bases in water, and when the roots had attained a suitable length 
the bulbs were transferred to the salt solution containing the respective 
growth substance. Root elongation was inhibited temporarily by indole- 
acetic acid and permanently by all the others. The roots of Allium and 
Narcissus thickened markedly just back of their growing points as a 
result of treatment with indoleacetic, indolebutyric, naphthaleneacetic, 


and naphthoxyacetic acids, and to a lesser extent in the tryptophan. 
Roots of Tiilipa showed little enlargement and no adventitious root 
formation. In Allium and Tulipa proliferation of the pericycle occurred 
with the formation of numerous root primordia in Allium. Neither true 
proliferation nor root primordia occurred in Narcissus. 

Responses of Fruits 

Parthenocarpy induced by growth-regulating substances was first 
accomplished by Gustafson (i8) in 1936 by applying known chemical 
compounds in lanolin to the pistils of a number of plants. The following 
year Gardner and Marth (16) reported the induction of parthenocarpy 
in American holly, Ilex opaca, by spraying the open blossoms with dilute 
aqueous solutions of several growth substances, including indoleacetic, 
indolepropionic, indolebutyric, and naphthaleneacetic acids. Gardner 
and Kraus (15) investigated the histological changes of the pistil of the 
holly as affected by indoleacetic acid and found that the development of 
the parthenocarpic fruits paralleled almost precisely that following 
pollination. The chief differences were that the stigmas of the sprayed 
fruits proliferated somewhat more than those pollinated, did not collapse 
and suberize as soon, and developed neither embryo nor endosperm in 
the ovules. 

Beal (4) appHed i per cent growth substance and lanolin mixtures 
shortly after anthesis to the cut surface of ovaries of Lilium regale from 
which the stigmas and styles had been removed by cutting squarely 
across the top of the ovary at the base of the style. Indoleacetic, naph- 
thaleneacetic, and naphthoxyacetic acids and a combination consisting 
of equal volumes of the three i per cent concentrations thoroughly mixed 
were used. 

Ovaries to which the three, as well as those to which the combination 
were applied, enlarged in length and diameter at approximately the 
same rate and attained nearly the same final size. Their length was as 
great as that of fruits resulting from pollination, but their diameter was 
always less. Neither apical tumors nor adventitious roots were formed. 

A transection of an ovary at 795 hours (33 days) after treatment 
showed well-developed carpel walls and ovules. The greater part of 
the growth had resulted from enlargement of cells, although a few cell 
divisions had occurred in some regions of both the carpels and ovules 
following treatment. Again neither embryos nor endosperm were formed. 

J. M. BEAL 163 

A naturally parthenocarpic variety of cucumber similarly cut at the 
base of the style and smeared with 2 per cent indoleacetic acid and 
lanolin mixture behaved rather differently according to Young (39). 
If treated at full bloom the tissues adjacent to the surface of appHcation 
remained alive but showed shght proliferation. Ovules were intermediate 
in size between those of controls and those given the prebloom treatment 
with indoleacetic acid. If treated about 4 days before full bloom, tissues 
of the nectary, floral tube, and neck proliferated to form a small apical 
tumor in which no vascular bundles or root primordia appear. Ovules 
developed to about half normal size, and seed coats became partially 

AppHcation of a 3 per cent indoleacetic acid and lanoHn mixture to 
the cut surfaces of partially mature bean pods which had had their tips 
removed resulted in the production of large vascular apical tumors 
and roots (20). The tissues composing these tumors were derived mainly 
from the proliferated exocarp and mesocarp of the pod. The endocarp, 
which in untreated pods is principally derived from proliferated epi- 
dermal cells, became somewhat more active in treated pods, but formed 
little or no part of the apical tumor. Vascular elements apparently were 
not differentiated from this tissue. The large apical tumors are built up 
from derivatives of cells near the treated surface, a far shorter distance 
than was the case in stems. The seeds aborted in at least half the apically 
treated pods; the larger the tumor the fewer the seeds. This appeared to 
result from the mobihzed materials that entered the pod being diverted 
to the tumor rather than to the seeds. 

Lateral Application to Stems 

Hamner and Kraus (20) made lateral applications of a 3 per cent 
indoleacetic acid and lanolin mixture to bean stems by drawing the 
mixture out as a thin thread which was then laid on the second internode 
to encircle it completely. Care was taken to avoid injury to the stems. 

Marked yellowing and swelling occurred 2 or 3 mm. above, below, 
and at the hne of application. The effects increased with time until by 
the end of a week or ten days the tumors were often 2 cm. or more long, 
spindle shaped, and markedly ridged over the vascular bundles. Roots 
in longitudinal rows emerged mainly between the swellings over the 
vascular bundles. 

The histological responses closely resembled those of stems decapitated 


and treated terminally. The pith showed practically no meristematic 
response, but the cells of the inner cortex, endodermis, primary phloem, 
cambium, and the rays flanking the phloem showed marked activity. 
The roots which developed were related to the rays in the same manner 
as those developed in apical tumors of decapitated plants. 

These responses indicate that almost any parenchymatous tissues may 
become meristematic when growth-regulating substances are applied 
to them. It is obvious also that the same general types of responses 
are incited by a large number of these substances, although there are 
marked differences in the degree or intensity of responses of the several 
tissues in relation to the specific substances. The difference in response 
with respect to the kind of substance employed is greater than it is to 
the concentration of a single substance applied over a rather wide range 
of the higher concentrations. It is, however, impossible to say just what 
the concentration of the substance which reaches any cell or group of 
cells may be when applications are made in the various ways listed. There 
is no doubt that the substances are soluble and that they diffuse into and 
among the cells of the plant, but the rate at which this may occur is 
difficult to determine. 

Thus it would appear that: 

1. In the presence of growth substances and proper food and nutrient 
supply, cells of recognizably differentiated tissue systems may dedif- 
ferentiate, become embryonic, and proliferate. From these derivatives, 
tissues quite distinct in type may be differentiated (for example, vascular 
bundles from phloem, endodermis, pith, etc.). 

2. In general not wholly new responses are evoked, because many of 
the tissue systems respond similarly to changes in nutrients and food 
supply, and to wounding and other environmental changes (for example, 
heahng of wounds, rooting of cuttings, delay of abscission). 

3. Although no genuine new tissues arise the nature of their distribu- 
tion and proportions and pattern may be profoundly affected. 

4. To this extent morphogenesis is brought under control, or may be 
manipulated, as is now done in so many instances in the agricultural 
field by the use of these compounds. 

5. The course of development apparently is changed, but more than 
a growth substance is required; foods and nutrients play important 
roles in the quantity and quality of response. 

6. Age of tissues, or perhaps more basically the chemical nature of 

J. M. BEAL 165 

the tissues correlated with aging, plays a major role in the type and 
quantity of response. 

7. It is as important to know the qualities of the tissues that are 
to respond as it is to know the growth substance to be employed, since 
to many of the substances the responses are very similar while to others 
they may be quite different, as for example to naphthaleneacetic acid 
and naphthylacetamide. Thus the growth-regulating substance can be 
regarded only as a releaser (or in part a controlling means) of response 
of the living cell, which still remains the critical entity, and not as the 
prime inciter itself. 

8. While in morphogenetic studies genetic capacity may control, yet 
growth-regulating substances determine the degree to which expression 
does or may take place. They are one of a complex group of factors, in- 
cluding light, temperature, food and nutrient supply, and other envi- 
ronmental conditions. Therefore growth regulator or Ergocrine is a 
more precise use of terminology than growth substance or even hor- 
mone, because many of the effects of these substances suppress certain 
physiological reactions as well as excite them. The same is true for the 
morphological responses and final visible changes as well. 


1. Bachofer, C. S., Botan. Gaz., 110:119 (1948). 

2. Bausor, S. C, Reinhart, W. L., and Tice, G, A., Am. J. Botany, 

27:769 (1940). 

3. Beal, J. M., Botan. Gaz., 99:881 (1938). 

4. , ibid., 105:25 (1943)- 

5. , ibid., 107:217 (1945). 

6. , ibid., 108:166 (1946). 

7. BoRTHwicK, H. A., Hamner, K. C, and Parker, M. W., ibid., 98:491 


8. BoYSEN Jensen, P., Avery, G. S., and Burkholder, P. R., Growth 

Hormones in Plants (McGraw-Hill Book Co., 1936). 

9. Brown, N. A. and Gardner, F. E., Phytopathology, 26: 708 (1936). 

10. Burstrom, H., Ann. Agri. Col. Sweden, 10:209 (1942). 

11. Carlton, W. M., Botan. Gaz., 105:268 (1943). 

12. Cooper, W. C, Plant Physiol, 10:789 (1935). 

13. Czaja, a. T., Ber. Deiitsch. Botan. Ges., 53:197 (i935)- 

14. Fischnich, O., Planta, 24:552 (1935). 

15. Gardner, F. E. and Kraus, E. J., Botan. Gaz., 99:355 (i937)' 

16. , and Marth, P. C, ibid., 99:184 (1937). 

17. Goldberg, E., ibid., 100:347 (1938). 

18. GusTAFSON, F. G., Proc. Nat. Acad. Sci. U. S., 22:628 (1936). 



19. Hamner, K. C, Botan. Gaz., 99:912 (1938). 

20. , and Kraus, E, J., ibid., 98:735 (1937). 

21. Harrison, B. F., ibid., 99:301 (1937). 

22. Kraus, E. J., ibid., 102:602 (1941). 

23. , Brown, N. A., and Hamner, K. C, ibid., 98:370 (1936). 

24. , and Mitchell, J. W., ibid., 101:225 (1939). 

25. Laibach, F. and Fischnich, O., Ber. Deutsch. Botan. Ges., 53:528 (1935). 

26. Levan, a., Heriditas, 25:87 (1939). 

27. Link, G. K. K., Wilcox, H. W., and Link, A. DeS., Botan. Gaz., 98:816 


28. Mitchell, J. W. and Whitehead, M. R., tbtd., 102:770 (1941)- 

29. MuLLisoN, W. R., ibid., 102:373 (1940). 

30. Murray, M, A. and Whiting, A. G., ibid., 109:13 (1947). 

31. Noirfalise, a., La Cellule, 48:309 (1940). 

32. Falser, B. A., Botan. Gaz., 104:243 (1942). 

33. Scott, F. M., ibid., 100:167 (1938), 

34. Smith, E. F., /. Agr. Res., 8:165 (^9^7)- 

35. Snow, R., Netv Phytologist, 34:347 (1935)- 

36. SwANSON, C. P., Botan. Gaz., 107:522 (1946). 

37. TuKEY, H. B., Hamner, C. L., and Imhofe, B., ibid., 107:62 (1945). 

38. Whiting, A. G. and Murray, M. A., Botan. Gaz., 107:312 (1946). 

39. Young, }. O., ibid., 105:69 (1943). 

40. Zimmerman, P. W. and Wilcoxon, F., Contrib. Boyce Thompson Inst., 

12:1 (1935). 

Comparative Effects of Growth Substances on 

Stem Anatomy 


IX/Tany different growth substances have been tested in attempts to 
determine the nature of their effect on plant growth (lo). It has 
been found that growth substances may have characteristically different 
effects on a plant, particularly on the anatomical structure of the stem. 
Thus, in studies carried out here, 2,4-dichlorophenoxyacetic acid (2,4-D) 
stimulated cell proliferation and lateral root formation (9); a-naph- 
thaleneacetamide induced cambial activity resulting in the formation 
of more xylem and in a thickening of the cell walls of the xylem and the 
phloem fibers (4) ; and jS-naphthoxyacetic acid was particularly effective 
in producing parthenocarpic tomato fruits (7). Alpha-naphthaleneacetic 
acid has been reported to prevent the preharvest drop of apples in the 
fall as well as to thin apples shortly after blossoming in the spring (3,8). 

In our studies of the anatomical structure of plants treated with 
growth substances the following problems have been given special at- 
tention: i) why do most dicotyledonous and only some monocotyledon- 
ous plants respond to 2,4-D; 2) what is the possible role of a-naphthalene- 
acetamide in delaying calcium deficiency symptoms; 3) and how does 
a-naphthaleneacetic acid function in the thinning of apples at one stage 
and in preventing them from dropping at a mature stage? 

Much work has been done on 2,4-D regarding its practical value as a 
weed-killer. It has recently been reported that some species of dicotyle- 
dons and monocotyledons responded to 2,4-D while others did not (2,6). 
The structure of the stem of young kidney bean plants treated with 2,4-D 
was reported by Swanson (9). In our investigations older plants were 
used (six internodes or more) belonging to the monocotyledonous and 


dicotyledonous plants. They were sprayed with .01 per cent aqueous 
solution of 2,4-D. 

All the plants were in a vegetative stage when treated. Samples 
of the stem of monocotyledonous plants were taken in the region that 
showed a response to the growth substance, such as swelling of the stems 
or the presence of lateral roots. The dicotyledonous plants were sampled 
at the second through the eighth internode from the stem tip. Prolifera- 
tion of cells was apparent in some plants soon after treatment, while in 
others several days elapsed before a response was evident. The dicotyle- 
donous plants usually made a greater anatomical response to 2,4-D than 
did the monocotyledonous plants. 

Tomato plants were sensitive to this growth regulator. Eighteen days 
after treatment lateral roots had emerged through the cortex. The cells 
of the phloem parenchyma, cambium, ray parenchyma, and pericycle 
proliferated to form root primordia. Proliferation was not limited to the 
interfasicular region as it was in plants such as cocklebur, for bands 
of active cells encircled the outer stelar region (Fig. 1,2). 

Fourteen days after treatment with 2,4-D cell division had occurred 
in some of the cortical cells of Peperomia; but the cells most sensitive 
to this treatment were those adjacent to the vascular bundles, par- 


Figure I. Fourth internode of tomato control plant. Figure 2. Fourth 
internode of tomato eighteen days after treatment with 2,4-D; band of lateral 
roots formed around stem. Figure 3. Fourth internode of control plant of 
Peperomia. Figure 4. Treated stems oi Peperomia responded to 2,4-D; prolifera- 
tion of cells and adventitious root formation was apparent. Figure 5. Second 
internode of cocklebur 97 hours after treatment with 2,4-D showing pro- 
liferation of cells in the interfasicular region of the stem. Figure 6. Eighth 
internode of cocklebur 25 days after treatment; lateral roots emerging through 
the cortex from the interfasicular regions. Figure 7. Cross section of a leaf 
25 days after treatment with 2,4-D; leaf swollen and root primordia dif- 
ferentiating. Figure 8. Stem tip of cocklebur 25 days after treatment with 
2,4-D; cells of leaves surrounding stem tip have proliferated, stem tip has 
broadened, and cells below apex have become necrotic. Figure 9. Stem tip of 
normal plant. Figure 10. Fourth internode of Coleus 35 days after treatment; 
root primordia formed from fasicular region of stem. Figure 11. Fourth 
internode of untreated stem of Coleus. Figure 12. Untreated plant oi Drucena 
showing cambial region. Figure 13. Lateral roots from stem of Dracena 24 
days after treatment with 2,4-D. 

^ 13 




>.■ *c 





S'^\^^^:o^m '^iT^r''/ 






ticularly those next to the phloem which were probably phloem paren- 
chyma cells and perlcycle cells. Thirty-two days after treatment roots 
developed from the phloem side of the vascular bundle were emerging 
through the cortex and epidermis (Fig. 3, 4). 

Ninety-six hours after treatment proliferation of cells was apparent 
in the interfasicular region of the second internode from the stem tip 
of cocklebur. Only a few scattered cell divisions were apparent in the 
cortex (Fig. 5). Twenty-five days after treatment roots had extended 
beyond the cortex (Fig. 6). Unlike tomato, where proliferation occurred 
anywhere in the cambium of the stem, lateral roots were formed only 
from the interfasicular region of the stem of cocklebur. This resulted 
in a misplacement of the vascular bundles in the stem perhaps due to 
the pressure of the developing roots extending through the cortex. 
The leaves and stem tips of cocklebur were also studied. The stem tip 
had enlarged to several times its normal size, the apex being broader 
and the surrounding leaves showing masses of proliferating cells (Fig. 
8, 9). Prohferating cells forming root primordia gave rise to the swellings 
of the veins and midrib of the leaves (Fig. 7). 

Coleus responded to 2,4-D in that large root primordia were formed 
from the fasicular region of the stele. The cambium, phloem parenchyma, 
and ray cells proliferated to form root primordia (Fig. 10, 1 1). Poinsettia 
also gave a marked response to 2,4-D, which appeared later than in the 
above species. Other species such as sweet potato, Oxalis, lambs'- 


Figure 14. Cross section from normal plant of Tradescantia. Figure 15. 
Cross section of stem of Tradescantia after treatment with 2,4-D; lateral roots 
appear to differentiate from the meristematic zone near the periphery of the 
stem. Figuie 16. Cross section of normal stem of Philodendron. Figure 17. 
Cross section oi Philodendron stem which failed to respond to 2,4-D. Figure 18. 
Cross section of normal quack-grass rhizome. Figure 19. Rhizome of quack 
grass treated with 2,4-D. Band of lateral roots encircled rhizome which 
differentiated from meristematic zone. Figure 20. Fourth internode of cockle- 
bur grown at a temperature of 7o°C. with complete nutrient solution. Figure 
21. Fourth internode of cocklebur grown with nutrient lacking calcium. 
Figure 22. Fourth internode of cocklebur grown in a nutrient lacking calcium 
but treated with a-naphthaleneacetamide. Figure 23. Fourth internode of 
cocklebur grown with a complete nutrient supply and treated with a-naph- 


quarters, pigweed, and Verbena likewise responded to 2,4-D; and the 
tissues responsible for proliferation and root formation were the same as 
those for the previous plants mentioned, namely the cambium, phloem 
parenchyma, ray cells, and pericycle. 

Comparing the monocor\ledonous plants with the dicotyledonous 
plants, the same tissues prohferated to form root primordia. Dracena 
developed a swelling at the base of the leaves which perhaps included 
several nodes and internodes. Upon examination of this region it was 
apparent that a cambial-like region was present in this plant (Fig. 12). 
Eishteen davs after treatment lateral roots ori^inatin? in the cambial 
zone and in the parenchyma cells on either side of it (Fig. 13) were 
extending through the cortex. 

Adventitious roots in stems of Tradescantia treated with 2,4-D were 
formed from the meristematic zone near the periphery of the stem 
(Fig. 14, 15). 

Philodendron failed to respond to treatment with 2,4-D. \'ascular 
bundles were distributed throughout the stem, the larger ones occupying 
the center. Over the phloem of each bundle was a cap of thick-walled 
fibers which were perhaps too differentiated to revert to an active stage 
of prohferation. .\lthough there were parenchyma cells in the stem 
which ordinarily responded in other species they failed to do so in 
Philodendron (Fig. 16, 17). 

.-Uthough the above ground stem of quack grass was not affected by this 
growth substance, the rhizome made a striking response. The parenchyma 
cells adjacent to the differentiating bundles proliferated and were re- 
sponsible for the formation of lateral roots (Fig. 18, 19), 

The anatomical responses of plants to 2,4-D were similar regardless of 
whether they were dicotyledonous or monocotyledonous (1,5). Pro- 
liferation of cells and root formation from the cambium, parenchyma 
cells, ray cells, and pericycle were common to both groups. 

From these results it has been concluded that whether a plant can 
be stimulated generally depends on whether there are cells in the stem 
which are immature enough to proliferate when treated with 2,4-D. 
These types of cells were more limited in amount in monocotyledons than 
in dicotyledons, which may explain why the monocotyledons treated 
with 2,4-D usually recovered. 

Another growth substance which had a quite different effect on the 
anatomical structure than 2,4-D was a-naphthaleneacetamide. Mitchell 


(4) reported its effect on cell structure in bean plants. With the appUca- 
tion of this growth substance there was increased cambial activity in 
the stem resulting in the formation of thick-walled xylem cells. A greater 
number of Hgnified cells were present as compared with the control. 

It was observed that stems of cocklebur grown without a calcium 
supply generally had smaller and thinner-walled cells. This was especially 
conspicuous in the xylem cells, xylem parenchyma cells, and pericycle 
fibers where there was decreased hgniiication. In fact vessels frequently 
collapsed because of lack of rigidity of the cell wall. Alpha-naphthalene- 
acetamide induced a thickening and hgnification of cell walls when 
sprayed on or apphed in lanolin to the plants grown without calcium. 
The first experiment was conducted at a temperature of 70° F. The plants 
were eight inches tall when the treatment was started. A series of cockle- 
bur plants was set up, some of which were given a complete nutrient 
solution, a nutrient lacking calcium, a complete nutrient and a spray 
treatment with a-naphthaleneacetamide, and a nutrient lacking calcium 
but also receiving an a-naphthaleneacetamide treatment. Thirty-six days 
from the start of the experiment severe symptoms were apparent in 
plants grown without calcium. At the same time plants grown without 
calcium but sprayed with a-naphthaleneacetamide showed no symptoms 
like curling of the leaves near the stem apex or a spotted appearance of 
the leaves. Plants given the other two treatments appeared normal. 

The anatomical structure of the fourth internode of plants given a 
complete nutrient supply showed the usual organization of the cells 
(Fig. 20). An active cambium differentiating phloem and xylem was 
apparent. Plants lacking calcium in the nutrient solution showed all 
cells to be smaller and thinner walled; the xylem elements were fewer 
and less hgnified; the vascular bundles were small with few vessels; 
and the cambium was less conspicuous (Fig. 21). 

Plants given a complete nutrient solution and treated with a-naph- 
thaleneacetamide showed an active cambium differentiating vascular 
elements (Fig. 23). Xylem elements, especially tracheids, were thicker 
walled than in the control, and increased thickening of the phloem fibers 
was also evident. Plants grown without calcium and treated with a- 
naphthaleneacetamide showed no deficiency symptoms thirty-six days 
after treatment. The fourth internode of these plants showed anatomical 
characteristics resembling those of normal untreated plants. The vas- 
cular bundles were shghtly smaller than in the control plants, but the 


cambium was just as active. There was a greater lignification of the 
vascular elements and the fibers, and the cells were much larger than in 
plants grown without calcium (Fig. 22). Except for the size of the 
vascular bundles, the structure of the control plants and of plants grown 
without calcium but treated with a-naphthaleneacetamide was very 

During the winter of 1948 the experiment was repeated under long- 
day photoperiod and at a temperature of 65° F. Twenty days after the 
start of the experiment deficiency symptoms were apparent in plants 
grown without calcium. Twenty-four days after treatment deficiency 
symptoms were also becoming apparent on plants grown without calcium 
but treated with a-naphthaleneacetamide. After forty-four days plants 
grown without calcium, and those grown without calcium but sprayed 
with a-naphthaleneacetamide showed severe calcium deficiency symp- 
toms. The plants were sampled for anatomical observations at this time. 
The fourth internode showed structural abnormalities typical of calcium 
deprivation in plants grown without calcium and plants grown without 
calcium but treated with a-naphthaleneacetamide (Fig. 24, 25, 26, 27). 
The experiment was repeated and similar results were ob'^ained. 

The question then arose as to why results similar to those obtained 
the previous late spring at 70° F. were not secured. Could the difference 
in temperature account for the difference in the effect of a-naphthalene- 
acetamide on delaying calcium deficiency symptoms? 

The experiment was again repeated in the greenhouse, except that 
it was carried out at a temperature of 75° F. After twelve days plants 
grown without calcium showed symptoms which rapidly became more 
severe. After sixty days the plants grown without calcium were dead 
and those grown without calcium but treated with a-naphthalene- 
acetamide showed no calcium deficiency symptoms. After seventy-five 
days only slight symptoms of calcium deficiency were apparent in plants 
grown without calcium but treated with a-naphthaleneacetamide. At 
this time all plants were sampled for anatomical observations. 

The anatomical structure of the fourth internodes of these plants 
corresponded closely to those of the first experiment at the warmer 
temperature. The stems of plants grown without calcium had small 
bundles and small cells, resulting in a decreased amount of lignification. 
Plants grown without calcium but treated with a-naphthaleneacetamide 
showed a structure more closely resembling the normal plant. 


Figures 24-27. Fourth internode of cocklebur grown at a temperature of 
65°C. Figure 24. Stem of plant grown with complete nutrient supply. Figure 
25. Stem of plant grown without calcium. Figure 26. Stem of plant grown 
without calcium and treated with a-naphthaleneacetamide. Figure 27. Stem 
of plant grown with calcium and treated witha-naphthaleneacetamide. Figures 
28-30. Second internodes of hemp. Figure 28. Stem of control plant. Figure 

29. Stem of plant given an aqueous spray of a-naphthaleneacetamide. Figure 

30. Stem of plant treated with a-naphthaleneacetamide in lanolin. Figures 
31-33. Fourth internodes of hemp. Figure 31. Stem of control plant; no 
evidence of thickening of the fibers. Figure 32. Stem of plant given aqueous 
spray of a-naphthalcneacetamide; fibers have become thick-walled. Figure 33. 
Stem of plant treated with a-naphthaleneacetamide in lanolin. Figures 34-36. 
Sixth internodes of hemp. Figure ^54. Stem of control plant; fibers have started 
to put on thick walls. Figure 35. Stem of plant given aqueous spray of a- 
naphthaleneacetamide; fibers have become thick-walled. Figure 36. Stem of 
plant treated with a-naphthaleneacetamide in lanolin; shows greater number 
and greater thickening of fibers. 


Sections of the stem were also prepared for microincineration. The 
mineral residue was recorded by means of photomicrographs to deter- 
mine the distribution of minerals. The greatest amount of mineral 
residue was present in stems grown with a complete nutrient solution 
and treated with a-naphthaleneacetamide; stems of plants grown with a 
complete nutrient supply had the next greatest amount; stems of plants 
grown without calcium but treated with a-naphthaleneacetamide had a 
mineral distribution closely resembhng the control plants; and stems of 
plants grown without calcium showed the least amount of mineral 
residue. When microchemical tests for calcium were made with 2 per 
cent sulphuric acid, a positive test was obtained for all four treatments. 
The exact role of a-naphthaleneacetamide in delaying calcium deficiency 
symptoms is not known. It may permit greater uptake of calcium from 
the soil or there may be a conversion of calcium from a relatively 
immobile to a mobile form. The available supply of calcium would then 
make possible normal growth of cells. 

Since a-naphthaleneacetamide induces cambial activity, a greater 
thickening of cell walls, and lignification, hemp plants were sprayed with 
an aqueous solution of a-naphthaleneacetamide and others received an 
application of this growth substance in lanolin to the fourth internode 
from the stem tip. The plants were in a vegetative condition when the 
treatments were made. Sixteen days after treatment the plants were 
sampled for anatomical studies. The second internodes of the controls 
and those treated with a-naphthaleneacetamide showed no thickening 
of the fibers (Fig. 28, 29, 30). The fourth internode of the control plants 
showed no thickening of the fibers (Fig. 31); whereas the stems treated 
with a-naphthaleneacetamide showed a thickening of the walls of the 
fibers (Fig. 32, 33). In the sixth internodes the controls and those treated 
with the grov/th substance had thick-walled fibers (Fig. 34, 35, 36). There 
appeared to be little difference in the number of fibers with these three 
treatments, but the wall thickening was greater in stems that were 
treated with the a-naphthaleneacetamide in lanolin. 

It has frequently been shown that chemicals can be used to set fruit. 
It was found that a 0.5 per cent mixture of /5-naphthoxyacetic acid could 
be used to set fruits satisfactorily, but that frequently the paste came in 
contact with ovaries resulting in misshapen fruit. Therefore the spray 
method was used at a concentration of 75 milligrams per hter. Cross 
sections of the nearly mature fruit showed the absence of seed in the 


fruits sprayed with /3-naphthoxyacetic acid. Hollow fruits sometimes 
occurred in the sprayed fruit, but occasionally these were also found in 
the pollinated ones. Young fruits were prepared for a study of the mineral 
residue in sprayed and pollinated fruits. After microincineration the 
ash residue in the sprayed and pollinated fruits were compared. There 
was a Hke mineral pattern in both sprayed and unsprayed fruits which 
agrees with reports made by others that the mineral composition as 
shown by analysis is similar when fruits are set either by pollen or with 

Alpha-naphthaleneacetic acid is used to thin apple blossoms and also 
to delay or prevent fruit-dropping at harvest time. The question was 
raised as to how this growth substance could have seemingly opposite 
effects. An investigation now under way shows that it has but the one 
effect of delaying dropping of fruit. The thinning of blossoms is the 
result of heavy nutritional dropping following heavier early sets induced 
by a-naphthaleneacetic acid. Therefore, the final result is fewer fruits 
when the acid is applied at the blossoming stage. 


1. Eames, a. J., Science, 110:235 (1949). 

2. Fifth Annual North Central Weed Conference Research Report. Project I. 

Use of herbicides in control of perennial herbaceous weeds. December, 
1948. Springfield, Illinois. 

3. Gardner, F. E., Marth, P. C, and Batzer, L. P., Proc. Amer. Sac. Hort. 

Sci., 37:415 (1940). 

4. Mitchell, John W., Botan. Gaz., 101:688 (1940). 

5. Murray, Mary A. and Whiting, A. Geraldine, ibid., 110:404 (1949). 

6. Proc. Fourth Annual Meeting of the North Central Weed Control Conference 

and Report of Research Committees. December, 1947. Topeka, Kansas. 
pp. 271-279. 

7. Roberts, R. H., and Struckmeyer, B. Esther, Proc. Amer. Soc. Hort. 

Sci., 44:417 (1944). 

8. Stebbins, T. C., Neal, A. L., and Gardner, V. R., ibid., 48:63 (1946). 

9. Swanson, Carl P., Botan. Gaz., 107:522 (1946). 

10. Zimmerman, P. W. and Hitchcock, A. E., Ann. Rev. Biochem., 17:601 

Formative Effects of 
Hormone-Like Growth Regulators 


FORMATIVE effects may be defined as changes in pattern from that 
normally resulting from the genetic constitution of the plant under 
the influence of particular environments. When the environment is 
more or less constant for a usual habitat, the size and shape of the plant 
and pattern of organs are said to be normal for a given species. Unusual 
environments involving temperature, moisture, light, or chemical sub- 
stances bring about different expressions of so-called normal character- 
istics. The results may be referred to as formative effects brought about 
by a given combination of influences. At this time we shall be concerned 
especially with chemical influences. 

There are three groups of chemical compounds which cause modifica- 
tions in form of plants. These are /?-naphthoxyahphatic acids (13), substi- 
tuted phenoxyaliphatic acids, and substituted benzoic acids (14). There 
are other less well-known synthetic compounds and even extracts of 
plant tissue which have formative influences. In addition to having a 
formative influence, the naphthoxy and phenoxy groups of compounds 
have many other hormone-like characteristics. For example, they cause 
cell elongation, cell division, curvatures, and induction of roots. The 
derivatives of benzoic acid cause formative effects with little or no cell 
elongation. 2,3,6-Trichlorobenzoic acid is an exception. 

There is need for a single term to cover all substances which have 
formative effects on plants. The word formagen was proposed by Zim- 
merman and Hitchcock (15) and used by King (5). Beal proposed the 
word telomorphic (2), but neither of these words has been widely 
accepted. I shall, however, use the word formagen at present as a matter 
of convenience. 



The formative influence is not apparent on parts of the plant present 
at the time the chemical is applied. Only those parts which grow after 
the plant is treated show formative effects. Old leaves, for example, 
cannot be reshaped (Fig. i). 

Molecular configuration and comparative activity for cell elongation involving 

the tomato plant as test object 



















I ^ 








^Inactive for formative effects. 

Figure I. A. Y-shaped tomato plant. Left branch treated near middle with 
/?-chlorophenoxyacetic acid 20 mg./g. of lanolin. Note modified new leaves 
and flowers of shoot on right. B. Enlargement of "A" showing parthenocarpy 
before flower opened. C. Tomato leaves. Upper row from one plant; lower 
row, modified shoots and leaves taken at random from plants treated with 
chlorophenoxyacetic acid compounds. 

Figure 2. Tomato shoots showing effect of 2,3,5-triiodobenzoic acid (TIB). 
A. Tomato stems to show effect of applying 5 mg. of TIB in 50 ml. of water 
to soil of a four-inch pot on June 5. Photograph was taken July 17. Left, 
control stem with normal flower cluster; middle, axillary flower cluster with 
an abnormally long heavy peduncle; right, two axillary flower clusters, one 
with an abnormally large flower and long peduncles, and the other with a 
short peduncle and unorganized yellowish flower tissue at the tip. B. Three 
tomato shoots. Left, control with a normal flower cluster and axillary leafy 
shoot; middle, one large flower cluster having also two leaves and long peduncle 
with several node-like structures; right, axillary flower cluster developed from 
vegetative tissue. The flower cluster has also a few leaves. The flowers are 
abnormally large and peduncle abnormally long. 



The principal modifications induced by active substances are changes 
in flowering habit, size, shape, pattern, and venation of organs. The blade 
of the leaf is usually reduced in area, and the veins converge toward the 
midrib. Clearing of the modified veins, mottling, and other symptoms 
often cause modifications which may be confused with virus diseases. 
Leaves often fail to separate from each other, forming large modified 
organs or cups. Flower buds become tubular where sepals fail to separate, 
and ovaries frequently develop into seedless fruit. Flowering habit and 
correlation of organs are modified especially by derivatives of benzoic 


Molecular configuration and comparative activity of substituted phenoxyali- 
phatic acids involving the tomato plant as test object 

























Phenoxyacetic acid 




2-Chlorophenoxyacetic acid 
3-Chlorophenoxyacetic acid 
4-ChIorophenoxyacetic acid 
2,4-Dichlorophenoxyacetic acid 
2,4,5-Trichlorophenoxyacetic acid 


0. 125 










2-Chlorophenoxyacetic acid 
Q!-(2-Chlorophenoxy)propionic acid 
Q!-(2-Chlorophenoxy)butyric acid 
4-Chlorophenoxyacetic acid 
a-(4-Chlorophenoxy)propionic acid 
Q:-(4-Chlorophenoxy) butyric acid 


0. I 

0. 125 
0. 125 








2,4-Dichlorophenoxyacetic acid 








a-(2,4-DichIorophenoxy) butyric acid 
2,4,5-Trichlorophenoxyacetic acid 










a- (2 , 4 , 5-Trichlorophenoxy) bu ty ric 


0. I 





acid (Fig. 2). All of the substances having formative influences also 
cause ovaries of tomato to develop without pollination (parthenocarpy). 
They vary in effectiveness and practical value. For example, i mg./l. 
of 2,4-dichlorophenoxyacetic acid (2,4-D) is as effective as 500 mg./l. of 
some of the derivatives of benzoic acid. Though not as effective as 2,4-D, 
a- (2-chlorophenoxy) propionic acid is recommended for practice because 
it does not modify leaves. 

Physiological activity of a specific type and for a specific species is 
associated with molecular configuration as a whole rather than any one 
part of the molecule. A few examples may be given: 2,4-D is one of the 
most active compounds known for many hormone-like responses, but 
2,6-D is practically inactive; 2,4-D and 4-chlorophenoxyacetic acid both 
have power to elongate cells and modify the leaves and other organs of 
tomato {Lycopersicon esculentum Mill.) plants. The latter compound 
causes a striking modification of Turkish tobacco {Nicotiana tabacum L.) 
leaves and Kalanchoe daigremontiana Hamet et Perrier plants while 2,4-D 
does not. 2-Chlorophenoxyacetic acid and 4-chlorophenoxyacetic acid 
cause cell elongation and modification of tomato leaves while the 
propionic and butyric homologs cause cell elongation but not modifica- 
tion of leaves. 2,4,5-Trichlorophenoxyacetic acid and higher homologs 
cause cell elongation but not modification of leaves; 2,4,6-trichloro- 
phenoxyacetic acid does not induce cell elongation but modifies leaves. 
More illustrations of this sort are shown in the accompanying tables. 

Physiological activity can be determined at the present time only by 
biological tests. Both molecular configuration and the genetic constitu- 
tion of the species are involved. Tomato and tobacco are closely-related 
species, but 2,4-D modifies only tomato, whereas 4-chlorophenoxyacetic 
acid modifies both. The mechanism of modification in the plant is com- 
plex, and we can only theorize on what combination of factors makes for 
activity or inactivity. 

The recent work of Burton (3) is welcomed as one of the first attempts 
to determine what happens to the structure of tissue to bring about these 
odd forms. Using the bean leaflet as a test object Burton worked out the 
normal structural developments and compared these with chemically 
induced modifications. It appears that the normal bean leaflet develops a 
lamina by the activity of a subepidermal marginal meristem, which 
produced four internal layers of plate meristem. The adaxial (upper) 
of these layers develops into the palisade layer and the other three 



produce the spongy mesophyll. The veins are initiated by divisions of 
rows of cells in the layer beneath the embryonic palisade. Many inter- 
cellular spaces (air) normally appear in the spongy tissue. 

Using three substituted phenoxy acids Burton found that these were 
more or less specific for given structural variations from normal. For 
example, 2-chlorophenoxyacetic acid inhibited the formation of inter- 
cellular spaces in the spongy tissue; 4-chlorophenoxyacetic acid inhibited 
the activity of the plate meristem (between the veins) and the veins 
became approximate with continuous parenchyma. 2,4-D brought about 


Molecular configuration and comparative activity of substituted phenoxyali- 

phatic acids involving the tomato plant as test object 

Xylenoxy compounds 

Cell Formative 

elongation effects 

3,5-Dimethylphenoxyacetic acid 
a-(3,5-Dimethylphenoxy)propionic acid 
Q:-(3,5-Dimethylphenoxy) butyric acid 
2,5-Dimethylphenoxyacetic acid 
a:-(2,5-Dimethylphenoxy)propionic acid 
Q:-(2,5-Dimethylphenoxy)butyric acid 
3,4-Dimethylphenoxyacetic acid 
a!-(3,4-Dimethylphenoxy)propionic acid 
Q:-(3,4-Dimethylphenoxy)butyric acid 
2,4-Dimethylphenoxyacetic acid 
a:-(2,4-Dimethylphenoxy)propionic acid 
Q:-(2,4-Dimethylphenoxy) butyric acid 

























a progressive modification of all leaves developed after the chemical was 
applied. The latter compound also caused various structural modifica- 
tions similar to those of both the other acids. Chlorenchymous tissue 
was usually confined to the margin, and cells without chlorophyll over 
vascular tissue gave the veins a transparent effect. Burton worked on 
only one species, the bean. It will be interesting to see how structures of 
other species respond to the same chemicals. 

Modification of flowering habit and correlation of organs induced with 
substituted benzoic acids are illustrations of special chemical influence. 
In 1942 Zimmerman and Hitchcock (14) reported that the pattern of 
leaves, the flowering habit, and correlation of organs were modified by 
means of 2,3,5-triiodobenzoic acid. They showed that flower clusters 



of tomatoes were induced to grow from axillary buds where leafy shoots 
normally appear and that terminal buds were replaced with flower 
clusters. The usual flower clusters also developed along the stem. The 
treated plants lost much of their apical dominance and produced an 
abnormally large number of axillary leafy branches. It was further 
pointed out that though benzoic acid was physiologically inactive, the 
molecule could be activated by the substitution of amino groups or 
halogen atoms in the ring (7,10,11,12), The positions 2, 3, and 5 of the 
nucleus appeared to be the most important for substitutions. None of 
the mono-substituted compounds tested was found active. 2,5-Diiodo- 
benzoic acid was active for modification and pattern of leaves but 
did not induce axillary flowers. 2,3,5-Triiodobenzoic acid induced forma- 
tive effects and also modified the position of flower clusters. 2-Amino- 
3,5-diiodobenzoic acid was inactive, but 2-chloro-3,5-diiodobenzoic acid 
was very active resembling 2,3,5-triiodobenzoic acid (11). 2,3,6-Tri- 
chlorobenzoic acid causes cell elongation, initiates roots, and modifies 
leaves. The molecular configuration as a whole rather than any part of 
the molecule appeared to determine physiological activity. 

Galston (4) attempted to initiate flowering of soybeans by the use of 
2,3,5-triiodobenzoic acid. Though he succeeded in increasing the number 
of flowers per plant from 32 on the control to 181 on the treated plant, 
Galston states that 2,3,5-triiodobenzoic acid does not possess florigenetic 
properties since it will not induce vegetative soybean plants to flower. 
He further stated that 2,3,5-triiodobenzoic acid caused morphological 
responses such as shortening of the internodes, loss of apical dominance, 
epinasty of young leaves, and so on, but that the chemical itself was 
without auxin activity. The latter conclusion perhaps was drawn from 
the fact that 2,3,5-triiodobenzoic acid is not active on the Avena 

Tumanov and Lizandr (8) found that 2,3,5-triiodobenzoic acid re- 
tarded growth of Perilla and caused formative effects. Alfalfa was more 
sensitive than Perilla. These authors found a difference in sensitivity dur- 
ing short and long days. The treatment caused a variation in the number 
and size of leaflets in alfalfa. Spraying with weaker solutions, 0.005 P^^ 
cent, in long days brought about increased yield of alfalfa seed. Flax and 
sunflower species showed notable changes in growth when treated with 
0.0 1 per cent solution. This concentration also caused peas to branch 
through stimulation of axial buds and fusing of leaflets. There was, how- 

p. W. ZIMMERMAN l8l 

ever, no definite sign that 2,3,5-triiodobenzoic acid could be considered 
as having florigenetic properties. 

Owen (6) treated a number of species with 2,3,5-triiodobenzoic acid 
and brought about distortions of leaves, stems, and flowers but failed 
to change any organ from the vegetative to the flowering stage. Avery 
and Johnson (i) say that the induction of flowers with 2,3,5-triiodo- 
benzoic acid in place of vegetative tissue in tomato strongly suggests 
that the substance plays the role of flower-inducing hormones in some 

The best support for the earlier findings of Zimmerman and Hitch- 
cock was published by Waard and Roodenburg (9). These workers found 
that the treatment with 2,3,5-triiodobenzoic acid caused the flower 
cluster to develop at the top of the plant while the vegetative shoot was 
suppressed. This was considered as a shifting of the correlative relations 
between the vegetative shoot and flower cluster. They also reported an 
increase in the number of initiated flower buds and the formation of 
axillary flower buds when the plants had very few leaves. In fact the 
illustration shows the flower buds arising from plants with only cotyledon 
leaves. They concluded that the chemical has the property of starting 
the process of flower formation and also that it is possible to shorten the 
vegetative juvenile stage of tomatoes. 

In contrast with the case in the substituted phenoxy acids, the para 
position is not an important location in the molecular configuration for 
active derivatives of benzoic acid. Not all possible substitutions have 
been made, but from the information at hand the halogen substitutions 
in the 2, 3, 5, and 6 positions make the molecule more active than the 
substitution of amino or nitro groups. For example, 2-amino-3,5-di- 
chlorobenzoic acid is inactive while 2-chloro-3,5-diiodobenzoic acid is 
very active for modification of leaves and induction of axillary flower 
clusters. 2,5-Dichlorobenzoic acid is especially active for inducing par- 
thenocarpic fruit and modified leaves but does not induce axillary or 
terminal flower clusters. 

Since the effects of 2,3,5-triiodobenzoic acid vary in summer and 
winter with the rate of growth of tomato plants, it is evident that the 
substance cannot work alone. It must depend upon materials made by 
the plant in order to cause vegetative tissue to produce flowers. It is 
assumed that plant hormones in general require supporting substances 
produced by the plant to cause well-known physiological responses. 



For example, the rooting response after treatment with indolebutyric 
acid is conditioned by the age of the experimental species, the environ- 
mental growing conditions, and the storage of material in the treated 

Repeated experiments with tomato plants verify the earlier reports 
(15) that 2,3,5-triiodobenzoic acid and 2-chloro-3,5-diiodobenzoic acid 
cause axillary buds to develop flower clusters instead of leafy shoots. 
Branches and main tomato shoots were caused to terminate in flower 
clusters appearing to supplant a shoot growing point. Treated tomato 


Molecular configuration and comparative activity of derivatives of benzoic 

acid involving the tomato plant as test object 





Benzoic acid 
2Todobenzoic acid 
3-Iodobenzoic acid 
4-Iodobenzoic acid 
2,4-Diiodobenzoic acid 
2,3,5-Triiodobenzoic acid 
3,5-Diiodobenzoic acid 
2-Iodo-3,5-dibromobenzoic acid 
2-Chloro-5-nitrobenzoic acid 
2-Amino-5-chlorobenzoic acid 
2-Bromo-3-nitrobenzoic acid 
2-Chloro-3,5-diiodobenzoic acid 
2,3,6-Trichlorobenzoic acid 


























plants also developed flower clusters in the usual places along the stem, 
but they frequently had fewer or more than the normal number of 
flower buds. In fact, internodal, axillary, and terminal flower clusters 
were similar and were characterized by extremes from short to long, 
heavy peduncles, and fasciated flowers mixed with small buds. Upon 
recovering from the chemical influence plants often produced abnormally 
large flowers (Fig. 3D). Some of the fasciated flowers when pollinated 
produced a circular ovary resembling a doughnut (Fig. 3C). Such ovaries 
had sepals both within and around the ovary. Under certain conditions 
the axillary peduncles had only yellowish cells at the tip, indicating 
unorganized flower cells. 

Figure 3. Tomato plants. A. Uppermost portion of a plant showing axillary 
flower clusters developed from vegetative tissue and terminal flower cluster 
supplanting the vegetative growing point. The plant was treated on December 
22 with 4 mg. of TIB in 50 ml. of water applied to the soil. Photograph was 
taken the following February 16. B. Uppermost portion of a tomato plant 
treated with a lanolin preparation of TIB 10 mg./g. of lanolin on December 22. 
Photograph was taken February 16. The picture shows axillary flower cluster 
developed from vegetative tissue. The cluster has one large circular flower and 
an abnormally large number ot associated smaller buds. C. Tomato cluster. 
Tubular peduncle with circular ovary induced with TIB 10 mg./50 cc. of 
water applied to soil January 11. Photograph was taken on March 8. Note 
sepals within the circular ovary which developed after pollination. D. Terminal 
and internodal flower cluster induced with 10 mg. TIB/50 cc. of water applied 
to pot January 11. Photograph was taken March 8. Internodal peduncle is 
branched and has one abnormally large flower. 

p. W. ZIMMERMAN 183 

Some but not all of the substituted phenoxy acids have formative 
effects when applied to Kalanchoe plants. Two striking differences ap- 
peared when 4-chlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic 
acid are compared. The former causes a change in correlation of organs, 
hyponasty of leaves, modification of leaf pattern, and pronounced mon- 
strosities at the terminal part of the plant. In contrast, 2,4-D causes 
epinasty of leaves and root formation of treated stem tissue but no 
monstrosities. Similar differences appeared when these two chemicals 
were tested on Turkish tobacco plants. When applied to tomato plants, 
however, the effects are nearly indistinguishable (14). Such cases as these 
make it appear that the molecular configuration of the substance must 
in some way interlock with the mechanism of the species to bring about 
a given response. The genetic constitution of the species — perhaps the 
gene — appears to be the determining factor. This is mentioned because 
tobacco and tomato are closely related species, and yet they respond 
differently when tested with these substances. 


1. Avery, G. S., Jr. and Johnson, E. B., Hormones and Horticulture (Mc- 

Graw-Hill, 1947). 

2. Beal, J. M., Botan. Gaz., 105:471 (1944). 

3. Burton, D. F., ibid., 109:183 (1947). 

4. Galston, a. W., Am. J. Botany, 34:356 (1947). 

5. King, G. S., Proc. Louisiana Acad. Sci., 10:35 (i947)- 

6. Owen, O., Nursery & Market Gard. Indus. Develop. Soc. Ltd. Exp. & Res. 

Sta. Ann. Kept., 32:65 (1946). 

7. Synerholm, M. E. and Zimmerman, P. W., Contrib. Boyce Thompson 

Inst., 14:39 (1945). 

8. TuMANOv, I. I. and Lizandr, A. A.,/. Bot. U.R.S.S., 3i(3):20 (1946). 

9. Waard, J. DE and Roodenburg, J. W. M., Proc. Koninkl. Nederland. 

A\ad. Wetenschap., 51:248 (1948). 

10. Zimmerman, P. W., Cold Spring Harbor Symposia Quant. Biol., 10:152 


11. , Torreya, 43:98 (1943). 

12. , Ind. Eng. Chem., 35:596 (1943); Boyce Thompson Inst. Plant 

Research, Professional Papers, 1:307 (1943). 

13. , and Hitchcock, A. E., Contrib. Boyce Thompson Inst.. 12:1 (1941). 

14. , ibid., 12:321 (1942). 

15. , ibid., 12:491 (1942). 

Practical Applications 
of Growth Regulators 

Vegetation Control on Nonagricultural Land 


UNWANTED plants are not exclusively a problem of the farmer; almost 
everyone has a vegetation control problem. Weeds in lawns, gar- 
dens, and vacant lots, undesirable woody and herbaceous plant growth 
on highway, utility, and railroad rights-of-way are a part of our daily 
existence. Industrial grounds, canals, ditches, lumber yards, oil-tank 
farms, and military installations all have their particular problems of 
vegetation control. For factors of safety, health, fire protection, and 
general efficiency of many operations, as well as for aesthetic values, 
vegetation must be confined to desired species or kept from growing 
beyond certain heights. 

In the past we have had to resort almost exclusively to mechanical 
methods of controlling plant growth. True, certain chemical weed-killers 
have been used to a limited extent for a number of years, but the 
discovery of the herbicidal value of the chlorophenoxyacetic acids has 
given us an entirely new concept of the possibihties of controlling 
vegetation by chemical means. I wish to discuss certain vegetation 
control problems, to point out how research on herbicides has helped 
in their solution, and to mention some remaining problems which you 
as research workers interested in plant-growth control must take the 
lead in solving. 

It has been my privilege to work with several public utilities in the 
eastern states on right-of-way vegetation control since 1945, soon after 
the first publications on 2,4-dichlorophenoxyacetic acid (2,4-D). Our 
first problem was one of formulation. It was soon found that the esters 
of 2,4-D were more consistent in their action than the salts, particularly 
on species with a relatively thick cuticle. Apparently the solubility of the 
esters in leaf wax is an important factor. In spite of the many reports of 


desirable results with certain additives to the salts of 2,4-D the esters 
are largely used today in right-of-way vegetation control because day 
in and day out they are the most effective formulations we have for the 
general run of species, regardless of rainfall subsequent to application. 
It must be remembered that in right-of-way control many crews must 
operate continuously over a period of several weeks and a formulation 
or a chemical must be versatile in its action. 

Brush regrowth from woody plants that have periodically been cut 
off, sometimes for many years, constitutes the chief problem on a utility 
right-of-way in areas of moderate to heavy rainfall. Experience has 
shown that spraying may be successfully carried out from about the time 
the leaves are fully developed until late summer. This is not in conflict 
with the general concept that plants should be in an active state of 
growth for best results with 2,4-D. Except under very dry conditions 
such woody plants are growing actively throughout the summer months. 
Experience has shown that spraying before the leaves have fully de- 
veloped will often result In Inferior kills. This appears to agree with the 
concept that 2,4-D Is most actively translocated from leaves to stems 
in association with carbohydrate movement. 

It soon became apparent in the early work with 2,4-D on woody 
plants that many species were highly resistant. In the summer of 1946 
an old mountaineer in West Virginia told me that the best blackberries 
he picked that summer were on the right-of-way that had been sprayed 
the year before. We had eliminated most of the competing woody species. 
Because blackberries and other members of the genus Rubus are so 
widespread the entire program for a time appeared discouraging. It takes 
no ecologlst to see that eliminating competing plants would enable a 
species hke blackberry to take over an area. 

Screening of a number of compounds related to 2,4-D on blackberry 
and other resistant species was carried out and 2,4,5-trichlorophenoxy- 
acetic acid (2,4, 5-T) was found to be specific for some of them, particu- 
larly members of the genus Rubus. During the past two years much of 
the woody plant control on rights-of-way has been carried out with a 
mixture of 2,4-D and 2, 4, 5-T. Admittedly this Is a shotgun treatment 
and at times one or the other component might prove best, but until 
we know more about how species respond and until the spray operators 
know more exactly the species with which they will have to contend the 
mixture appears to be desirable. Not all undesirable woody species are 

K. C. BARRONS 189 

readily killed by this combination of chemicals, but they do provide a 
practical measure of control. Perhaps further work will uncover com- 
pounds specific for those plants that are somewhat resistant to 2,4-D 
and 2,4,5-T. 

Although some use has been made of low-volume, high-concentrate 
sprays of the esters of the chlorophenoxyacetic acids in oil, most of the 
right-of-way work is conducted with more dilute sprays applied by power 
rigs mounted on trucks. On the average better results have been obtained 
with larger volumes applied under sufficient pressure to wet stems and 
foliage on the inside of dense growth. Furthermore, a little drift from 
a high concentrate spray can cause considerable damage to desirable 
plants adjacent to rights-of-way areas. A little drift from a more dilute 
spray carries less toxicant and is less likely to cause difficulty. Low- 
volume spraying of right-of-way vegetation does have a place where 
limited access by truck and limited water supply makes the use of 
high volumes applied by power rig impractical. 

Spray operators have learned that they must pay attention to wind 
velocity and frequently skip certain stretches of the right-of-way ad- 
jacent to sensitive crops. Most of the instances of damage to desirable 
plants adjacent to rights-of-way can be traced to carelessness. 2,4-D 
and 2,4,5-T are not materials to be sprayed indiscriminately and everyone 
using them must recognize the hazards before he begins. Areas adjacent 
to especially sensitive crops must often be omitted from the spraying 
program for that season even though the wind is blowing in the opposite 
direction at the time of application. There is always the possibihty of 
dust on the leaves of the foliage at the time of application blowing onto 
the sensitive crop. The fact that some esters of 2,4-D are volatile has 
frequently been pointed out as a possible cause of the reaction of crops 
adjacent to sprayed areas. An analysis of the problem of damage to 
plants off the right-of-way indicates that spray drift is the chief cause 
of trouble and the blowing of dust a likely cause in some cases. Although 
volatility may be a factor the writer feels that it has been overempha- 
sized in relation to the other factors. 

Although the chief aim of utihties to date has been to control, and 
insofar as possible to erradicate, woody plants on the right-of-way much 
nongrass herbaceous vegetation has been killed by the spraying program. 
From the ecological standpoint it is interesting to note that many 
rights-of-way which were formerly infested with brushy growth now 


are covered with a dense grass sod after two years of spraying. Apparently 
grass and grass seed were present in sufficient amounts to permit it to 
become established once the competing woody plants and tall-growing 
herbaceous species were eliminated. Where species predominate which 
are stunted but not killed by the available herbicides a mixed grass 
and shrub community can apparently be maintained without the woody 
plants growing excessively large. 

During the present season there were many thousands of miles of 
utility rights-of-way sprayed with the chlorophenoxyacetic acids. This 
is definitely not a one-season program. One treatment will not result in 
the eradication of many plants. The general practice at present appears 
to be to spray woody regrowth up to ten feet tall during the summer 
and then make a second application the next year. Cost of each of these 
sprays in many cases is no more than the cost of a single cutting with 
scythes or brush hooks. After two consecutive years of spraying a third 
year may be skipped and in some cases the fourth year. It appears that 
a continuous maintenance program will require respraying at intervals 
of about three years, depending, of course, on rainfall and species 
composition. The utilities that have been in this program the longest 
now conservatively estimate that they are cutting the cost of maintaining 
their rights-of-way in half. 

An interesting recent development in vegetation control is the appli- 
cation of 2,4-D and 2,4,5-T to the bark of woody plants and to cut 
surfaces of stumps (2). Absorption varies with the species and no doubt 
with many other conditions; however, good kills have been obtained 
from application during all seasons of the year. Dormant season applica- 
tion offers the possibility of extending operations to make more con- 
tinuous use of labor and also may make possible the use of these herbicides 
on rights-of-way in areas where highly sensitive crops preclude summer 
foliage spray application. 

Highway departments have much the same problem with respect to 
woody vegetation, and their methods of operation are similar. They must 
use even greater care, however, with respect to plants on adjoining 
properties because so often highways transverse populated areas. Much 
of the utility right-of-way spraying is in mountainous country where 
there are few crops near by. In addition to spray-gun operations for 
woody plants some highway departments are using spray booms to 
advantage, treating the entire area from the edge of the road to the 

K. C. BARRONS 19^ 

fence line. Where tall-growing or otherwise obnoxious weeds are present 
2,4-D and sometimes 2,4,5-T can be used to good advantage for their 

Railroads like the highways have a brush problem, which heretofore 
has been handled almost entirely by mechanical cutting. Like the high- 
way departments they must give a great deal of consideration to sensitive 
crops adjoining their right-of-way, but some railroads, especially those 
running through the eastern mountains, are applying sprays of 2,4-D 
and 2,4,5-T from on-track equipment with successful results. Roadbed 
treatment is primarily with herbicides of the soil-sterilant or contact 
type. 2,4-D has been used for roadbeds in only a few cases where nongrass 
species predominated. 

The control of water hyacinth in Florida and Louisiana with 2,4-D 
is a notable example of the success of this compound in solving an old 
problem. Tremendous sums have been spent in mechanical chopping 
of water hyacinth in canals. Now airplane application of relatively 
small amounts per given area are being successfully used. Visitors in 
south Florida during the past two years have seen visual evidence of 
the success of this clean-up campaign. Lotus is another emergent aquatic 
that has been successfully controlled with 2,4-D particularly by the 
Tennessee Valley Authority as a part of its mosquito-control activities. 

The vegetation-control problems of various industries are so varied 
that they cannot be discussed individually. Undesirable woody growth 
and tall-growing herbaceous plants can frequently be brought under 
control or eradicated on industrial grounds by the proper use of the 
chlorophenoxyacetic acids. This is also true of the tremendous areas 
given over to our military establishments. 

The use of 2,4-D for weed control in lawns and recreational turf 
areas is almost too well known to warrant comment. It is of interest that 
white clover and other legumes that are sometimes undesirable com- 
ponents of turf may be more readily killed by 2,4,5-T than by 2,4-D. 
In this connection the writer has frequently observed a greater response 
from semiresistant plants, such as white clover, during hot weather than 
during the cool seasons of the year. 

Poison ivy, one of our most undesirable plants in recreational areas 
is also more susceptible to 2,4,5-T than to 2,4-D. The difference between 
these two chemicals is often the difference between a high percentage 
of eradication and a temporary reduction in top growth. 


New chemicals other than the chlorophenoxyacetic acids are becoming 
available for vegetation control purposes. Possibly one of the most 
interesting of these is sodium trichloroacetate (sodium TCA) which has 
a herbicidal effect on many grasses (i). It also appears to be promising 
for killing cacti and palmetto. 

Although relatively large amounts of this chemical are required for a 
high degree of grass-kill smaller amounts have a practical growth-con- 
trolling effect. For example, on northern species such as quack and blue 
grass twelve to fifteen pounds per acre of sodium TCA applied when the 
infloresence is first emerging has eliminated flowering and retarded 
growth for several weeks. When applied as a spray with 2,4-D many 
nongrasses can be killed and the growth of grass controlled in one 
operation. This technique appears valuable wherever mechanical mowing 
cannot be carried out, such as along ditches and around highway guard 
rails. Combined with the phenolic contact sprays sodium TCA has 
shown much promise for general weed control on railroad beds. 

The action of this chemical, which I am taking the liberty of calling 
a growth-regulating substance, is little understood. The immediate 
foliage-burning effect appears to be independent of the systemic effect 
which with quack grass occurs only after root absorption takes place. 
In my own work far more grass killing has resulted when the chemical 
was applied to the surface of newly plowed ground than when grass 
foHage was sprayed. Obviously soil moisture relations are important 
with a chemical that is largely absorbed through roots. At times con- 
siderable dormancy of grass buds occurs following treatment without a 
lethal effect. It is hoped that more physiologists will study the action of 
this new herbicide. It seems hkely that many of the variations in field 
results are related to the physiologic condition of the grass as well as to 
soil moisture and rainfall. 

No doubt we are on the threshold of many further developments in 
chemical vegetation control. The recent report on maleic hydrazide 
(3) indicates that growth control without lethal action has definite 
possibilities for many kinds of plants. Such chemicals could be of great 
value to highway departments who must maintain turf at a reasonable 
height and also to public utilities who have a tremendous tree-trimming 
problem. Needless to say such chemicals would be of interest to all of 
us who have a weekly session with the lawn mower. 

Many problems in vegetation control remain unsolved. We need 

K. C. BARRONS ^93 

growth-regulating substances more efficient than 2,4-D or 2,4,5-T for 
the control of many species such as ash, smilax, and certain oaks and 
maples. Although existing chemicals have some effect on cattails, bracken 
fern, and horsetail we need something more efficient for their control. 
Translocation of 2,4-D and 2,4,5-T to the root systems of some weeds 
is inefficient, as is the case with milkweed and leafy spurge. Better 
herbicides or improved knowledge of how to use our present ones is 
needed for such plants. We need chemicals with specific growth-con- 
trolling action but without lethal effect for use on trees and shrubs 
as well as on grasses and certain herbaceous plants. There is need for a 
chemical for the control of all plant growth on railroad beds and other 
areas where no vegetation is wanted. Possibly a nonselective herbicide 
of the growth-regulating type is not beyond the realm of possibility. 


1. Barrons, K. C, Down to Earth, 4(4) :8 (1949). 

2. Barrons, K. C. and Coulter, L. L., ibid., 4(2) :4 (1948). 

3. ScHOENE, D. L. and Hoffman, Otto L., Science, 109:588-90 (1949). 

Differential Responses in Crop Plants 


ONE of the most important characteristics of 2,4-dichlorophenoxy- 
acetic acid is its selective action. Although the reasons for this 
selectivity are not fully understood, it has been the basis for a wide 
practical use. Selective action becomes a distinct advantage because it 
permits the spraying of certain weeds growing in tolerant crops. Studies 
of the practical application of selective herbicides are necessarily con- 
cerned with the reaction of the crop as well as the weeds. 

Research in this phase of investigation soon showed that not only were 
there differential responses of certain species classed as weeds and others 
grown as crops but that there were different reactions among crop plants 
and, even further, that varieties and strains of the same crop might 
show varying tolerance or resistance to applications of herbicides. Later 
it was learned that the same variety or strain may be influenced in 
reaction by environmental conditions. It is the purpose of this paper 
to discuss such differential responses among field-crop plants, particularly 
the small grains, flax, and corn. The different phases of the problem will 
be discussed separately by reviewing the pertinent literature and pre- 
senting short summaries of new information from unpublished experi- 
ments of the writer and co-workers. 

Selective Action of 2,4-D 

Species and varietal differences. — Norman (39) points out that selectivity 
is only an apparent one based on different degrees of susceptibility to 
a particular dosage appUed in a particular way, and Crafts (10) states 
that even tolerant plants succumb provided the concentration is high 

Editor's Note: Paper No. 2503 of the Scientific Journal Series, Department of 
Agriculture, University of Minnesota, St. Paul, Minnesota. 


enough. This conception is unquestionably true. Nevertheless, the dif- 
ferences in response of plants within certain limits of dosage, time of 
application, and environment are large enough to make possible the 
killing or severe injury of certain undesirable species in the presence of 
crop plants. Generally the monocotyledons have been more resistant 
to 2,4-D than the dicotyledons, but there are sevefal notable exceptions 
in each group. Among the monocotyledons certain strains of the bent 
grasses and buffalo grass have proved susceptible. Among the leguminous 
crops Buchholtz (4) found red clover more tolerant than alfalfa, and 
Willard and Shaw (48) found Ladino white clover and common white 
clover more tolerant than alfalfa, red clover, sweet clover, alsike clover, 
and lespedeza although all species could be classed as relatively susceptible. 
Because of their susceptibility to 2,4-D, the spraying of legumes with 
this herbicide has commonly been considered too hazardous, but thou- 
sands of acres of flax, also a dicotyledon, have been sprayed successfully. 

Marked varietal differences in response to 2,4-D both in mono- 
cotyledons and dicotyledons have been observed by many investigators. 
Dunham and Tandon (16) found large varietal differences in tolerance of 
flax to 2,4-D. An application of four times the amount that reduced the 
yield of Crystal and B5128 did not injure Redwing. Similar or even 
larger differences exist in corn. Buckley (7), Holden et al. (26), Jugen- 
heimer et al. (27), Lee (31), Leng and Slife (33), Miller (37), Rossman 
and Staniforth (41), and Viehmeyer (46) found wide differences in re- 
sponse to 2,4-D among inbreds, among single crosses, and among double 
crosses. Elder and Davies (18) reported varietal differences in sorghum. 
Derscheid et al. (12) found no significant reduction in yield among nine 
oat varieties but did find differences among barley varieties. Seven 
varieties of spring wheat responded in a similar manner to 2,4-D accord- 
ing to Helgeson et al. (25). Sexsmith (42) noted no differences among 
six varieties. Albrecht (2) concluded that bent grass strains vary con- 
siderably in their tolerance to injury from 2,4-D. 

Some environmental effects. — Differences in reaction to 2,4-D may be 
very marked among varieties and strains but they are influenced by the 
dosage, the time of application, and environmental factors. Dunham 
and Robinson (15) sprayed ten varieties of flax with 4 oz. of triethanola- 
mine and 1.3 oz. of butyl ester per acre and reported that all varieties 
responded aUke at these low dosages. Dunham (13) states that varieties 
of flax differ most widely in their response to 2,4-D: (a) when the ester 

R. S. DUNHAM 197 

is used, (b) when rates of amine or sodium salts are heavier than recom- 
mended, and (c) when apphcations are made in bud and bloom stage. 
There is evidence also that both temperature and sunlight are influencing 
factors (28,34,47). Arakeri and Dunham (3) studied the relation of some 
environmental factors to the pre-emergence treatment of corn with 
2,4-D and concluded that out of all the factors studied, the most 
important were water, soil type, pH, and organic matter content of the 
soil; less important was time of application, and least important were 
depth of planting and dosages. 

Stage of growth and selectivity. — The stage of growth of a plant at the 
time it is sprayed has been recognized as an important factor by many 
investigators. Commonly it has been reported in terms of height. More 
recently an attempt has been made by Dunham et al. (14) to describe 
the stage of small grains and flax in terms of morphological development. 
The "tiller," "shooting," and "boot" stages of wheat and the cotyledon, 
true leaf, and stem elongation stages of flax are illustrated. 

Derscheid (11), in summarizing the abstracts of 27 investigators who 
worked with 2,4-D on spring wheat, oats and barley, states: "These 
data indicate that all three crops are less tolerant at early 3-leaf and 
5-leaf stages of growth than after they have become fully tillered. 
The most susceptible period, however, is when heads are emerging from 
the boot." Elder (17) reviewed the data on winter wheat and states that 
it "is more resistant to 2,4-D in early spring when fully tillered or in 
early joint stage and more susceptible when treated soon after planting 
in the fall months. The boot to heading stage is a susceptible period." 
Dunham et al. (14) state that information relative to flax is not so 
clearly established as for the small grains, but apparently the most 
susceptible time is from bud formation to bloom. They also advise the 
farmer to avoid spraying when the stem is rapidly lengthening. 

There is increasing evidence that susceptibility is closely related to 
periods of rapid growth in the plant. Conflicting results with corn when 
described in terms of the height of the plant can be explained on the 
basis of rapidity of growth. Lee (30) reports that: "Experiments con- 
ducted in both 1947 and 1948 indicate that small corn is more susceptible 
to damage than larger corn. This year (1948) in Indiana large corn, 
12 inches or more in height, was more easily damaged than small 
corn. The reason was apparently because of the diff"erence in growing 


Paatela (40) made intensive studies of the relation of increase in height 
of flax to its susceptibility to methoxone and 2,4-D, In experiment i, 
flax plots sown on the same day were sprayed over a period of six weeks 
as they reached seven stages of development. Thus treatment I was 
made when the flax was in the cotyledon stage; II, when 4.6 cm. tall; 
III, 7.8 cm. tall; IV, 12.5 cm. tall; V, 17.1 cm. tall; VI, in bud; and VII, 
in flower. 

In experiment 2, stages I, II, V, and VI were obtained by sowing the 
flax at successive dates. All plots were sprayed on the same day. Since 
the date of spraying was July i, growth conditions were favorable for 
more rapid growth of flax in all stages as compared to experiment i. 
This fact was determined by height measurements of unsprayed flax 
for a period of six days following the spraying of the remaining plots. 
Injury was markedly greater in experiment 2 than in experiment i, with 
one exception, even though the flax was in the same stage of development 
in each case. The one exception was the flax sprayed in the cotyledon 
stage. Data illustrating this result were selected in cooperation with 
Paatela and are presented in Table i. 

In a more recent study at Minnesota, Paatela and Dunham grew three 
varieties of flax under conditions conducive to slow growth in one 
instance and rapid growth in the other. The following treatments were 
used: i) Flax was grown 3-4 inches tall with 12 true leaves at approxi- 
mately 50° F. and sprayed with (a) methoxone and (b) amine salt of 
2,4-D. One half the population remained in the cool room; the other half 
was removed to the greenhouse at time of spraying. 2) Flax was grown 
to the same stage as in i at high (approximately 85° F.) temperatures 
in the greenhouse. One half the population remained in the greenhouse; 
and the other half of the population was removed to the cool room at 
time of spraying. Response was measured by the bending and twisting 
of the stems, a characteristic reaction of flax to the growth-regulating 
herbicides. Both the number of afl"ected plants and the degree of epinasty 
were recorded. The number of plants multiphed by the degree of bending 
as indicated by a scale of o to 3 was divided by the total number of 
plants. The resulting decimals are the data reported in Table 2. Dif- 
ferences appear small when expressed in decimals until comparisons are 
made. Thus 6 oz. of methoxone on Dakota started in the warm room 
and moved to the cool room caused 8 times as much bending as when 
the plant was grown continuously in the cool room (0.8 and o.i). It is 



apparent that all three varieties showed the greatest response when 
started in the warm room and transferred to the cool chamber and the 
least, even at high dosages, when started in the cool chamber whether 
they remained there or not. Varietal differences are also apparent in this 


Comparison of increase in height of untreated flax and yield of seed from flax 
sown the same day and sprayed at four stages of growth (Experiment i) versus 
untreated flax and flax sown on four dates and sprayed on the same day 

(Experiment 2). 

Increase in 



Yield of 




= 100) 

AFTER treated 

Stage of plants at 

PLANTS were 

Sodium salt 




OF 2,4-D 


Cotyledon (Exp. i) 
( " 2) 


I 10 


4.6 cm. (Exp. i) 
5-3 " ( " 2) 






17. 1 cm. (Exp. i) 

19.2 " ( " 2) 





Bud (Exp. i) 
" ( " 2) 





*Morpholine salt of 2,4-D. 

An experiment now in progress at Minnesota by Shulstad, Dunham, 
and Heggeness indicates that the rate of increasing height differs among 
flax varieties; that this rate differs for the same variety when sown at 
ten-day intervals; and that the order of varieties ranked according to 
rate of increase in height varies with different planting dates. The 
experiment has not been completed, but there is also evidence that 
varieties rated on the basis of tolerance to 2,4-D under a given set of 
growing conditions do not necessarily maintain that order under a 
different environment. 

It appears from all the evidence available that rapidity of growth is an 
important factor in determining the susceptibility or tolerance of flax 




Response of Minerva, Dakota, and Redwing flax to applications of methoxone 
and 2,4-D made under variable growing conditions. 






Started in cool room 

Remained in cool room 

6 oz. methoxone 

0. 1 

0. 1 


6 oz. 2,4-D 

0. 1 



12 oz. methoxone 

0. 1 


12 oz. 2,4-D 


0. 1 

18 oz. methoxone 

0. 1 


18 oz. 2,4-D 

0. 1 

0. 1 

Started in cool room 

Removed to warm room 

6 oz. methoxone 

0. 1 



6 oz. 2,4-D 




12 oz. methoxone 



12 oz. 2,4-D 



18 oz. methoxone 



18 oz. 2,4-D 



Started in warm room 

Moved to cool room 

6 oz. methoxone 




6 oz. 2,4-D 

1 .0 



Started in warm room 

Remained in warm room 

6 oz. methoxone 

0. 1 



6 oz. 2,4-D 




* (Number of plants affected) x (scale of to 3 for degree of bending) divided 
by (total number of plants). 

to 2,4-D. Although varietal differences exist, they are not constant 
under all conditions. 

Dunham et al. (14) have pointed out the association of rapid growth 
and stage of plant. Thus, the small grains commonly grow slowly until 
well tillered, a stage relatively tolerant. The following period of jointing 
or shooting is one of rapid elongation under favorable growing conditions 
and is generally a more susceptible stage. Likewise in flax the period 
of stem elongation is commonly avoided in spraying for, under favorable 
conditions, it is one of rapid growth. 

Numerous investigators have reported reduced yields from plants 

R. S. DUNHAM 201 

sprayed when flower development is active. Thus the boot and heading 
stage of small grains, the bud and bloom stage of flax, and the tasseling 
stage of corn have been singled out as particularly susceptible periods. 
It is questionable whether rapidity of growth or development is very 
closely associated with susceptibility in these stages. It is quite probable 
that spraying at these times affects the flower development so that the 
injury is directly expressed in yield of seed. 

Morphological responses of plants to 2,^-D. — The reaction of crop 
plants sprayed with 2,4-D is often characteristic. Morphological changes 
as described in the literature are as follows: 

Wheat exhibits club-shaped spikes, irregular arrangement of 
spikelets, branched rachis, 2 spikelets per rachis node, fused 
glumes, thickened culm, and chlorosis. (See 20,21,32,42). 
Oats show onion leaf, blasted spikelets, late tillering, and 
interference with heading. (See 8,23,44). 
Barlev shows extended internodes on rachis, round inter- 
nodes, 2, 3, or more kernels at each rachis node in 2-row 
varieties, naked kernels, multiple awns, spikelet groups in 
2-row resembling 6-row type (i). 

Flax shows bending and twisting of stem, twin bolls, fused 
leaves, swollen stems, excessive branching, death of the cen- 
tral stem, and chlorosis. (See 16,45). 

Corn shows stalk curvature, stalk brittleness, lodging, fascia- 
tion of brace roots, and onion leaf (See 6,7,22,31,43). 

These abnormalities are not necessarily permanent. Frequently plants 
recover from them with no detrimental eff'ect on yield (6,7,25,36,44,45). 
Delay in maturity, and reduction in height are commonly mentioned 
in the literature as eff"ects of 2,4-D application. Tandon (45) points 
out that the delay in maturity of sprayed flax is greater at the final 
bloom stage than when ripe and that at practical rates of application 
this delay in maturity "is not of much practical consequence since 
the sprayed flax was not more than 2 days later than the check." 
Commenting on the frequent reports of serious delay in flax, Dunham 
has pointed out that two possible explanations for these contradictory 
results cannot be tested because of insufficient pertinent information in 
the reports submitted. Delay as reported may have been measured at 
bloom, rather than at maturity. Flax ripening too late in the season 


may prolong the process abnormally and any delaying effect of treat- 
ment might be exaggerated (13). Tandon (45) made fortnightly measure- 
ments of height in seven varieties of flax sprayed with 0, 4, 8, and 16 oz. 
of sodium salt, amine salt, and ester of 2,4-D. He concluded that up to 
8 ounces there is little reduction in height, and as the crop advances 
towards maturity even the little reduction noticed at earlier stages 
is practically eliminated. Paatela (40) has also reported that before the 
final height (of flax) was recovered, the average height of treated plants 
increased (more than the untreated) and the increase was greatest for 
plants treated with the highest concentration. 

Chemical and physiological responses of plants to 2,^-D. — The diff"er- 
ential response of crop plants to applications of 2,4-D may be expressed 
in factors affecting quality. Most of the work reported has dealt with 
protein content of wheat and with the oil content and iodine number of 
the oil in flax. Helgeson et al. (25) and Mitchell and Linder (38) report 
increases in the protein content of sprayed wheat, but the increase 
was associated with a reduced yield, an association that would normally 
be expected. Corns (9) obtained an increase in protein of barley when 
yields were reduced by more than five bushels. Erickson, Seely, and 
Klages (19), however, report an increase in protein of wheat without 
a corresponding decrease in yield. 

The effect on the oil content and iodine number of the oil in flax 
has been investigated by Tandon (45), Klosterman and Clagett (29), 
and Paatela (40). Recent studies of the writer and co-workers will be 
outlined briefly. Tandon found a distinct differential response among 
seven varieties to the amine and sodium salts apphed at 4, 8, and 16 oz. 
per acre. The ester of 2,4-D at these rates reduced both oil percentage 
and iodine number in all varieties tried except the oil in B5128 at 4 oz. 
per acre. Paatela reported a reduction in oil up to 2.3 per cent when the 
flax was sprayed with the morpholine salt of 2,4-D in the bud stage and 
a reduction in the iodine number when treated in the cotyledon stage. 
Square yard samples were obtained by Klosterman and Clagett from 
sprayed and unsprayed portions of four fields. The spray was applied at 
0.175 pounds of 2,4-D acid per acre in the form of the alkanolamine salt. 
The variety Dakota was grown on two fields and Minerva and Sheyenne 
on one field each. Stages of growth varied between fields at time of 
spraying. No significant differences were found in oil percentage; the 
iodine number of sprayed Dakota was significantly higher than that of 

R. S. DUNHAM 203 

the unsprayed, while that of unsprayed Minerva was higher than that 
of sprayed. The authors conclude that this formulation at the dosage 
used "is not detrimental to the value of the flax crop." 

Further investigation has been completed recently by Dunham and 
Robinson. Koto flax sown in rod rows was sprayed in the 2-inch stage, 
the prebud stage, the late bud stage, and the full bloom stage with 
4, 8, and 24 oz. of the sodium salt, the amine salt, and the ester of 
2,4-D. The data for the sodium salt are reported in Table 3. It is clear 
that the relatively tolerant Koto variety was adversely affected by the 
treatment since differences in percentages of oil were significant at the 
I per cent point and in iodine number at the 5 per cent point according 
to the t test. 

Largest reductions in oil percentage resulted from spraying in prebud 
and late bud stages, the first stage representing the approximate end of 
vegetative growth. Differences from the 4 oz. treatment may not be 
significant except in late bud and full bloom stages, but there is a reduc- 
tion in all instances. 

Likewise the iodine number was adversely affected in general. The 
two exceptions among 12 paired comparisons are the 4 oz. and 24 oz. 
applications at prebud. 

In the 1948 variety test at Minnesota the percentage of oil in Minerva, 
Victory, and B5128 was reduced 2.37 per cent, 0.56 per cent and 0.91 
per cent respectively with only 1.3 oz. of 2,4-D acid supplied as butyl 
ester per acre. 

To the pure-seed producer the effect of 2,4-D on germination of seeds 
produced on sprayed plants is of vital concern. Buchholtz (5), Derscheid 
(12), and Goodwin et al. (24) report the viability of oats unhurt. Elder 
(17) reports no injury to winter wheat seed. Helgeson et al. (25) stated 
that "germination of grain (hard red spring and durum wheats) was 
reduced by ester treatment in the boot stage only." Dunham and 
Robinson sprayed corn at five stages of growth and oats and barley at 
two stages each without injuring germination of the resulting seed. 
Tandon's (45) data show "that not even those varieties (of flax) which 
were susceptible to 2,4-D in other respects showed any reduction in the 
viability of seed." Further work at Minnesota found this to be true 
only when the flax was sprayed before late bud stage. Marth et al. 
(35,36) report no injury to the viability of Kentucky bluegrass or 
timothy seed from treated plants. 




Oil content and iodine number in Koto flax sprayed with sodium 2,4-D at 

various dosages and dates. 

Amount of 

Stages of flax 





Oil per cent 

2 inches 

4 oz. 

32 -39 




































T,ate bud 



















Full bloom 


























S. E. unsprayed plots 


Iodine number 

2 inches 
























164. 1 







Late bud 









161 .3 







Full bloom 





















S. E. unsprayed pic 




Differences in percentages of oil were significant at the i per cent point and 
in iodine number at die 5 per cent point according to the t test. 


This paper discusses the response of crop plants especially small grains, 
flax, and corn to the applications of growth-regulator herbicides, par- 
ticularly 2,4-D. The work of numerous investigators is cited, and origmal 
research not yet published is presented. The following statements sum- 
marize the discussion. 

R. S. DUNHAM 205 

i) The selective action of 2,4-D makes possible the killing or severe 
injury of certain undesirable species in the presence of crop plants. 
Differences in reaction to 2,4-D occur among species and among varieties 
and strains of the same species. 

2) These differences may be influenced by dosage, time of application, 
and environment. 

3) The rapidity of growth during the vegetative phase of plant de- 
velopment and/or the stage of fiower development at the time of 
application are important factors influencing the results from using 
2,4-D or methoxone. 

4) The morphological reaction of crop plants is often characteristic. 
Some abnormalities resulting from spraying with 2,4-D are listed. It is 
pointed out that plants frequently recover completely from such initial 

5) Increase in the protein content of wheat and barley and reduction 
of the oil content and iodine number of the oil in flax have been reported. 
Varietal differences have been found to this reaction in flax. The 
viability of seed from crop plants sprayed with 2,4-D at stages before 
bud has not been injured. 


1. Aberg, Ewert and Denward, Thore, Atin. Royal Agri. Coll. of Sweden, 

14:366 (1947). 

2. Albrecht, H. R., /. Am. Soc. Agron., 39:163 (1947). 

3. Arakeri, H. R. and Dunham, R. S., Uni. of Minn. Tech. Bull. 190 


4. BucHHOLTZ, K. P., Res. Rep. ^th Ann. N. Cent. Weed Cont. Conf, Sect. Ill, 

No. I (1948). 

5. , ibid., Sect. Ill, No. 10 (1948). 

6. , ibid., Sect. Ill, No. 60 & 61 (1948). 

7. Buckley, G. F. H., ibid., Sect. Ill, No. 62 (1948). 

8. Carder, A. C, ibid., Sect. Ill, No. 12 (1948). 

9. Corns, W. G., ibid.. Sect. Ill, No. 16 (1948). 

10. Crafts, A. S., Bull. Dept. Agri. Calif, 35:34 (1946). 

11. Derscheid, Lyle a., Proc. ^th Ann. Meet. N. Cent. Weed Cont. Conf, 

21 (1948). 

12. , Stahler, L. M., and Kratochvil, D. E., Res. Rep. ^th Ann. N. 

Cent. Weed Cont. Conf, Sect. Ill, No. 11 (1948). 

13. Dunham, R. S., Proc. ^th Ann. Meet. N. Cent. Weed Cont. Conf, 228 (1948). 

14. , Crim, R. F., and Heggeness, H. G., Uni. of Minn. Agri. Ext. 

Pamph. 168 (1948). 

15. , and Robinson, R. G., Res. Rep. ^th Ann. N. Cent. Weed Cont. 

Conf, Sect. Ill, No. 40 (1948). 


i6. , and Tandon, R. K., Minn. Farm & Home Set'., V:6 (1948). 

17. Elder, W. C, Res. Rep. $th Ann. N. Cent. Weed Cont. Conf., Sect. Ill 


18. , and Davies, Frank F., ibtd.. Sect. Ill, No. 80 (1948). 

19. Erickson, L. C, Seely, C. I., and Klages, K. H., /. Am. Soc. Agron., 

40:659 (1948). 

20. Foster, J. Roe, Res. Rep. ^th Ann. N. Cent. Weed Cont. Conf., Sect. Ill, 

No. 25 (1948). 

21. Friesen, H. a., ibid.. Sect. Ill, No. 29 (1948). 

22. FuELLEMAN, R. F. and Slife, F. W., ibid.. Sect. Ill, No. 64 & 65 (1948). 

23. GoDBouT, Eugene, ibid.. Sect. Ill, No. 15 (1948). 

24. Goodwin, H. F., Slife, W., and Fuelleman, R. F., ibid.. Sect. Ill, No. 9 


25. Helgeson, E. a., Blanchard, K. L., and Sibbitt, L. D., Bimonthly 

Bull, N. Da\. Agr. Exp. Sta., X: 166 (1948). 

26. HoLDEN, C. A., Jr., Brooks, J. S., and Elder, W. C, Res. Rep. ^th Ann. N. 

Cent. Weed Cont. Conf., Sect. Ill, No. 67 (1948). 

27. Jugenheimer, R. W., Slife, F. W., and Fuelleman, R. F., ibid., Sect. 

Ill, No. 68 (1948). 

28. Kelly, Sally, Am. J. Botany, 35:810 (1948). 

29. Klosterman, H. J. and Clagett, C. D., Bimonthly Bull., N. Da/{. Agr. 

Exp. Sta., XI:96 (1949). 

30. Lee, Oliver, C, Proc. $th Ann. Meet. N. Cent. Weed Cont. Conf., 18 


31. , Res. Rep. ^th Ann. N. Cent. Weed. Cont. Conf, Sect. Ill, No. 73 


32. Leggett, H. W., ibtd.. Sect. Ill, No. 21 (1948). 

33. Leng, E. R. and Slife, F. M., ibid.. Sect. VIII, No. 7 (1948). 

34. Marth, p. C. and Davis, F. F., Botan. Gaz., 106:463 (1945). 

35. Marth, P. C, Toole, V. K., and Toole, E. H., /. Am. Soc. Agron., 

39:426 (1947). 

36. , ibid., 39:780 (1947). 

37. Miller, John H., Unpublished data, U.S.D.A., University Farm, St. 

Paul (1948). 

38. Mitchell, John W. and Linder, R. J., Res. Rep. ^th Ann. N. Cent. Weed 

Cont. Conf, Sect. VIII, No. 9 (1948). 

39. Norman, A. G., J. Am. Soc. Agron., 40:111 (1948). 

40. Paatela, Juhani, Valtion Maatalouskpetoiminnan Jtdl^nsuja, 131 (1949). 

41. RossMAN, E. C. and Staniforth, D. W., Flant Physiol., 24:60 (1949). 

42. Sexsmith, J. J., Res. Rep. $th Ann. N. Cent. Weed Cont. Conf, Sect. Ill, 

No. 33 (1948). 

43. Shafer, N. E. and Wolfe, H., ibid.. Sect. Ill, No. 77 (1948). 

44. Shaw, W. C. and Willard, C. J., ibid., Sect. Ill, No. 7 (1948). 

45. Tandon, R. K., Agron. J., 41:213 (1949). 

46. Viehmeyer, Glenn, Res. Rep. ^th Ann. N. Cent. Weed Cont. Conf, Sect. 

Ill, 78 (1948). 

47. Weaver, R. J. and De Rose, H. R., Botan. Gaz., 107:509 (1946). 

48. Willard, C. J. and Shaw, W. C, Ohio Agr. Exp. Sta., Farm & Home 

Res. Bull., 255:188 (1948). 

Growth Substances in Relation to the Production 

of Tree Fruits 


THE propensity of many of the plant growth substances to influence 
abscission of flowers, fruits, and leaves has been used to considerable 
practical advantage in horticulture, particularly in relation to tree fruits. 
Strangely enough, the growth substances may act not only to delay 
abscission but also to implement it, and both phenomena have found 
horticultural application. The answer to this seeming paradox is by no 
means clear, although it is perhaps to be explained by the mode of 
abscission encountered, which differs in different kinds of plants and in 
different organs of the same plant. This thought will be referred to in 
more detail as this discussion develops. 

This review of accomplishments with plant hormones will be restricted 
to tree fruits and will treat of control of mature fruit drop and of fruit 
thinning and its opposite corollary, fruit set, with brief mention also of 
several problems which call for investigation. A mere review of hterature 
would seem inadequate to the occasion if it did not treat of the subject 
objectively, attempting to trace the advances made to the present 
status of accomplishment, pointing out the failures and the present Wind 
spots in our understanding, and developing some rationale for the ap- 
proach to new achievements in the light of our present knowledge, 
limited as it is. 

Control of Preharvest Fruit Drop 

Apples. — The possibiUty of using growth substances to control the 
drop of apple fruits was suggested by the observed effect of these 
compounds in causing the persistence of leaf petiole stubs on mis- 


cellaneous cuttings that had been treated to stimulate rooting. Other 
instances were also noted of delayed abscission of various treated organs, 
particularly floral parts. Since the first report of control of apple drop in 
1939 by Gardner, Marth, and Batjer (16) there have been not less than 
seventy-five scientific publications dealing with hormone chemicals in 
relation to fruit drop. As a result of these various reports hormone sprays 
have become a standard orchard practice for apples and pears in most 
sections where these fruits are grown, in many cases being applied by 
airplane over large acreages. It should be noted that at the time the idea 
was conceived plant-hormone chemicals were being produced in minor 
quantities and consequently were sold at prices that would make their 
use for orchard spraying appear to be fantastic. It has been the history 
of most synthetic biological compounds, however, that chemical know- 
how coupled with sufficient demand has resulted in great price reduc- 
tions. Accordingly, a plant investigator should not discard, untried, his 
ideas for practical applications simply because of the current price of the 
compound under consideration. 

Little would be served by attempting to review all of the papers 
relating to apple drop control. The first detailed report of the original 
work (17) established the effectiveness of a-naphthaleneacetic acid 
(NAA) and its amide at concentrations of 5 to 10 ppm. Many of the 
subsequent reports served to confirm the early findings and to extend 
the results to additional varieties and conditions. Omission of specific 
mention of each of these numerous papers in no way reflects on their 
importance and helpfulness. A number of reports have served to demon- 
strate the usefulness of various carriers for the hormone compounds and 
also methods of application. Still others have given techniques for 
screening compounds for effectiveness and have furthered the knowledge 
of penetration and movement of these substances within the tree. 

Despite efforts to find more effective or more adaptable compounds 
for drop control, NAA and its amide and salts remain the most useful. 
In searching for more effective compounds Batjer and Marth (3) found 
that 2,4-dichlorophenoxyacetic acid (2,4-D) applied to Winesap apples 
extended the effective period greatly beyond that of NAA, but that it 
did not take effect as quickly. The results were so exceptionally good on 
Winesap that many other varieties were subsequently tried by Batjer 
and Thompson (4) and Harley et al. (19,20). Unfortunately, the only 


apple varieties reported thus far to be appreciably affected by 2,4-D 
sprays are Winesap, Stayman Winesap, Kendall, and Bonum, the last 
two being minor varieties. Apparently the differential effect of 2,4-D 
on different species of plants, which is the basis of its usefulness as an 
herbicide, extends even to varieties. 

Those workers interested in attempting to explain the differential 
herbicidal eflect of 2,4-D might well ponder the results of Edgerton and 
Hoffman (14), who introduced a solution of this compound into the 
transpiration stream of Mcintosh apple trees. Numerous attempts to 
control fruit drop of Mcintosh variety by 2,4-D sprays have been 
uniformly unsuccessful, but a very definite effect was secured by the 
injection method. Although abscission of both fruit and leaves was de- 
layed, no injury to the trees was reported. 

While sprays of 2,4-D are effective in controlling drop of a few 
varieties, its use is not without hazard in apple and pear orchards. For 
best results it should be applied earlier than NAA; but the earlier it is 
applied the more danger there is of damaging holdover effects which may 
be expressed in deformed leaves and shoots the next spring. Marsh and 
Taylor (27) described some severe damage to susceptible varieties of 
apple from 2,4-D residue in a central spray system which had been 
utilized for weed control and subsequently used for a late spraying 
of the trees with summer oil. The persistence of the effect of 2,4-D is 
sometimes remarkable. Moon, Regeimbal, and Harley (32) reported 
a case in which Stayman Winesap, sprayed in August, 1946, showed 
appreciable drop control in the following year's crop picked in October, 

Citrus Fruits. — Preharvest drop of oranges and grapefruit, unlike that 
of apples and pears which in general occurs during the few weeks prior 
to time of picking maturity, may straggle along for many weeks. This 
situation is due in part to the fact that citrus, not being a starchy fruit, 
has no definite physiological maturity stage. Once ripe enough to eat, 
its picking may be, and usually is, delayed for weeks or even months 
in order to catch a favorable market. Thus the cumulative loss from 
droppage may be appreciable, rarely amounting to less than 10 per cent 
of the crop and often much more. Certain varieties, such as Pineapple 
and Temple oranges, may also exhibit a wave of heavy droppage in the 
latter part of their season, in which most of the fruit drops within a few 


days' time. Tangerines, on the other hand, adhere tenaciously, and it 
is only with difficulty that mature fruit of any stage can be pulled intact 
from the tree. 

The response of citrus fruits to sprays of naphthaleneacetamide is 
quite in contrast to the response of apples. Gardner (15) found that 
10 ppm. of this compound had no effect on Pineapple oranges but that 
100 ppm. reduced drop markedly if applied early in the harvest season 
(November) and was still effective 12 weeks later when the fruit was 
harvested in February. When applied in January even this high concen- 
tration was without effect. Most citrus growers would prefer to take 
the chance that they will harvest their crop ahead of heavy droppage 
rather than invest in the cost of such a concentrated spray, and for this 
reason, in part, the naphthalene compounds are not used for citrus. 

In California, Stewart and Klotz (45) reported appreciable reduction 
in the preharvest drop of Valencia and Washington Navel oranges from 
2,4-D sprays applied in early summer. Marsh grapefruit, in experiments 
by Stewart and Parker (46), also responded but apparently not as 
satisfactorily as oranges. At the highest concentration (225 ppm.) some 
of the young grapefruit on the trees at time of spraying (not the mature 
crop for which the sprays were applied) developed quite abnormally, 
having cylindrical shapes, thick rinds, many prominent rudimentary 
seeds, and even navels. The oranges responded similarly at high con- 
centrations (45). From numerous reports by Stewart and his coworkers 
it appears that low concentrations, 5 ppm. for example, are nearly as 
effective as 25 ppm., at which concentration and above occur increasing 
injury to the tree, abnormal fruits, and abscission of young fruits of the 
new crop. This reduction in the quantity of fruit might well account 
for the slightly larger fruit sizes of oranges reported by Stewart (44). 

In Florida, Gardner (unpublished results), using the sodium salt of 
2,4-D on several citrus varieties in October at 10 ppm,, did not secure 
as outstanding control of drop as that reported in California. The 
Pineapple oranges picked 12 weeks after spraying dropped half as much 
fruit during that period as the controls. The sprays on Valencias and on 
Marsh grapefruit were somewhat less effective and, in the case of grape- 
fruit, the effect did not persist as long as with oranges. "Seedling 
oranges," which approach a varietal status in Florida because of the 
high incidence of nucellar seedlings, failed completely to respond to the 
2,4-D. It appears that here again is an instance of differential varietal 


susceptibility, as in the case of apples. A great deal more work needs to 
be done under Florida conditions and with Florida varieties to establish 
the most effective safe concentrations and times for application. In the 
Florida drop control experiments the sprays have thus far been applied 
in the late fall months not only because this is near the beginning of the 
drop period, but also because the trees are not flushing new growth and 
do not normally do so until February and can therefore withstand a 
higher concentration without injury than at other periods. Moreover, 
sulphur sprays and dusts for rust mite control are commonly applied 
at this time, and it appears entirely feasible to include 2,4-D at no 
extra expense other than its insignificant cost. The results thus far 
indicate, however, that the presence of wettable sulphur moderately 
reduces the effectiveness of the 2,4-D, although it is possible that this 
difficulty may be overcome simply by increasing the amount used. The 
low cost of this material and the possibihty of including it in pest-control 
sprays are important assets favoring its wide adoption for citrus fruits. 
Pears. — Passing reference has already been made to the drop of pears, 
but since this fruit responds to hormone sprays so readily, it appears 
desirable to review briefly the present status of its drop control. Summer 
pears for the fresh fruit market are harvested prior to full maturity, 
and thus a drop problem is usually not of great moment. Canners, on 
the other hand, need to have the fruit more nearly ripe, and the delay 
in picking for this purpose and for increased size results in appreciable 
amounts of grounded fruit. Apparently Strickland ei al. (47) in Aus- 
tralia were the first to report pear drop control by hormone sprays. 
Their treatments with 20 ppm. of naphthaleneacetamide in three appli- 
cations have since been shown to be much more extravagant than 
necessary. Davey and Hesse (12) obtained appreciable control in Bartlett 
variety witn both NAA and its amide at 5 and 10 ppm. They make no 
mention of injury from NAA at the higher concentration, although 
Batjer et al. (6) reported a yellowing and premature drop of considerable 
foHage from this concentration. These last investigators obtained effec- 
tive drop control of Bartletts with 2.5 ppm. of either NAA or 2,4-D. 
Concentrations of 2,4-D higher than 2.5 ppm. (5 and 10 ppm.) caused 
yellowing of old foliage, injury to buds, and malformation of new fruit 
and foUage in the following season proportional to the increase in 
concentration. Overholser et al. (35) mentioned that Bosc variety, as 
well as Bartlett, responds to NAA; but these workers did not try 2,4-D. 


Peaches and Apricots. — Peaches and apricots delayed in picking to 
attain proper maturity for canning are subject to considerable drop in 
certain areas. Hesse and Davey (21) found that the Stewart apricot re- 
sponded to both NAA and its amide but that with Elberta peaches the 
response was so slight as to hold no commercial advantage. Effective 
sprays for peach drop control would be a boon to the canning-peach 

Effect of preharvest sprays on fruit maturity. — Space will not permit a 
review of the work on the effect of growth substance sprays on fruit 
maturity, but the problem is of such importance in fruit storage that at 
least brief mention should be included here. It now seems quite certain 
that growth substances can and do hasten maturity of apples and pears on 
the tree and affect their subsequent storage life. The extent of this effect 
depends on the compounds used and their concentration, as well as on 
the length of delay in harvest made possible by their use. Citrus consti- 
tutes a notable exception, perhaps unfortunately, for in this case a 
hastening of maturity might have important advantages in marketing. 
The results of Blondeau and Crane (9) in hastening the maturation of 
CaHmyrna figs from a normal 120-day period to 60 days by sprays of 
2,4,5-trichlorophenoxyacetic acid indicate that important advantages 
in time of ripening might be gained with certain fruits. 

Some Factors Influencing Effectiveness 

Methods of application. — Dust applications, assuming equal effective- 
ness, have advantages over spray applications on the basis of lower 
labor costs and more rapid coverage, particularly important in case of 
large acreages. Hoffman, Edgerton, and Van Doren (23,24) reported 
that under favorable dusting conditions NAA incorporated in a talc 
dust was equivalent in drop control of Mcintosh apples to roughly 
the same quantity of the compound applied as a spray. Southwick 
(39,40), on the other hand, using the same concentrations and the same 
variety, did not find dusts to be equal to sprays. Unfavorable conditions 
for dusting are more apt to occur than for spraying, and this may explain 
the lack of complete agreement. In subsequent work Southwick's results 
with dusts were more nearly the equal of sprays (41). Marth, Batjer, and 
Moon (28) have also compared dusts with sprays, using Stayman Winesap 
as the test variety, and reported comparable results. The uncertainty 


of adequate coverage inherent in the dusting operation probably accounts 
for its minor usage in commercial hormone control of fruit drop. 

Tukey and Hamner (49) and also Marth et al. (28), using a highly 
concentrated solution of hormone in hand aerosol bombs, found this 
method of application to be effective. Obviously its usefulness is re- 
stricted to small trees, although Hamner and Rasmussen (18) found that 
a concentrated oil solution of NAA applied as a vapor by a commercial 
fog machine (Todd Insecticidal Fog Applicator) could be used success- 
fully on standard-size trees. The uncontrolled drift of the fog with even 
slight air currents is the chief limiting factor. 

Since the first trials in 1944 the apple and pear growers of the Pacific 
Northwest have made increasing use of airplane applications. Naph- 
thaleneacetic acid dissolved in an oil emulsion in high concentration is 
applied by low-flying planes equipped to disperse the material in minute 
droplets which are forced down through the trees by air turbulence 
created by the planes. This method of application has obvious advantages 
in covering large acreages quickly and, according to tests conducted by 
Thompson and Batjer (48), it is quite effective in fruit drop control, 
although apparently not the equivalent of a thorough, conventional 
spray application. 

There is no question but that thorough coverage with the applied 
growth substance is important for maximum drop control regardless of 
the method of application. While there is some transmission effect 
through the tissues for short distances, at least in the case of NAA, such 
effects are quite limited. Batjer and Thompson (5), carefully applying 
this compound by hand to fruit stems and cluster bases and to the sub- 
tending foliage only, found that this foliage was the chief means of 
transmitting the effect to the point of fruit abscission. There was no 
evidence of transmission, however, from a completely sprayed spur 
to unsprayed spurs nearby on the same branch. It is entirely possible 
that in the case of 2,4-D the transmission effects take place over much 
greater distances in the case of the few varieties on which it is effective. 
The ability to affect abscission by transmission over considerable distance 
within the plant tissues will be an important characteristic of new 
compounds destined for fruit drop control. 

Varietal differences. — While all apple varieties are apparently affected 
by NAA and its amide, the response varies greatly depending on the 


variety. In general the hormone sprays are more effective on early- 
maturing types than on late varieties, but there are exceptions to this 
generahty. It is probable that in early varieties not only are tissues of 
the stem and also the leaves in a more reactive condition, but also that 
the sprays are appHed during periods of higher temperatures than in 
the case of late varieties. 

Another factor which may account for the observed varietal differences 
in response of apples is the variation in the mode of the abscission process. 
McCown (30) stated that varieties differ in the order in which the 
various tissues in the abscission zone begin their process of abscission 
and that those varieties in which the pith abscission is delayed until 
after the bark tissues show signs of separation respond most readily to 
hormone sprays. In the case of Mcintosh, a variety on which hormone 
sprays are generally reported as being effective for a period of only 10 
days, he pointed out that abscission in the pith tissues begins very early, 
by the time there is noticeable striping of the fruit, and he questioned 
whether hormones could be very effective after this time. This observa- 
tion may also explain the general lack of appreciably longer response of 
Mcintosh to a second spraying, as has been reported by Batjer and Marth 
(2), Murphy (34), and others, although with some varieties this pro- 
cedure is effective. 

The existence of varietal differences in apples in their response to 
2,4-D has already been noted, most varieties being quite indifferent to 
this compound in both tree and fruit reactions. Edgerton (13) has pub- 
lished some results comparing the effect of NAA and its methyl ester 
and also 2,4-D and its methyl and amyl esters on apple petiole drop and 
fruit drop in three varieties. The petiole drop test consists of cutting 
uniform leafy shoots from the trees and placing their bases in water, 
cUpping off the leaf blades, spraying the petioles with the growth- 
substance solutions, and recording subsequent petiole abscission over a 
period of days. Edgerton found that the petioles of Stayman Winesap 
and Winesap varieties responded to 2,4-D and its esters, thus agreeing 
with the observed response in fruit drop control with these two varieties. 
Mcintosh, on the other hand, failed to respond to these compounds in 
both fruit and petiole abscission. On Stayman Winesap and Winesap 
the order of effectiveness of the various compounds on petiole drop 
agreed quite well with the observed order of effectiveness in control 
of fruit drop. It would appear from the limited evidence thus far 


available that the petiole-drop test may be a simple and useful means 
not only of screening varieties for response to a particular compound 
but also of screening new compounds for effectiveness in controlling 
drop of a particular kind of fruit. 

Temperature. — It is recognized that hormone responses in general are 
favored by high temperatures and impeded by cold. Without any 
precise information available, it was thought that temperatures lower 
than 70°F. at the time of spraying were unfavorable for drop control 
even though a temperature rise took place later on. In this connection 
the work of Batjer (i) is of considerable interest. He found that NAA 
sprays applied at midday with the temperature at approximately 8o°F. 
were consistently more effective than sprays apphed in the early morning 
of the same day with the temperature approximately 20 degrees lower. 
From a practical standpoint the difference in control was not great, and 
there was some indication that the unfavorable influence of the lower 
temperatures might be compensated for by using a higher concentration 
of the hormone. The work of Overholser et al. (35) is in complete 
agreement on the point of temperature effects at time of spraying. 
Batjer, in attempting to study the temperature relationship more closely, 
used the apple petiole technique in which the leaf blades were removed 
and the petioles sprayed at a controlled range of temperatures. The 
subsequent rate of abscission of the petioles was recorded at a uniform 
temperature for all lots. The results indicated that above 72°F, tempera- 
ture effects were negligible. Below 72°F. the control of abscission was 
increasingly poorer with lowering of the temperature. 

Fruit Thinning and Fruit Set 

As all fruitgrowers know, thinning by hand is a tedious and expensive 
operation. Thinning is frequently necessary, however, in order to secure 
fruit of marketable size and to insure regular annual bearing with certain 
varieties that are prone to overcrop one year and fail to set the next. 
Numerous studies have been made to establish a practical thinning pro- 
cedure by use of certain caustic sprays. The results have been variable 
because the process is dependent on kiUing or injuring only a portion 
of the flowers and young fruits, causing them to abscise. The procedure 
is not without hazard since the margin of safety is narrow before injury 
to the fohage and twigs occurs. Early thinning is also complicated by the 
risk that frost may subsequently kill additional flowers or fruit and thus 



the crop is over thinned. Hormone sprays, while not a panacea for all 
of these hazards, do offer the possibility of thinning without injury to 
the young foliage since their action is not necessarily a caustic one. 

A discussion of the role of growth substances in fruit thinning must 
necessarily consider their effect on fruit set since one process is the 
opposite of the other, and the concept of thinning by means of these 
compounds was the outgrowth of the unsuccessful attempts with tree 
fruits to increase fruit set. For the purpose of this discussion, fruit set is 
considered to be the net resulting crop after all abscission, from flowering 
through the June drop has occurred. 

The writer is not aware of any well-substantiated case in tree fruits 
of an improvement in fruit set by means of growth substances. This 
statement does not apply to the few cases of those fruits that can be 
stimulated to set parthenocarpically and where an improved set is the 
result of such stimulation coupled perhaps with faulty or complete lack 
of pollination. On the other hand, there are many recorded failures of 
the hormone chemicals to improve set and instances in which fruit set 
was actually decreased. 

The success with growth substances in controlling mature fruit drop 
naturally led to trials to increase crop production by preventing the 
abscission of young fruits. Gardner, Marth, and Batjer (17) recorded the 
failure of NAA and its amide, applied at petal fall, to improve the set 
of several apple varieties but made no mention of any resulting decrease 
in set. Burkholder and McCown (10) found that NAA sprays at 10 ppm. 
applied to Starking at full bloom reduced the number of clusters that 
held fruit past the June drop by 15.1 per cent, and at 50 ppm. the reduc- 
tion was 77.7 per cent; the amide at 50 ppm. reduced the set by 34.0 
per cent. Severe epinasty and leaf scorch accompanied the use of 50 
ppm. of NAA but no injury attended the amide. If the thought of using 
either of these substances as intentional fruit thinners occurred to these 
workers at the time, it was not mentioned. 

With certain citrus varieties one might logically expect an improve- 
ment in set with growth substances since many varieties are apparently 
able to set parthenocarpically even without the benefit of applied stimu- 
lation. Pomeroy and Aldrich (50), however, made extensive trials on 
Washington Navel oranges and Marsh grapefruit without success. They 
used several growth substances, both naphthalene and indole compounds, 
at various concentrations and applied in different ways. Naphthalene- 


acetic acid at relatively high concentrations reduced the set rather than 
increased it. Stewart and Heild (44) using 2,4-D also noted a reduction 
in number of orange fruits set, although not necessarily a reduction in 
number of harvested boxes because the fruits remaining grew to larger 
size. The lack of response of citrus varieties in setting additional fruit 
under hormone stimulation is especially puzzling in view of its naturally 
parthenocarpic tendency. 

Failure of the hormones to prevent the abscission of young fruits is 
not restricted to the fruits mentioned. Negative results are frequently 
deemed of such little interest that they are never published. In Florida 
the Haden mango blooms profusely but sheds its small fruits to the 
degree that satisfactory crops are infrequent. The June drop of avocados 
often converts a seemingly heavy set to a very light crop. Gardner and 
others in Florida (unpublished results) used various hormone chemicals 
on these two fruits without benefit. Smith (38), in the hope of controlling 
the immature drop of pecans, found no effect on shedding from applica- 
tions of indoleacetic acid or naphthaleneacetamide at 50 ppm, Naph- 
thaleneacetic acid at this concentration, however, gave a definite increase 
in the shedding of young nuts. 

If one were to generalize from the above record it would be concluded 
that with tree fruits hormone treatments have either been without effect 
on fruit set or, in the higher concentrations, have resulted in reduced set. 
Schneider and Enzie (36,37) were apparently the first to utifize the 
abscission-promoting effect of these compounds on young fruits with 
the specific intent of fruit thinning. They reported that NAA sprays 
at 100 ppm. on apples nearly eliminated the crop on all varieties tested, 
but with marked injury to leaves and growing points. At 10 ppm. the 
effect was more moderate on both scores. Naphthaleneacetamide at 
80 ppm. performed well in thinning without damage to foliage. Indole 
derivatives, however, were of no value. Hoffman, Southwick, and Edger- 
ton (25,39) and also Batjer and Thompson (7) reported on the use 
of the sodium salt of NAA for thinning apples and in general found it 
a promising material although subject to variable results — overthinning 
in some instances and underthinning in others. This may be an expression 
of the variable state of the trees or of the conditions of spraying rather 
than an inherent shortcoming of the compound itself. It would appear 
that more attention should be given to naphthaleneacetamide as a 
thinning spray rather than to the acid or the sodium salt despite the 


higher concentration of amide required. The absence of any deforming 
epinastic effects from the amide recommends its use where tender new 
growth is concerned. In the meantime, the apple industry is making 
increased use of NAA and its sodium salt for apple thinning and will 
probably continue to do so until more effective and more consistent 
materials are found. 

In the case of peaches it is of interest to note that Southwick et al. 
(40) found no thinning effect from either the sodium salt of NAA up to 
40 ppm. or its methyl ester up to 20 ppm. in the sprays. Murneek and 
Hibbard (33) also reported the use on peaches of the sodium salt as a 
thinning spray in the range of 5 to 40 ppm., but the conditions of their 
experiment were such as to make interpretation of the results somewhat 
difficult. In one test there appeared to be an actual increase in set over 
the controls, but the counts were made before drop was completed. 
The data of another test appeared to show a slight reduction in set with 
the use of hormone sprays although, without the benefit of any statistical 
evaluation, it is questionable that the difference is significant. It should 
be recalled that applications of the naphthalene derivatives have also 
been shown to be without effect on the preharvest drop of peaches. 

Growth Substance Effect in Relation to Stage of Development 

From the previous discussion it would appear that the available 
hormone chemicals offer a possible solution to problems involving a 
delay of the preharvest abscission, whereas in the case of flower and 
young fruit abscission the same compounds offer no hope of increasing 
fruit set and in fact, in many instances, will promote fruit shedding. The 
reason for this seemingly different action of growth substances at these 
different stages of development is of fundamental importance in the 
approach to any problem involving abscission. Admittedly the question 
calls for more investigation, but with little information available some 
speculation may be permissible. 

Type of abscission. — In studying the abscission of apple fruits both 
MacDaniels (26) and McCown (30) found that the anatomical changes 
accompanying the preharvest drop differ in character from those of the 
early drop of young fruits and flowers. Early drop according to these 
workers is associated with a definite preformed abscission layer resulting 
from secondary cell division. In late drop the changes are characterized 


chiefly by alterations in the cell walls in an abscission zone. It must be 
assumed that the action of the hormones in the late drop is to delay the 
weakening of the cell-wall material so that separation of the cells is 
slowed, but that these compounds do not prevent the secondary cell 
division which results in the formation of an abscission layer observed 
in the case of early drop. The fact should not be overlooked that growth 
substances may also delay apple leaf abscission, particularly in instances 
where the leaf blades have been removed as in the petiole test for hor- 
mone activity. Delay of leaf fall in apple orchards has also been observed 
in cases where 2,4-D was used. No description has been found in the 
literature of the mode of abscission of apple leaves, but in certain other 
plants the process appears to be a combination of cell-wall changes in 
certain tissues and abscission layer formation in other tissues. 

Both early and late abscission delayed. — The possibility exists that there 
is actually no fundamental difference in the response of the tissues to 
growth substances in early and late abscission. Persistence of flowers, 
or at least of their petals, for an appreciable period following a hormone 
spray is a common observation. The usual occurrence following the 
spraying of young fruits, particularly with a rather high concentration, 
is a noticeable delay in their abscission, even though the subsequent drop 
is increased. Thus growth substances may have a direct eifect in delaying 
the weakening of the cell walls regardless of the type of abscission in- 
volved. The increased drop of young fruits when abscission is resumed 
might be explained on the basis of competitive antagonism between the 
apphed compounds and the natural auxins produced by the developing 
fruit which provides a steady supply to prevent abscission just as the 
leaf blade provides its petiole with an anti-abscission auxin. This is 
highly speculative but not without some basis of experimental evidence. 

Problems Needing Solution 

To date the record of the research worker in solving many of the 
fruitgrowers' problems by means of growth substances is not outstanding 
in relation to the seeming possibilities. It is true that control of pre- 
harvest drop has been rather thoroughly studied and the fruit industry 
has adopted these sprays as a regular practice and to great advantage. 
The use of hormone sprays for fruit thinning, an unexpected outgrowth 
from the attempts to improve set, is also gaining in orchard use. There 


are, however, a number of important problems that would appear to 
be amenable to application of growth substances, on which little or no 
progress has been made. Some of these should be mentioned here. 

Parthenocarpy. — The subject of parthenocarpy is ably reviewed in a 
separate paper in this symposium, and it is not the intent to encroach on 
that discussion here other than to point out that for our most important 
tree fruits, such as apples, peaches, pears, and the citrus varieties, no 
practical progress has been made in stimulating parthenocarpy. The 
work of Crane and Blondeau (9,11) with the Calimyrna fig may consti- 
tute a partial exception to this general statement. While most figs 
are naturally parthenocarpic, this variety is almost completely non- 
parthenocarpic in the sense that it does not hold its fruit to maturity 
unless pollinated, although it should be noted that considerable de- 
velopment of the syconia takes place without pollination. In the work 
of these investigators sprays of indolebutyric acid at 1,500 ppm. (9) 
and, of more practical significance, 2,4,5-trichlorophenoxyacetic acid 
at concentrations as low as 10 ppm. (11) caused the fruit to complete 
its development and at a greatly accelerated rate. The need for more 
work on parthenocarpic stimulation in tree fruits is real, in view of the 
number of self-sterile varieties and frequent unfavorable pollination con- 
ditions which result in poor set. There is also the possibihty that by 
proper stimulation parthenocarpic fruit might be superior in some 
respects to fertilized fruit. The problem is intimately tied in with the 
general problem of improving fruit set. It should be pointed out, 
however, that the parthenocarpic response is a different reaction from 
simply the prevention of abscission, for it is possible to hold ffowers 
and young fruits on the tree for appreciable periods, but without 
stimulation of ovary development. 

Delay in flowering. — The greatest hazard in most sections in the pro- 
duction of many deciduous fruits, particularly those that tend to bloom 
early in the spring, is freezing temperature at flowering time. A delay 
of even 10 days in blossoming might save many crops from freezing 
damage. The inhibiting effect of many of the hormone chemicals on 
lateral leaf bud development is well known. Unfortunately flower buds 
are not as subject to inhibition by these compounds as are leaf buds. 
Winklepeck (51) applied a spray of NAA at 125 ppm. to peach trees 
just as the flower buds were swelling and beginning to break and reported 
that the sprayed trees arrived at full bloom two weeks later than the 


controls. No mention was made of injury from the sprays. Mitchell 
and Cullinan (31) failed to confirm these promising results with peaches 
by using sprays applied in the spring. While the leaf buds were retarded 
the flower buds were either injured by the high concentrations of 
the growth substances or were not delayed in opening. Hitchcock and 
Zimmerman, working with peaches and several other fruits, reported 
(22) that a more effective time to apply the growth substances is during 
the preceding summer when differentiation of the flower buds takes 
place. Their report was most encouraging and did not emphasize any 
injurious effects from the high concentrations of the potassium salt 
of NAA used at this time of year. From subsequent work on peaches 
Marth, Ha vis, and Batjer (29), using the same compound at the same 
concentration range and same time of year, concluded that injury to 
buds and even to branches is so severe as to preclude use of this compound 
as a possible orchard treatment. Moreover, the delay in flowering was, 
at the best, only two days; and according to these workers, it was 
probably associated with injury rather than an inhibiting effect of the 
sprays. Usually the critical test of the feasibility of any suggested orchard 
practice is whether or not fruitgrowers adopt it. Growth substance 
applications for delay of flowering in fruit trees have not reached that 
stage, but certainly the importance of the problem warrants more 

Breaking of dormancy. — In determining the southern limits of decidu- 
ous fruit culture probably no factor is more important than the chilling 
requirements necessary to terminate the rest period of the trees. During 
mild winters at the present southern limits of these fruits the chilling 
requirements may not be satisfied, with the result that the trees and their 
potential crops suffer from delayed foliation. It is not unlikely that 
dormancy involves a hormone relationship. The work of Bennett (8) 
points to a high auxin content in buds and shoots of pear trees as the 
dormant condition is entered, and a progressive disappearance of the 
auxin as the rest period is gradually broken. The growth-inhibiting 
effect of applied synthetic hormones on vegetative buds, particularly 
the effect of these compounds in maintaining dormancy of tubers, 
further supports a relationship between hormones and rest period. 
Obviously here is a problem in which it would be desirable to accomplish 
the opposite of prolonging dormancy. It is possible that to accomplish 
this end compounds may be necessary that have quite a different physio- 


logical action from what we now think of as hormone activity. It is 
scarcely necessary to point out how important it would be to have a 
ready control over dormancy, being able both to impose and terminate 
it, and how such control could extend southward the culture of many 
deciduous trees. 


1. Batjer, L. p., Proc. Am. Soc. Hort. Set., 40:45 (1942). 

2. , and Marth, P. C, ibid., 38:111 (1941). 

3. , Science, 101:363 (1945). 

4. Batjer, L. P. and Thompson, A. H., Proc. Am. Soc. Hort. Sci., 47:35 


5. , ibid., 51:77 (1948). 

6. , and Gerhardt, Fisk, ibid., 51:71 (1948). 

7. Batjer, L. P. and Thompson, A. H., ibid., 52:164 (1948). 

8. Bennett, J. P., (Unpublished) Joint Symposium, Amer. Soc. Plant 

Physiol, and Am. Soc. for Hort. Sci., (Chicago, 1947). 

9. Blondeau, Rene and Crane, Julian, Science, 108:719 (1948). 

10. Burkholder, C. L. and McCown, M., Proc. Am. Soc. Hort. Sci., 38:117 


11. Crane, Julian C. and Blondeau, Rene, Plant Physiol., 24:44 (1949). 

12. Davey, a. E. and Hesse, C. W., Proc. Am. Soc. Hort. Sci., 40:49 (1942). 

13. Edgerton, L. J., ibid., 49:42 (1947). 

14. , and Hoffman, M. B., ibid., 51:67 (1948). 

15. Gardner, F. E., Proc. Fla. State Hort. Soc, 54:20 (1941). 

16. , Marth, P. C, and Batjer, L. P., Science, 90:208 (1939). 

17. , Proc. Am. Soc. Hort. Sci., 39:415 (1939)- 

18. Hamner, C. L. and Rasmussen, E. J., ibid., 49:78 (1947). 

19. Harley, C. p.. Moon, H. H., Regeimbal, L. O., and Green, E. L., 

ibid., 47:39 (1946). 

20. Harley, C. P., Moon, H. H., and Regeimbal, L. O., ibid., 50:38 (1947) 

21. Hesse, C. O. and Davey, A. E., ibid., 40:55 (1942). 

22. Hitchcock, A. E. and Zimmerman, P. W., ibid., 42:141 (1943). 

23. Hoffman, M. B., Edgerton, L. J., and Van Doren, A., ibid., 40:35 


24. , ibid., 42:203 (1943). 

25. Hoffman, M. B., Southwick, F. W., and Edgerton, L. J., ibid., 49:37 


26. MacDaniels, L. H., ibtd., 34:122 (1936). 

27. Marsh, Ray S. and Taylor, Carlton F., ibid., 49:59 (i947)- 

28. Marth, P. C, Batjer, L. P., and Moon, H. H., ibid., 46:109 (1945). 

29. Marth, P. C, Havis, Leon, and Batjer, L. P., ibid., 49:49 (1947)- 

30. McCowN, M., ibid., 36:320 (1939). 

31. Mitchell, John W. and Cullinan, Frank P., Plant Physiol, 17:16 


32. Moon, H. H., Regeimbal, L. O., and Harley, C. P., Proc. Am. Soc 

Hort. Sci., 51:81 (1948). 


33. MuRNEEK, A. E. and Hibbard, A. D., ibid., 50:206 (1947). 

34. Murphy, Lyle M., ibid., 40:42 (1942). 

35. Overholser, E. L., Overley, F. L., and Allmendinger, D. P., ibid., 

42:211 (1943)- 

36. Schneider, G. W. and Enzie, J. V., ibid., 42:151 (1943). 

37. , ibid., 45:63 (1944). 

38. Smith, C. L., ibid., 44:119 (1944). 

39. Southwick, F. W., Edgerton, L. J., and Hoffman, M. B., ibid., 49:26 


40. , Hoffman, M. B., and Edgerton, L. J., ibid., 51:41 (1948). 

41. Southwick, Laurence, ibid., 40:39 (1942). 

42. , ibid., 42:199 (1943). 

43. , ibid., 44:109 (1944). 

44. Stewart, W. S. and Heild, H. Z., Calif. Citrograph, 34:284 (1949). 

45. Stewart, W. S. and Klotz, L. J., Botan. Gaz., 109:150 (1947). 

46. Stewart, W. S. and Parker, E. R., Proc. Am. Soc. Hon. Sci., 50: 187 


47. Strickland, A. G., Kemp, H. K., and Beare, J. A., Fruit World and 

Mar\et Grower (Melbourne, Australia), 42:7 (1941). 

48. Thompson, A. H. and Batjer, L. P., Proc. Am. Soc. Hort. Sci., 47:44 


49. Tukey, H. B. and Hamner, C. L., ibid., 46:102 (1945). 

50. Pomeroy, C. S. and Aldrich, W. W., ibid., 42:146 (1943). 

51. Winklepeck, R. L., Hoosier Horticulture, 20:152 (1939). 

Use of Growth Substances in Tropical Agriculture 


SYNTHETIC plant hormones have found extensive use in tropical agri- 
culture. From the point of view of progress in research, however, 
the accent has been far too much on use and not enough on the under- 
lying fundamental principles. In the long run a balance between theo- 
retical and practical knowledge must exist. It is true that at times trial 
and error methods can yield spectacular results. Yet, sooner or later 
"bugs" develop which can be corrected only after theoretical knowledge 
has caught up and the principles involved are more fully understood. 

Tropical crops and plants make highly worthwhile subjects for studies 
in fundamental plant physiology. Often physiological principles are 
apparent in tropical plants while they are obscure in plants growing 
under the different environment of the middle latitudes. It is no wonder, 
therefore, that new principles are often brought to light by those working 
in the tropics. Fitting, working in the tropics on orchids, was first to 
reahze the existence of hormone-like substances in the development of 
the ovary, and, as a result of these observations, to use the word hormone 
for the first time in plant physiology (57). It was Bouillenne and Went 
(5), again working in the tropics on tropical plants, who conceived the 
idea that substances now known as auxins promote the initiation of 
adventitious roots on cuttings. 

The enormous potentialities of plant physiological research in the 
tropics is not sufficiently realized by those responsible for investing in 
research. If money is invested in tropical research it is usually done with 
immediate material benefits in view. However understandable this is, 
there is a great need for the establishment of permanent institutions in 
the tropics where plants are studied for the sole purpose of "extending 
the horizons of our intellect" as Bronk (6) has so aptly put it. 


In discussing the tropical uses of plant hormones, I will attempt to do 
so by including the underlying principles as far as they are known at 
present. They could be classified under the following headings: Fruiting, 
Rooting, and Weeding. 


Growth-regulating substances are known to affect tropical fruit crops 
in a variety of ways. They may promote the flower initiation of the plant. 
This can be done directly, as in the pineapple, or indirectly, as in litchi. 
The synthetic hormones may also regulate the fruit growth and develop- 
ment after the flowers have been initiated naturally, as is exemplified by 
the fig. Finally, after the fruit has been formed by natural processes, 
hormones may delay its abscission from the tree and improve its keeping 
quaUties. This is illustrated by the orange. Each of these effects will be 
dealt with below. 

Crop control in the pineapple. — The pineapple plant produces in its 
lifetime only one fruit. Flowering, under natural conditions, starts in 
the fall and continues through the winter (Fig. 2). Approximately six 
months after flower formation the fruit is ready for harvest. The age 
at which a pineapple plant begins to flower depends upon its variety and 
the external conditions. On the average, when a pineapple plant during 
the flowering season reaches an age of 1 8 months it is capable of producing 
a marketable fruit. Some varieties Hke the Puerto Rican Cabezona take 
occasionally as long as 5 years before they flower. Such slow varieties 
can be brought into earlier production by treatment with synthetic plant 
hormones (48). These hormones will also cause flowering in very young 
plants too immature to flower in the natural season. 

In the tropics the growing of plants is not restricted by frost and 
similar factors which so drastically curtail crop production in our middle 
latitudes. There is no reason, therefore, why a crop like the pineapple 
cannot be made to yield beyond its natural season, or even throughout 
the entire year. In the pineapple industry several methods have been 
in use for several decades by which growers have succeeded in extending 
the harvest season. 

It began when it was accidentally discovered in the Azores that smoke 
will force the plants into early flowering (46). Older growers in Puerto 
Rico still remember how tents were erected over the rows under which 
a smoky fire was made during the night. An investigation of the active 

Figure I. 1 he application of synthetic phuit liormones in a Hawaiian 
pineapple plantation. The hormones force all plants uniformly into flower, 
thus extending the harvest season and making picking operations more efficient. 




components of the smoke showed that its effect is due to unsaturated 
hydrocarbons, principally ethylene (35). Acetylene also was found to be 
active. These unsaturated hydrocarbons found a widespread use in the 
pineapple industry. At present their use has declined considerably, as 
they are being replaced by synthetic plant hormones. 

In 1939 it was discovered that naphthaleneacetic acid (NAA)* could 
force pineapple plants into flower (10). This compound has been accepted 
by many growers as the best of the flower-inducing agents which are 




S 60 
I 50 
u! 40 
>* 30- 

1 I"' — I 1 1 r 

J J A S N 

N D J F M A M 



Figure 2. Experiment showing flower induction in pineapples of the Red 
Spanish variety in Puerto Rico. Each month 125 plants were treated with 
5 different concentrations of naphthaleneacetic acid (NAA), 5 cc. of which was 
poured in the center of the plant. The amount of NAA per plant which gave 
maximal flower induction is shown above each point of the curve; one treat- 
ment was washed out by rain. The course of normal flowering is indicated by 
the control curve. The results show that by the use of NAA nearly 100 per 
cent flowering can be obtained throughout the entire year. 

*Editor's Note: In this paper as originally submitted, the abbreviations for 
naphthaleneacetic acid and indoleacetic acid were NA and lA. For consistency, 
these have been changed to NAA and lAA, respectively. 


available at present. In Hawaii especially, NAA is used extensively. 
Under current commercial practice approximately 25 grams of the 
sodium salt are applied per acre. This is sprayed on by large ground 
equipment using spray booms 50 feet long (Fig. i). The cost of the 
chemical is only about fifty cents an acre at present, while the total 
cost of treatment is roughly five dollars an acre. Only one treatment is 
required for flower induction. 

The advantages of forced flower induction in the pineapple industry do 
not lie exclusively in an extension of the harvest season. The much 
improved uniformity and regularity of flowering and consequently of 
fruit production is of equal importance. Prior to the introduction of the 
practice of forcing by chemical means, the earhest and the latest fruits 
produced in a field during one season might be months apart. This 
entailed repeated harvests for one and the same field. At present, with 
chemical flower induction, the entire field is forced into flower at once, 
and therefore all fruits are ripe at the same time. Thus the entire crop 
of one specific field can be harvested with a single operation. Still 
another advantage of the use of chemical flower-inducing agents is that 
they increase the yield of the fruit per acre, as a larger percentage of the 
plants is forced into flower than without treatment. 

Systematic hormone treatments have now made planned harvesting 
a reality. These treatments are so made that when the harvesting in one 
field has been completed the crew with its trucks and other machinery 
moves on to the next. The frantic rush, so characteristic of most perish- 
able fruit crops, has thereby largely been eliminated. 

Chemical flower induction not only makes it possible to determine the 
date of the harvest, but also to estimate with reasonable accuracy the 
tonnage a field will produce. A glance at Figure 3 shows that the more 
leaves a plant has the larger the fruit it will produce. Since after flower 
formation has taken place the number of leaves on the plant does not 
further increase, it is possible, by the use of graphs such as given in 
Figure 3, to predict at the time flower-inducing treatments are made the 
average weight of the fruit that wifl be produced. Even though much 
remains to be desired one may say that for the pineapple, crop control 
is more complete than for any other fruit crop. 

Flower induction with NAA has certain drawbacks. Part of these 
have already been overcome. Thus, the compound causes a peduncle 
which is more slender than usual. This may result in a loss of fruit due 



to falling over. The application of )S-naphthoxyacetic acid, applied after 
the fruit development is well under way, has corrected the difficulty (28). 
Another effect of the use of synthetic hormones applied in the course of 
fruit development is a delay in fruit maturation, causing an increase in 
fruit weight (11,28). This double hormone treatment, one for flower 
initiation and a later one for strengthening the peduncle and increasing 






CO 20-1 



-J 10 

SPRAYED WITH 010 MG NA /PLANT (00005 %) 
ON AUGUST 16,1946 ^ -^ ^ " 

HARVEST JAN- 17, 1947 







5 LBS 


Figure 3. Regression curve showing the relation between the number of 
leaves of a pineapple plant and the weight of the fruit it produces. The data 
were obtained on 100 plants of the Smooth Cayenne variety growing on 
Vieques Island (P.R.) which were forced into flower by an aqueous spray of 
0.0005 per cent NAA. The average number of leaves was 25.0 ± 0.53; and 
the average fruit weight 3.5 ± 0.07 lbs. The broken lines indicate the standard 
error of estimate. 

the fruit weight, has been adopted as a standard practice in several of the 
large plantations. 

A drawback which has not yet been overcome is that the use of 
NAA on pineapples of the Cayenne (Hawaiian) variety causes a reduction 
in the number of slips which the plant produces. These slips are branches 
which develop from lateral buds located on the upper part of the 
peduncle just under the fruit. They are important as replanting material. 
Not all pineapple varieties, however, suffer from a severely reduced 


slip production as a result of NAA treatment. It is entirely possible that 
among the auxins a compound, or combination of compounds, may be 
found which does cause flower initiation, and yet does not suppress the 
lateral bud development. 

Crop control in the pineapple is not complete without a means 
whereby it is possible to prevent precocious flowering. This has been 
accomplished by the application of large quantities of auxins. Theo- 
retically this is understandable as it seems to be a general rule that 
auxins stimulate at low concentrations, while they inhibit these same 
reactions at high concentrations (27). From a practical standpoint this 
principle has not worked out satisfactorily for the control of undesired 
flowering in the pineapple. It is not impossible that growth inhibitors 
such as maleic hydrazide (36) might supply the solution to this problem. 

As a result of the rapid progress in chemical growth regulation our 
fundamental concepts of which characteristics in a plant are desirable 
and which are not must undergo considerable change. Prior to chemical 
growth regulation it was desirable for a plant to flower readily during the 
natural flowering season, as this insured a high average annual yield per 
acre. At present, with cheap and efficient chemical growth regulation 
at our disposal, we would prefer plants which would not flower at all, 
except after chemical treatment. This would do away with the problem 
of precocious flowering, making planting throughout the entire year 
possible, thereby permitting fruit production on a commercial scale 
throughout the entire year. The Cabezona variety of Puerto Rico 
approaches these qualifications. It produces flowers readily after treat- 
ment with minute quantities of synthetic auxins, yet does not flower 
abundantly in the natural season (48). 

Physiology of Jlower formation in the pineapple. — The pineapple is the 
only plant in which the application of known compounds will cause 
flower formation. Since, with the exception of ethylene and acetylene, 
these compounds belong to the group of the auxins, a study of the 
physiology of flowering is of more than local interest. Auxins are not 
species specific, and experience has taught that when an auxin elicits a 
certain reaction in one plant species it is likely to do likewise in most 
other species. Thus, when auxin causes pineapples to flower, it is most 
likely that in other plant species also it is involved in the process of 


What do we know about the physiology of flower induction in the 
pineapple? In the first place we know that as minute amounts as 50 
micrograms of synthetic auxins per plant will cause the vegetative 
growing point of the pineapple to change promptly into a floral apex 
(48). We also know that auxin exists inside the pineapple plant, and that 
the highest concentrations of native free auxin are found in the growing 
point (54). This auxin has been identified as indoleacetic acid (22). The 
immediate precursor of this indoleacetic acid in the pineapple plant is 
indoleacetaldehyde (23), which is stored in considerable quantities in the 
bases of the youngest leaves (54). Since these leaf bases are located in 
close proximity to the apex, it is likely that free auxin of the growing 
point originates from the precursor in the leaf bases of the youngest 
leaves. It is also known that the pineapple plant contains enzyme 
systems which convert tryptophan into indoleacetaldehyde and indole- 
acetic acid. Furthermore from the pineapple leaf an enzyme system has 
been isolated which inactivates indoleacetic acid (23). It is clear, there- 
fore, that the pineapple possesses an active auxin metabolism. This is 
again manifested by the curious geotropic flower induction of the 
Cabezona variety. When the vegetative plant is put on its side, it will 
not only right itself by a normal geotropic process, but in addition it 
will go into the flowering stage completely out of season and without the 
benefit of treatment with chemicals. The phenomenon has been in- 
terpreted as being controlled by the native auxin (50). 

Under natural conditions flowering is brought about by a drop in 
night temperature during the winter (51). Photoperiodic effects seem 
of minor consequence in the pineapple. At present experimental data 
are lacking which link temperature and auxin induced flower induction. 
It has been suggested that this link may be found in the plant's organic 
acid metabolism (51). Also lacking is a link between these two types of 
flower induction and the induction by unsaturated hydrocarbons. No 
evidence was found that ethylene treatment increases the auxin level of 
the plant; Cooper (12) found no change, while Carl Leopold (personal 
communication) found a slight but consistent decrease in the free auxin 

Conclusions on the physiology of flower formation in the pineapple. — 
From the evidence available one is confronted with the facts that, on the 
one hand, an active auxin mechanism exists in the plant and that auxin 


will bring about flower initiation. Yet, on the other hand, an increase 
in the auxin level per se does not seem to be necessary for flower forma- 
tion in the pineapple. 

One might therefore conclude that it is conceivable that ethylene 
treatment increases the response of the tissues to auxin, thereby bringing 
about an increase in the physiological activity of the auxin in the plant. 
This assumption does not seem unlikely when viewed in the light of a 
recent discovery on the role of auxin in cambial growth. In the January, 
1949, issue of the Va\blad voor Biologen C. Reinders-Gouwentak (34) 
discloses that when auxin is applied to dormant branches of Fraxinus 
growth of the cambium is promoted along the entire length of the 
branch, provided, however, that this branch has been treated previously 
by ethylenechlorohydrin. Auxin by itself had only a slight effect on 
cambium growth in the immediate vicinity of a cut surface, while 
ethylenechlorohydrin by itself was entirely ineffective in this respect. 
Apparently the ethylenechlorohydrin changed the metabolism of the 
branch, making it highly responsive to auxin. Similarly, ethylene might 
bring about metabolic changes in the vegetative apex of the pineapple, 
making the tissue more responsive to the auxin it contains, thereby 
bringing about flower formation. 

Indirect flower induction in litchi. — In the Hawaiian Islands litchi trees 
flower and fruit so irregularly that it is often believed that this crop has 
few economic possibilities. It was found, however, that in litchi too, 
flowering may be induced by treatment with growth-regulating chemi- 
cals. In contrast to the pineapple where auxins promote flower formation 
directly, in the litchi flower formation is due to the suppression of lush 
young shoots by auxins. A concentration of 50 ppm. of NAA applied 
before October immediately stops vegetative development (37). As a 
result of such treatments 88 per cent of the trees flowered, as compared 
to only 4 per cent in the controls. This indirect type of flower induction 
in litchi is probably due to a building up of nutrients and growth factors 
which normally would have been translocated away to the rapidly grow- 
ing vegetative branches. This eflect is comparable to flower induction 
by girdling of juvenile citrus trees (19), and of the rotenone-producing 
Peruvian Lonchocarpus (14). Here as well as in litchi trees flower induc- 
tion apparently is also the result of the accumulation of nutrients and 
growth factors. 

Shortening of the ripening period of the fig. — Although most commercial 


fig varieties are parthenocarpic, the Calimyrna fig requires pollination 
by a specific wasp. It has been shown that when the mature pollen- 
receptive inflorescences are sprayed with suitable auxin preparations, 
the Calimyrna fig also can be forced into producing parthenocarpic 
fruit (2). This in itself might not be of great physiological interest were 
it not for the fact that some of the auxins reduce the normal ripening 
period of the fruit so drastically that the average 120 day period is cut 
in half. This was the first example where auxins cause a drastic reduction 
in ripening time of fruit; recently it has also been shown in apples and 

The case assumes further interest if the active compounds are examined 
(3,16). 4-Chlorophenoxyacetic acid will cause ripening of the fruit with- 
out materially speeding up the ripening period. Except for the absence 
of seeds parthenocarpic fruit produced with this chemical resembles that 
produced by normal pollination. It is entirely possible that this treat- 
ment will replace the cumbersome caprification by the wasp. Quantities 
of approximately 100 grams of 4-chlorophenoxyacetic acid per acre, 
at 40 to 60 ppm., are required. 

When an extra chlorine atom is added to the molecule so that 2,4- 
dichlorophenoxyacetic acid is formed, a compound of considerably lower 
activity than the original 4-chlorophenoxyacetic acid is obtained. The 
activity of this dichloro compound is so low that it was originally thought 
to be inactive. Recently it was found that it can bring about partheno- 
carpy in the fig in concentrations in excess of 250 ppm. (40). When still 
another chlorine atom is added to form 2,4,5-trichlorophenoxyacetic 
acid, a compound of unusual physiological activity is obtained. Not 
only does it cause parthenocarpic fruit setting at 10 ppm., but in addition 
it hastens the ripening period. As is shown in Figure 4, curve A, it does 
so by omitting the rest period between the initial and the final stage 
of rapid fruit growth. The trichloro compound also will accelerate 
ripening when appHed to pollinated Calimyrna fruit (Fig. 4, curve B), 
and to naturally parthenocarpic fruit of varieties such as the Black 
Mission. Why such closely related chemicals have such widely different 
effects, which, however, are more quantitative than qualitative, is not 
known at present. 

The double sigmoid growth curve C of Figure 4 reflects two periods 
of rapid fruit growth separated by a rest period. Many explanations have 
been offered for the occurrence of this rest period. In view of its successful 



elimination by treatment with a suitable auxin, it would seem justified 
to conclude that a low auxin level is the causal agent (i6). 

Increased fruit ripening in the banana. — Speeding up of fruit ripening 
by auxins has also been observed in bananas after they have been removed 
from the plant; a thorough spraying with .02 to .05 per cent of 2,4- 
dichlorophenoxyacetic acid replaces the customary ethylene-induced 

Figure 4. Experiment showing acceleration of fruit ripening of the Cali- 
myrna fig by treatment with an aqueous spray containing 25 ppm. of 2,4,5- 
trichlorophenoxyacetic acid (2,4,5-T). Normal fruit growth is shown by the 
solid curve. The first part of this curve is due to growth of the fruit (syconium) 
prior to pollination, which took place about June 30. Without pollination 
or suitable auxin treatment fruits drop off shortly after this time. Treatment 
at this time of the nonpollinated fruit causes growth represented by curve A. 
Pollinated fruit treated on July 31 immediately came out of the rest period 
and followed curve B. (3). 

ripening (30). The effect may be attributed to the known stimulatory 
effect of auxins on the amylolytic enzyme system in plants (29). 

Uses of auxins on citrus fruit. — Preharvest drop of citrus fruit is a 
serious problem, and when it was shown (20) that NAA can control 
a similar drop in apples, attempts were made to reduce drop in citrus 
by similar means. These attempts all met with failure until 2,4-D was 


tried (41). This compound persists longer in the plant than NAA which 
may account for its effectiveness in citrus. Concentrations of about 
8 ppm. of 2,4-D applied as a drenching aqueous spray to the trees have 
reduced preharvest drop in Valencia and Navel oranges (41), as well as 
in grapefruit (42). 

Further beneficial effects of 2,4-D were discovered. Thus, it turned 
out that treated fruit after storage kept better than the nontreated con- 
trols because less black button developed. In addition the fruit stems of 
treated plants showed less die-back (43). The storage life of lemons also 
could be increased by 2,4-D treatments of the fruit before storage. 

One of the difficulties often associated with insecticidal oil emulsion 
sprays on citrus has been an increased drop of leaves and immature 
fruit. It was now found that the addition of 2,4-D to these emulsions 
could check this drop. 2,4-D is now added to oil emulsions for a dual 
control program (43) ; not only does it check the fruit and leaf drop, 
but it reduces fruit stem die-back, and during storage cuts losses due to 
black button. An ester form of 2,4-D sprayed on at 4 ppm. in terms of the 
final emulsion is most efficient. Related chlorinated phenoxyacetic acids 
seem to have effects comparable to 2,4-D; 2,4, 5-T may even be more 
effective (39). 


After the discovery that auxins promote root formation on cuttings 
(45) general use has been made of this principle. One might say that 
auxins have been tried on almost every plant for which vegetative 
propagation might offer advantages. The results of all these trials, which 
include many tropical species, have been summarized in extensive tabular 
form (1,30,31,44). It is intended to discuss here first an example of the 
use of growth substances in the vegetative propagation of a tropical 
crop, to be followed by some fundamental aspects of the process of 
root formation as worked out on a tropical plant in the tropics. 

Root formation on cacao cuttings. — The use of synthetic plant hormones 
for the promotion of root formation on cuttings as applied in the tropics 
can perhaps be illustrated best by its use in the vegetative propagation 
of cacao in Costa Rica. In order to appreciate the importance of cacao 
in the economy of Central American agriculture a few words must be 
said about the banana culture with which the cacao culture is closely 
allied. When the banana industry came into being in this area at the 


beginning of the present century the plants grew well anywhere in the 
coastal lands. In 19 10 a wilt was reported which started to invade 
plantations especially in the moist areas with acid soils. By 1920 this 
wilt, caused by Fusarium oxysporum cubense, had become a major prob- 
lem which finally drove the industry from the original plantations (32). 
Thus, new jungle had to be cleared for new banana plantations and new 
crops had to be found to occupy the abandoned banana lands. Cacao 
proved to be one of several crops suitable for replanting the old banana 
lands. In order to make culture profitable it is necessary that the planta- 
tions consist of trees which combine a high yield of high quality fruit 
with a reasonable resistance against diseases. This is only practical at 
present by the vegetative propagation of parent plants of proven out- 
standing performance in these respects. In cacao this propagation is 
done most profitably by cuttings, which brings us back to our starting 

When cuttings are taken from a cacao tree without any special pre- 
cautions, and then planted in a propagator, they will wilt and fail to 
form roots. The reason for this is that the bark of the cacao tree contains 
mucoid material. When the cutting is taken from the branch this will 
ooze out and plug the xylem vessels, thus causing the death of the cutting 
by water starvation. When the cut stem ends are kept in water before 
being transferred to the propagator much of this trouble can be avoided. 
This is an example of how a seemingly minute detail may spell success 
or failure of a technical process. 

There are many other conditions which have to be observed for the 
successful rooting of cacao cuttings (8,9,18). Without auxin treatment, 
however, root formation will still be unsatisfactory. When, however, 
cuttings are treated with a suitable auxin prior to being placed in the 
propagator, the following benefits result: roots are formed faster; a larger 
percentage of the cuttings forms roots in a shorter time; and the cuttings 
are more easily transplanted because under auxin treatment a compact 
root system composed of many short roots results. These are the reasons 
that synthetic plant hormones have become an integral part of propaga- 
tion by means of cuttings. 

The most widely used of the synthetic auxins for this purpose is 
indolebutyric acid (IB). It is used either alone or in combination with 
other auxins such as NAA (24). In practice it is most frequently formu- 
lated as a powder. The moistened ends of the cuttings are dipped into 

Figure 5. Coffee cuttings showing the necessity of leaves for root formation. 
All cuttings were treated at the base by dipping into 50 per cent ethyl alcohol 
containing 2 mg. of indolebutyric acid (IB) per cc. They were taken from 
I year old plants and photographed 50 days later; the defoliated cuttings 
had regenerated some small leaves at that time. The experiments took place 
in Puerto Rico. 


this powder before being placed in the propagator. The author has 
found the alcohol dip method (13) also highly practical. The cutting 
ends are dipped in 50 per cent alcohol containing a relatively high 
concentration of a suitable auxin, such as 2 mg. of IB per cc. 

Recently attention has been called to the effectiveness of some of 
the chlorinated phenoxy acids (25). In view of the striking results 
that this class of synthetic auxins gave in the citrus and fig industry 
in the regulation of fruit production, it is not unlikely that they may 
find application in some cases where IB or NAA failed to induce roots. 

Some physiological aspects of auxin action in cuttings. — Cuttings of 
many plant species have responded to auxin treatment, yet many remain 
that as yet have not produced roots. A number of woody species such 
as Hevea, coffee, and mango will form roots when the cuttings are taken 
from young parent plants but fail when they are taken from older trees. 
The reasons for failure to root are not well understood at present. It 
would seem that it is not due to lack of auxins but rather to lack of 
proper cofactors. Auxins will act only when cofactor requirements are 
satisfied, or in other words, when the tissue is in reactive condition. 
There are many examples of this in auxin physiology. Auxins will cause 
tumor formation only when the tissue has been prepared by Bacillus 
tumefaciens (see 47). Auxin will cause cambial growth only if the tissue 
has been made reactive to it; this can be done by treating dormant 
branches with ethylenechlorohydrin (34). In cuttings too, auxin will 
cause root formation only when the tissue has been put in a responsive 
condition. What this involves has been analyzed in Hibiscus cuttings 
(52) and will be briefly discussed. 

It is known by horticulturists that the presence of leaves on cuttings 
greatly increases the chances of successful root formation (15). This 
effect of leaves is demonstrated on coffee cuttings in Figure 5. It will 
be seen that even though both the leafy and the defoliated cuttings 
received the same auxin (IB) treatment, only the leafy cuttings re- 
sponded with root formation. This effect is further elaborated in Figure 
6 for Hibiscus cuttings. In these the terminal bud was removed in order 
to avoid complications with the naturally produced auxin. Without 
leaves and without auxin (IB) treatment no roots were formed, as 
indicated by point A in Figure 6. Without auxin treatment an increase 
of the number of leaves left on the cuttings is also ineffective for root 
formation (curve AB). Defoliated cuttings treated at the base by dipping 



them in 50 per cent alcohol containing 2 mg. of IB per cc. also failed 
to root (point C). However, when this auxin treatment was given to 
leafy cuttings, roots were formed in proportion to the number of leaves 
present on them (curve CD). Leaves, then, provide factors which 
together with auxin cause root formation. At present we have also 
learned something about the chemical nature of these factors. 

In order to investigate which factors were contributed by the Hibiscus 
leaves to the cutting, defoliated Hibiscus cuttings were treated at the 
base with a number of compounds which conceivably might be auxin 
cofactors. This was followed by the usual IB treatment. One of the first 
compounds to be tried as a possible cofactor was sucrose. It has been 
known for years to stimulate root formation on cuttings (17), and was 
indeed found active in the defoliated, IB-treated ////^/Va/^ cuttings (52), 
Yet the number of roots thus formed was still far below that formed 



Figure 6. Graph showing how root formation on Hibiscus cuttings depends 
upon both auxin and the presence of leaves. AB shows that without auxin 
leaves are ineffective. CD shows that in auxin treated cuttings (IB treatments 
as in Figure 5) the number of roots formed increases with the number of 
leaves left on the cutting. F shows that the effect of leaves in auxin treated 
cuttings can be replaced by treatment at the base with 4 per cent sucrose and 
0.1 per cent ammonium sulfate; sucrose and 10 ppm. arginine has a similar 
effect. AE (dotted line) shows that without auxin treatment sucrose and 
ammonium sulfate are ineffective, even in the presence of leaves. (52). 


on leafy cuttings. Next it was found that ammonium sulfate, given 
together with sugar, would increase the number of roots of IB-treated 
defoliated cuttings so much that it was equal to that of IB-treated leafy 
cuttings. This is represented by point F in Figure 6. The same treatment 
given to leafy cuttings did not further increase the number of roots 
formed (curve FD). 

Since it is unlikely that leaves would contribute ammonium sulfate 
to the cutting, a search was made for more likely substances. Among the 
most promising of these is arginine. When tested in the presence of 4 
per cent sucrose 10 ppm. of arginine would produce an effect equivalent 
to 1,000 ppm. of ammonium sulfate (52). Since both arginine and sucrose 
are natural constituents of the plant it seems reasonable to assume that 
one of the functions of the leaf is to provide cuttings with these com- 
pounds, which then together with auxin cause root formation. 

Arginine has recently also been recognized as a co-factor for auxin 
action in the elongation of the Avena coleoptile (4); it has a function as 
a high energy phosphate acceptor and accumulator. This would make 
its action as a cofactor to auxin understandable because auxin itself 
appears to be intimately connected with the phosphate metabolism of 
the plant, possibly with the transfer of high energy phosphate. 


With respect to dollar volume of sales, 2,4-D for weed control un- 
doubtedly constitutes the most important use of a synthetic plant hor- 
mone. In 1948 an estimated 16,000,000 pounds of 2,4-D compounds 
were manufactured and sold at an average price of $0.75 per pound 
(21). This compound is most frequently used as a selective herbicide on 
crops belonging to the family of the Gramineae. Consequently, it is 
finding its major use in the tropics in the culture of sugar cane (55). 
The aerial portions of sugar cane are practically completely resistant 
to 2,4-D sprays (49), but its roots are quite sensitive (7). 

Against susceptible tropical sugar cane weeds such as Commelina and 
Ipomea, 2,4-D is highly eflfective. The small dosages necessary to kill 
these weeds make low-volume applications of 2 to 4 gallons per acre 
feasible. When the acreage involved is large enough such low-volume 
sprays can be best applied by plane. Most efficient of these sprays is 
2,4-D in an ester form dissolved in oil, which, however, can be flown on 
safely only at times when there is a minimum danger of drift, and, as an 


added precaution, when there are no susceptible crops near by. From 
the point of view of drift damage to susceptible crops dust formulations 
of 2,4-D are the most dangerous, and within the continental United 
States the Civil Aeronautics Commission will not grant licenses for 
2,4-D dust applications. In the tropics dusts are still used. In areas with 
dusty soils there is considerable danger of 2,4-D drifting to adjacent 
areas even though the substance may be applied in an aqueous form. 
The finely pulverized soil serves as a vehicle which carries the absorbed 
2,4-D to localities where it is not desired. 

The exclusive use of 2,4-D as a selective herbicide brings with it the 
danger that the weed population which is effectively eradicated by it 
will be replaced by a group of plant species resistant to it (53). Thus it 
was observed in sugar-cane fields in Puerto Rico that the easy-to-kill 
Comtnelina was being replaced by the more resistant Poinsettia hetero- 
phylla and highly resistant grasses such as Digitaria. 

2,4-D is used also as a so-called pre-emergence spray. In sugar-cane 
technology this term is used with reference to the emergence of the 
weeds, while in the middle latitudes it usually refers to the crop emer- 
gence. For pre-emergence purposes the 2,4-D is applied in such large 
dosages that it prevents the germination of weed seeds, including those 
of grasses. Amounts up to 5 pounds of 2,4-D per acre are applied. 
Generally speaking no damage is done to the sugar cane if this amount 
is not exceeded. Whether or not damage is done to the crop by pre- 
emergence applications of 2,4-D depends greatly upon a combination of 
soil properties and rainfall. These conditions are quite well understood 
at present due to the work done at the Hawaiian Sugar Planters Experi- 
ment Station (7). 

The principle upon which pre-emergence weed control in sugar cane 
depends is that the 2,4-D stays in the surface layers of the soil. When this 
condition is realized the 2,4-D will prevent weed seeds from developing, 
while it will not interfere with the cane roots which are growing below 
the zone in which 2,4-D is present. The shoot of the cane which is 
relatively Insensitive to 2,4-D will penetrate through the 2,4-D layer 

The Hawaiian investigators showed (7) that under the influence of 
rain 2,4-D moves downward through the soil as a concentration front 
comparable to those seen in chromatographic columns. In some soils 
this 2,4-D front moves very slightly even after much rain, while there 


are other soils in which the 2,4-D concentration front moves downward 
to a considerable extent with only a slight amount of rain. Between these 
extremes a number of intermediate soil types exist. It is obvious that 
when conditions exist which will bring the 2,4-D front within the zone 
of growing sugar-cane roots, the latter are killed and the crop is lost. 
Such conditions may occur after heavy rains in soils which fix 2,4-D 
rather well; or again, they may occur after light rains in soils which hold 
2,4-D only poorly. Ideal conditions for pre-emergence use of 2,4-D are 
those in which the herbicide remains fixed to the upper soil layers, thus 
preventing weed growth without interference with the deeper cane 

Relatively little definite knowledge is available on why 2,4-D is such 
an effective herbicide; neither do we know upon which properties its 
action as a selective herbicide rest. It is clear, however, that 2,4-D is 
an auxin and as such is translocated within the plant. It can reach the 
sensitive meristematic regions of the plant through normal channels 
because during the time of transport it is not toxic to the plant and for 
this reason does not interfere with its own translocation as is so often 
the case with some of the more violent plant poisons. After it arrives at 
the site of action, 2,4-D is not easily inactivated like the native auxin, 
indoleacetic acid. This persistence inside the plant is also in all probability 
a contributing factor to the high efficiency of 2,4-D as an herbicide. 
The depletion of carbohydrate reserves associated with the effect of 
2,4-D on plants is not believed to be the primary cause of its phyto- 
toxicity (33,38). Interference with the aerobic respiration seems not 
unlikely as a primary cause. Germinating seeds (26) as well as bacteria 
C56) which require free oxygen for their respiration are smothered by 
2,4-D, while those organisms capable of anaerobic respiration are not 
affected to any significant degree by it. For a more extensive discussion 
on tropical weed control the reader is referred to (52) ; for tropical weed 
species, to (58) ; and for the reasons for the physiological activity of 2,4-D 
as a herbicide, to (57). 

Concluding Remarks 

In conclusion one can say that the outstanding uses of synthetic 
plant hormones in the tropics are: the use of naphthaleneacetic acid 
in the pineapple industry, making crop regulation possible to an extent 
unequaled in other fruit crops; the use of indolebutyric acid in the whole- 


sale propagation of cacao, making it possible to plant thousands of acres 
of young plants all with a proven record of high yield and quality 
coupled with disease resistance; the use of 2,4-dichlorophenoxyacetic 
acid as a weed killer in the sugar-cane industry, making it possible to 
eradicate weeds which could not be conquered by hoe and machete. 

One may well ask what the future will bring. Here one has to dis- 
tinguish between the immediate future and the years a generation or 
more beyond that. In the immediate future we may expect that an 
increasing number of uses will be found for plant hormones in the tropics. 
Tropical agriculture is expanding as an inevitable result of an increased 
world population. Plant hormone technology is also rapidly advancing. 
In addition, fundamental knowledge is slowly but surely increasing in 
the auxin field. 

The outlook for the remote future seems less bright. Here no specific 
reference is made to plant hormones but to new applications of plant 
physiological research in general. These applications, so new that one 
cannot imagine them at present, depend invariably upon results obtained 
in fundamental research, that type of research which is undertaken with 
the sole aim of extending the horizons of our intellect. One could liken 
the relation between applied and fundamental research to the relation 
between logging operations and reforestation. The forests planted today 
will yield forest products tomorrow. On examining what fundamental 
research, of the type we are discussing, is being carried on today, we 
cannot be over-optimistic as far as plant physiology is concerned. In 
the tropics less fundamental research than ever is going on, even though 
technological research there is flourishing. In the middle latitudes, once- 
great European sources of knowledge have completely ceased to be 
productive. In our own country, despite a veritable boom in technological 
research, truly fundamental research in plant physiology is limited to a 
relatively few institutions. There is danger, indeed, that in our great 
desire for technological progress we are neglecting to nourish the source 
of it all, thereby slowly starving the goose that laid the golden eggs. 


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13. , ibid., 44:533 (1944). 

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16. Crane, J. C. and Blondeau, R., Proc. Am. Soc. Hort. Sci., 54:102 (1949). 

17. Curtis, O. F., Am. J. Botany, 13:549 (1918). 

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Crops Techn. Comm /j (1940). 

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23. , ibid., 20:367 (1949). 

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Inst., 11:143 (1940). 

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(May II, 1948). 

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30. Mitchell, }. W. and Marth, P. C, Growth Regulators (University of 

Chicago Press, 1947). 

31. Mitchell, J. W. and Rice, R. R., USDA Miscell. Public. No. ^g^ (1942). 

32. Powell, }. S., Agriculture in Costa Rica (Pan American Union, Washing- 

ton, D. C, 1943). 

33. Rasmussen, L. W., Plant Physiol., 22:377 (i947)- 

34. Reinders-Gouwentak, C, a., Va\blad v. bioL, (Dutch) 29:9 (1949). 

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39. Stewart, W. S., Citrus Leaves, 28(1 1):6 (1948). 

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50. , and Cruzado, H. J., Am. J. Botany, 35:410 (1948). 

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Growth Substances 
in Vegetative Development 

The Role of Growth Substances in Vegetative 
Development as Exemplified in Tissue Cultures 


GROWTH substances and their manipulation have played a con- 
siderable role in the development of plant tissue cultures as such, 
and in the control of their development and differentiation. Indeed 
Haberlandt's theory of wound hormones, leptohormones, and other 
growth substances arose originally out of his attempts to explain the 
difficulties encountered by himself and others in their attempts to 
establish plant tissue cultures. 

Vegetative tissues of most if not all plants, when excised and placed 
on a moist substratum whether nutritive or not, will undergo a consider- 
able initial enlargement due chiefly to hyperhydric increase in size of 
the surface cells. There is in general very little polarity to this enlarge- 
ment if the fragment is small and if it is taken from a relatively un- 
differentiated region such as the pith, the cortical parenchyma, the 
interior of tubers or fleshy roots, etc. Larger or better differentiated 
masses may show some tendency to localized or even tubercular en- 
largement, but this is exceptional in the initial phases. This initial 
expansion is largely a physical response due to alteration in stresses, 
in the respiratory processes of the traumatized and exposed surfaces, 
in osmotic patterns, and so on. It has nothing whatever to do with 
normal proliferative growth. Indeed, tissues of some plants, such as 
those from mature tubers of Helia7rthiis tiiberosa, do not ordinarily show 
even this initial enlargement. These unresponsive tissues are now 
thought to be almost devoid of residual auxins, and the more usual 
initial enlargement is believed to be due, in part, to the activity of 
auxins which are present in most excised tissues. The ratio of available 


auxin to exposed surface is supposed to account for the much more 
widespread and more pronounced enlargement obtained with relatively 
large explants. 

Where present, this initial phase is commonly followed by the for- 
mation of an irregular cambium immediately below the surface, often 
involving cells which have undergone some enlargement.This cambium 
cuts off cells to the outside which are rapidly suberized. There is 
usually little or no new internal tissue formed. The cells of the interior 
of the fragment become woody, chlorophyll is formed in the outer- 
most layers if the explants are exposed to light, and the fragment, 
although it may enlarge to as much as six or eight times its original 
volume, finally settles into a static condition in which it may remain 
alive and green but not growing for several months. If small fragments 
have been used enlargement is generally quite regular, the original 
outlines of the explant being recognizable after many months. Large 
fragments may give rise to localized, tubercular growths along with 
nongrowing or necrotic areas. If such a fragment is divided into two 
or four pieces and the parts are replanted on fresh substratum, the cut 
surfaces will undergo a similar series of changes but will again come to 
rest. The presence or absence of externally supplied nutritive substances, 
sugars, salts, and the like, apparently plays no role in this phase of de- 
velopment, which is entirely dependent, except for the moisture 
required, on residual suppUes within the explant. It is sometimes pos- 
sible to continue this subdivision through five or six passages, but it 
is not possible to establish continuously growing strains. This de- 
velopmental pattern has been observed repeatedly since the days of 
Haberlandt and Rechinger. It is quite consistent and has led more than 
one early investigator to make premature claims to having established 
tissue cultures, before it was realized that a few such passages are not 
sufficient to establish the ability of such cultures to grow continuously. 
Neither Gautheret (1934 — Salix) nor myself (1939 — tomato and beet) 
has been free from this fault. 

In 1939, however, Gautheret and Nobecourt independently showed 
that, if a growth substance such as indoleacetic acid or naphthalene- 
acetic acid is added at a suitable concentration to a complete nutrient 
either at the time of the initial excision or as a substratum for the first 
subcultures (that is to say after completion of the initial residual 
growth) when growth becomes dependent on external materials the 


pattern can, in many cases, be radically changed. The degree and nature 
of the change depend on the concentration and activity of the growth 
substance used, as well as on the tissue involved. At low concentrations 
the tendency to initial hyperhydric enlargement is only sHghtly accen- 
tuated. The activity of the cambium, however, is greatly enhanced. 
Instead of being relatively superficial, the cambium tends to develop 
at a much greater depth in the fragment and to involve not a single 
layer but a considerable region. Instead of only a few superficial cork 
cells and deeper lignified masses, there is a prohferating region which is 
continuous both tangentially and radially, the new-formed cells tend 
to remain for a considerable time thin-walled, and there is formed an 
erratic but nevertheless clearly defined phloem on one side and xylem 
on the other. The initial arrangement of these layers may vary with 
the region from which the tissues were excised, but by gradual re- 
arrangement the phloem always comes finally to occupy the superficial 
position. In addition the enlarged outer hyperhydric cells frequently 
undergo disoriented divisions resulting in the formation of nodular 
masses which, when they become sufficiently large, develop their own 
peripheral cambium and proceed as distinct centers of growth. Isolated 
areas of meristematic activity also often arise deep within the mass, 
especially in the neighborhood of necrotic areas. These localized centers, 
either superficial or deep-seated, give rise to the tuberculate and often 
loose and friable character of many rapidly growing plant tissue cultures. 
Subcultures can be made easily from such masses without important 
trauma. Indeed new growths frequently arise where a few cells have 
been accidentally dropped in manipulating the larger masses. 

Two facts about these cultures appear at this stage of our ignorance 
to be of special importance. First, these cultures are still relatively 
disorganized, the only evidence of polarity being the radial polarity 
exemplified in the xylem-cambium-phloem orientation. In the second 
place the whole development of these cultures and the contrast between 
the static surviving masses discussed first and these rapidly growing, 
highly active disorganized masses has resulted from and is dependent 
on the presence of a certain amount of auxin in the substratum. Just 
how this auxin functions, aside from retarding differentiation (depo- 
sition of cell wall material) and enhancing mitosis (deposition of 
cytoplasmic proteins), is not at present clear even in a general way. 

If the concentration of growth substances is increased somewhat more 


the superficial hyperhydric response is further accentuated. The sub- 
surface cambial activity is initially not much changed, but instead of 
continuing unmodified for long periods these dividing regions are 
quickly organized into large numbers of well-defined root primordia. 
There are usually no stem growing points, although Nobecourt has 
reported the repeated, sporadic, and unexplained appearance of stem 
growing points and leaves on his carrot cultures. They are rare or 
entirely wanting in other laboratories and may indicate no more than 
a difference in varieties of carrot used. Such rooted cultures are, of 
course, quite useless as tissue cultures. They do, however, give us im- 
portant leads in the study of auxin function. These cultures are not 
merely differentiated, but this differentiation possesses a definitely polar 
orientation. Moreover it is clear that here large numbers of roots have 
arisen without the presence of leaves or similar tissues from which 
rhizocahnes in the classic sense could arise. The function of the hypo- 
thetical rhizocahne has been taken over, in dramatic manner, by either 
the heteroauxin itself or, secondarily, by substances arising in undif- 
ferentiated stem or root tissue under the influence of externally supplied 

If, finally, the concentration of growth substance is still further in- 
creased, both polarity and organization are completely lost, and there 
remains only an exaggerated and pathological hyperhydricity. Enormous 
superficial cellular vesicles are formed, giving rise to a rapid and isodia- 
nietric initial enlargement of the explant. These vesicles no longer appear 
to be capable of cell division so that the initial rapid mechanical enlarge- 
ment is followed shortly by necrosis and death. This behavior pattern 
at high auxin concentration is probably one of the facts responsible for 
the weed-kiUing action of such substances as 2,4-D, although excessive 
prohferation at lower auxin concentrations and, in the absence of in- 
creased nutrient supplies, subsequent nutritive exhaustion is undoubt- 
edly another major factor in weed killing. 

These matters of cell size, cell number, and cell arrangement, of 
organization versus disorganization, of organization versus gross expan- 
sion, of continued function versus necrosis, and similar contrasting 
aspects of behavior can all be shown in tissue cultures to be under the 
control of growth substances. This is most clearly defined in the Jerusalem 
artichoke {Helianthus tuberosa) but is also demonstrable in varying 
degrees in tissue cultures of willow, hawthorn, grape, carrot, and many 


Other plants. The responses observed in these plants are cellular, gen- 
eralized in character, and bear little apparent resemblance to the controls 
which auxins mediate in plants in nature, although these cellular ac- 
tivities are undoubtedly primarily responsible for the more specific 

Much more specific responses can, however, be studied in tissue 
cultures of certain plants. If roots of dandelion, chicory, or related plants 
are cut up and the bits placed on moist sand or on a simple nutrient 
they will form buds on the upper surface and roots on the lower. This is 
true of almost any explant, no matter how small, if it will survive at all. 
Addition of auxin to the substratum in progressively increasing con- 
centrations results in a progressive blocking of bud and root formation 
together with a considerable increase in the mass of callus formed on the 
surface. If auxin is applied locally to the upper surface of an explant in 
the form of an agar block, the blockage of bud formation and increase 
in callus development can be shown to center around the appHed auxin 
and to become weaker with distance therefrom. The same result can 
in part be produced by implanting a preformed bud instead of using an 
agar block. The presence of the bud not only blocks the development 
of neighboring buds but also induces formation of a vascular strand which 
will connect the implant either with roots that may already be present 
or with newly formed roots. It does not, however, modify the amount 
of callus formed. This induction of vascular strands will take place 
through a cellophane sheet. Every indication is that it is mediated by 
some substance or substances of the general nature of the auxins. 

This kind of approach has been extended by a double use of the tissue 
culture approach. Kulescha has shown by direct Avena tests that plant 
tissue cultures contain auxin in concentrations which are directly pro- 
portional to their rate of growth in culture and their degree of resem- 
blance to tumors. Normal tissues which will not grow in culture without 
added auxin have little or none present. Tissues which have been 
habituated by prolonged cultivation on an auxin medium so that they 
are no longer dependent on external supplies show moderate levels of 
auxin content, while sterile crown gall tissues which grow in culture at 
a rapid rate are found to have a high auxin content. If, now, each type 
of tissue is grafted into the surface of slices of chicory, the inhibition of 
buds and the extent of callus formation induced parallel the observed 
auxin content of the implanted tissues. Normal tissue implants induce 


little or no alteration in the callusing and budding behavior, while crown 
gall tissues completely suppress budding for some distance and bring 
about, per contra, an exuberant callus formation. Gautheret and Camus, 
and de Ropp, claim that the new callus itself possesses tumor quaUties, 
but this should be re-examined. 

From what I have said I think it is clear that the question of the role 
of auxins in the vegetative development of tissue cultures has not yet 
received the investigative attention that it deserves. Techniques for 
such studies do exist, however, and it is to be hoped that they will be 
utilized in the near future. 

Factors Influencing the Growth of Plant Embryos 


THE growth of plant embryos has been the subject of many in- 
vestigations during the last half century. It is a field of great 
theoretical interest particularly because of the opportunity that is pro- 
vided for observing the development of adult structures from the cells 
that are initially the least differentiated. Two major problems have been 
considered in this connection. First, how is the embryo fed, which of 
the surrounding tissues contribute directly to its support, and what is 
the nature of the food material it receives? Second, to what extent is the 
pattern of embryonic growth autonomous, what is the role of the other 
seed parts in the control of embryonic development, and by what 
mechanisms is this control exerted? Solutions to these problems have 
been sought through both histological and physiological investigations, 
and by use of both natural and artificial conditions of growth. 

A histological study by Nutman (8) deals with the evidence for the 
formation of growth-promoting substances in the developing rye kernel. 
He observed within the embryo sac and neighboring tissues a series of 
discontinuous growth phases associated with characteristic degeneration 
in certaiii parts of the developing fruit. Thus, soon after fertilization 
the synergids degenerate while simultaneously the antipodals divide; 
then when the antipodals degenerate, the endosperm nuclei begin to 
divide near the degenerating tissue, a characteristic nucellar strip under- 
goes a new development, and the embryo enlarges greatly. As the 
nucellar strip, in turn, is absorbed, the aleurone layer of the endosperm 
is formed. Finally, during the same general period a strict time associa- 
tion may be observed between the appearance of the stem, root, and 

Editor's Note: Paper No. 408 from the Department of Genetics, University of 


coleoptile primordia and the local degeneration of the endosperm in 
the regions of embryonic differentiation. The author postulates that 
hormones are liberated from the degenerating tissue, and that they 
influence nuclear divisions, cell enlargement, and tissue differentiation. 
Among other studies of this general problem have been those of 
Brink and Cooper (2). Their approach has been to observe a variety of 
cases of arrested seed development, and to attempt from an analysis of 
these to arrive at an understanding of the important aspects of normal 
development. They conclude that in the angiosperms the endosperm 
typically plays an important part in the nutrition of the young embryo. 
The endosperm, because of its genetic constitution, seems well suited 
to compete successfully with the integuments which are at the start 
bigger than the embryo or endosperm, and which are themselves 
actively enlarging. The aggressive endosperm is able to establish itself 
as a physiologically dominant tissue in the seed and simultaneously to 
perform for the young embryo certain nutritional functions of which the 
embryo is not yet capable. 

The endosperm is not, however, always of prime importance, as shown 
by Cooper and Brink (3) in their study of the common dandelion. This 
plant is an autonomous apomict, in which no fertilization occurs, either 
of the egg or of the central nucleus. In contrast to a related sexual 
species with the typically precocious endosperm, there appears to be 
with this material no regular interdependence of the embryo and endo- 
sperm. Many of the seeds show embryos that are much more advanced 
than the corresponding endosperms. One regular feature in the common 
dandelion is, however, the presence in the ovule, at the time the embryo 
starts developing, of an extensive amount of stored food material. 

These examples are but a few of many studies which have shown that 
the very young embryo is not independent but must rely for its nutri- 
tion on the presence of some other tissue, be it endosperm or a functional 
substitute for it. The actual compositions of the materials used by the 
embryo have not been determined, but indirect evidence points to the 
importance both of hormones and of substances of direct nutritional 

The control of the growth of older embryos, especially in the stages 
just prior to maturation, is also a question of interest. During the later 
portion of embryonic growth, embryos are generally autotrophic with 
respect to any special growth substances. They are often capable of 


immediate germination, as shown by experiments with excised embryos 
in vitro and by germination tests on unripe seeds. The interest in the 
tissues surrounding the embryo now centers on the question of why an 
embryo able to germinate does not do so, and why instead it continues 
to grow embryonically until it is fully mature. This question is considered 
in a recent review paper by Evanari (5). A number of factors have been 
found which may prevent premature germination. These include high 
osmotic pressure, unfavorable pW, and a variety of chemical substances 
such as ethylene, organic acids, unsaturated lactones, aldehydes, essential 
oils, and alkaloids. They occur in all parts of plants, including the seed 
coat, endosperm, and the embryo itself. Their effects are mostly non- 
specific, although there are variations in the strength of the inhibition 
response and in the subsequent reaction of the posttreatment scedhng. 
The second approach to the whole problem of plant embryo growth is 
that of experimentation with artificial media, that is, the cultivation 
of plant embryos in vitro. It is generally true that any plant embryo, if 
old enough at the time of excision, may be successfully cultivated on a 
medium containing only water, agar, minerals, and sugar. Qjnversely, 
it has been found that the embryos of all species which have been tried, 
if young enough when excised, will fail to grow on any artificial medium 
now known. The experiments to be discussed here concern embryos 
between these two age limits. Van Overbeek, Blakeslee, and Conklin 
(11,12) found that growth of immature Datura embryos was benefited 
by the addition to the medium of an arbitrary mixture of growth factors 
including glycine, a number of vitamins, and several other organic 
nutrients. These supplementary substances, however, did not aid in 
the growth of embryos which were less than about .5 mm. long when 
excised. These still failed to develop. The further addition to the medium 
of unautoclaved coconut milk, a convenient source of endosperm, made 
it possible to grow undifferentiated embryos as small as .1 mm. long. 
Further evidence led to the conclusion that there are two factors present 
in coconut milk: a heat stable factor (probably auxin) causing growth 
but not differentiation, and a heat labile factor which induces differentia- 
tion. The material responsible for the latter effect has not been identified, 
but Blakeslee and Satina (i) have since found that a factor of comparable 
activity is present in malt extract. Haagen-Smit and co-workers (6), 
testing these findings on other material, found that coconut milk was 
not a limiting factor for the growth of excised maize embryos. Embryos 


more than .3 mm. in length did not require coconut milk for continued 
growth in litro, while smaller embryos failed to develop even if the 
medium were supplemented with coconut milk. This study indicates 
that the requirements for embryo growth var\' among different plant 

Curtis (4) found that the growth of embryos of many orchid species 
was better and more regular when peptone or nucleic acid were present 
in the medium. Nucleic acid from yeast was also shown by Kent and 
Brink (7) to be favorable for the growth of barley embryos which were 
too voung to develop in its absence. The components of the substance 
which are active in this case have not been determined, but they are 
known to be heat stable. 

Sanders and Burkholder (9). working with Datura embryos, concen- 
trated their attention on the effects of various amino acids, and they 
showed that casein hydrolysate or a mixture of 20 amino acids markedly 
improved the growth and differentiation of the two species studied. The 
addition of individual amino acids and mixtures of a few generally 
resulted in much poorer growth than that occurring on the more com- 
plete mixture. Some of the individual amino acids or small groups of 
them caused modifications in the proportions of the embryos, increasing 
or decreasing the cotyledon size and increasing the number of root 
primordia. and sometimes premature root growth resulted. The amino 
acids in the medium thus appeared to influence both the growth and 
differentiation oi Datura embryos. Spoerl (10), in his studies on orchid 
embryos, tested the effects of 19 amino acids as nitrogen sources, and 
found that most of them inhibited growth. Arginine. however, sup- 
ported good growth of embryos from unripe seeds, while aspartic acid 
proved to be a satisfactory nitrogen source for older embryos. 

In a studv of the effect of casein hydrolysate on the growth of im- 
mature Hordeitm embryos, Ziebur and co-workers (13) found that they 
could not duplicate the effect of this product with a mixture of merely 
amino acids. Casein hydrolysate has a clear-cut. two-fold effect on 
barlev embryos: it prevents germination, and it supports embr^^onic 
growth. It was found that both parts of this effect could also be pro- 
duced bv supplementing the medium with amino acids, inorganic phos- 
phate, and sodium chloride, all of which are components of commercial 
casein hydrolysate. Table i compares the effects of casein hydrolysate 
and of its three components, used singly and all together. The embryos 




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on the control medium did not continue their embryonic growth, but 
germinated in about three or four days, forming seedhngs which are 
abnormally small and spindling. This type of growth is indicated when 
the shoot height is large and the percentage of dry matter is small 
(around 10 per cent). If i per cent of casein hydrolysate is added to the 
medium, very young embryos fail to germinate and older ones are 
retarded so that the shoot height is reduced relative to the control; the 
wet and dry weights are also reduced, but the percentage of dry matter 
is high. The effect of the amino acid mixture containing phosphate 
and sodium chloride is indistinguishable from that of the casein hydrol- 
ysate. Each component alone, however, fails to duplicate the effect 
of the complete mixture. The added phosphate reduces the height 
slightly, while the amino acids and sodium chloride are more effective 
in this respect. Note that, in general, the shoot height is reduced on 
media with high osmotic pressure. The percentages of dry matter of 
embryos grown on media containing each of the three supplements 
singly are intermediate between those of the control and the complete 

A similar experiment, using the components in combinations of two, 
showed that none of the three possible pairs reproduced the results 
given by the casein hydrolysate, although the combination of amino 
acids and sodium chloride with an osmotic pressure of about nine 
atmospheres caused almost complete suppression of germination. Experi- 
ments with high concentrations of sucrose or mannitol demonstrated 
that the inhibition of germination could be attributed to the high osmotic 
pressure of the medium, as shown in Table 2. On all three media of high 
osmotic pressure the shoot height is low and the percentage of dry matter 
is high. It may be concluded then that the germination-inhibiting effect 
of casein hydrolysate is due to its osmotic pressure, for which the amino 
acids and sodium chloride are mainly responsible. The rapid embryonic 
growth which takes place on the casein hydrolysate medium is greater 
than that occurring on the media supplemented with sucrose or mannitol; 
the wet weight of the embryos on the casein hydrolysate medium is the 
greatest, and the dry weights on the casein hydrolysate and sucrose 
media, respectively, are greater than on the mannitol medium. Mannitol 
is nutritionally inactive for barley embryos. It is thought, therefore, 
that these weight differences result from the fact that casein hydrolysate 
and, to a lesser extent, the additional sucrose serve as nutrients as well 








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as osmotic agents. The experiments with pairs of components lead to 
the further conclusion that the nutritional effect of casein hydrolysate 
may be attributed to the amino acids and phosphate. The results as a 
whole suggest that one is dealing here with another case where the 
determination of the amount and the type of development is not mainly 
dependent on one specific growth requirement of the embryo, but may 
result from an interplay of both nutritional and physical factors. 

In summarizing this discussion it will be realized that the field of 
plant embryo growth is relatively undeveloped. A number of workers 
have had a variety of results with different materials, and it is difficult 
to draw from their data any conclusions as to the important factors which 
in general influence the growth of plant embryos. Histological studies 
have contributed an understanding of the probable functions of the 
various seed parts in supporting and controlling the growth of young 
embryos, but the exact chemical nature of the substances involved is 
not known. Experiments on the inhibition of seed germination have 
led to the discovery of a variety of ways in which the development of 
older embryos may be controlled in nature. In vitro studies of the 
embryos of a number of different species have shown that at early stages 
embryos require for their nutrition not only minerals and sugar, but 
also in some cases vitamins, amino acids, nucleic acid, and unknown 
materials occurring in coconut milk or malt extract. And the pattern 
of growth followed by an embryo may be influenced not only by the 
known growth hormones, but also by amino acids and such other factors 
as osmotic pressure. Future research in this field should include more 
of the exploratory work just reported, but especially should attempts 
be made to relate the effects of an apparently diverse group of factors 
all of which play an influential role in the growth of plant embryos. 


1. Blakeslee, a. F. and Satina, Sophie, Science, 99:331 (1944). 

2. Brink, R. A. and Cooper, D. C, Botan. Rev., 13:423 (1947). 

3. Cooper, D. C. and Brink, R. A., Botan. Gaz., 111:139-153 (1949). 

4. Curtis, J. T., Am. Orchid Soc. Bull., 16:654 (1947). 

5. Evanari, M., Botan. Rev., 15:153 (1949). 

6. Haagen-Smit, a. J., Siu, R., and Wilson, Gertrude, Science, 101:234 


7. Kent, Nancy and Brink, R. A., ibid., 106:547 (^947)* 

8. Nutman, p. S., Ann. Botany, N. S. 3:731 (1939)- 


9. Sanders, Mary E. and Burkholder, P. R., Proc. Nat. Acad. Sci. U. 5., 
34:516 (1948). 

10. Spoerl, Edward, Am. J. Botany, 35:88 (1948). 

11. van Overbeek, J., Cold Spring Harbor Symposia Quant. Biol., 10:126 


12. , CoNKLiN, Marie E., and Blakeslee, A. F., Am. J. Botany, 

29:472 (1942). 

13. ZiEBUR, Nancy Kent, Brink, R. A., Stahmann, M.A., and Graf, L. H., 

37:144-148 (1950). 

Growth Substances and the Formation 
of Buds in Plant Tissues 


THE analysis of developmental processes of plants, particularly in 
relation to the role of growth substances, is beset by difficulties of 
many kinds, but perhaps mainly from two sources. One is the continuous 
change in composition and rates of synthesis of growth factors in the 
tissues, and another is the complex influences of each part on the growth 
of other parts and on the development of the plant as a whole. In fact, 
from comparisons based on mere inspection of normal and patho- 
logical plant materials, one may conclude that normal development, as 
contrasted with simple growth, must depend more directly on correlative 
processes, often of an inhibitory nature, than on the actual synthesis of 
new cell materials. For experimental purposes, therefore, it seems 
necessary to start with simple materials with limited capacities for 
autotrophic growth and difl^erentiation. 

This report deals primarily with the chemical induction of organs, 
especially of buds, in parenchyma tissue and excised segments of stems 
and roots grown in vitro on media of known chemical composition. Some 
conclusions drawn from these experiments which may have a general 
bearing on growth and morphogenesis in plants will also be discussed. 
The intimate connection between this topic and the general problem 
of the correlative action of growth substances in plants will be apparent. 

Experiments with tobacco callus cultures. — During the period 1937- 
1940 several investigators (3,8,13,12,10) obtained evidence of different 

Editor's Note: This work was supported in part by the University Research 
Committee on funds from the Wisconsin Alumni Research Foundation, and in 
part by a grant-in-aid from the American Cancer Society upon recommendation 
of the Committee on Growth of the National Research Council. 


kinds which provided indirect support for the view that auxin may act 
as a coenzyme. If this is true, it means that auxin must act in one or 
perhaps several, but nevertheless definite, metaboUc systems. Further- 
more, from the fact that quantitative growth responses to appUed 
indoleacetic acid are obtained only over a restricted range of concentra- 
tions, it may be deduced that other components of this "auxin reaction" 
system must also be present in relatively limited quantities in the plant. 
It was of interest, therefore, to determine what this system and its other 
components might be. A possible experimental approach to this problem 
became available with the development by White (14) of callus tissue 
cultures which could be grown indefinitely on a medium of known 
composition. White found that callus of the hybrid Nicotiana glauca X 
N. langsdorffii grown in submerged cultures would differentiate to pro- 
duce numerous buds (15), and one of us found (9) that the addition of 
indoleacetic acid (lAA) or naphthaleneacetic acid (NAA) to the culture 
medium would completely prevent the initiation of buds. Even low 
concentrations (o.i mg./l. or less), which did not retard the rate of 
increase in fresh or dry weights of the tissues, effectively prevented 
bud formation, whereas higher concentrations inhibited also the rate 
of growth of the callus. In short, the tobacco tissue cultures have a very 
sensitive response to lAA and are a favorable material for the testing 
of various organic compounds which might normally interact with auxin. 
On the above assumption of an auxin reaction system, such compounds 
should counteract the inhibiting effect of lAA on bud formation and 
at the same time should increase the growth of the callus in media with 
added lAA. 

Substances were selected for testing which are, or might be, required in 
relatively large amounts in respiration. Tests were first carried out with 
the four-carbon dicarboxylic acids, malic and succinic acids. The results 
were negative. Such effects on bud formation as were obtained by the 
addition of these acids could be ascribed entirely to lowering of the 
pH of the medium. A careful study of the relation of pH to bud forma- 
tion (16) showed that the optimum was in the range from 4 to 4.5 (9). 

The effects of increased concentrations of phosphate and sucrose were 
tested with positive results. The data from one experiment are sum- 
marized in Table i. It may be seen that about half the callus pieces form 
buds in the control nutrient solution and that none form buds in the 
cultures with added lAA. With increased KH2PO4 and sucrose contents, 


on the other hand, bud formation occurs also in the presence of lAA 
and up to about the same extent as in the control cultures without added 
lAA. In these experiments the concentration of Fe2(S04)3 was also 
increased to maintain available iron in the medium. Control series with 
increased Fe2(S04)3 alone were negative, so that the effect on bud 
formation is ascribed to the KH2PO4. * 


Effect of indoleacetic acid, and changes in concentrations of KH2PO4, 
Fe2(S04)3, and sucrose on growth and bud formation in tobacco callus in vitro. 
Initial pH adjusted to 5.0. Cultures started 12/22/41. Final measurements 


Control 4FE4P 4FE8P 4FE8P2S 

Indoleacetic acid mg./l. 

1. 00 1. 00 1. 00 i.o 

No. of cultures 

17 17 17 

18 18 17 



Mean fresh weight in mg. 

54 42 65 

42 47 49 



Mean dry weight in mg. 

3.4 2.6 4.1 

2.6 2.9 3.1 

' 31 


No. of cultures forming buds 


3 8 9 



Per cent forming buds 

53 47 

17 45 53 



4 Fe = Fe2(S04)3 10 mg./l.; 4 P and 8 P = KH2PO4 50 and 100 mg./l., 
and 28= sucrose 40 g./l. 

The effectiveness of phosphate in counteracting the inhibitory effect 
of added auxin suggested that organic phosphates might be limiting 
components of the auxin reaction system. Since an important influence 
of phosphate in respiration is through its role in purine metabolism, it 
seemed desirable to test phosphorylated adenine derivatives. None were 
immediately available and, therefore, adenosine in combination with 
inorganic phosphate was first tried. The results of one experiment are 
shown in Figure i . It may be seen that either adenosine or NAA added 
singly reduces the growth of the callus, but the two added in combination 
give as good or better growth than the controls. However, no buds were 
obtained on either control or treated tissues in this experiment. In 

♦Recently R. W. Howell has compared the effects of NaH^PO^ and KH^O^ 
on bud formation in tobacco stem segments. He finds both equally active so that 
the effect of the salts may be ascribed definitely to the {ll^O~) radical rather 
than to the accompanying cation. 





T 1 1 1 1 r — I 1 



Figure i. Effect of concentration of adenosine on the growth of tobacco 
callus in vitro with and without addition of 0.25 mg./l. NAA. Exp. #14 
started 12/24/43. Harvested 3/16/44. Data of 32 cultures for each treatment. 

later experiments increases were obtained in the number of callus tissues 
forming buds as illustrated in Table 2. 

The addition of adenine similarly was found to stimulate the formation 
of buds, and adenine is, in fact, more active than corresponding concen- 
trations of adenosine (Table 3, compare also Fig. 7). 

The above results show that the inhibiting effect of auxin on bud 
formation is reversibly counteracted and that the growth rate of callus 
in the presence of added lAA is increased by the addition of adenine or 
adenosine and phosphate. The results suggest, therefore, that auxin may 
exert its eflect on growth through an action In one or more phosphoryla- 
tion systems. 

Experiments tvith tobacco stem segment cultures. — The effects obtained 
with treatments of lAA, adenine, and its derivatives on callus cultures 
have been confirmed and the results considerably extended by experi- 
ments with excised stem segments of tobacco. 

Young stems of variety Wis. #38 were surface sterilized, sectioned, 
and cultured on White's nutrient medium as described by Skoog and 
Tsui (11). This material was found to give clear-cut and reproducible 



Effect of adenosine on bud formation in tobacco callus cultures. (White's 
medium, 3/4 strength. Experiment started 9/23/43, measurements made 












Number of cultures 
Number forming buds 
Per cent forming buds 

















*Cultures in the last column received increased concentrations of the following 
components of the basic medium: KH^PO^, 5 times (62.5 mg./l.); Fe2(S04)3, 3 
times (7.5 mg./l.) ; and sucrose 2 times (40 g./l.). 

responses to treatments with adenine and lAA or NAA. It was, therefore, 
chosen for detailed studies of the effects of the above compounds on 
growth and organ formation. These include measurements of effects of 
added growth substances and nutrients as well as quantitative analyses 
of associated changes in composition of various constituents and nutrients 
within the tissues. The work is still far from complete, but the results 
reported here are based on our present experience with about ten 
thousand cultures. 

Representative results of the effects of the different treatments with 
relatively high adenine and low lAA concentrations on growth are shown 
in Figure 2, and results of anatomical studies, carried out by Dr. Sterling, 
on these tissues at successive stages of their growth are summarized by 
the diagrams in Figure 3. 


The effect of adenine sulfate on bud formation of tobacco calli obtained in 

first transfer (on 6/18) from stem internode segments cultured for 29 days. 

Cultures started in liquid media 6/18/48. Measurements 7/21/48 

Concentrations of Number of Number of calli Number of 
adenine sulfate calli observed forming buds buds formed 

o (control) 27 o o 

2.5 mg./l. 30 4 4 

5 mg-/l- 30 6 10 

10 mg./l. 21 7 14 

No root formation occurred. 






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Growth of control segments. — The ability of the stem segments to 
grow on the basal medium is a function of their size and of the vigor of 
the plants from which they were taken. Pieces 3x2x2 mm. or less in 
size ordinarily remain alive for several weeks but do not grow appreciably 
unless they are supplied with lAA. Larger pieces (7x5x2 mm.) which 
were selected as standard material for these and all subsequent experi- 
ments form masses of callus at their basipetal ends within two to four 
weeks in culture. After longer periods (6 to 10 weeks) a few pieces (5 per 
cent on the average) may form a bud or root or both, but growth is 
restricted mainly to the formation of callus, which starts from the cambial 
region of the basipetal surface. This callus may be subcultured on the 
same medium with added lAA, but, at least in most cases, neither the 
original stem tissue nor the first transfer of callus will continue to grow 
on the basic medium without lAA. 

Effects of adenine treatments. — When adenine is added to the medium 
the formation of callus is less than in controls, but a very striking forma- 
tion of buds occurs. These buds grow out most abundantly along the 
lateral edges and especially from the apical halves of the segments with 
relatively lower auxin content, but also to some extent from the basipetal 
halves and attached, newly formed, callus. As indicated in Figure 3, 
Dr. Sterhng has found that they originate from parenchyma formed by 
the cambium and from both external and internal phloem regions. The 
number of pieces forming buds in a given treatment and the number of 
buds per piece both increase as functions of the concentration of adenine 
in the medium up to about 50 mg./l. Higher concentrations are often 
toxic. With optimal concentrations, generally between 90 and 100 per 
cent of the segments form buds. The effects of concentration of adenine 
and time of culturing are illustrated in Figure 4. In this experiment the 
highest average number of buds per segment was 2.7 after twenty-eight 
days. In an experiment with tissues from field-grown plants, however, 
an average of about 20 buds per segment and extremes of 35 to 40 buds 
per segment have been obtained. The number of buds obtained is not 
necessarily the maximum number that can be produced by the tissue, 
because as one or a few buds start to develop, they produce auxin in 
sufficient quantities to raise the concentration in the original stem tissues, 
and thus tend to retard or prevent further bud formation. The buds 
which form early may develop several leaves, but, unless root formation 
occurs, the buds are incapable of continued growth. If they form roots 



and are transplanted, however, they grow into normal mature plants. 
Effects of lAA or NAA treatments. — Even though there are important 
quantitative differences in growth responses to lAA and NAA, the 
general effects of these substances in the present experiments are closely 
enough alike, so that they can be considered together. The addition of 
either lAA or NAA to the medium markedly stimulates the growth of 





a=. 20 





EXP. 76 
5/2/4 9 












• - — 




10 20 30 


Figure 4. Effect of concentration of adenine sulfate on bud formation in 
tobacco stem segments. Exp. # 76 started 5/2/49. Curves in order from the top 
represent treatments with 40, 20, 10 and mg./l. adenine respectively. 
Initial pH 4.0, KH2PO4 concentration 37.5 mg./l. 

the tissues through both enlargement of existing cells and the formation 
of callus over wider areas of the segments. 

Significant increase in callus growth and decrease in bud formation 
may be obtained with as little as 0.02 mg./l. and maximum effects with 
concentrations in the range from i to 10 mg./l. These substances also 
induce the formation of roots as indicated in Figures 2 and 3, which effect 
is comparable to that known to occur in cuttings. 

Effects of adenine plus lAA or NAA. — Combined treatments with 


ad^'nine «Tk3 l.VA or NAA W»c! to madced enlarjfemeru and cell division 

in rhe tiicajcs of the <i«Tn se^rmcnts paitioulariy of ihe pith and superficial 
oortioal layers. How^exer, :he relative grox^th of the different tissues 

deycixls on the relatix-e concentrations of the two substances. 

"Dje evtent of or^jan formation i? also dependent on both the concen- 
trations aj>d proportions of adenine and auxin. For example in the 
treatments i^jo^'n in Ficfure 2. with high adenine and relatively low I.\.\, 
tiftere is x'er\- rapid {growth of the oripnal tissues and of new parenchyma, 
bat t^crc is no organ formation, \\ith higher concentrations of I.\.\, 
loot fonnation occurs even though it is much less than would be obtained 
fiom dte same ooncentrations of lAA in the absence of adoiine. The 
resahs obtaaned %ith high adenine and only minute concentrations of 
l.V\ are iUustratcd in Figure 5. Here the formation of buds is restricted 
to the acropetal ends of the segments and roots usually form later at the 
bases of the buds and/or at the basipetal ends of the stem segments. 

In generaL therefore the results indicate that under the conditions of 
these experiments the tx'pes of growth and organ formation u-hich occur 
are determined b^- the concentrations of auxin and adenine supplied in 
die nutrienr medium. Relatively high auxin concentrations £avor tlic 
fo rma riran of roots and prex'ent the formatitwi of buds. High adenine 
concentrations fax'or the formation of buds and decrease the extent of 
root formation. With both substances added together in proper con- 
centrations it is possible to get marked stimulation of growth widiout 
organ formation. 

Corr^arathf experiments wjih horse radish and carrot root segments. — ^The 
results obtained with tobacco stem segments indicate tliiat the morpho- 
genctic response to treatments ■with adenine and L\-\ must var\^ with the 
tissue conyosition- Hence considerable variabilir\' in responses is to be 
expected between different organs of the same plant and particularly 
between different species. Tests "were, therefore, carried out with horse 
radish and zi — ■ — "" which can be conveniently^ culturedL The 
former mateLr..i_ ^-^t ^1. because it has a marked capacity to fonn 

--'•—"'"-,■• ''■ :\^ and : ;_: reason has been used cxtcosivciy in work 
(7j. A - ^7 re 6, omtnJ segqieats of hocse 

^"-odoced oa the average ooe 

. rie the number of bods was 

- : TTie xwesence of L\.\ e\'en 
. ^ . . _. __. - : ; tnted the fotmatioo of 

Figure ::- F rte-.-rs or .ikiiniiiie r.iiuie jjia ^\,\ jh .pn^w^zL -luc jrgan loxmaCLon 
ia tobacco srem segments. Exp. =C started q, z8, 48. Photagrapiieii aiier 
30 davs. r. Controi; 2. .Adenine suifete 40 mg., L; 3. L\A 0.02 mg., L; 4- 
Adenine sui£ite 4.0 mg. L plus L\i\ cuox mg.. 1- 

Figure 5. Effects of treatments with liigh adenine combined with very 
low lAA concentrations on bud formation in tobacco stem segments. Exp. 
#42 started 9/17/48. Photographed after 115 days. Treatments from left 
to right: Control; adenine sulfate 40 mg. 1.; do. plus 17/I. lAA; do. plus 
57/1. lAA. Initial pH 4.0; KH2PO4 12.5 mg./l. 



buds and in all cases markedly retarded the appearance of buds. How- 
ever, when adenine was supplied together with lAA in the same con- 
centrations as of each substance above, bud formation was restored, in 
this experiment, to the same value as in the treatments with adenine 
alone. These results are in general agreement with the data for tobacco, 
even though the inherent capacity to form buds was greater and the 

"6 25 








EXP 61 

STARTED 1/12/49 




20 30 40 


Figure 6. Effects of treatments with adenine sulfate and lAA on bud 
formation in horse radish root segments. Exp. #61 started 1/12/49. hiitial 
pW 4.0; KH2PO4 37.5 mg./l. 

capacity to form roots directly from the original tissue was less in the 
horse radish. These differences are strikingly correlated with the relative 
auxin contents of the tissues of the two species (Tables 5 and 6), 

Carrot tissues responded differently. The growth of the original seg- 
ments, depending on their size and morphological make-up probably 
as well as on the strain, was either stimulated or inhibited by low con- 
centrations of lAA in different experiments, and was stimulated by higher 
concentrations. The highest concentrations (10 to 100 mg./l.) induced 


root formation. Adenine had relatively little effect on growth when 
supplied alone, but it completely counteracted the inhibiting effect, 
when it was obtained, from low concentrations of lAA. No bud forma- 
tion resulted from treatments of the segments. However, in subcultures 
of the callus produced on the segments, roots were formed in response to 
lAA treatments, and a few transfers formed buds, but not exclusively in 
cultures with adenine treatment. In tissues from all three species, there- 
fore, a very definite interaction between adenine and lAA was demon- 
strated, but the quantitative requirements for a particular type of 
growth or organ formation were very different. 

Specificity of adenine for bud formation.— In the case of auxins we know 
that a large group of related compounds with certain structural features 
in common have similar effects on cell elongation and also on root 
formation. It was of interest, therefore, to determine the specificity of 
adenine in its effect on bud formation. As shown by the curves in Figure 
7 adenine derivatives such as adenylic acid and adenosine are active. 
Also guanine, though it acts more slowly, may be nearly as effective as 
adenine. However, xanthine and the pyrimidine, uracil, are either com- 
pletely inactive or have a very low activity. Preliminary tests with 
cytosine indicate that this compound may possess some activity. On the 
other hand, the buds which were produced in tissues treated with 
cytosine appeared so much later than from treatments with adenine, that 
the effect of this substance may be less direct. The results are as yet 
fragmentary but suggest that the entire purine nucleus together with 
either substituted NH2 groups in the 2 or 6 position, or else the absence 
of keto groups in both these positions is required for high activity. 

Arginine, which has been reported to substitute for adenine in the 
phosphate energy transfer in invertebrate tissues, has also been shown 
to promote the growth of Avena coleoptiles and pea stem segments 
(1,4). It also promotes growth of tobacco tissues under the conditions 
of our experiments, but, in our limited experience with the tobacco 
tissues, arginine has a general growth-promoting effect which is quite 
distinct from the effect of the active purines. The specificity of adenine 
and structurally related compounds for bud formation may well be 
compared with the specificity of lAA, and other compounds with auxin 
activity, for root formation. Still, neither group of compounds can be 
designated as specific organ-forming substances, since both are un- 
questionably essential for growth of all cells and tissues. Furthermore, 



u 25 








10 20 30 40 

Figure 7. Relative activities of different purine derivatives and uracil on 
bud formation in tobacco stem segments. Exp. #69 started 3/15/49. Initial 
pH 4.0, KH2PO4 12.5 mg./l. Curves: i. adenine sulfate; 2. guanine; 3. adenylic 
acid; 4. nucleic acid; 5. adenosine; 6. xanthine; 7. uracil; and 8. control. 
All substances added in 0.00025 n^ol^r cone, except nucleic acid which was 
85 mg./l. 


even though they may be hmiting factors for the formation of buds and 
roots respectively under normal conditions as well as under a given set 
of experimental conditions, it is obvious that different conditions could 
be selected, where organ formation would be limited by other factors. 
Effects of inorganic nutrients. — While White's nutrient medium was 
originally devised for continued culture of excised roots, it contains all 
necessary ingredients required for continuous growth of tobacco callus 
cultures, but certain quantitative modifications in mineral components 
result in more rapid growth rates of the tissues. It is a question, therefore, 


Effect of adenine sulfate added to the nutrient medium at different /?H values 
on the formation of buds in stem segments of tobacco. Exp. 39. Started 8/5/48 





Adenine sulfate 


pW OF 

No. OF 






IN MG./l. 






























to what extent changes in mineral composition might also affect the 
capacity of the tissues to form organs. So far only a few of the many 
possible variations in nutrient composition have been tested in tobacco 
stem segments. Thus, a marked effect on pYi on bud formation in response 
to added adenine has been established. As indicated in Table 4, adenine 
is effective in inducing bud formation in the range from pW 4 to pW 7 
which was tested, but acid pW is highly favorable. Since acid pW also 
favors bud formation of callus tissue in submerged cultures without added 
adenine, it may be assumed that the pW effect is not entirely, if at all, 
on the adenine uptake by the tissues. 

Similarly, increases in phosphate supplies in the medium increase the 
effectiveness of the adenine treatments. As shown in Figure 8, a threefold 
increase in phosphate content (from 12.5 mg./l. to 37.5 mg./l.) causes 
about twice the number of buds to be formed per segment treated with 
40 mg./l. adenine. However, a ninefold increase in phosphate is evidently 



above the optimum requirement and no better than the standard 

Variations in potassium concentrations as stated above appear to have 
no effect on bud formation. Different NO3" levels have been tested only 
in callus cultures not suppHed with adenine. No significant effect on the 
capacity of the tissues to form buds was obtained in these experiments. 
Other components may have some effect but have not been studied in 

Interactions of I A A, adenine, ribose, and phosphate.- — Since the meta- 
boUcally active nucleotides contain ribose as well as phosphate combined 
with adenine it was of interest to test effects of ribose on growth and 











10 20 30 40 


Figure 8. Effect of phosphate concentration on bud formation in tobacco 
stem segments treated with adenine sulfate. Exp. #32. Started 7/17/48. 
Initial pW 4.0. Curves: i. control without adenine, KH2PO4 12 mg./l.; 
2. adenine sulfate 40 mg./l.; KH2PO4 12.5 mg./l.; 3. same as 2 except KH2PO4 
37.5 mg./l.; 4. same as 2 except KH2PO4 1 12.5 mg./l. 



organ formation. So far our results on ribose are restricted to tests on 
growth of callus supplied with adenine. lAA, and different levels of 
(AosfAate singly and in different combinations. Interestingly enough, 
in the lower ccmcentration range ribose may have a growth-promoting 
^ect, but even in concentrations as low as lo mg./l. it may be strongly 
inhibitor\-. as is I.\-\. The tv^o substances together exert an even stronger 
inhibitor)' effect on growth. However, in the presence of adenine this 
iahibidoa is counteracted in all cases, and. in fact, v^ith low ribose 
concentrations more rapid growth, as measured by average increases in 
fresh and dr\' weights of the tissues, have been obtained than from the 
addition of adenine or adenine and lAA in the absence of ribose. 
However, the additional increase in growth obtained from ribose in the 
presence of adenine and L\.\, has so far been too small to be definitely 
significant. In spite of this, the results obtained with combinations of 
any two or all three components of the nucleotides strongly suggest a 
close relationship between them and auxin in their effect on growth. 

Analyses of tissue composition. — .\nalyses of tissue composition includ- 
ing "free" and bound auxin, adenine, phosphate fractions, and changes 
in these components with treatments leading to different t\'pes of growth 
and organ formation are in progress. In general it may be said that the 
readily extractable auxin content in the tobacco stem segments decreases 
rapidly from the original level, and a gradient with relatively higher 
concentrations present in the basal half than in the apical half is estab- 
lished. A return to higher auxin levels occurs after buds have formed 
and, therefore, becomes most marked at first in segments treated with 
adenine. However, rapid growth of the segments themselves is also 
associated with increased auxin content. Representative data are shown 
in Table 5. The effects are particularly marked in horse radish segments 
(Table 6} whose auxin content is lower than in tobacco at the start and 
higher after buds have been formed. These changes in auxin content, 
therefore, run parallel with the growth and n,'pe of organ formation 
which occur. Low auxin levels are associated with bud formation and 
high auxin levels with root formation. It is probable, therefore, that the 
number of buds forming on a segment is limited by the auxin production 
in the buds which first grow out. Due to difficulties in obtaining quanti- 
tative estimates of auxin concentrations in carrot tissues no reliable 
data have yet been obtained on this species. 

From the results presented above, it might be assumed that adenine 




Auxin content in tobacco stem segments cultured on media with adenine and 
lAA. Expt. C. Cultures started 0, rS 48. Tissues extracted 2 hours with ether 










28 DAYS 







Adenine sulfate 


15. s 


1 -f 





(4omg. I.) 
Indoleacetic acid 







(0.04 mg., I.) 
Indoleacetic acid 

(0.04 mg.. I.) and adenine 
siilfate (40 ms. l-"* 






10. I 

is absorbed and combines with phosphate in tlie tissues and that the 
content of organic phosphate would be affected by the external supplies 
both of L\-\ and adenine. Experiments by Holm (6) have established 
that treatments with L\.\ tend to increase the total phosphate content 
of the apical halves of the tobacco stem segments over that in controls, 
but tend to decrease the phosphate content of the lower halves. Addition 
of adenine, on the other hand, has relatively little effect on the phosphate 
content in the apical halves but tends to increase the concentration in 
the basal halves. Adenine supplied in combination with. LAA leads to 

T.\BLE 6 

Auxin content of horse radish root segments cultured on media with adenine 

and I.A\. Exp. 6^. Started 2. 24. 49: mitial pH 4.0, Phosphate 37.5 mg.< 1. 

Tissues extracted 2 hours with ether 


-extr.\ctable acxin 






weight .vfter: 


14 DAYS 

29 DAYS 





Adenine sulfate (|o mg., 1.) 



12. I 

LA\ (0.02 mg.y l.) 


2. 1 


Adenine sulfate (40 mg., 1.) 

and LA\ (0.22 mg. I.' 

I .a 

■- i 



increases in the phosphate content of the segments and particularly 
counteracts the lowering in phosphate content which is apt to result 
from addition of lAA alone. However, the main increases in phosphate 
content obtained by the treatments have been in the inorganic fraction. 
Significant increases in organic phosphate fractions, therefore, either 
do not occur or the compounds formed are too labile to be detected in 
spite of all attempts with the several methods which were employed. 
In any case the results point to a sensitive equilibrium between auxin, 
adenine, and phosphate contents in the tissue, according to which 
phosphate uptake may be increased or decreased by addition of either 
one of the two organic compounds, depending on the relative supply 
of the other. Typical results of the phosphate content and distribution 
for different treatments and at successive stages of growth are shown in 
Figure 9, and the proportions of the total phosphate present in different 
fractions at the start and after 14 days of growth are shown in Table 7. 
Further evidence on the localization of the phosphate in the tissues is 
being obtained by isotope analysis. Data on adenine content and carbo- 
hydrate fractions are still too fragmentary to be interpreted. 

Discussion and conclusions. — The evidence obtained on growth and 
organ formation in tissues cultured in vitro demonstrate that under 
the conditions of these experiments the relative growth of different 
tissues and the type of organ formation which will occur depend on 
the composition of the nutrient medium. Generally the application 
of lAA or NAA leads to rapid growth of parenchymatous tissues, to 
root formation, and to suppression of bud formation, whereas the appli- 
cation of adenine leads to bud formation. Clearly, however, these 
substances are not specific either for the formation or growth of par- 
ticular organs. Both are required for all types of growth. The responses 
elicited by their application to the tissues depend on the proportions 
and concentrations in which they are supplied as well as on the propor- 
tions and concentrations of these and other essential nutrients present at 
the start or supplied during the growth period, through synthesis or 
from external sources. 

In regard to the specific actions of auxin and adenine in growth 
the only new information provided by the present experiments is the 
definite interaction of these substances and other components of the 
nucleotides. This strongly supports the original assumption that auxin 
acts as a coenzyme. If we accept the known function of adenine phosphate 





I 5 



DAYS 4 8 12 18 2328 4 8 12 18 2328 4 8 12 18 2328 4 8 12 18 23 28 




















DAYS 4 8 12 18 2328 4 8 12 18 23 28 4 8 12 18 2328 4 8 12 18 23 28 

Figure 9. Effects of treatments with adenine sulfate and lAA on total, 
inorganic, and organic phosphate contents in tobacco stem segments. Exp. 
#G started 6/19/49. Initial pH 4.0.; KH2PO4 37.5 mg./l.; Adenine sulfate 
40 mg./l.; lAA 0.04 mg./l. A. Upper halves of segments; B. Lower halves 
of segments. Shaded portion of columns are inorganic fractions. White portions 
are organic fractions. (Data from L. G. Holm.) 











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complexes as mediators in energy transfer reactions in cellular syntheses, 
it appears that auxin also must be concerned in these phosphorylation 
systems. It seems, however, that its role in morphogenesis can hardly 
be restricted to the regulation of the rate of a single reaction leading to the 
formation of a single final product, but it must at least include the regula- 
tion of the relative rates of coordinated synthetic reactions so as to 
modify both the quantities and proportions of several products. 

The correlative and morphogenetic actions of auxin which are well 
established in bud inhibition and root formation must be extended to 
include the formation of buds. For even though the addition of lAA 
in our experiments results in suppression rather than promotion of bud 
formation, the marked effect of auxin on cell proliferation together with 
its properties of high physiological activity and unique mode of transport 
must be decisive in furnishing new cells, in setting the stage at which 
these become differentiated into primordia, and in enabling the latter 
to develop as organized units. 

The plan of the present work has been based in part on the early 
observations indicating that the conditions leading to induction of bud 
formation are similar to the conditions required for the subsequent 
growth of the buds. Some results have been obtained which show that 
separate factors may become limiting in these two processes. Neverthe- 
less, the general conclusion from all the results is that the correlative 
mechanism which determines the relative growth of organs, and hence 
the final form of the plant, also functions at the level of cellular dif- 
ferentiation into tissues and primordia. 

Keeping in mind the fact that in our present state of ignorance, growth 
of any kind is far too complex a phenomenon to be interpreted in detail, 
the formulation of simple points of view on the nature of growth and 
developmental processes may be useful in guiding further inquiry. For 
example, the concept of a growth substance regulating the rate of growth 
of tissues has been extremely fruitful. It has been a prerequisite step in 
obtaining the evidence, which is now accumulating, to show that sub- 
stances of many kinds may be preferentially produced or concentrated 
in certain tissues or organs and that any one of a number of such 
substances may become a limiting factor for growth. However, recently 
Sachs' old concept of organ-forming substances has been revived. It 
has appeared in various new forms (2,5), but to the general effect that 
a specific substance is required for the formation and/or growth of each 


kind of plant organ, that such morphogenetic substances are syntP,esized 
only in certain cells, tissues, or organs, and/or that they are produced at 
specified stages of development. The results we have obtained are in 
disagreement with such concepts. On the contrary our findings suggest 
that both organ formation and subsequent development are brought 
about by quantitative changes in amounts and interactions between 
nutrients and growth factors which are essential for growth of all cells, 
so that the pattern of development is determined by the relative supplies, 
through synthesis, transport, and accumulation of these materials at 
particular loci. On this basis, the morphogenetic capacities of a given 
cell or tissue are limited not only by its genetical potentialities for 
syntheses but more often by its morphological environment, that is, 
by its particular position in the structurally complex plant body. This 
concept demands that normal growth of cells must lead to a unified 
general pattern of development in all plants of comparable genetic 
constitution, but it permits infinite variation in details. 

As a working hypothesis, it lacks the conciseness of concepts invoking 
the participation of a specific substance for each step in morphogenesis, 
but it offers equal opportunities for the separation of individual growth 
factors. In addition, it provides an over-all plan for both physiological 
and morphological experimentation on correlation phenomena, which, 
we believe, must control structural organization at all levels in the 
development of plants. 


1. Bonner, James, Am. J. Botany, 36:323-332 (1949). 

2. BouiLLENNE, R. and BouiLLENNE, Walrand M., Pwc. 6th Int. Congr. 

of Exp. Cytology, 591-596 (1949); and International Symposium on 
Morphogenesis (Strasbourg, July, 1949). 

3. Commoner, Barry and Thimann, K. V., /. Gen. Physiol, 24:279-296 


4. Galston, Arthur W. and Hand, Margery E., Archives of Biochemistry, 

22:434-443 (1949)- ' 

5. Gautheret, Roger J., Rev. Cyt. Cytophysiol. Vegetales., 7:45-215 (1944). 

6. Holm, LeRoy, G., A study of phosphorus compounds in relation to 

growth of plant tissues. Thesis, University of Wisconsin, 1949. 

7. Lindner, R. C, Botan. Gaz., 100:500-527 (1939). 

8. Schneider, Charles L., Am. J. Botany, 25:258-270 (1938). 

9. Skoog, p., ibid., 31:19-24 (1944). 

10. , Schneider, C. L., and Malan, Peter, ibid., 29:568-576 (1942). 

11. Skoog, F. and Tsui, Cheng, ibid., 35:782-787 (1948). 


12. Thimann, K. v., Skoog, F., and Byer, A. C, ibid., 29:598-606 (1942). 

13. Thimann, K. V. and Sweeney, B. M.,J. Gen. Physiol., 21 :i23-i35 (1937)- 

14. White, P. R., Am. J. Botatiy, 26:59-64 (1939). 

15. , Bull. Tor. Hot. Club, 66:507-513 (1939). 

16. Zarudnaya, Katerina, The effect of pH on growth of tobacco callus 

tissue cultures. M.A. thesis, Johns Hopkins University, 1945. 

The Development of Stems and Leaves 


THIS paper is not intended as a general review of the field of leaf 
growth or stem growth factors other than auxin. It will be an 
attempt to correlate a limited number of otherwise unrelated facts and 
experimental evidence, special stress being laid on work carried out in 
the Kerckhoff Laboratories at the California Institute of Technology in 

Leaf Growth. — The problem of plant form is usually considered to 
lie within the domain of the discipline of morphology. Whereas in its 
inception this was purely descriptive, during the last half century, and 
especially since the articles by Sachs on "Stoff und Form" (22), an 
experimental morphology has been developed too. If we restrict our 
considerations to leaves we can divide the analysis of their form in two 
broad groups. 

On the one hand the shape of a leaf can be considered as a basic 
concept, much as the atom is the basic unit for a chemist. The idealistic 
morphology, of which Troll (30) is a recent exponent, considers form 
as something absolute which may be modified by external conditions 
but which essentially can not be broken down into constituent elements. 

On the other hand leaf form can be envisaged as being the resultant 
of all factors contributing to the length, width, and thickness of the 
organ. Such a point of view does not consider form as a category. Most 
investigators who have to deal with form — experimental morphologists 
and geneticists — use this approach, and it is the only possible approach 
for the physiologist. Modern exponents of this point of view are Huxley 
(13) and Sinnott (26). 

Experimental morphology uses the latter approach, and in explaining 
the differences obtained as a result of particular treatments the prevailing 


physiological concepts are embraced. When the differential effects of 
plant nutrition became known, especially through the work of Klebs, 
differences in form were generally attributed to differential nutrition 
(see 8). Thus the earlier explanations based on specific growth substances 
(22,3) were more or less abandoned. With the increase in our knowledge 
of plant hormones, they again assume a central part in morphogenetic 

With the knowledge gained in the last ten years about leaf growth 
factors it is interesting to develop a unified view of leaf shape, which can 
account for many of the variations we observe within a single plant, 
differences between closely related genetic races or effects of external 
conditions, effects of viruses and other diseases, or teratological phe- 

As a basis for the following analysis we assume that petiole, vein, 
mesophyll, and stipule growth are independent of each other. This has 
been shown experimentally in transplantation experiments with peas, 
in which these parts on the scion were independently influenced by the 
stock (36,38). The work of D. Bonner has shown that mesophyll growth 
is controlled by substances such as adenine which are not primarily 
concerned with stem and vein growth (4,5). There are certainly many 
other growth factors for mesophyll (21), but for the following considera- 
tions it is sufficient that adenine specifically increases mesophyll de- 
velopment in young radish and pea leaves without affecting vein growth. 
On the other hand, it is possible to increase vein growth without 
mesophyll development by applying auxin to leaves (40). In general it 
seems that petiole and vein growth are influenced by the same factors 
which increase stem elongation. Thus there is a marked separation in 
physiological response of stem, petiole, and vein growth on the one hand, 
and mesophyll growth on the other, a difference based upon a different 
set of growth factors for each type of tissue. For the sake of convenience 
in this paper the vein growth factors will be named caulocaline, and 
the mesophyll growth factors will be summarized under the name of 
phyllocahne without further reference to their chemical nature. Caulo- 
caline and phyllocaline thus arc purely physiological names, that is 
functional, not chemical. 

For the above reasons we should consider leaf form as a synthesis of 
two separate tendencies which are independently controlled by separate 
sets of growth factors: a tendency to hnear development of veins, and to 

F. W. WENT 289 

surface development of mesophyll. By following these tendencies 
throughout the angiosperms we find that there are two major leaf types 
which behave differently upon changes in proportions of caulocaline 
and phyllocaline. These are the palmate and the pinnate leaves. To the 
latter type belongs also the parallel-veined leaf. In a pinnate leaf a 
decrease in the amount of phyllocaline results in a narrowing of the 
distance between the veins, which thus make a smaller angle with the 
midrib. This results in a narrower leaf. Yet the veins can retain the same 
length if the same amount of caulocaline is available. In a palmate leaf, 
on the other hand, the major palmate veins retain the same angle, but 
a decrease in phyllocaline results in lobing. 

Morphology. — There are a few outstanding examples of differential 
development of leaves which are mentioned in all morphology text 
books. As a first case we will discuss the form differences between sub- 
merged and floating leaves in certain water plants. If we start with the 
monocotyledons we see there that leaves of Sagittaria, Alisma Plantago, 
A. nutans are all linear if they grow under water, which is the case in the 
young leaves and in older plants which are grown in streaming water. 
Under those conditions these species can hardly be recognized one from 
another, but the first leaves which reach the surface of the water and 
either float on it or stand up above it develop a pronounced blade by 
widening of the apical part of the leaf. In successive leaves this widening 
increases until the typical form is reached, but the plant reverts to its 
linear type of leaves as soon as it is submerged again. In Potamogeion 
natans and P. heterophyllus the same difference between narrow sub- 
merged leaves and wide floating leaves occurs. In dicotyledonous plants 
with palmate leaves the submerged leaves are not linear themselves, 
but the leaf is divided into linear segments such as in Cabomba, Lim- 
nophila, and Batrachium. In those leaves mesophyll development is 
practically lacking. The floating leaves, however, are entire and have 
good mesophyll development so that the whole space between the veins 
is filled. In both of these cases linear leaves or linear lobes must be 
attributed to a lack of mesophyll growth factors, whereas the vein 
growth factors are normally present. Another tempting conclusion would 
be that in these plants the mesophyll growth factors diffuse out into the 
water just as they diffuse out of germinating peas if these are submerged 

*How general this phenomenon is of leaf reduction in submerged leaves, is in- 


In land plants a similar phenomenon may be observed. Palmate leaves 
often have different degrees of division in the same plant. In general the 
older leaves are more divided than the younger ones up to a certain age 
when the division decreases again. In this case there also seems to be a 
differential between vein growth and mesophyll growth; the larger the 
quotient vein/mesophyll the deeper incised the leaf will be. Good 
examples of such a series in one plant are given by Krenke (16, figures 
1 12-120). Another extreme case is Begonia carolinaefolia (7). The figures 
of Krenke (15, figures 17-19) for Broussonetia papyrifera, are also good 
examples of differential growth. Goebel (8, page 37) already gives a 
scheme explaining various leaf forms by differential development of the 
various regions of the leaf primordia. \^elenovsky (32) also mentions in 
general: "Es geschieht auch nicht selten dass die Spreite an der Rippe 
teilweise oder ganz an einer oder an beiden Seiten verschwindet." 

In the case of bud scales (8,32), we find nice transition series between 
typical scales completely lacking in leaf blade and normal leaves. Since 
the bud scales are considered as transformed stipules rather than leaves, 
we can suppose that at the time of their development there were different 
amounts of stipule and leaf growth factors present. This conclusion 
is strengthened by the observation that under certain conditions organ 
primordia which normally would have developed into scales can give 
rise to more or less normal leaves (8, page 428). Such observations are 
frequently made on the second bud scale of pea seedlings. A quantitative 
expression of such differences (heteroblastic development) was recently 
attempted by Ashby (2), who did not present a physiological explanation, 
but only a graphical or mathematical presentation. 

Differential vein-mesophyll development is common in leaves on 
flowering branches. Often leaf shape changes when the flowering condi- 
tion is attained. Typical and often quoted examples are the difference 
in leaf form on flowering and vegetative branches of Hedera helix and 
of Campanula rotundifolia. In most other plants the leaf implanted just 
below a flower stand has reduced mesophyll development. 

Light. — The effect of light on growth is complex. In the first place 

dicated by the following quotation from Velenovsky: "Die Spaltung des unter- 
getauchten Blattes in lineale Abschnitte ist bei den dikotylcn Aiten eine ver- 
breitete Erscheinung." J. Velenovsky, Vergleichende Moiphologie der Pflanzen 
(Prague, 1905), 413. 

F. W. WENT 291 

light is necessary for the formation of various growth factors and the 
production of carbohydrates, but in the second place the light also 
determines the reaction of the tissues to the available growth factors 
either directly or through activation or destruction of growth factors. 
Let us first consider peas. When grown in near darkness, pea seedlings 
develop small etiolated leaves, but when the cotyledons are cut off 
even this small amount of leaf growth stops. It Ukewise can be almost 
completely inhibited by complete darkness. This can be interpreted as 
meaning that phyllocaline is stored in the cotyledons and is activated 
in the leaves by light. 

In squash a similar behavior can be observed. In darkness seedlings 
do not produce leaf blades, but with sufficient illumination leaves grow 
out. Squashes differ from peas in that they require over 100 times as 
much light to have their phyllocaline activated. 

In mature plants the phyllocaline is no longer stored in the cotyledons 
but comes from the mature leaves where it is formed under the influence 
of light (9). Actually in recent experiments (14) it could be shown that 
light is required both on the mature leaves and on the leaf primordia for 
leaf growth in squash plants; illumination of either one alone is insuffi- 
cient to produce leaf growth. 

These facts can be generalized. Seedlings generally do not expand their 
leaves when grown in darkness. Many seedlings germinating in a dark 
forest produce only rudimentary leaves, but their stems are decidedly 
elongated. Thus climbing plants reach light before they have exhausted 
their cotyledons in useless leaf growth when they germinate in places 
where there is not enough light to attain the compensation point. 

Helianthits plants produce leaves of different shapes when grown in 
different degrees of shading. In full light the plants are vigorous and 
have large heart shaped leaves. In light shade the plants reach approxi- 
mately the same size, the leaves being only slightly smaller. But with 
further decreasing light intensity the leaf size decreases much more than 
the length of the stem. Not only is this true, but the leaf form of the 
shade plants also is different from that of the sun plants. The leaves of 
the former are elliptical, and the lateral veins make much sharper angles 
with the midrib than in the sun leaves. This indicates that mesophyll 
growth is much more reduced than vein growth in the shade. In the 
heaviest shade the first leaves are still approximately normal in size; 
apparently the storage of leaf growth factors in the seed is sufficient to 


make the first leaves develop, but the successive leaves become smaller 
and smaller. 

Both the synthesis of the phyllocaline precursor and its transformation 
into the active material require light. On the other hand the caulocaline 
complex is relatively independent of light. Auxin production can occur 
and continue for considerable periods of time in complete darkness, 
and another factor of the caulocaline complex is formed in the root 
system in darkness. Therefore we should expect the following effects: 
shade does not decrease stem elongation but prevents leaf development, 
and strong light causes expansion of leaves. 

Recent work (10) has shown the effect of photoperiod on leaf shape. 
This is more fully described by Ashby (2). 

Nutrition. — Much work has been done on the effect of various nutri- 
tional deficiencies on the growth of plants. In many plants it is easy 
to distinguish between the various inorganic deficiencies since the lack 
of each element has certain specific effects on the plants. These effects 
are partly expressed as mottling or other color characteristics of leaves. 
In certain cases, however, pronounced differences in development are 
produced also. The latter interest us most in connection with our 
subject. One of the most typical results of zinc deficiency is the so-called 
Httle leaf phenomenon (6). In tomatoes, citrus, and a number of other 
fruit trees the leaves which develop when the plants are grown with 
insufficient amounts of zinc have characteristic small and narrow leaves 
which ultimately will affect the whole growth. This effect on leaf 
growth may also be produced to some extent by nitrogen deficiency. 
In Helianthus and tomato plants narrow leaves or leaflets are formed when 
an insufficient amount of nitrogen is present in the culture solution. The 
effect of boron deficiency is different and is confined especially to the 
growing part, which ultimately dies. In Helianthus again potassium 
deficiency greatly reduces growth in length, although the leaves are 
still wide and heart-shaped. Similar differences in the leaves of Pota- 
mogeton were described by Pearsall and Han by (20). Thus it becomes 
evident that the nutrition experiments also give clear indications of 
differential leaf, vein, and stem growth. 

Viruses.— h general analysis of the effects of viruses on plants shows 
that they can be divided roughly into three groups of symptoms: 

I. Mottling of leaves and stems, due to chlorophyll defects, in severe 

F. W. WENT 293 

cases causing necrosis of tissues. In the second place growth defects 
result which can be traced to deficient photosynthesis. 

2. Phloem necrosis, which prevents further translocation of food and 
growth substances, resulting in stunted growth of all organs, high carbo- 
hydrate content of leaves, etc. 

3. Developmental deficiencies, in which one or more growth processes 
are affected. This third group is the most interesting from our standpoint, 
and within it we can recognize different types. 

A. Retarded growth of stem, accompanied in the later stages 
by increased lateral bud development, leading to witches'- 
brooms. This is typical for deficient auxin supply. In the first 
place the lack of auxin prevents normal elongation of the 
main shoot, and secondly the lateral buds can grow out since 
they are no longer inhibited by auxin. Similar effects may be 
observed in dwarf varieties of plants, where dwarfing is due to 
excessive auxin destruction (31). Thus we are led to assume 
that such virus effects are due to auxin deficiency caused by 
the virus, and perhaps also to destruction of auxin {Primus 
virus 3, Saccharum virus 3, and Delphitjium virus i are good 
examples of this case). 

B. Retarded mesophyll growth. The early symptoms of this 
type of virus are narrow leaves. In severe cases the mesophyll 
may be practically lacking so that only the midrib remains. 
This would be a typical case where the virus specifically 
affects phyllocaline without influencing vein growth. As an 
example may be quoted Cucumis virus i on Spinacia, tomato, 
etc. and the shoestring virus of tobacco {Nicotiana virus i). 

C. Leaf curling due to retarded vein growth. In a few cases 
this may be accompanied by unchecked stem growth, but 
more often stem elongation is inhibited as well. In these cases 
mesophyll growth is more or less normal so that the whole 
leaf bulges and is corrugated. Examples are Rubus virus 3, 
Gossypium virus i, and Nicotiana virus 10. 

Thus if we summarize the effects of viruses on growth phenomena, 
we are led to assume that vein growth, mesophyll growth, and stem 
growth are unrelated phenomena, which may be specifically affected 


by one or another virus. The effects of the viruses are ahvays growth 
retardations of any of these processes, suggesting that the viruses act 
by destroying or inactivating specific growth factors or by decreasing 
their production. Any combination of these effects may be found in 
various virus diseases. Doubtless a closer analysis of the virus effects 
would reveal many more specifically affected growth factors, so that 
observations in this field may give many clues for the existence of 
further specific growth factors. 

Genetical EwV/e/zc^.— Practically the same disturbances in leaf form 
and size which result from virus attack can be found as hereditary 
malformations. Both in tobacco (deformis, see 12) and in tomato (wiry, 
see 17) forms with linear leaves are known in which the lack of mesophyll 
is due to single recessive genes, and which look remarkably like shoe- 
string-virus diseased plants. Since all these abnormal leaves look much 
alike, it is logical to assume that all the agents affect the same basic 
growth process, causing the disappearance of phyllocaline. In other 
pinnate leaves, such as Antirrhinum (23), genes cause narrowing of leaves, 
whereas in palmate leaves a similar gene gives rise to laciniate forms 
(for example, Chelidonium) . 

Sirks (27) has investigated the inheritance of leaf size in eight varieties 
of Vicia Faba, measuring both width and length of individual leaflets. 
He interprets his results by assuming a factor (G) for general leaf growth 
(with various allelomorphs), which acts in conjunction with additional 
factors for width (W) and length (B + T). Thus he definitely considers 
leaf growth as caused by independent factors for growth in width and 

in length. 

Such examples can be augmented by citing the work of de Winton 
and Haldane (41) on Primula, in which 5 or 6 leaf form characteristics 
were recognized, or the work of Shull (25) on Capsella Bursa- Pa starts, 
or the many investigations on cotton (see, for example, Stephens, 29). 

It is interesting to note that mutations which seem to exert their 
effect by decreasing the amount of special hormones are common to a 
number of plants. In addition to the phyllocaline-destroying genes could 
be cited the auxin-destroying genes, which cause dwarfing and often 
excessive branching. 

Morphogenetic Effects of Certain Growth Substances.— Zimmerman (43) 
has described the effect which many substances with an auxin-like 
structure have on leaf development. Since then many other chemicals 

F. W. WENT 295 

have been developed which produce similar effects. There is no general 
correlation between growth-promoting effects and effects on reduction 
of leaf blade development (see for example, Zimmerman's paper on 
page 176). The effect of 2,4-dichlorophenoxyacetic acid on tomato or 
cotton leaves is much like that of shoestring virus or the genes discussed 
above. Since all these widely different agents cause the same final 
results, it must be concluded that they all affect the same basic, and 
probably simple, process, like the phyllocaline-caulocaline balance sug- 
gested in the previous paragraphs. 

Taxonomic Evidence. — In a large number of genera, species are dis- 
tinguished by leaf characteristics, and again here narrower or broader 
leaves or laciniate and hardly incised leaves characterize different species. 
Edgar Anderson's analysis (i) is an example of how taxonomists and mor- 
phologists move hand in hand towards a physiological explanation. He 
made a comparison between the closely related species Nicotiana Langs- 
dorffii and N. alata and found them to differ in 1 1 genetic coefficients. 
Among these the leaf-vein angle was one, a sharp angle being correlated 
with narrow leaves and narrow corolla lobes. 

In Sidalcea the basal leaves are crenate or crenately incised. Most 
species [S. malvaejiora, S. oregana) have the upper leaves palmately 
twice cleft into linear divisions. But in other species the upper leaves 
are not parted or divided. In Ranunculus or Delphinium some species 
are also characterized by leaves which are divided into narrow segments 
whereas other species have almost entire leaves. 

Teratological Evidence. — In any book on teratology a host of examples 
can be found in which a normally broad-leaved plant bears exceptional 
narrow leaves, or where palmately divided leaves are found on otherwise 
entire-leaved plants.* 

This enumeration has stressed the differential effects of light, nutri- 
tion, viruses, genes, growth substances, normal and abnormal develop- 
ment on caulocaline and phyllocaline production or action. Yet this does 
not mean that leaf growth is that simple. When we know more about the 
factors controlling leaf growth we undoubtedly will find that more 
are involved. In the genetic analysis more than two sets of gene pairs 
were necessary to account for the observed differences in leaf form. With 
only two sets of leaf growth factors involved, leaf form would be much 
less variable. Yet it seems that many of the most common variations in 

*M. T. Masters, Pflatizen-Teratologie (Leipzig, 1886), 482, 483, 516, 517, 518. 


leaf form can be accounted for by the two-factor scheme of phyllocaHne- 
caulocaHne in their relative proportions. 

Stem Growth. — Very little new evidence has been collected during the 
last few years concerning the role of factors other than auxin in the 
elongation of stems. That such other factors exist was assumed as soon 
as the work with auxin started (33) and was further supported by subse- 
quent experiments (34,35)37)- Schneider (24) also found that in addition 
to auxin and sugars some other factor controlled growth of Avena 
coleoptile sections. In a few experiments Went and D. Bonner (39) 
were able to replace this factor with pea diffusate or yeast extract, but 
these experiments could not be repeated consistently. Therefore all 
our knowledge about caulocaline, as this factor has been named, is 
circumstantial. In this respect it rather resembles florigen, with which 
it also has in common the fact that it moves only through continuous 
vascular connections. When a stem is cut and grafted on another base, 
growth is resumed only after the graft has "taken" or in other words has 
new vascular tissue between the graft partners (11). This was also found 
in the case of the transmission of the flowering stimulus (42). 

Synthesis of caulocaline takes place in roots, and in exceptional cases 
in stems (for example, Asparagus 18). In branches of deciduous plants and 
in seeds it can be stored in limited amounts, but otherwise stem elonga- 
tion occurs only when such stems are connected through vascular ele- 
ments with roots. Therefore stem cuttings of nondeciduous plants do 
not start to grow until new roots have appeared. Similarly detached 
buds grown under sterile conditions start to elongate only after roots 
have been formed on them (21,28). This is even partly true for Asparagus 
stem tips, whose growth is much speeded up when roots regenerate on 
them (18). 

Summary. — Growth of leaves can be differentiated into vein growth, 
under the influence of the same factors as stem growth (caulocaline), 
and mesophyll growth, controlled by different growth factors (phyllo- 
caUne) among which adenine has been identified. Difl^erences in leaf 
form can often be interpreted as being due to differential vein and 
mesophyll growth. Thus it seems that the form of submerged leaves, of 
shade leaves, of many virus-infected leaves, of laciniate and other 
genetically controlled leaf forms is due to lack of mesophyll growth 
factors. Also in other cases, where for example, virus has caused decreased 

F. W. WENT 297 

vein growth, the leaf shape can be explained by the phyllocaline- 
caulocaline balance. Hardly any new data concerning caulocaline have 
been collected. Parallelisms between caulocaline and florigen are pointed 
out in that both are translocated inside the plant only through intact 
vascular connections, and both have defied most of the attempts at 


1. Anderson, E. and Ownbey, R. P., Ann. Missouri Botan. Garden, 26:327 


2. AsHBY, E., New Phytologist, 47:153 (1948). 

3. Beijerinck, M. W., Botan. Zeit. 46:1, 17 (1888). 

4. Bonner, D. and Haagen-Smit, A. J., Proc. Nat. Acad. Sci., 25:184 (1939). 

5. , and Went, F. W., Botan. Gaz., 101:128 (1939). 

6. Chandler, W. H., ibid., 48:625 (1937). 

7. Doposcheg-Uhlar, J., Flora, 102:24 (191 1). 

8. Goebel, K., Organographie der Pflanzen (Jena, 1928). 

9. Gregory, F. G., Ann. Botany, 42:469 (1928). 

10. Harder, R. and von Witsch, H., Ja/irb. wiss. Botan., 89:354 (1940). 

11. Hayward, H. E., and Went, F. W., Botan. Gaz., 100:788 (1939). 

12. Honing, J. A., Genetica, 5:455 (1923). 

13. Huxley, J. S., Problems of Relative Groivth (New York, 1932). 

14. Juhren, M. C. and Went, F. W., Am.]. Botany, 36:(8), 552 (1949). 

15. Krenke, N. p., Phenogenetical Variability (Moscow, 1933), p. 11. 

16. , ibid., (Moscow, 1935), p. 529. 

17. Lesley, }. W. and Lesley, M. M., /. Heredity, 19:337 (1928). 

18. Loo, S., Am. J. Botany, 32:13 (1945). 

19. Masters, M. T., PJlanzen-Teratologie (Leipzig, 1886). 

20. Pearsall, W. H. and Hanby, A. M., New Phytologist, 24:112 (1925). 

21. DE Ropp, R. S., Ann. Botany, 9:369 (1945). 

22. Sachs, J., Arb. Bot. Inst. Wiirzbtirg, 2:452, 689 (1880, 1882). 

23. ScHiEMANN, E., Zeitsch. Ind. Abst. Vererb., 41:53 (1926). 

24. Schneider, C. L., Am. J. Botany, 25:258 (1938). 

25. Shull, G. H., Verh. naturf. Verein Briinn., 49:?' (191 1). 

26. SiNNOTT, E. W., Science, 85:61 (1937). 

27. SiRKS, M. J., Proc. Koninkl. Akad. Wetenschap. Amsterdam, 32:1066 (1929). 

28. Skoog, F., Am. J. Botany, 26:702 (1939). 

29. Stephens, S. G.,J. Genetics, 46:313 (1945). 

30. Troll, W., Biol. Zentr., 49:43 (1929). 

31. van Overbeek, J., Plant Physiol., 13:587 (1938). 

32. Velenovsky, J., Vergleichende Morphologic der Pflanzen (Prague, 1905), 

33. Went, F. W., Rec. trav. bot. neeri, 25:1 (1928). 

34. , Proc. Koninl^l. Al^ad. Wetenschap. Amsterdam, 38:752 (1935). 

35. , Plant Physiol., 13:55 (1938). 

36. — , Am. J. Botany, 25:44 (1938). 


37. , ibid., 26:109 (1939a). 

38. , Botan. Gaz., 104:460 (1943). 

39. , and Bonner, D. M., Arch. Biochem., 1:439 (1943). 

40. , and Thimann, K. V., Phytohortnones (New York, 1937). 

41. DE WiNTON, D. and Haldane, J. B. S., /. Genetics, 27:1 (1933). 

42. WiTHROw, A. P. and Withrow, R. B., Botan. Gaz., 104:409 (1943). 

43. Zimmerman, P. W., Torreya, 43:98 (1943). 

.Growth Substances 
in Reproductive Development 

Chemical Regulation of Sexual Processes in Fungi 


IN 1880 Sachs (61) advanced the hypothesis that the differentiation, 
development, and the proper function of vegetative and sexual organs 
of plants depend upon the activity of specific chemical substances. 
Shortly thereafter chemical correlative mechanisms were postulated to 
explain a number of well-known sexual phenomena in fungi: conjugation 
between smut spores (13), antheridial production in Saprolegriia (10), 
hyphal fusions (70), and so on. None of these, however, claimed a more 
definitive basis than deductive speculation. 

In the seventy years which have elapsed since the publication of 
Sachs's work numerous authors have contributed to the greater under- 
standing of the role of chemicals in the regulation of sexual processes 
in fungi. These contributions fall naturally into two categories dealing 
respectively with i) the chance effects of various chemical substances 
upon the sexual process as measured by the quantitative and/or quali- 
tative effect upon the final product of sexual reproduction, and 2) the 
effects of specific chemical substances secreted by a plant and playing 
indispensable roles in the regulation of its own sexual process or of those 
of another and compatible individual of the same species. The first type 
of regulation may be considered as a biological accident, whereas the 
second type of regulation constitutes an integral and necessary part of 
the mechanism of the plant's vital reproductive process, and the chemical 
regulators, originating and exerting their physiological role within the 
plant, may be considered as true hormones. 

The first category will be reviewed briefly here while the second will 
be given more thorough attention with considerable detail of those all 
too few cases where the broad outlines of the chemical correlative 
mechanisms have been elucidated. 


Extra- specific chemical regulation of sexual processes. — Among the earlier 
works on the relationship to sexual reproduction of chemical substances, 
nutrients, or other compounds in the medium, are those dealing with 
the factors influencing sexuality in the water molds, members of the 
aquatic order Saprolegniales. The works of Klebs (25,26,27), Kauffman 
(24), Pieters (42), and Coker (15) established the general pattern of 
environmental conditions necessary for the production of sexual organs 
and traced the effects of many variants in the chemical environment of 
the plant. 

The first of a large number of papers, continuing to the present time, 
on the interspecific effects of metabolites on sexual reproduction ap- 
peared in 1903 when MoUiard (37) demonstrated that the relative 
abundance of ascocarps oi Ascobolus was greatly increased by the presence 
of certain bacteria. Heald and Pool (23) described a similar stimulation 
of the sexual process in Melanospora pampanea by the metabolic products 
of certain species of Fusarium and Basisporium. In the latter case the 
effective agent was shown to be a complex organic acid. Sartory (62, 
63,64) demonstrated the effectiveness of bacteria in association with a 
yeast and with two species of Penicillium in promoting sexual activities 
in these fungi. Dodge (17,18) observed the beneficial effects of certain 
bacterial associates on the fruiting of Ascobolus magnificus and also 
pointed out the probable role of the plant's own metabolic activities 
on the sexual process. The formation of perithecia of Thielava hrasicola 
was likewise found by McCormick (36) to be greatly increased by 
extracts of other fungi. The number and size of perithecia of Venturia 
inequalis were shown by Wilson (71) to be greatly increased by a heat- 
labile component of the filtration-sterihzed brei of Penicillium sp. 

Robinson (59), in an extensive investigation of growth and sexuality 
in Pyronema confiuens, demonstrated the close dependence of sexual 
reproduction on the chemical nature of the medium and the essential 
role of light in initiating the immediate conditions for the formation of 
sexual organs and for the development of ascocarps. Comparable work 
on a number of species oi Phytophthora by Gadd (20), Lester-Smith (35), 
Ashby (4,5,6,7), and Leonian (31,32,33), complicated by uncertainties 
as to the pattern of sexuality obtaining in the genus, finally resulted in 
the demonstration of beneficial effects upon the production of oogonia 
and oospores by extract of peas and by extracts of numerous unicellular 
green algae. The influence of C/N ratio upon sexual vigor in Mucor 


heitnalis and Phycomyces Blakesleeamis was investigated by Schopfer (65) 
and Ronsdorf (60) respectively. In P. B/af{esIeeafms phloroglucin com- 
pletely suppressed sexual activity while histamine greatly augmented 
the production of zygospores. The only demonstrated instance of a 
significant effect upon the sexual process of a fungus by a mammalian 
sex hormone is that described by Plumb and Durrel (43) in which 
oestrin or theelin was shown to suppress completely the production of 
gametangia and zygospores in Rhizopus nigricans. 

The abundance of perithecia of Melanospora destruens was shown by 
Hawker (8,21,22) to be greatly increased by the presence of contaminat- 
ing colonies of Botrytis, Fusarium, Gleosporiiim, Sclerotinia, and Peni- 
cillium. Lentil extract also stimulated perithecial formation; this effect 
was proved to be due to thiamin and biotin, each of which alone would 
support vegetative growth in a synthetic medium while both were 
required for sexual activity. Inositol, which was shown to have no effect 
on M. destruens, was shown by Raper (45,47) to increase the intensity 
of the sexual reaction in matings of Achlya ambisexualis in an agar 

The requirements for sexual reproduction in Phycomyces Bla\esleeanus 
was the subject of an extensive investigation chiefly by Schopfer and 
Robbins and their associates during the decade 1932-42. Thiamin or 
its pyrimidine component is required for vegetative growth but other 
accessory substances were considered necessary for zygospore formation 
(66,67,68). Such factors were obtainable from numerous natural sub- 
strates including malt, oats, potatoes, and agar (52,53,54). The activity 
in potatoes consisted of two mutually augmentative factors, Zi and Z2, 
the former adsorbed on Norite, the latter not adsorbed on Norite (56). 
Factor 7i was identified as hypoxanthine (58) and was replaceable by 
guanine (57), a closely related purine-base compound. The contention 
that specific accessory substances were required for zygospore formation 
was disputed by Leonian and Lilly (34) who claimed that sexual sterility 
resulted from nutritional inadequacies. Zygospores were formed in a 
synthetic medium containing thiamin, sugar, and only one of a number 
of amino acids (aspartic acid being the best one). Succinic acid increased 
the abundance of zygospores while ammonium nitrate completely sup- 
pressed their formation; the two effects were partially antagonistic. 

The most completely elucidated interspecific stimulation of sexual 
processes is that of Zygosaccharomyces sp. described by Nickerson and 


Thimann (40,41). This species in pure culture gave only 8 to 12 per cent 
conjugation while in cultures contaminated with Aspergillus m'ger the 
incidence of conjugation was increased to 65 to 75 per cent. This effect 
could be readily duplicated by filtrates of the culture fluid in which 
the contaminating Aspergillus had grown. The yeast itself was shown to 
contain the active substance but in low quantity. The incidence of 
conjugation in slant cultures was shown to be much higher in the thin 
end of the slant and this could be correlated with the higher percentage 
of dead cells in this region as shown by staining with Methylene Blue. 
This points to the possible role of diffusible substances originating from 
moribund or dead cells in aging cultures in the normal reproductive 
process of the yeast. The stimulating activity of the filtrate of A. ?iiger 
proved to be organic and to consist of two fractions, an organic acid and 
a member of the vitamin B complex. Each alone was active but together 
gave activity greater than the sum of their single activities. The stimu- 
latory effects of these two substances could be duplicated by glutaric 
acid and riboflavin. Whether these two compounds are identical to 
the active substances secreted by A. niger and contained in the cells 
of Zygosaccharomyces has not been determined. 

In Table i is presented a summary list of the demonstrated cases 
of interspecific stimulation of sexual processes and of the few cases where 
known chemical compounds exert a significant effect on sexual reactions 
. in the fungi. 

Intra- specific chemical regulation of sexual processes. — The first demon- 
stration of the initiation and coordination of sexual reproductive processes 
by diffusible, autosecreted substances was given by Burgeff in 1924 (14) 
for Mucor mucedo. In matings of (+) and ( — ) strains of this species 
on an agar medium a restraint area was formed along the line of inter- 
mingling of the two myceha and into this region only a few hyphae 
penetrated from each mycelium. At the tips of these hyphae corraloid 
swellings developed before any contact was established and there were 
produced upon them what appeared to be progametangia. This was 
considered to be the initial stage in the sexual reaction and since it 
occurred before contact of (+) and ( — ) hyphae it indicated the presence 
and activity of diffusible chemical messengers. To test this hypothesis 
Burgeff performed the classical experiment of placing a block of agar 
containing (-|-) hyphae on the surface of a ( — ) mycelium growing upon 
an agar medium with a permeable collodian membrane interposed be- 



Extra-Specific Chemical Regulation of Sexual Processes in 


Species affected 



Identity Authority 

Ascobolus sp. 


Pilobolus sp. 



Penicillium sp. 


Fusarium spp. 





Theilavia brasicola Other fungi? 


Green Algae 

Apothecia -j- 
Perithecia + 

Zygospore + 

Apothecia + 

Sporulation -\- 
Perithecia -f- 
Perithecia + 
Oogonia -|- 

Organic acid 

Venturia inequalis Penicillium sp. Perithecia + 


Rhizopus Nigricans Mammals 

Zygospores + 
Zygospores — 

Zygospores — 










(63, 64) 








Spp. Fusarium, Perithecia + 

Botrytis, etc. 
Lentils Perithecia + 

? (8, 21, 22) 



Oogonia + i-inositol 

Potato, Oats, Zygospores + 
Malt, Agar, etc. 

Zygosaccharomyces Aspergillus Conjugation + 


Zi, Hypoxan- 

Z2, ? 

1. Glutaric 


2. Riboflavin 

(45. 47) 
(57. 58) 

(40, 41) 


tvveen the two compatible mycelia. Corraloid swellings, the zygophores 
or sexual organ primordia, formed on the ( — ) mycelium opposite new 
growth across the membrane from the agar block containing the (+) 
hyphae. Thus activation without contact at a distance, or telomorphosis, 
was proven. Also in a number of cases compatible zygophores on opposite 
sides of the membrane were seen to grow towards each other; this he 
termed zygotropism. Thus the initiation of the formation of sexual organs 
and their correlated development were proved to be the result of 
hormonal action. 

These results were confirmed and in some cases extended by a number 
of workers on M. mucedo and other heterothallic members of the 
Mucorales. Verkaik (69) claimed that only the (+) strain could be 
induced to initiate progametangia in membrane matings and that there- 
fore only the ( — ) strain secreted a stimulatory substance. Ronsdorf (60) 
and Kohler (28), working with P. B la J^esleeanus and M. mucedo respec- 
tively, found that zygophores could be induced on the mycelia of both 
strains and that at least two substances must be involved; one from each 
strain acting upon the mycelium of the opposite and compatible strain. 
The former worker attempted unsuccessfully to extract the active 
substances from the mycelia of both strains. The activity of histamine 
in augmenting the sexual reaction intensity in matings of (+) and (— ) 
strains was interpreted as indicating a close chemical relationship be- 
tween this substance and the sexual hormones secreted by the plants. 

Krafczyk (29,30), in experimental work on Pilobolus crystallinus, 
postulated much more extensive activity of sexual hormones than had 
been claimed by previous workers. The mutual attraction of vegetative 
hyphae of the two sexual strains before the formation of zygophores, the 
initiation of sexual organ primordia, the coordinated enlargement of the 
progametangia while in contact at their tips, and the delimitation of the 
gametangia by septation of the progametangia he considered to be under 
hormonal control, that is, hormonal control of the entire sexual process 
until the time of gametangial differentiation. 

In none of the Mucors, however, has sufficient evidence been obtained 
to postulate either the number of substances involved, the loci of their 
origins, or their specific activities in correlating the various phases of the 
sexual reproductive process. 

In 193 1 a paper by Moreau and Moruzi (38) touched off an interesting 
controversy. These workers claimed the production of perithecia as the 


result of diffusible hormones acting between compatible strains of 
Neitrospora in the absence of hyphal contacts. Two compatible strains 
inoculated in the opposite ends of a U-tube were alleged to produce 
perithecia although no mycelial growth bridged the connecting tube 
between them. Extensive efforts to duplicate these results were made 
by Dodge (19) and Aronescu (2,3) who, by genetical methods, clearly 
demonstrated that whenever perithecia appeared it was the result of 
association of nuclei (hence of mycelia) of both compatible strains. 
Further evidence for hormonal induction of perithecia by Moreau (39) 
was unconvincing. 

The refutation of Moreau's extensive claims, however, by no means 
indicates that hormones are not involved in the sexual process of 
Neurospora and related forms. Two observations by Backus (9) would 
indicate that they might well be. The first is that conidia placed on 
medium or even on a glass surface over which the vegetative mycelium 
of the compatible strain has grown will not germinate although they 
retain their ability to fertihze compatible ascogonia. The second, lateral 
branches arise from the trichogyne only at points normal to the shortest 
distance to compatible conidia, microconidia, or hyphae, and that such 
lateral branches grow directly to the fertilizing element. 

A case similar to the latter has been described by Zickler (72) in 
Bombardia, in which the tips of the trichogynes are strongly attracted 
by and grow to masses of microconidia, or spermatia. That this is a 
chemotropic response was shown by i) a capillary filled with an extract 
of spermatia and introduced into the agar in the vicinity of compatible 
trichogynes caused directional growth of the trichogynes toward and 
into the tip of the capillary, and 2) an agar block soaked in the filtrate 
of liberated spermatia had the same directional effect on the growth of 
trichogyne tips. Boiling had no adverse effect on the activity of the 
extract and filtrate. 

It is only in a few species of the aquatic phycomycetous order 
Saprolegniales, however, that sufficiently detailed information has been 
obtained to approach a definition of the over-all hormonal coordinating 
mechanism of sexual processes in the fungi. As early as 1881 de Bary 
(10) suggested that a chemical substance secreted by the oogonial initial 
induced the formation of antheridial hyphae and exerted over them a 
chemotropic effect. Kauffman (24) induced antheridial production in a 
species normally lacking the male sexual organs by adjusting the content 


of inorganic salts in the medium; he interpreted these results as affecting 
the conditions under which de Bary's postulated substance would be 
secreted or effective. Couch (i6) suspected specific chemical substances 
as correlative in the mating of male and female strains of Dictyuchus 
monosporus on the basis of the following observations: i) oogonial initials 
and antheridial hyphae were formed on reacting mycelia at locations far 
removed from the region of actual contact; 2) the directional growth 
of antheridial hyphae to the oogonial initials; and 3) the marked stimula- 
tion, including sexual organ formation, in intergeneric matings with 
Thraustotheca primoachlya. He devised experiments, including the effects 
of extracts and filtrates, membrane matings, and matings in which 
certain of the partners were not in actual contact, but the results were 
in each case negative. 

Bishop (11,12) found definite evidence for the activity of diffusible 
hormones in Sapromyces Reinschii of the Leptomitaceae. He demon- 
strated the production of antheridial hyphae and oogonial initials on 
male and female mycelia before contact and the induction of greatly 
increased branching of male hyphae under the influence of the filtrate 
of a female culture. These effects as well as the directional growth of 
antheridial hyphae to the exact distal ends of oogonial initials he at- 
tributed to diffusible hormones. No attempt, however, was made to 
define the minimal mechanism necessary to explain these effects. 

The hormonal mechanism regulating the sexual process has been more 
completely worked out by Raper and associates in two heterothallic 
species of Achlya, A. ambisexiialis and A. bisexualis, than elsewhere 
in the fungi. The various phases of the sexual process up to and including 
the differentiation of oogonia and the delimitation of oospheres (eggs) 
has been shown to be initiated and coordinated by specific diffusible 
hormones (44). A number of lines of evidence indicated hormones as 
the coordinating agents: i) the unvarying sequence of events and the 
pattern of temporal relationship between the successive stages; 2) the 
different and characteristic stages of the sexual progression attained in 
the reciprocal interspecific matings between the two heterothallic species; 
and 3) the effects of variations in the nutrient content of the medium 
upon the sexual reaction (45). Conclusive proof of the hormonal mecha- 
nism as well as the means of determining the number of hormones in- 
volved, the loci of their secretion, and their specific activities has been 


furnished by distance reactions (telomorphotic reactions). These reactions 
include among others initiation of and correlation of organ development 
before contact of the two sexual strains in matings in water and on agar, 
matings with permeable membranes interposed between the two part- 
ners, and the effects of filtrates (46,48,49). 

No less than six distinct hormones are now known to be involved 
in the coordinative mechanism: four secreted by the male and two 
by the female. The hormone mechanism as it appears on the basis of 
information currently available is given in Table 2.* 

The entire sexual reaction is initiated by the simultaneous secretion 
of three substances by the vegetative male and female plants: hormone 
A by the female, hormone A', an augmenter of the activity of hormone 
A, and an inhibitor by the male plant. This complex of hormones induces 
and regulates the formation of antheridial hyphae on the male plant, 
the number of male sexual organ primordia being a logarithmic function 
of the concentration of hormone A and a linear function of the concen- 
tration of hormone A' (48,49,50). The quantitative production of 
antheridial hyphae depends also on a number of physical factors includ- 
ing temperature, hydrogen-ion concentration, electrolyte concentration 
(48), and under certain restricted conditions, light intensity (50). Hor- 
mone A is known to be indispensable to the reaction, but whether this is 
also true of hormone A' has not been determined since the only means of 
determining the effect of hormone A' is the quantitative reaction of the 
same plant which secretes it (49). 

The antheridial hyphae, beginning shortly after their initiation, secrete 
hormone B which induces the initiation and controls the development 
of the oogonial initials, the female sexual organ primordia, on the female 
plant. The oogonial initials during the period of active growth and until 
the time of their delimitation secrete at least one hormone, hormone C. 
If only one hormone is involved at this stage it has two distinct effects: 
the attraction, that is, the directional growth of the antheridial hyphae 
along the concentration gradient to the source of the secretion, the 
oogonial initial; and the deUmitation of a short segment of the tip of the 
antheridial hypha as the male gametangium or antheridium, but only 

*Two subsequent papers by the author, Bot. Gaz., 112: 1-24 (1950) and Pioc. 
Nat. Acad. Sci. (in press), deal respectively with a newly discovered, J-secreted 
hormone and with the roles of sexual hormones in homothallic species. 

































2 < 












I— ( 

I— I 





) CO 

' 9 


' ^ 








1— t 



H ^ 

H •-' 

_.o >< 


5 t c^ 

























after it has become applied to the oogonial initial or some other soHd 
surface. It is quite probable that hormone C is in actuality a complex of 
two or more distinct substances. 

Once the antheridia have become delimited they secrete yet another 
substance, hormone D, which causes the oogonium to be delimited 
by the formation of a transverse septum across the base or stalk of 
the globular initial, and the reorganization of the protoplasmic contents 
of the oogonium to form a number of uninucleate, spherical gametes 
(oospheres or eggs). Again in this stage more than a single hormone may 
well be involved. 

The entire sexual reaction, except for the transfer of nuclei in fertiliza- 
tion, progresses in a normal manner between certain compatible partners 
separated by a permeable membrane. In other matings (with or without 
an interposed membrane) the reaction stops at earlier phases correspond- 
ing in each particular case with the specific action of one of the several 
hormones. Interspecific or even intergeneric matings between hetero- 
thallic species and various homothallic forms likewise proceed to definite 
end points in the sexual progression, each characteristic for the specific 
mating and again to stages corresponding to the specific actions of single 
hormones. Actually there is considerable evidence that the coordination 
of the sexual processes in the homothallic forms is similar, excepting 
hormone specificities, to that of the male plus female mechanism in the 
heterothallic forms (50). 

Isolation and chemical identification has not as yet been possible 
for any of the hormones involved in the sexual reaction of Achlya. 
The physical and chemical characteristics of hormone A have been 
worked out in considerable detail, and enormous enrichment of the 
active compound has been achieved: from 1,440 liters of culture fluid 
of the female plant of ^. bisexualis, 37 per cent of the initial activity was 
concentrated to 0.0002 grams. This material was active in inducing 
antheridial hyphae on male plants of A. ambisexualis in a concentration 
of less than io~^^ (51). The nutritional requirements of and environ- 
mental factors affecting hormone A production have been intensely 
studied but much more information is needed (1,48). The chemical and 
physical properties of hormone A' have been subjected only to pre- 
liminary investigation. Isolation and/or identification of any of the 
hormones oi Achlya will depend on the availability of far larger quantities 


of raw filtrates than can be produced with the faciHties commonly 
available in the academic microbiologist's laboratory. 

Although there are many instances in which control of sexual reactions 
in fungi by means of hormones or hormone-like substances have been 
described or indicated, surprisingly little is actually known of the specific 
roles of such chemical agents. In all too few cases have studies of such 
effects been carried beyond the initial observations through the intensive 
explorations requisite to an understanding of the correlative mechanism. 
Perhaps the information now available from both fungi and algae, 
although fragmentary and incomplete, may stimulate work in this 
biologically interesting, and perhaps in time practically important, field 
of botanical study. 


1. Allen, Carlene M., Thesis, Univ. of Chicago. (1947). 

2. Aronescu, Alice, Mycologia, 25:43 (1933). 

3. , i^id., 26:244 (1934). 

4. AsHBY, S. P., Trans. Brit. Mycol. Soc, 13:86 (1928). 

5. , ibid., 14:18 (1929). 

6. , ibid., 14:254 (1929). 

7. , ibid., 14:260 (1929). 

8. AsTHANA, R. p. and Hawker, L. E., Ann. Botany, 50:325 (1936). 

9. Backus, M. P., Bull. Tor. Bot. Club, 66:62 (1939). 

10. DE Bary, Anton, Beit. z. Morph. und Physiol, d. Pilze, 4, (1881). 

11. Bishop, Harlow, Thesis, Harvard University (1937). 

12. , Mycologia, 32:505 (1940). 

13. Brefeld, Oscar, Bot. Unters. ii Schimmelpilze, 5:1 (1883). 

14. BuRGEFF, H., Bot. Abhandlungen, 4:5 (1924). 

15. CoKER, W. C, The Saprolegniaceae (University of North Carolina Press, 


16. Couch, J. N., Ann. Botany, 40:848 (1926). 

17. Dodge, B. O., Bull. Tor. Bot. Club, 39:139 (19 12). 

18. , Mycologia, 12:115 (1920). 

19. , Bull. Tor. Bot. Club, 58:517 (1931). 

20. Gadd, C. H., Ann. Roy. Bot. Card. Peradeniya, 9:47 (1925). 

21. Hawker, Lilian E., Ann. Botany, 50:699 (1936). 

22. , ibid., NS 3:657 (1939). 

23. Heald, F. D. and Pool, V. W., Nebraska Agr. Exp. Sta. Rep. 22:130 (1909), 

24. Kauffman, C. H., Ann. Botany, 22:361 (1908). 

25. Klebs, G.,Jahr.f. Wiss. Bot., 32:1 (1898). 

26. , ibid., 33:513 (1899). 

27. , ibid., 35:80 (1900). 

28. Kohler, F., Planta, 23:358 (1935). 

29. Krafczyk, H., Ber. Deutsch. Bot. Gesell., 49:141 (1931). 

30. , Beit. z. Biol. d. PJlanzen, 23:349 (1935). 


31. Leonian, L. H., Phvtopath., 21:941 (1931). 

32. ,7. Agr. Res., 51:277 (1935). 

33. • , Botan. Gaz., 97:854 (1936). 

34. , and Lilly, V. G., Am. J. Botany, 27:670 (1940). 

35. Lester-Smith, W. C, Ann. Roy. Bat. Gard. Peradenyia, 10:243 (1927)' 

36. McCoRMicK, F. A., Conn. Agr. Exp. Sta. Bui/., 269:539 (1925). 

37. MoLLiARD, M., C. Rend. Acad. Sd., 136:899 (1903). 

38. MoREAu, F. and Moruzi, C, C. Rend. Acad. Set. Paris, 192:1475 (1931). 

39. Moreau, F., ibid., 206:369 (1938). 

40. Nickerson, W. J. and Thimann, K. V., Am. J. Botany, 28:617 (1941). 

41. , ibid., 30:94 (1943). 

42. Pieters, a. J., ibid., 2:529 (1915). 

43. Plumb, G. W. and Durrel, L. W., Science, 2:386 (1933). 

44. Raper, John R., Science, N.S. 89:321 (1939). 

45. ■ , Am. J. Botany, 26:639 (i939)- 

46. , ibid., 27:162 (1940). 

47. , Mycologia, 32:710 (1940). 

48. , Am. J. Botany, 29:159 (1942). 

49. , Proc. Nat. Acad. Sci. U. S., 28:509 (1942). 

50. , Unpublished Notes. 

51. , and Haagen-Smit, A. J.,/. Biol. Chem., 143:311 (1942). 

52. RoBBiNS, W. J., Botan. Gaz., 101:428 (1939). 

53. , Am. J. Botany, 26:772 (1939). 

54. , ibid., 27:559 (1940). 

55. , Botan. Gaz., 102:520 (1941). 

56. , and Hamner, K. C, ibid., 101:912 (1940). 

57. , and Kavanagh, F., Proc. Nat. Acad. Sci. U. S., 28:4 (1942). 

58. , Proc. Nat. Acad. Sci. U. S., 65 (1942). 

59. Robinson, W., Ann. Botany, 40:245 (1926). 

60. RoNSDORF, L., Planta, 14:482 (1931). 

61. Sachs, J., Arb. Bot. Inst. Wiirzberg, 452 and 689 (1880). 

62. Sartory, a., C. Rend. Soc. Biol., 72:558 (1912). 

63. , ibid., 79:174 (1916), 

64. , ibid., 83:1113 (1920). 

65. ScHOPFER, W. H., Bull. Soc. Bot. Geneve, 2nd ser., 20:149 (1928). 

66. , Bull. Soc. Bot. Suisse, 40:87 (1931). 

67. , ibid., 41:73 (1932). 

68. , C. Rend. Soc. Phys. Hist. Nat. Geneve, 57:45 (1940). 

69. Verkaik, C., Proc. K. Acad. Wetensch. Amsterdam, 33:656 (1930). 

70. Ward, M., Ann. Botany, 2:319 (1888). 

71. Wilson, E. E., Phytopathology, 17:835 (1927). 

72. Zickler, H. Cited in Hartmann, M., Die Sexualitdt (Fischer, Jena, 1943)1 

P- 385- 

The Sexual Substances of Algae 


UNION of two gametes to form a zygote is a widespread phenomenon 
among algae, especially those belonging to the grass green algae 
(Chlorophyta) and the brown algae (Phaeophyta). Depending upon 
the alga, there is a union of two motile flagellated gametes, or a union 
of a small motile gamete and a large immobile nonflagellated gamete, 
or a union of two nonflagellated gametes one or both of which move 
in an amoeboid manner. 

Sexuality among algae was first recognized by Nathan Pringsheim 
(17) in 1858 in connection with his study of reproduction in Oedogonium, 
a green alga in which a small motile male gamete (the antherozoid) 
swims to and unites with a relatively large immobile egg lying within 
the female sex organ (the oogonium). Within a few years it was suggested 
that the swimming of antherozoids towards an egg is not a matter of 
chance but is a chemotactic response to substances secreted by the egg. 
This assumption was confirmed in the classical studies of Pfeffer (16) 
where he showed that antherozoids of pteridophytes exhibit a positive 
chemotactic response to a difl'usion gradient of malic acid and certain 
other organic acids. In this connection it is interesting to note that he 
reports negative results with gametes of the green algae Ulothrix and 

Subsequent studies on sexual substances of algae have, in the main, 
been upon heterothallic (dioecious) species where each thallus produces 
but one kind of gamete. Some of these studies have been upon collections 
taken from field to laboratory; other studies have been upon clones 
grown in vitro in the laboratory. Irrespective of the source of material, 
the most satisfactory algae for study of sexuality are those in which both 
male and female gametes are motile and biflagellate. When one mixes 


motile gametes of opposite sex there is an immediate aggregation of the 
gametes in cknnps of ten to a hundred or more. This striking reaction, 
which takes place in less than five seconds, is so clean-cut that there is 
never any doubt as to whether there is a positive or a negative sexual 
response. When there has been a positive clumping reaction the fusion 
of biflagellate gametes can be demonstrated by killing the swarmers and 
noting the presence of quadriflagellate zygotes. In rare cases (1,15) 
the clumping is due to many gametes of one sex swarming about a 
single gamete of opposite sex. In most cases it is thought, and it is 
definitely known in certain cases (7,18), that there are numerous gametes 
of both sexes in a clump. 

Up to a certain concentration of gametes the size of the clumps 
formed after mixing those of opposite sex is correlated with the con- 
centration, but beyond this concentration there is no further increase in 
number of gametes in a clump. When mixed in optimum concentration 
the number of gametes in a clump is not the same from species to species 
and may consist of two, less than half a dozen, ten to twenty, or a 
hundred or more. For this reason one can distinguish between different 
intensities of sexual reaction. 

The first demonstration of excretion of sexual substances from algal 
gametes was by Jollos (3) and was by means of differences in intensity 
of sexual reaction in Dasydadiis, a heterothallic marine green alga. Since 
it was impossible to distinguish between male and female gametes on the 
basis of size or morphology those of opposite sex were arbitrarily desig- 
nated as plus and minus. When Jollos mixed plus gametes of low intensity 
with minus gametes of high intensity he obtained a stronger clumping 
reaction than that from mixing plus and minus gametes of low intensity. 
He then placed plus gametes of high intensity in a dish, allowed them to 
swim there for a couple of hours, and then filtered off the water. Plus 
gametes of low intensity were placed in the filtrate and kept there for 
a couple of hours. When these plus gametes were mixed with minus 
gametes of low intensity the clumping reaction was stronger than that 
with untreated plus gametes. The same change in reaction was induced 
in minus gametes of low intensity. Jollos interpreted the increased sexual 
intensity induced in gametes of low intensity as being due to an absorp- 
tion of sexual substances excreted into the water by gametes of strong 
intensity. These experiments also show that both male and female 
gametes excrete sexual substances. Geitler (2) has also shown that 


zoogametes of both sexes of the green alga Tetraspora lubrica excrete 
sexual substances. He placed gametes of one sex in water from which 
gametes of opposite sex had been removed by centrifuging. This was 
soon followed by a clumping of the introduced gametes, but not by 
their fusion in pairs. 

The most striking of all results with algae immediately after being 
taken from field to laboratory are those reported by Moewus (13) for 
Mo?iostroma. This heterothallic marine green alga has a blade-like thallus 
one cell in thickness and one in which each fertile cell produces a number 
of biflagellate gametes. A zygote formed by union of two biflagellate 
gametes enlarges greatly and then has a division of its protoplast into 
32 haploid quadriflagellate zoospores. Half of the zoospores develop 
into female thalli and half develop into male thalli. Moewus placed 
fertile portions of a thallus in a small amount of water, added quartz 
sand, ground in a mortar, and then filtered. The effect of the sexual 
substances in the filtrate upon germinating zygotes was studied and 
found to be the same with filtrates from either plus or minus thalli, 
but to vary according to dilution of the filtrate. Mature zygotes placed 
in an undiluted filtrate formed 64 (instead of 32) small biflagellate 
swarmers when the zygote germinated. These swarmers fused in pairs 
shortly after liberation. Mature zygotes placed in filtrates diluted 1:2 
or 1:4 produced 32 large biflagellate swarmers. When liberated these 
neither fused in pairs nor reacted to plus or to minus gametes. Mature 
zygotes placed in a filtrate diluted 1:10 produced 32 large quadriflagellate 
swarmers which also showed no sexuality. 

In another series of experiments mature zygotes were placed in various 
dilutions of the filtrate until the beginning of cleavage of their contents 
and then transferred to undiluted filtrate. Zygotes first placed in dilu- 
tions of 1 :2 or 1 :4 and then in undiluted filtrate produced 32 biflagellate 
swarmers that fused in pairs when liberated. Those first placed in a 
dilution of 1:10 produced 32 quadriflagellate swarmers that also fused 
in pairs when liberated. These experiments with extracts obtained from 
fertile portions of Monostroma show that asexual swarmers can absorb 
sexual substances from the surrounding water and function as gametes. 
However, there are certain unanswered questions as to the manner in 
which the sexual substances affect the swarmers. One question is the 
manner in which a filtrate containing sexual substances extracted from 
a female plant, or those extracted from a male plant, causes half the 


swarmers from a germinating zygote to function as female gametes and 
half to function as male gametes. Another question is the correlation 
between concentration of sexual substances in the extract and the number 
of flagella borne by swarmers from a germinating zygote. 

Algae which can be grown in vitro are the most satisfactory for study 
of sexuality because certain of the external factors affecting the forma- 
tion of sexual substances can be controlled. To be suitable for study in 
pure culture the alga must be one which i) grows rapidly in culture; 
2) is heterothallic and with both kinds of gametes motile; 3) produces 
gametes readily; and 4) produces them in abundance. Many algae which 
can be grown in pure culture fail to meet all of these requirements. For 
example, Chlorella and Scenedesmus grow rapidly in culture but never 
reproduce sexually. On the other hand, such gamete-producing algae 
as Ulva, Enteromorpha, and Cladophora grow slowly when cultured in 
the laboratory and do not produce gametes readily. Among the algae 
meeting all the foregoing requirements are the siphonaceous algae 
Protosiphon and Botrydium, and various unicellular Volvocales including 
Chlamydomonas and Polytoma. 

The nature of sexuality has been studied most extensively in Chlamy- 
domonas. When grown in a liquid culture-media this alga is in a motile 
unicellular state. When grown on a .moist substratum, as on agar, 
Chlamydomonas forms a palmella stage in which all cells of the culture 
are without flagella and he embedded in a common gelatinous matrix. 
When palmella cultures are flooded with water or with inorganic 
nutrient solutions the cells develop flagella within an hour or two, escape 
from the gelatinous matrix, and swim about in the hquid. Students of 
sexuality in Chlamydomonas have found it far more convenient to 
culture it in the palmella stage and then induce motiUty than to obtain 
motile cells from liquid cultures. 

Many, if not all, species of Chlamydomonas do not have a division of 
cell contents to form gametes. Instead, any cell can function as a gamete 
if it contains sexual substances in sufficient concentration. In palmella 
cultures of species isolated and grown in the laboratory at Stanford the 
cells do not contain a sufficient amount of sexual substances until the 
cultures have attained a certain age, and this age is not the same for all 
species. At first cell division in these cultures is at a rapid rate, later the 
rate becomes slower and slower. It is thought that here the sexual 
substances accumulate at a constant rate, but that in most strains rapidly 


dividing cells do not accumulate these substances in sufficient concen- 
tration before the amount is halved or quartered by division to form 
two or four daughter cells. 

The most extensive studies on sexual substances of Chlamydomonas 
are those of Moewus on three interfertile species, C. eugametos, C. 
Braunii, and C. dresdenensis. These three species and mutants derived 
from them are collectively known as the eugametos group. When a 
palmella culture of any member of the series is flooded with water in 
light the cells become motile, but if this is done in darkness they do not 
become motile. Transfer of motile cells from light to darkness is soon 
followed by a loss of motihty. On the other hand, Moewus (8,11) finds 
that both cells which have become immobile in darkness and palmella 
cultures flooded in darkness do become motile when various sugars are 
added to the liquid. Although members of the eugametos group can be 
made motile in darkness by means of sugars the cells do not react sexually 
when mated with sexually functional cells of opposite sex. 

When palmella cultures standing in darkness are flooded with the 
filtrate from a culture standing in light and containing sexually functional 
cells the eff'ect is different. Here the cells in darkness become both motile 
and sexually functional (8). This is due to the fact that the cells in 
darkness have absorbed both motility-inducing and sexuality-inducing 
substances excreted by cells swimming in light. However, a filtrate from 
an illuminated culture will only induce sexuality when applied to a 
culture of the same sex. That is, a filtrate from a female culture will 
induce sexuality in a female culture but not a male culture. The reverse 
is true for filtrates from male cultures. 

Having shown that sexual substances of the eugametos group are 
formed only in light, Moewus then proceeded to determine more pre- 
cisely the conditions under which they are formed and the nature of the 
substances. He soon found that for both male and female cells the sexual 
substances are formed only in light from the blue end of the spectrum. 
Sexual substances are formed in light of 4358 and 4961 A, but there is 
no formation of them in light of 5461, 5770, 5791, 5890, or 6430 A (10). 

The effect of light upon sexual substances excreted from motile 
sexually functional cells was also studied. This was done by obtaining a 
filtrate containing the sexual substances, exposing the filtrate to light 
for a specific time, flooding cells in darkness with the illuminated filtrate, 
and then noting whether or not this procedure induced sexuality in 


darkness. When C. eiigametos is treated in this manner and the filtrate 
exposed to red, yellow, or green light for several hours there is no loss of 
the capacity to induce sexuality in darkness. The effect of blue or 
violet light is different. A filtrate from a male culture exposed to blue 
or violet light retains, for less than 30 minutes, the ability to induce 
sexuality in male cells in darkness. When the exposure lasts for more than 
30 minutes the filtrate loses the capacity to activate either male or 
female cells. Filtrates from female cultures are unable to induce sexuality 
in female cells after exposure to blue or violet light for more than 30 
minutes, but exposure of female filtrates to blue or violet light for 75-90 
minutes causes a change into substances capable of making male cells 
function sexually. Filtrates illuminated for more than 90 minutes are 
unable to induce sexuality in either sex. 

Although red light is incapable of causing a formation of sexual 
substances in C. eiigametos it is capable of causing a formation of a 
precursor (P) of them. When Moewus (9) made either male or female 
cells motile in darkness by means of glucose and then illuminated with 


red light (5890 A) there was an excretion of the precursor into the water. 
When a filtrate from such a culture was exposed to blue light for 20-30 
minutes it contained a substance (9S) capable of making female cells 
sexually functional. This disappeared after illumination for 30 minutes, 
but when illumination was continued for 30-40 minutes (a total illumina- 
tion of 60-70 minutes) the filtrate contained the male sexual substance 
(cf S). The male substance disappeared after illumination for 10 minutes 
and neither it nor the female reappeared with further illumination. 
Illumination for approximately 100 minutes resulted in the formation 
of an end substance (E). When written as a formula the series of changes 
may be expressed as follows: 

P -^ 9S -^ cTS -^ E 

In the change from precursor to end substance there is a gradual 
decrease in percentage of precursor in the filtrate and a corresponding 
increase in the percentage of end substance. When the change has 
progressed to a certain stage the ratio between the two results in the 
female sexual substance, and a further progressive change in the ratio 
results in the male substance. Moewus (9) demonstrated this by testing 
the cllect of various combinations of precursor and end substance in 
inducing sexuality of male and female cells in darkness. For C. eiigametos 


he found that a mixture of three parts precursor and one part end 
substance made female cells sexually functional, and that one part 
precursor and three parts end substance made male cells functional. 
Mixtures in the ratio of i:i, 2:2, 4:1, and other ratios were without 
effect on either male or female cells of this species. Later Moewus 
(10,12) found that the ratios are not the same for all members of the 
eiigametos series. According to the species or variety the ratio between 
precursor and end substance necessary to make female gametes sexually 
functional may be 95:5, 85:15, 75:25, or 65:35. In this series male 
cells are, respectively, made functional in mixtures of precursor and 
end substance in the ratio of 5:95, 15:85, 25:75, and 35:65. For each 
member of the series the ratio for female cells is the reciprocal of that 
for male cells: that is, 95:5 and 5:95, 85:15 and 15:85, 75:25 and 25:75, 
65:35 and 35:65. 

Moewus' studies on behavior of cells in light of various wave lengths 
and in darkness show that sexual substances present in members of the 
eiigametos group are formed in light and disappear shortly after removal 
from light. Disappearance of sexual substances of the eiigametos group 
may be due to a breakdown in the absence of light but this seems im- 
probable because certain other species do not have a loss of sexual 
substances when transferred to darkness (see p. 325). It seems more 
probable that sexual substances formed in hght by members of the 
eiigametos group have the substances diffusing out from the cells almost 
as rapidly as they are formed. Since there is no new formation of sexual 
substances after transfer to darkness and outward diffusion of sexual 
substances continues after this transfer, the cells soon reach a state 
where they do not contain a sufficient concentration of sexual substances 
to cause gametic union. 

The climax of work on sexual substances of the eiigametos group came 
with the demonstration that they are all formed by degradation of the 
carotinoid pigment, protocrocin (5,6). A molecule of protocrocin breaks 
down into two molecules of picrocrocin and one of crocin (Table i). 
Each of the two molecules of the carotinoid picrocrocin breaks down 
into a molecule of glucose and a molecule of the carotinoid safranol. 
The molecule of crocin breaks down into two molecules of the sugar 
gentiobiose and one molecule of aV-crocetin dimethyl ester which, in 
time, becomes transformed into /ra/z^-crocetin dimethyl ester. Genetic 
analysis by Kuhn and Moewus (4) and by Moewus (14) has shown that 









• w^ 







J c 
CO '-s 
< 3 



















► u 









► .2 




C '-' 
.-< to 

4-1 U 
lU , 

O ^- 

U 4-1 

■^ .S 

► .2 









■^ -D 


each step in degradation of protocrocin is brought about by a different 
gene and that at least certain steps in the series are each due to a specific 

Of the substances produced from protocrocin Moev^-us (cf) identifies 
the precursor (P) present in a filtrate from cells grown in red light as 
m-crocetin dimethyl ester, and the end product (E) resulting from 
illumination of the filtrate with blue Hght as trans-ciocetin dimethyl 
ester. This identification is based upon the fact that a cis/trans mixture 
in the same ratio as a P/E mixture also induces sexuahty in cells made 
motile in darkness. When placed in an ascending series according to 
intensity' of sexuaUr\' the five sexual types discovered among the euga- 
metos group show the following range of cis/trans differences (Table 2). 

T.\ELE 2 
Cis/trans ratios for the five sexual types of the Chlamydomonas 

eugamentos group 

Type Type Type Type Type 


cisj trans ratio 
female cells 
cis/trans ratio 
male cells 




95 '5 


For female cells differences in the cis/trans ratios of the five sexual 
types approximate ven.' closely the ratios in the Bergmann -Niemann 
series of (2:1), (2:3), (2x3:1), (2x3^:1), (2x3':!;, (2x3*:!), ar. : : .- r. .t 
cells they approximate the trans/ cis ratios (see Table 3). 

Since cis/ tram mixtures induce sexuahty in darkness it might be 
argued that these are the only substances concerned with gametic 
union. Moewus (12) holds that this is not the case and that there are 
both gamete-attracting substances ''^amones) and sex-determining sub- 
stances ftermonesj. He holds that cis trans mixtures are ganK>nes and 
that picrocrocin and safranol are termones. The gamone (g^-'nogamone) 
of a female cell alwavs contains more cis- than /razij-crocetia dimethvl 
ester, whereas the reverse is true for the gamone (androgamone) of a 
male cell. 

To prove that cis, trans mixtures (gamones) are not the only substances 
concerned in sexuaht}', Moewus (12) took a filtrate from a heterothallic 


female culture grown in red light and exposed it to blue light until it 
contained the end substance (trans-crocetm dimethyl ester). This filtrate 
did not induce sexuality in darkness but an addition to it of a cis/ trans 
mixture of the proper proportion did induce sexuality in darkness. 
From this he argues that the filtrate contains a sexual substance in 
addition to the sexual substance {cis/ trans mixture) inactivated by blue 
light. This argument seems inconclusive when one takes into considera- 
tion the fact that cisj trans mixtures in distilled water also can induce 
sexuality in darkness. 


Table showing the correlation between cis/ trans ratios of the five sexual types 

of the Chlamydomonas eugametos group and Bergmann-Niemann ratios 

Ci si trans ratio 


trans/ cis ratio 











2 X 3:1 
2 X 32:1 
2 X 3^:1 





Moewus obtained more convincing evidence for the presence of 
termones in his study of homothallic strains. When taken from darkness 
and exposed to light for an hour the cells of a homothallic clone become 
motile, aggregate in clumps, and fuse in pairs. Moewus (12) placed 
homothallic clones in darkness, flooded them with a solution of picro- 
crocin and allowed them to remain in darkness for 20-25 minutes. 
These cultures were then taken to Hght but there was no gametic union 
of the cells even after illumination for an hour or more. However, when 
the cells had been standing in light for an hour and were tested against 
functional male and female gametes they reacted sexually with the 
male but not with the female gametes. The picrocrocin makes all cells 
in a homothallic culture function as female gametes. Solutions with a 
concentration of from 8 x 10^ to 8 x lo^^ molecules of picrocrocin per zc. 
produce this effect, but those with a concentration greater than 8 x \o^'^ 


do not. When treated in similar manner with safranol all cells of a homo- 
thallic clone functioned as male gametes. This takes place in safranol 
solutions with 5 x 10^ to 5 x 10® molecules per cc. but not in solutions 
of higher concentration. 

From the foregoing experiments Moewus concludes that picrocrocin, 
or a closely related compound, is the female sex-determining substance 
(gynotermone) and that safranol is the male sex-determining substance 
(androtermone). As already noted, picrocrocin is derived from proto- 
crocin; and safranol, in turn, is derived from picrocrocin, a change 
brought about by a specific enzyme (14). Female cells of heterothallic 
species lack this enzyme and so have no conversion of the picrocrocin 
into safranol. Male cells of heterothallic species produce this enzyme 
and so have a conversion of all the picrocrocin into safranol. Homothallic 
species have a conversion of only a part of the picrocrocin into safranol 
and so have both picrocrocin and safranol within their cells. Cells with 
more picrocrocin than safranol are female; those with more safranol 
than picrocrocin are male. 

Cultures of members of the eugametos group have not been available 
to others studying the sexuality of Chlamydomonas and so there has 
been no confirmation or extension of the striking results reported for 
this group by Moewus. However, studies on sexuality in other species 
of Chlamydomonas do afford a certain amount of data for comparison 
with the results reported for the eugametos group. 

Plus and minus clones of seven heterothallic strains, none of which is 
interfertile with any other, and several homothallic strains have been 
isolated at Stanford University. The heterothallic strains include C. 
Reinhardi Dang., C. intermedia Chodat., C. minutissima Korshikov, and 
a strain superficially resembling C. minutissima but not interfertile with 
it. One of the homothallic strains has been identified as C. Snowiae 

The behavior of the Stanford strains with respect to light and darkness 
is quite different from that of the eugametos group. When motile 
sexually functional cells of Stanford strains are taken from light to 
darkness the cells remain motile and sexually functional for several 
hours, and in certain cases for a day or two. When palmella cultures of 
both homothallic and heterothaUic Stanford strains are grown in hght 
and then kept in darkness for 24 hours before flooding, the cells become 
motile within an hour or two after flooding. If plus and minus clones 


of heterothallic strains are treated in this manner and mixed in darkness 
there is a fusion in pairs to form quadriflagellate zygotes. 

There are two alternative hypotheses to account for the different 
behavior in darkness of the Stanford series and of the etigametos series: 
Light is not essential for the formation of motility and sexuality 
inducing substances in the Stanford series; or light is essential for their 
formation in the Stanford series but in darkness these substances diffuse 
out from the cells at a much slower rate than in the eugametos series. 

A demonstration of the necessity of light in the formation of sexual 
substances in the Stanford series involves heterotrophic cultivation of 
them in darkness for many cell generations. Thus far the heterothallic 
C. Reinhardi is the only member of the Stanford series successfully 
cultured in darkness and only when the carbon source is sodium acetate. 

PreHminary experiments with this species showed that when palmella 
cultures are grown in darkness for several days and then flooded only 
a small percentage of the cells become motile. If these motile cells are 
mixed in darkness with sexually functional cells of opposite sex brought 
from light there is no sexual reaction. On the other hand if cultures 
grown in darkness are flooded and exposed to whitelight from a fluorescent 
lamp all of the cells become motile in about half an hour, but for an hour 
or so afterward they show no sexual reaction when mixed with functional 
gametes of opposite sex. After approximately two hours' exposure to 
light there is a formation of typical clumps sixty or more seconds after 
mixing with functional gametes of opposite sex. With further illumina- 
tion the time before the appearance of typical clumps after mixing 
becomes shorter and shorter, until they are formed within less than five 
seconds. The number of clumps in a mixture also shows the increased 
intensity of sexuaUty as exposure to Hght continues. In the earliest 
sexual responses after exposure to light there are only a few clumps after 
mixing with gametes of opposite sex, but in subsequent tests after further 
illumination there is a progressive increase in number of clumps. In these 
experiments the criterion for a development of sexual substances after 
exposure to light has been a formation of clumps after mixing with 
functional gametes of opposite sex. This was confirmed by killing the 
motile cells and finding quadriflagellate zygotes in the mixture. 

The foregoing experiments give support to the second of the two 
hypotheses to account for differences in behavior of the Stanford and 
the eugametos series in darkness. Namely, light is essential for formation 


of sexual substances in the Stanford series, but in darkness these sub- 
stances do diffuse from the cells at a much slower rate. 

Unpublished results of studies by H. C, Wendlandt at Stanford 
show that there is a quantitative relationship between white light from 
a fluorescent lamp and the development of sexual substances by C. 
Reinhardi. This was done by comparing the time required for the 
appearance of sexuality in cultures grown in darkness and then exposed 
to white light of i, lo, 25, and 50 foot-candles' intensity. The time for 
development of sexuality is not the same in all cultures exposed to light 
of any given intensity, but when the average time is taken for experi- 
ments in sextuplicate at each intensity the results are consistent (Table 
4). There is no appearance of sexuality in cultures illuminated at i foot- 

Average number of minutes exposure to white light of increasing intensity re- 
quired for appearance of sexuality in cultures of plus strains of Chlamydomonas 
Reinhardi grown in darkness when mixed with sexually functional minus cells 

Number of clumps 
in an area of 

0.5 SQ. MM. 









2 to 4 

4 to 8 

8 to 20 

more than 20 







candle, even if this is continued for more than 224 hours. In the other 
light intensities the time interval for the formation of sexual substances 
decreases as intensity of illumination is increased, and for each intensity 
there is a progressive strengthening of the sexual reaction as illumination 
is continued. Differences in time for sexual substances in plus and minus 
strains of C. Reinhardi are insignificant. 

The effect of light of different wave lengths upon the formation of 
sexual substances has also been studied in various members of the 
Stanford series. This was done by pouring nutrient agar into Petri 
dishes, allowing it to gel, inoculating the surface with material from a 
culture grown in light, and culturing for two weeks in blue light 
(4357 A) and in red light (6150-6900 A). When these palmella cultures 
were taken to the dark room and flooded the cells became motile and 


proved to be sexually functional when tested against the appropriate 

One possible source of error in the foregoing experiments is that 
the inoculum was from cultures growing in light and that the cells 
grown in red and in blue light may have contained a small amount of 
sexual substance because they were but a few cell generations removed 
from parent cells grown in light. To eliminate this possibility, C. 
Reinhardi was cultured in darkness and material from the culture used 
as the inoculum for a new culture grown in darkness. Repetition of this 
for five successive transfers resulted in cultures in which all cells were 
undoubtedly hundreds of cell generations removed from a parent cell 
grown in light. Plus and minus cultures of C. Remhardi grown in darkness 
through five successive transfers were then exposed under combinations 
of Corning glass filters giving light of the following wave lengths 

00 o 

4200-4600 A (blue), 5300-5700 A (green), 5900-6400 A (orange), and 


6200-6800 A (red). After exposure for 20 hours the cultures were 
returned to the dark room and flooded. In all cases the cells became 
motile in darkness and fused to form zygotes when mixed with functional 
gametes of the opposite sex. 

The ability of C. Reinhardi to form sexual substances in red, orange, 
and green light shows that for this species the sexual substances cannot 
be the combinations of cis- and /raw^-crocetin dimethyl ester that 
Moewus reports for the eiigametoi group. 


1. Berthold, C, Mitt. zool. Stat. Neapal, 2:401 (1881). 

2. Geitler, L., Biol. Zentr., 51:173 (1931). 

3. JoLLOs, v., ibid., 46:279 (1926). 

4. KuHN, R. and Moewus, P., Ber. dent. Chem. Ges., I},'.^^'] (1940), 

5. , and Jerchel, D., ibid.., 71:1541 (1938). 

6. KuHN, R., Moewus, P., and Wendt, C, ibid., 72:1702 (1939). 

7. Lerche, W., Arch. Protistenl^., 88:236 (1937). 

8. Moewus, P. ibid., 80:469 (1933). 

9. , Jahrb. wiss. Bot., 86:753 (^93^)' 

10. , Biol. Zentr., 59:40 (1939). 

11. , Arch. Protistenl{., 92:485 (1939). 

12. , Biol. Zentr., 60:143 (1940). 

13. , ibid., 60:225 (1940). 

14. , ibid., 60:597 (1940). 

15. Pascher, IK., Jahrb. wiss. Bot., 75:551 (1931)' 

16. Pfeffer, W., Unters. Bot. Inst. Tubingen, 1:363 (1884). 

17. Pringsheim, N., Jahrb. wiss. Bot., 1:1 (1858). 

18. Smith, G. M., Am. J. Botany, 33:625 (1947). 

Growth-Regulating Substances in Relation to 
Reproduction of Some Horticultural Plants 


DURING the past third of a century I have been interested in the 
physiological aspects of sexual reproduction of the higher plants, 
and for about a quarter of a century my major research project has 
been, and still is, on the "Physiology and Reproduction of Horticultural 
Plants." In this activity quite naturally I have been led recently into 
studies of certain theoretical aspects of the function of plant hormones 
and the practical application of so-called synthetic growth regulators 
with special reference to the production of horticultural crops. 

The physiology of sexual reproduction has been till recently, a much 
neglected field of investigation, as one may judge from inspection of any 
textbook of plant physiology. The relatively recent discoveries of the 
striking effects of the photoperiod and temperature, however, have 
created much interest in this phase of the functional hfe of plants (93,62). 
Now the naturally occurring auxins and even more the synthetic growth 
regulators have come under consideration in flower and fruit develop- 

Personally, I dislike the widespread use of the term auxin whose 
original meaning was that of a catalytic substance (hormone) bringing 
about growth by cell elongation. Recently it has been used to designate 
not merely one of the three originally discovered auxins, heteroauxin, 
but practically all synthetic growth regulators employed in experimental 
work (68). Surely there are many more native hormones in plants than 
indoleacetic acid. One cannot conceive that as vital and complicated 
a process as the formation and development of flowers, seeds, and fruit, 
with their diverse structures and manifold physiological functions. 


would involve or be regulated by only one or merely a few auxins. 

With your forebearance I shall refer to the naturally occurring form- 
and growth-controlling substances as hormones and to synthetic chemi- 
cals used for this purpose as growth-regulating substances. This despite 
the fact that, considering the variability in molecular structure, the 
synthetics per se do not always, and probably rarely, regulate growth 
and development but most likely only activate or catalyze in some 
manner a particular native hormone in plants (21,77). 

It is my intent to discuss briefly certain salient aspects of sexual 
reproduction with special emphasis on the possible involvement of the 
growth regulators. Plants with which we have largely worked and those 
phases of reproduction of greatest present concern to us perforce will 
be stressed. 

Flower Initiation 

Evidence is being accumulated that the initiation of floral primordia 
is activated by a special flower-forming hormone. It seems to be produced 
in the leaves, as in photoperiodism, whence it moves to the apical 
meristems. The formation of this hormone directly in the meristematic 
regions is not excluded, as with some temperature effects. While no one 
has yet succeeded in isolating this hormone it has been tentatively 
named florigen (13). Comparatively recently the idea has been advanced 
by Gregory (28) that only a florigenic precursor is formed in the leaves 
under particular environmental conditions and translocated to the meri- 
stems where the flower hormone is finally synthesized. Even an anti- 
florigenic substance, possibly found in leaves on vegetative shoots, has 
been suggested (48). 

After considering most of the experimental evidence indicating the 
probability of a flower-forming hormone, up to 1938, Cholodny (14) 
concluded that not a special but the already known native plant growth 
substance (heteroauxin), under certain conditions of distribution or even 
absence, most likely causes the transition of terminal meristems from the 
vegetative to the flowering condition. Though the situation probably 
is not as simple and direct as this (37,38), considerable evidence has 
been accumulated recently that certain synthetic growth regulators can 
initiate or inhibit flower production, depending on the concentration 

Clark and Kerns as early as 1942 (15), Cooper (16), and subsequently 

A. E. MURNEEK 33^ 

van Overbeek (70,71) reported that flowering in the pineapple, a rather 
abnormal parthenocarpic fruit, can be induced with certain synthetic 
growth regulators. About 50 milliliters of naphthaleneacetic acid (NAA) 
or 2,4-dichlorophenoxyacetic acid (2,4-D), at a concentration of 5 parts 
per million, when applied in the whorl of leaves near the apical meristems, 
resulted in practically all instances in the formation of floral primordia. 
Too high a concentration of NAA, however, retarded the time of 
flowering as long as 4 to 8 months (15). Through further tests by van 
Overbeek this treatment has been put to large scale commercial use. 
Till a few years ago this was the only instance where flower initiation was 
controlled by a growth regulator. The following currently published 
information has further bearing on this point. 

By spraying leaves of Xanthium plants with indoleacetic acid (lAA) 
and NAA solutions Bonner and Thurlow (6) were able to suppress 
initiation of floral primordia during the induction (short-day) period. 
The same results were secured when only 2 leaves of this plant were 
submerged nightly during the dark period in a NAA solution of a 
concentration as low as i mg. per Hter. In this experiment (6) and a 
parallel one (7), 2,4-dichloranisole, an antagonist of auxin, counteracted 
the inhibition of the above chemicals on formation of flowers. Moreover 
when 2,4-dichloranisole or triiodobenzoic acid were apphed to leaves 
of long-day vegetative plants, flowerlike buds were produced. Leopold 
and Thimann (46) were able to increase by as much as 35 per cent the 
number of flowers in barley by applying a weak solution of NAA through 
the cut surface of leaves. It is of interest to note that relatively high 
concentrations of the same substance inhibited flower formation. Since 
both flowering and growth were promoted by relatively low concentra- 
tions of NAA, the authors conclude that "these evidences indicate that 
the growth hormone, auxin, is not necessarily opposed to the functioning 
of the proposed flowering hormone, but rather influences it in a manner 
qualitatively similar to its influence on growth." 

It would seem desirable to know whether NAA in this instance, while 
stimulating growth in general, did not retard the multiplication of apical 
cells in the vegetative meristems thus leading to the formation of 
reproductive tissues. It has been shown that many synthetic growth 
regulators may delay or inhibit cell division in the meristem (3,5). 
In investigations of this kind cytological and chemical studies of terminal 
meristematic regions during the earliest stages of induction would seem 


to be in order (78). Attention should be called here to the reduction of 
boron deficiency symptoms, necrosis of the apical meristems of plants 
that have been made reproductive by short photoperiods (91,82). 
Perhaps it may be considered as circumstantial evidence that, with the 
onset of reproduction, terminal meristematic growth is not only cur- 
tailed but usually inhibited. 

Flower Development 

Flowers having been initiated, their further development and function 
would seem to depend upon many internal and external environmental 
factors. Anyone who has made a study of the growth of flowers is aware 
of the fact that drastic changes in the environment, such as, for example, 
marked shift in the photoperiod or temperature, may cause conspicuous 
changes or even abortion of flower buds, flowers, or parts thereof. 

The following stages in flower inception, development, and function 
may be recognized (55) : i) Terminal meristems or genetically determined 
loci where the floral hormone is received or synthesized. This is a stage of 
"ripeness to flower" established physiologically. Commonly there are 
far more such meristematic points than there are available hormone or 
other indispensable substances. Many terminal meristems thereby are 
eliminated early as flower producers. Devernalization and dedifl'erentia- 
tion are well-established phenomena (72,63). 2) Early floral development. 
If a relatively large number of flowers have started to develop, as seems 
to be frequently the case, most of them may be eliminated because of 
competition for supply of building material or specific catalytic sub- 
stances or hormones. 3) Late floral development. Though reaching 
anthesis, not all flowers are normal or can function properly (that is, 
participate successfully in fertilization). Though appearing well de- 
veloped, they may be abnormal in essential morphological and histo- 
logical structures. Usually plants produce an enormous number of flowers 
of which only a fraction form seeds (23,47,65). 

There is practically nothing known at present about the function 
of hormones during various stages of flower development. Though their 
multifarious forms and structures are determined genetically, most likely 
hormones of various kinds participate in the production of specific 
floral organs and tissues. 

A. E. MURNEEK 333 

Gametogenesis and Fertilization 

Several years ago in work on problems of the relation of nutrition 
to reproduction of the tomato, it was found that as a result of fertilization 
(gametic union) and formation of zygotes not only tissues accessory 
to the embryo and structures immediately subtending the flower (42,35) 
are stimulated in development, but that metabohsm throughout the plant 
is accelerated to some extent. This was evidenced by increased absorption 
of soil nutrients, and assimilation of carbon dioxide (51). It has been 
confirmed with several other plants (56,94,19,4). 

Subsequently two phases of the observed stimulation during the 
general period of flowering and fruit setting were recognized, one 
occurring about the time of synapsis and the other, a more important 
one, during syngamy (54,55,52,57). Stimulation at synapsis, postulated 
from general observation and gross analyses of the effects, was later 
fully established by a former student and colleague of mine (95). It 
appears to be initiated within the partly developed flower buds con- 
comitant with chromosome conjugation in the micro- and macrospore 
mother cells. In the staminate corn flowers the stimulation reaches a 
peak about 7-12 days after synapsis. The second stimulation, originating 
from gametic union in the embryo sac, about the time of fertilization, 
attained a maximal effect in corn some 15 days after pollination (95). 

Undoubtedly responsibility for the two periodic stimulations of growth 
associated with sexual reproduction lies with a hormone or hormones. 
Evidence, though not fully conclusive, indicates that it is not due to an 
increased production of the already well-known heteroauxin. From 100 
kilograms of 15-day old corn kernels furnished by us, Haagen-Smit et al. 
(34) extracted all the free Athena- testable hormone, of which only 9 per 
cent was 3-indoleacetic acid. What is the rest? Contrary to the effects 
of indoleacetic acid, the crude extracts of immature corn kernels, 
applied in lanolin paste, were highly active in the production of partheno- 
carpic tomatoes and peppers. Moreover, Stanley McLane, working in 
our laboratory, found that in the culture of excised immature corn em- 
bryos (10-20 days after polhnation) the addition of 3-indoleacetic acid, 
comparable in concentration to that found in extracts from 15-day old 
corn, inhibited growth, while a water extract of corn, without the natural 
indoleacetic acid removed, doubled growth of embryos. 

Shall we call these unknown hormones synapsin and syngamin re- 


spectively? Wittwer (95) found that the growth activity in extracts 
from corn pollen and immature grains is not destroyed by prolonged 
heating. According to McLane, syngamin seems to be insoluble in ether 
at pH 5.4, passes through a collodion membrane, and can be removed 
in part by treating corn extracts with activated charcoal. 

Fruit Setting 

The present evidence seems to point to the following sequences in 
the function of hormones in relation to fruit setting. Pollen germinating 
on the stigma either produces a hormone (32) or through secretion of an 
enzyme liberates a hormone from inactive combinations in the style and 
ovary (98,50). The male gametophyte, therefore, has at least two func- 
tions, fertilization of the egg and causing the ovary to grow. The latter 
action seems to be closely associated with prevention of abscission of the 
young fruit. 

Van Overbeek (68) believes that not only the carpels but also the 
ovules are controlled in their initial growth by a hormone originating 
in the microgametophyte. By injecting NAA or indolebutyric acid 
(IB) into the ovaries oi Melandrium and Datura not only parthenocarpic 
fruit were secured but they contained enlarged ovules with seed coats 
although no true embryos {66). He suggests that, with some plants at 
least, even division of the polar nucleus and the egg cell, before fertiliza- 
tion and triple fusion, may be brought about by the presence of pollen 
tubes in the style. Gustafson (32) has shown that pollen extracts when 
applied to pistils may cause them to develop into parthenocarpic fruit, 
while Laibach (45) and Thimann (84) found that pollen contained 
hormones. However, the greatest hormone effect on the fruit absciss 
layer probably comes after fertilization from the ovules (1,50), since 
the hormone content of ovules and developing seeds is exceedingly 
high (31). 

The disclosures by these studies, but probably even more the discovery 
by Gustafson (30) that several synthetic growth regulators will induce 
fruit development without pollination, and by Gardner and Cooper (28) 
that premature dropping of apples can be retarded by spraying with a 
dilute solution of NAA, have been of considerable value in horticulture. 
Moreover, it has created much interest leading to further experimental 
work on the use of growth regulators in horticultural practice. 

Through the tests by Zimmerman and Hitchcock (99) about 30 

A. E. MURNEEK 335 

organic chemicals so far have been found effective in fruit setting. Of 
these the naphthoxy and substituted phenoxy acids are especially potent 
for this purpose. Many-seeded fruit evidently respond more readily 
to stimulation in development by synthetic growth regulators than do 
fruit containing a single seed. Desirable concentrations and the time and 
mode of application have been studied extensively, particularly with 
plants with which the greatest success has been secured so far. 

Tomatoes. — When tomatoes are grown in the greenhouse during 
cloudy weather and/or subnormal temperature, or outdoors when the 
nights are relatively cold, the number and size of the fruit can be 
increased by the use of IB, NAA, and 4-chlorophenoxyacetic (CIPA) 
acid, some of their homologues, and a number of other synthetic growth 
regulators (41,58,61,97). The flowers are usually sprayed when in full 
bloom or, preferably, after pollination and fertilization have occurred. 
Pollen tube growth and fertilization, as noted before, in their aggregate 
effect on growth of the ovary supplement the effects of synthetic growth 
regulators on carpel development. There does not seem to be any 
appreciable commercial value although there is a great deal of talk about 
the production of seedless tomatoes. 

Tomato flowers are receptive to stimulation of this kind several days 
after pollination. If whole plant spraying is practiced, which has been 
shown to be even more effective than flower-cluster spraying (58, 61), 
the spray must be so directed as to cover only that portion of the plant 
containing mostly open flowers and young fruit. It has been observed 
(58,61) and pointed out by Roberts and Struckmeyer (73) that spraying 
tomato buds with growth regulators five or more days before pollination 
gave a poor set and fruit of relatively small size. 

Hemphill (40) has recently completed a thorough investigation on 
the time of application of three synthetic growth regulators at the 
concentrations most desirable for increase of the tomato crop. When 
applied eight days before anthesis pollen ceased to develop and collapsed 
in the anthers, while spraying four days before full bloom often caused 
premature pollen germination. The ovules were also detrimentally af- 
fected when a growth regulator was applied as early as the bud stage. 
They either ceased to develop and the embryo sacs disintegrated, or 
those that did grow were greatly retarded in development. Seed forma- 
tion was usually prevented. These results are in considerable agreement 
with those obtained by Britten (10), who sprayed NAA on developing 


maize caryopses. What is equally important, they throw considerable 
light on the results observed when NAA is used for the reduction of 
fruit set of the apple and some other fruit crops. 

Green beans. — Abnormally hot weather at the time when string beans 
are setting fruit may reduce the yield of pods considerably. This can 
be overcome to an appreciable extent by either spraying or dusting the 
plants with a desirable growth regulator. It may be combined with a 
necessary insecticide. Maturity of the pods, as judged by size is also 
increased by the treatment (59,96,26). The beneficial effect apparently 
is brought about by stimulation in growth of the carpels (pods) and an 
increase in chlorophyll content of the leaves. Probably the abscission 
layer between fruit and pedicel is also strengthened. The seed number in 
some cases may be reduced but, where the proper concentration is 
employed, it is usually about the same. 

Fruit Thinning 

Although we have tried repeatedly to increase the set of stone and 
pome fruits by the use of synthetic growth regulators, the results have 
invariably been negative. Instead of an increase, there has commonly 
been a reduction. This response, too, has been found under certain 
circumstances to have practical value as will be seen forthwith. 

Burkholder and McCown (12) reported that reduction in the crop 
of apples, when the set is excessive, may be brought about by spraying 
the trees in full bloom with NAA. This has been confirmed by others 
(18,75,79), some of whom found that exact timing of the apphcation, 
as on the first day of full bloom, is not required. 

During the past four years we have conducted extensive field investiga- 
tions on thinning apples and peaches with NAA, with special reference 
to the time of application and concentration to be used (60,64). The 
results, still largely unreported, show that this growth regulator is an 
efficient substance for reduction of the number of fruits on an apple or 
peach tree. For apples, the best time of application seems to be one to 
two weeks after full bloom. The concentration of the spray material to be 
used is 10-30 ppm. depending on the variety and whether it is an annual 
or biennial bearer. Varieties that are difficult to thin chemically may 
require two applications of the spray. Peaches may be thinned success- 
fully with NAA, applied about one month after bloom, at a concentra- 
tion of 30-40 ppm. for the light setting varieties and 40-60 ppm. for 

A. E. MURNEEK 337 

the heavy setters. By using still higher concentrations it is possible to 
remove the apple crop completely if that be required. This rather late 
thinning by means of a spray is more desirable than spraying in full 
bloom at which time it is difficult, often impossible, to tell what the 
crop is apt to be. 

Here then we have another illustration that the same growth regulat- 
ing substance may have opposite effects depending on the concentration 
used. At a relatively weak concentration NAA may initiate flower pro- 
duction, foster the formation and growth of fruit and prevent fruit 
abscission, while at higher concentrations it can stop fruit development 
and cause their drop. 

How can NAA accomplish these rather striking results in fruit thinning 
without harming the tree or the remaining fruit? Some Hght may be 
shed on this question by considering the natural sequences in embryo, 
endosperm, and fruit development of the peach and apple. 

Embryo Development and Fruit Growth 

The peach and other stone fruits require the presence of an embryo 
for their growth. The same is true of the apple and other pome fruit, 
which usually contain several seeds. Parthenocarpy is a rare phenomenon 
with the genera Primus and Malus. Tukey (85) found that the peach 
fruit develops in three stages: i) There is a rapid increase of the pericarp, 
including nucellus and integuments, for about forty to fifty days after 
full bloom. During this stage growth of the embryo does not parallel 
that of the pericarp. It remains embryonic, in a kind of arrested develop- 
ment. Note should be taken of the fact that development of the endo- 
sperm usually precedes that of the embryo (27). 2) During stage two 
the embryo grows rapidly to a maximal size. The duration is five to 
forty days depending on the variety, that is, how early the fruit ripens. 
Increase of the pericarp is at a relatively slow rate. 3) The pericarp or 
fruit increases rapidly in size to the time of fruit ripening. In very early 
varieties stage three is initiated while the embryo is in a period of rapid 
growth, with the result that it fails to reach full size (aborts), the nucellus 
and integuments collapse, the fruit ripens rapidly and drops from the 
tree. This cyclic type of growth seems to be characteristic of other 
cultivated drupe fruit. 

Tukey (87) artificially destroyed embryos in the peach at various 
periods. Killing in stage two of development resulted in an abrupt check 


of fruit growth and prompt abscission. When the embryo was destroyed 
between stages two and three the fruit failed to reach full size and 
ripened quickly. Destruction of the embryo in stage three was followed 
by increased fruit growth and somewhat earlier ripening. From these 
and other studies (88) it is quite evident that the embryo has a definite 
bearing on fruit formation. Naturally occurring hormones undoubtedly 
play a role in this relationship. Most probably they regulate the metabo- 
lism and nutrition not only of the embryo but also of the fruit. Much 
evidence that hormones are important factors in the development of 
young fruit comes from the successful use of various synthetic growth 
regulators in production of parthenocarpy (32). Moreover, it has been 
found in several instances that seeds of developing fruit are rich in 
hormone content (22,36). 

The situation in the apple as regards the relative period and rate of 
development of the embryo, endosperm, nucellus, and integuments is 
similar to that of a drupe fruit (24,80,76). But since the bulk of the apple 
is made up of accessory tissues (torus), it does not show cyclic stages of 
growth (89). A fairly good correlation, however, exists between seed 
(embryo) number and size of apples during early stages of their develop- 
ment (39). Later on, before ripening, this relationship seems to vary 
considerably depending primarily on the size of the crop and the avail- 
able food supply (74). Seeds do, of course, have a local effect on growth 
of the pericarp (86) and torus (74). 

By means of artificial culture of embryos of various ages, isolated from 
their natural environments and associated tissues, information has ac- 
cumulated as to their nutrient requirements (74,67,69). In most instances 
it has been found that in addition to the necessary inorganic substances 
and certain organic nutrilites, an embryo factor is indispensable. This 
has been discovered in yeast (44, 92, 43), tomato juice (43), malt 
extracts, and a number of other plant-tissue extracts and substances. 
Older embryos, being autotrophic, do not seem to require this factor. 
Van Overbeek (67,69) has successfully used a liquid endosperm, coco- 
nut milk, for the culture of young Datura and other embryos. At 
least two important factors probably are present in coconut milk: a 
thermolabile one, which causes embryos to grow rapidly, and a heat 
stable factor that inhibits root development of the embryo. 

Embryo culture is of considerable aid in obtaining plants from certain 
self- and cross-sterile matings and in the production of seedlings from 

A. E. MURNEEK 339 

aborted embryos. Eyster (25) has reported that when flowers were 
sprayed immediately before or shortly after pollination with a weak 
solution of naphthaleneacetamide, highly inbred self-sterile plants of 
several species produced viable seeds. 

Endosperm and Its Role 

In detailed studies of embryo, seed, and fruit development considerable 
emphasis has recently been placed on the endosperm (9). This 3n inter- 
callary tissue between the new and old sporophyte provides the medium 
suitable for the growth of young embryos. Usually its development 
precedes that of the embryo. A failure in the necessary nuclear division 
or function of the endosperm as a rule results in failure of embryo 
growth. The endosperm increases rapidly during the early stages of seed 
development and is digested and absorbed by the growing embryo. Its 
role seems to be entirely nutritive. There is usually little or no endosperm 
when the seed is mature, excepting where it has assumed the secondary 
function of a storage organ. 

The important activity of the endosperm in nourishment and de- 
velopment of the embryo of angiosperms has been summarized by 
Brink and Cooper (1940) which may be paraphrased as follows: Since 
the female gametophyte is exceedingly small in size and no endosperm 
is formed till after fertilization, the ovule contains little or no reserve 
food. The new sporophyte would be thwarted in development were it 
not for the fact that fertilization initiates not only growth of the endo- 
sperm but stimulates to active expansion the adjoining maternal tissues 
(pollination as noted before, does it also). Originating as uninucleate 
structures the embryo and endosperm must compete for food supply 
with the adjoining well-established tissues. Success of the young seed 
would seem to depend primarily on the endosperm as a nutritive agent 
of the embryo to assist it in estabhshing its dominant position in the 
ovule and ovary. Double fertilization appears to be the method con- 
ferring upon endosperm the necessary power through physiological ad- 
vantages of the hybrid condition. If during early development the 
endosperm fails to remain dominant the surrounding nucellus or integu- 
ments may outgrow it withholding or diverting the food supply from the 
embryo. This results in starvation of the embryo and collapse of the 
seed — the so-called somatoplastic sterility (17). 

Should one consider farfetched the suggestion that the synergids, 


and possibly even the so-called "X-bodies" that flank the egg before 
and immediately after fertilization, may have a similar function to 
endosperm but more specifically in nutrition of the egg cell? 

Doubtless in the endosperm-embryo relationship hormones have an 
important function. Voss (90) observed that isolated embryos of corn 
require for their development a naturally occurring hormone, which 
could not be replaced by indoleacetic acid. During germination a hor- 
mone from endosperm evidently was translocated into the embryo. 
This has been verified more recently by Guttenberg and Lehle-Joerges 
(33) who detected in all parts of the embryo a special hormone that 
originated in the endosperm. 

Fruit Thinning Again 

Now to turn once more to fruit thinning by means of a growth 
regulator. In this connection we must consider the state of development 
of the seed at the time the spray is applied and the possible effect of 
NAA on the embryo and endosperm. According to Tukey (85) and 
Harrold (36) microscopic embryos and normal endosperm are present 
in the peach about the approximate time the growth regulator is applied 
for thinning purposes. Five- to ten-celled embryos and a many-celled 
endosperm are present in the embryo sac of the apple up to two weeks 
after fertilization. 

In both stone and pome fruit (peach and apple), with which we are 
concerned in this discussion, endosperm development after fertilization 
and triple fusion precedes the division of the zygote nucleus (36,11). 
It is most important to note here that Bryant (11) observed that, 
depending on the kind of pollen used in fertihzation, the endosperm 
nuclei seem to vary considerably in number during this early stage of 
seed development. Growth of the endosperm usually lagged under self- 
pollination and when a less efficient pollen was used. This reminds one 
of the differences in endosperm production, as described by Cooper 
and Brink (17) in distant crosses oiNicotiatia, Petunia, and other species, 
wherein, however, the endosperm nuclei divided most rapidly after 
self-fertilization and more slowly in hybridization, resulting in collapse 
of the young seeds. This may have a considerable bearing on the dif- 
ferential abscission of fruit as a result of spraying with NAA. 

That the apple embryo and endosperm when microscopic may be 
sensitive is indicated by additional circumstantial evidence. As a result 

A. E. MURNEEK 34^ 

of an extensive survey of fruit setting of the Delicious apple, Gardner 
et al. (29) reached the conclusion that during the critical period of one 
to two weeks after full bloom the young fruit apparently is very sensitive 
and may abort easily if the environment, primarily temperature and 
sunlight, is not favorable. Of even greater interest is the contention 
that certain fungicides, used for scab control at this early period, may 
also have a profound effect on fruit set of this variety. 

Luckwell (49) has made a seasonal study of the hormone content 
of apple seeds in relation to endosperm development. The concentrated 
ether extracts from whole seeds and separately from embryos and endo- 
sperm-nucellar tissues (older seeds) were tested for their potency to 
produce parthenocarpic tomato fruit. The hormone concentration was 
about 18 times as high in endosperm-nucellar tissues as in the embryo. 
And since the nucellus constituted only a minor fraction of the sample, 
the author thinks that the hormone came chiefly from the endosperm. 
During the period of the usual postfertihzation abscission of apples 
(53,20) the hormone content was of a relatively low concentration. 
Luckwell expresses the view that the hormone found in endosperm 
seems to prevent fruit abscission. Does it mean that a hormone released 
by endosperm, in preventing abscission, would thereby help to maintain 
or increase the food supply to the seeds and fruit? 

Naphthaleneacetic acid, at the relatively high concentration used for 
apple and peach thinning, most likely disturbed the endosperm and 
possibly also the embryo in their physiological relationships during 
the critical period of early seed development. This effect may be a 
direct one or, less likely, through stimulation of excessive growth of the 
pericarp. Studies by Swanson et al. (83) on ovule abortion in Tradescantia, 
as disturbed by a 2,4-D spray, suggested that the younger the ovule the 
more harmful was the spray and that endosperm was inhibited in rela- 
tively mature seeds. Ovule collapse followed disintegration of the endo- 
sperm and the chalaza. Still later the nucellus and integuments collapsed. 

Preliminary observations by us of apple fruit thinned by means of 
NAA indicated invariably an effect on the seed, endosperm, and nucellus, 
which had collapsed. This probably was the cause of abscission of large 
numbers of the young fruit. Very probably the specimens that abscissed 
were most sensitive to the treatment due to the presence of fewer 
seeds or seeds with less developed endosperm because of partial pollina- 
tion or pollination with less compatible pollen. Fruit borne on spurs 


in relatively weak positions as regards food supply may also be more 
sensitive in this respect (39). 

When still higher concentrations of NAA are employed during ad- 
vanced development of the apple fruit (20 to 50 days after bloom) the 
seed collapsed, fruit growth either ceased completely or was greatly 
retarded, but abscission did not always follow. As yet we have not been 
able to conduct a detailed study of the histological changes in fruit as a 
result of NAA appHcation. Whatever the mechanism responsible for 
thinning may turn out to be, this synthetic growth regulator (NAA) has 
given us a powerful tool to control the size of the crop of certain fruit. 

Control of Preharvest Drop of Fruit 

Mention should be made, briefly at least, of the well-established 
practice of using a weak solution of NAA to retard the preharvest drop 
of apples, pears, and a few other fruits (28). The effect evidently 
expressed itself through a delay of the natural changes in the abscission 
layer between the fruit and its pedicel coincident with ripening, which 
lasts, from a NAA spray or dust, for about 10 days. 

Batjer and Thompson (2) have demonstrated that 2,4-D is even more 
active in its lasting effects for apple drop prevention, but for some 
unknown reason it seems to work mainly on the Winesap group of apples. 
Stewart et al. (81) have discovered that 2,4-D can prevent also the 
abscission of citrus fruit. 

Our tests (63) point to the fact that 2-methyl-4-chlorophenoxyacetic 
acid is almost as potent as 2,4-D for length of delay of the preharvest 
drop of apples and that 4-chlorophenoxyacetic acid stands in a position 
between the above two chemicals and naphthaleneacetic acid. 


This somewhat sketchy paper should serve as a brief summary of the 
present status of studies on the function of hormones in sexual reproduc- 
tion of some crop plants. Special emphasis has been placed in this 
discussion on the use of synthetic growth regulators in connection with 
certain cultural practices of horticultural plants. Objections have been 
raised to the widespread employment of the term auxin in designating 
both naturally occurring hormones and synthetic substances used both 
experimentally and in crop production as growth regulators. 

From flower initiation to delay of fruit abscission during the preharvest 

A. E. MURNEEK 343 

period, growth regulators have been found effective tools in the control 
of development of certain tissues and organs. Their practical application, 
in several instances, has outrun our present scientific knowledge. As a 
result of further experimental work additional and even greater usefulness 
undoubtedly will be found for these catalytic substances in seed and 
fruit production. Progress in this field certainly would be hastened if 
more information were available on the detailed physiology of plant 
reproduction, with particular reference to the crucial phases. 

Heretofore the main emphasis in analyses of sexual reproduction has 
been placed on the embryo and mechanisms leading to its formation 
and growth. Attention is now being called to the importance of the 
endosperm and associated tissues in seed and fruit development. 

Information has been presented which shows how the same synthetic 
growth regulator (naphthaleneacetic acid) may have quite opposite 
effects in sexual reproduction, depending on the concentration used 
and the time of application especially in relation to seed and fruit 
development. A relatively weak aqueous solution may initiate flowers, 
promote fruit set, and retard fruit abscission in some plants, while 
one of higher concentration will inhibit or delay flower induction, 
curtail or prevent seed development, stop or retard fruit growth, and 
cause abscission. 

Though probably of considerable importance in the functional life 
of plants, only a beginning has been made in studies of the over-all 
effects on metabolism of certain phases of reproduction, with reference 
to absorption of soil nutrients, carbohydrate assimilation, and other 
major plant activities. Hormones of various kinds, undoubtedly, have 
roles here just as in detailed tissue growth and development. 


1. Avery, G. S. et al.. Am. J. Botany, 29:765-772 (1942). 

2. Batjer, L. p. and Thompson, A. H., Proc. Am. Soc. Hort. Sci., 47:35-38 


3. Bauser, S. C, Am. J. Botany, 27:769-779 (1940). 

4. BiDULPH, O. and Brown, D. H., ibid., 32:182-188 (1945). 

5. Block, R., Contrib. Boyce Thomps. Instil., 9:439-454 (1938). 

6. Bonner, J. and Thurlow, J., Botan. Gaz., 101:613-624 (1949), 

7. Bonner, J., ibid., no: 625-627 (1949), 

8. Brink, R. A. and Cooper, D. C, ibid., 102:1-25 (1940). 

9. , Botan. Rev., 13:423-541 (1947). 

10, Britten, E. J., Am. J. Botany, 34:211-218^(1947). 


11. Bryant, L, R,, A^. H. Agr. Exp. Sta. Tech. Bull, 6i (1935). 

12. BuRKHOLDER, C. L. and McCown, M., Proc. Am. Soc. Hort. Set., 38:117- 

120 (1941). 

13. Cajlachjan, M. H., Hormonal theory of plant development (Moscow, 1937). 

14. Cholodny, N. G., Phytohormones (Kiev, 1938). 

15. Clark, H. E. and Kerns, K. R., Science, 95:536-537 (1942). 

16. Cooper, W. C, Proc. Am. Soc. Hort. Sci., 41:93-99 (1942). 

17. Cooper, D. C. and Brink, R. A., Genetics, 25:593-617 (1940). 

18. Davidson, J. H. et al., Mich. Agr. Exp. Sta. Quart. Bull., 27:352-356 


19. Dearborn, R. R., Cornell Agr. Exp. Sta. Mem., 192 (1936). 

20. Detjen, L. R., Del. Agr. Exp. Sta. Bull., 143 (1926). 

21. Dettw^eiler, C, Planta, 33:258-277 (1943). 

22. DoLLFUS, H., ibid., 25:1-25 (1936). 

23. DoRSEY, M. J., Genetics, 4:417-488 (1919). 

24. Elssmann, E. and Veh, von R., Gartenbauwiss., 6:1-59 (1931). 

25. Eyster, W. H., Science, 94:144-145 (1941). 

26. Fisher, E. H. et al., Phytopath., 36:504-523 (1946). 

27. Gray, G. P., Mich. Agr. Exp. Sta. Techn. Bull., 136 (1934). 

28. Gardner, F. E. and Cooper, W. C, Botan. Gaz., 105:80-89 (1943). 

29. Gardner, V. R. et al., Mich. Agr. Exp. Sta. Spec. Bull., 358 (1949). 

30. Gustafson, F. G., Proc. Nat. Acad. Sci., 22:628-636 (1936). 

31. , Am. J. Botany, 26:189-194 (1939). 

32. , Botan. Rev., 8:599-654 (1942). 

33. Guttenberg, H. and Lehle-Joerges, E., Planta, 35:281-296 (1947). 

34. Haagen-Smit, a. J. et al.. Am. J. Botany, 33:118-120 (1946). 

35. Harder, R. and Springorum, B., Biol. Zentrbl., 66:147-165 (1947). 

36. Harrold, T. J., Botan. Gaz., 96:505-520 (1935). 

37. Hatcher, E. S. J. and Gregory, F. G., Nature, 148:626 (1941)- 

38. Hatcher, E. S. J., ibid., 151:278-279 (1943). 

39. Hei5jicke, a. }., Cornell Agr. Exp. Sta. Bull. 393 (1917). 

40. Hemphill, D. D., Mo. Agr. Exp. Sta. Res. Bull., 434 (1949). 

41. HowLETT, F. S., Proc. Am. Soc. Hort. Sci., 39: 217-277 (1941). 

42. Katunsky, V. M., Compt. Rend. (Dol^lady) Acad. Sci. USSR., 12:343-346 


43. Kent, N. and Brink, R. A., Science, 106:547-548 (1947). 

44. LaRue, C. D., Bull. Tor. Bot. Club., 63:356-382 (1936). 

45. Laibach, F., Ber. d. Bot. Ges., 50:383-390 (1932). 

46. Leopold, A. C. and Thimann, K. V., Am. J. Botany, 36:342-347 (1949). 

47. Loehwing, W. F., Botan. Rev., 4:581-625 (1938). 

48. Lona, F., Lavori Bot. Torino, 8:285-311 (1947). 

49. LucKWELL, L. C, Hort. Sci., 24:32-44 (1948). 

50. MuiR, R. M., Am. J. Botany, 29:716-720 (1942). 

51. MuRNEEK, A. E., Mo. Agr. Exp. Sta. Res. Bull., 90 (1926). 

52. , Plant Physiol. 7:79-90 (1932). 

53. , Mo. Agr. Exp. Sta. Res. Bull. 201 (1933). 

54. , Science, 86:43-47 (1937). 

55. , Growth, 3:295-315 (1939). 

56. , and Wittvver, S. H., Proc. Am. Soc. Hort. Sci., 40:201-204 (1942), 

A. E. MURNEEK 345 

57. MuRNEEK, A. E., Science, 98:384-385 (1943). 

58. , et al., Proc. Am. Soc. Hort. Sci., 45:371-381 (1944). 

59. MuRNEEK, A. E., ibid., 44:428-429 (1944). 

60. , and HiBBARD, A. D., ibid., 50:206-208 (1947). 

61. MuRNEEK, A. E., ibid., 50:254-262 (1947). 

62. , and Whyte, R. O., Vernalization and Photoperiodism (Waltham, 


63. Murneek, a. E., Unpublished records. 

64. , and HiBBARD, A. D., Unpublished records. 

65. Nielsen, C. G., Botan. Gaz., 104:99-106 (1942). 

6G. Overbeek, J. VAN et al.. Am. J. Botany, 28:647-656 (1941). 

67. , ibid., 29:472-477 (1942). 

68. Overbeek, J. van, Ann. Rev. Biochem., 13:631-666 (1944). 

69. Overbeek, J. van et al., Am. J. Botany, 31:219-224 (1944). 

70. Overbeek, }. van, Science, 102:621 (1945). 

71. , Botan. Gaz., 108:64-73 (1946). 

72. Purvis, O. N. and Gregory, F. G., Nature, 155:113-114 (1945). 

73. Roberts, R. H. and Struckmeyer, B. E., Proc. Am. Soc. Hort. Sci., 

44:417-427 (1944). 

74. Sax, K., Maine Agr. Exp. Sta. Bull., 298 (1921). 

75. Schneider, G. W. and Enzie, J. V., Proc. Am. Soc. Hort. Sci., 45:63-68 


76. Sen, p. K., Ann. Rpt. East Mailing Exp. Sta., 1936:137-141. 

77. Skoog, F. et al., Am. J. Botany, 29:568-576 (1942). 

78. Sinnott, E. W., Botan. Gaz., 99:803-813 (1938). 

79. Stebbins, T. C. et al., Proc. Am. Soc. Hort. Sci., 48:63-66 (1946). 

80. Steinegger, p., Ber. Schweiz. Bot. Ges. 42:285-339 (1933). 

81. Stewart, W. S. et al., Botan. Gaz., 109:150-162 (1943). 

82. Struckmeyer, B. E. and MacVicar, R., Botan. Gaz., 109:237-249 (1948). 

83. Swanson, C. p. et al.. Am. J. Botany, 36:170-175 (1949). 

84. Thimann, K. v., Gen. Physiol., 18:23-34 (1934). 

85. TuKEY, H. B., Proc. Am. Soc. Hort. Sci., 30:209-218 (1933). 

86. , Science, 84:513-515 (1936). 

87. , Botan. Gaz., 98:1-24 (1936). 

88. , and Lee, F. A., ibid., 101:818-838 (1940). 

89. TuKEY, H. B. and Young, J. R., ibid., 104:3-25 (1942). 

90. Voss, H., Planta, 30:252-285 (1939). 

91. Warington, K., Ann. Botany, 47:429-457 (1933). 

92. White, P. R., Arch. Zellfor., 42:602-620 (1933). 

93. Whyte, R. O., Crop production and environment (London, 1946). 

94. Wittwer, S. H. and Murneek, A. E., Proc. Am. Soc. Hort. Sci., 40:205- 

208 (1942). 

95. Wittwer, S. H., Mo. Agr. Exp. Sta. Res. Bull., y]\ (1943). 

96. Wittwer, S. H. et al., Proc. Am. Soc. Hort. Sci., 47:285-293 (1946). 

97. , ibid., 51:371-376 (1948). 

98. Yasuda, S., Mem. Fac. Sci. and Agr. Tohoku Univ. IV, 27:1-51 (1939). 

99. Zimmerman, P. W. and Hitchcock, A. E., Contrib. Boyce Thomps. Inst., 

12:321-343 (1942). 

The Induction of Flowering with a Plant Extract 


IT IS agreed by workers who have studied the physiology of blossoming, 
especially as related to photoperiodism, that plants contain a chemical 
substance which induces flowering (3). This concept is based particularly 
upon grafting experiments in which a plant in flower or a leaf from a 
flowering plant will induce a nonflowering plant to blossom when the 
two are grafted together under the proper experimental conditions. No 
report of a successful extraction of the blossom-inducing chemical has 
been seen although the name florigen has been proposed for it by 
Cajlahjan (i). 

Several years ago Struckmeyer (4) reported that there is reduced 
cambial activity and increased maturation of tissues at the time sexual 
reproduction is initiated. Thus it appears that the blossom-inducing 
substance causes maturation of tissues in contrast to the proliferation 
of cells produced by the numerous growth substances (5), some of which 
are used as weed killers (2). Since florigen appears to have a physiological 
effect opposite to the so-called hormones, it was presumed that its 
extraction should require an unlike procedure, or at least an unusual 

In 1946 a solvent was found which gave promise of being useful in 
obtaining florigen. The first successful extraction and subsequent blossom 
induction of the short-day plant cocklebur {Xanthium echinatum) with 
its extract was accomplished in December of that year. The fifty-second 

Editor's Note: Published with the permission of the Director of the Agricul- 
tural Experiment Station. Tlie work reported here was supported in part by a 
grant from the Wisconsin Alumni Research Foundation. A verbal report of this 
work in inducing flowering with plant extracts was made before the meeting of 
the American Society of Plant Physiologists at Cincinnati, Ohio, September 10, 


plant to be treated produced about the same degree of blossoming as 
occurs on plants after exposure to one or two long dark periods (short 
days). Subsequent trials with crude extract were 8 to lo per cent success- 
ful. In January, 1948, small, colorless, isotropic granular particles were 
obtained from the extract. When these were dissolved in fresh solvent 
and applied to vegetative plants of cocklebur in a long-day environment 
in March, all of the 15 plants which were treated formed staminate 
blossoms comparable in development with those on plants which had 
been given one or two short photoperiods (Fig. i). The macroscopic 
blossom buds appeared after five to eight weeks. This amount of time is 
typical of the period usually needed to produce blossoming from the 
stimulus of a single long dark period. Seven of the 15 plants bore 
pistillate blossoms similar to those on plants which have received two 
short photoperiods. 

The extract was effective at as high a dilution as one part to ten 
thousand. The effect from the solution was also additive to photoperiod. 
Four plants given one short photoperiod and also sprayed with the 
solution made a greater blossom development than those with only one 
short photoperiod. A total of 43 cocklebur plants have been induced 
to flower by treating them with plant extract. 

Other effects from applying the plant extract were to reduce cambial 
activity and to induce maturation of tissues comparable to that resulting 
from photoperiod treatments which bring about a like degree of blossom- 
ing. The extract also inhibits callus formation in wounded areas of stems. 
It has not yet been determined if a substance of a like physiological 
activity would have a similar effect upon some animal tumors. 

The following procedure is used to obtain florigen particles. Soak a 
small sample of fresh or frozen leaves taken from plants in flower in the 
least practicable amount of a highly refined, odorless insecticide base 
such as Shell Dispersol, for an hour or longer. This solvent is a non- 
aromatic oil fraction recovered from kerosene and having an IBP of 
387° F. and an FBP of 485° F. Squeeze out the solvent, remove and 
discard the aqueous phase if any is present, and filter. A yellowish 
pigment which seems not to interfere with florigen extraction or activity 
is present in the extract from most species. Deep freezing at 0° F. or 
below is used to initiate separation of the particles. This progresses 
slowly for several hours or even days. The particles in extracts from some 
plants, for example, white sweet clover or sweet corn, dissolve at room 

Figure I. Tips of cocklebur plants with older leaves removed. A, Non- 
flowering plant. B, Fruiting plant (induced by short days). C, Partial fruiting 
induced by one long dark period. D, Nearlv normal fruiting induced by plant 
extract in addition to one long dark period. E, Partial truiting trom two long 
dark periods. F, Partial fruiting following treatment with plant extract. 

R. H. ROBERTS 349 

temperatures. The florigen particles are recovered by filtering or decant- 
ing, and evaporating the remaining solvent at a temperature below 60° C. 

The particles are highly insoluble in such solvents as water, alcohol, 
acetone, xylol, benzol, ether, ethylacetate, petroleum ether, ethanola- 
mine, polyethylene glycol, and dioxane. This may be an explanation 
of why attempts by numerous workers to obtain florigen have been 
unsuccessfijl. The particles are poorly soluble in Dispersol. Consequently, 
repeated extractions of the leaves yield added amounts of particles. 

No particles have been obtained from extracts of nonflowering plants 
such as those growing in a photoperiod unfavorable to flowering or 
from those plants which are not known to flower, as for example, some 
varieties of sweet potato. Particles of a like physical appearance have 
been obtained from 23 species of dicotyledons including Cuscuta 
(Dodder) and Monotropa (Indian Pipe), and from six monocotyledons. 
All flowering plants chosen for extraction have yielded particles. Tests 
are under way to determine if these will have interspecific effects on the 
induction of blossoming. Preliminary experiments directed towards char- 
acterization of the extracted particles are also under way. 

A solution of the florigen particles in Dispersol can be applied to 
plants being used in tests of flower induction by wetting the foliage 
with it. Plants for a preliminary test of induction should be selected 
from those with a systemic flowering habit, at least until more is known 
of the factors determining the induction of plants which flower only 
terminally. (Plants of the latter type, as Klondyke Cosmos, do not 
become induced to blossom by grafting.) A plant should not be expected 
to respond beyond its genetical potentialities. 

The limited action of the extract in inducing blossoming of cocklebur 
comparable to that from only one or two long dark periods may indicate 
that the present material may be a precursor rather than the actual 

For several months after September, 1948, the induction studies 
were interrupted by the accidental introduction of a volatile ester of 
2,4-dichlorophenoxyacetic acid into the greenhouses. In March, 1949, 
16 plants of 19 treated with extract became partially induced, again 
to about the same degree as plants given one or two long nights. In 
all 131 plants have been partially induced with plant extract. 

Interest has shifted for the present from the induction of blossoming 
to the nature of the extracts which have been obtained. By varying 


extraction conditions six crystalline substances have been secured. These 
are being termed florigens as they have been extracted from flowering 
plants and are not obtained from nonflovvering plants. Partial charac- 
terization under the direction of Dr. Ben Aycock of the department of 
Organic Chemistry of the University of Wisconsin indicates that they 
are mineral salts of fatty acids. Poor solubility makes identification and 
the determination of physiological activity slow, but observations to 
date indicate that information, which may answer the question of the 
nature of the mechanism of photoperiodism, will be obtained from a 
knowledge of the chemical nature of the extracts now available. 


1. Cajlahjan, M. Ch., Compt. Rend. {Dol^ady) Acad. Set. USSR, 18:607 


2. Crafts, A. S., Plani Physiol., 21:345 (^946)- 

3. Melchers, G. and Lang, A. Biol. Zentralb., 67:105 (1948). 

4. Struckmeyer, B. Esther, Botan. Gaz., 103:182 (1941). 

5. Zimmerman, P. W., Torreya, 43:98 (1943). 

Fruit Development as Influenced by 
Growth Hormones 


THE production ot fruits is ordinarily associated with pollination 
and fertilization, and it is almost axiomatic that if there is no 
pollination there is no fruit setting. In many parts of the country 
orchardists, to insure fruit setting, have gone as far as to distribute 
beehives in their orchards during the blossoming period. In spite of this 
common relationship between fruit setting and pollination and fertiliza- 
tion we find that there are numerous instances in which fruits are pro- 
duced without fertilization, and not even pollination is necessary in 
some cases (lo). These fruits are seedless either because of lack of fertiliza- 
tion or because the young embryos aborted leaving only tiny nutlets 
as a reminder of fertihzation. Some of our so-called seedless grapes are 
of the latter type (19), whereas the navel orange is illustrative of the 
former situation, where not even pollination is necessary (25). 

Most seedless or parthenocarpic fruits, as they are technically called, 
are probably produced as a result of pollination without fertilization. 
It was as a result of the production of seedless fruits in infertile crosses 
in Oenothera that the writer became interested in the subject. It was 
reasoned that if the mere pollination stimulated the ovary to grow into 
a fruit there must be transferred a stimulus either from the pollen or the 
pollen tube to the ovary, and accordingly, several experiments were 
set up to test the idea, Laibach (15) and Thimann (23) had found that 
pollen contained growth hormones or auxin. If pollination without 
fertilization can cause the ovary to develop into a fruit, and pollen con- 
tains auxin, why not supply synthetic hormone directly to the pistil.? 
That was done, and in 1936 (5) the writer succeeded in producing seedless 


fruits of the tomato and many other plants by applying synthetic growth 
hormones to the pistil. The general procedure was to mix the hormone 
or growth-promoting substance with lanolin, which was applied to the 
cut surface of the style of unopened flower buds after the anthers had 
been removed. To insure diffusion of the chemical into the ovary the 
style was shortened, and when the style was not too long the stigma 
was cut to allow easy penetration. The growth-promoting chemicals 
used in this early work were phenylacetic, indoleacetic, indolepropionic, 
and indolebutyric acids. Concentrations of chemicals were quite high, 
sometimes as high as 2 per cent in lanolin, but 0.25 per cent concentra- 
tions were also used. 

In 1934 Yasuda (28) had produced a few near-normal sized cucumbers 
by injecting into the young ovaries of the cucumber flowers water 
extracts of cucumber pollen. As far as is known this was the first success 
in producing full-sized fruits without seeds by chemical treatment. 
Other investigators (10) had previously attempted to initiate fruit 
development by treating the pistil with pollen extracts, but the growth 
produced was limited. 

In 1937 Gardner and Marth (3) produced parthenocarpic fruits in 
Ilex opaca and the strawberry with hormone treatment. In the same 
year Hagemann (11) produced seedless fruits in the gladiolus. Since that 
time numerous investigators have grown parthenocarpic fruits as a 
result of treating the pistils with growth hormones, but there have been 
many failures too. No success has been obtained with such plants as 
apples, pears, cherries, and peaches. 

A practical use of this method, which was not anticipated in 1936, 
has also been made. Among the investigators most responsible for this 
may be mentioned Howlett (12), Strong (22), Roberts and Struck- 
meyer (20) and Murneek and Wittwer (18). During the dark months of 
December, January, and February, the setting of tomatoes grown in 
greenhouses in the northern states is very much reduced due to the 
poor development of pollen. By spraying the blossom buds with any 
one of a number of growth-promoting substances the setting has been 
increased as much as one hundred per cent, and the fruits are of normal 
or even larger size. Some investigators have added a spreader to the 
solution, which enables the chemical to remain spread over the surface 
of the blossom buds after the water has evaporated, but others have 
dissolved the chemical in alcohol and made dilutions of this concentrated 

Figure i. John Baer tomato fruits. The two central fruits were produced 
by pollination and the two end ones by treating the cut style of an unopened 
flower bud with 2 per cent indolebutyric acid in lanolin. 

Figure 2. A cross section of a parthenocarpic tomato fruit produced by 
treating cut style of an unopened flower bud with indolebutyric acid. The 
locules are well developed, but the ovules have not developed into seeds. The 
gelatinous material found in normal seeded fruits fills the locules of this 
parthenocarpic fruit. 

Figure 3. Fruits of crook-neck summer squash. Left: Parthenocarpic fruit 
produced by the appHcation ol indolebutyric acid to the style of flower bud. 
Right: A normal seeded fruit produced by pollination. 

Figure 4. Buttercup Squash. The fruit at the lower left was produced by 
pollination and has an abundance of seeds. The other two are seedless and were 
produced by treating the pistil with 2 per cent naphthaleneacetic acid in 

Figure 5. Fruits of Maryland Mammoth Tobacco. Left: Two normal truits 
produced by pollination. Center: The ovary of a freshly opened flower. 
Right: Two parthenocarpic fruits produced by injecting a solution of 0.2 per 
cent potassium indoleacetate into the ovary through the pedicel. 

Figure 6. Crook-neck Summer Squash. At the extreme left is a normal 
fruit. All the others were produced by cuttmg oif the apical end of the ovary 
in the flower bud stage and smearing the cut surface with 5 per cent indole- 
butyric acid in lanolin. The larger ones at bottom and right had more of 
the ovary left and some o\ules. 


solution. Mitchell et al. (17) have used carbowax in a concentration of 
0.5 per cent with good results. Concentrations of growth substances 
have ranged from a few parts per million to 300 ppm., but part of 
this difference in concentration has been due to the type of chemical 
used. Wittwer (26) has found that 25 to 30 ppm. of p-chlorphenoxy- 
acetic acid gave satisfactory results. Not only has it been found that 
hormone spray increases the setting of tomatoes in the greenhouse during 
the winter, but it has also been found that if the first two or three 
clusters of flower buds on plants grown outdoors in early spring are 
sprayed the setting will be much increased. The cold of the early spring is 
extremely unfavorable for tomato setting, perhaps because of poor 
pollen development, but the pistils are perfectly capable of developing 
into fruits if stimulated by growth-promoting substances. The chemicals 
used to increase fruit-setting in the tomato are numerous, but the ones 
most commonly used are indolebutyric, naphthaleneacetic, and naph- 
thoxyacetic acids as well as several derivatives of phenylacetic acid as 
p-chlorphenoxyacetic, 2,4-dichlorphenoxyacetic, and 2,4,5-trichlor- 
phenoxyacetic acids. In passing it should be stated that in spraying 
tomato buds or flowers either in the greenhouse or in the field the 
resulting fruits are not always parthenocarpic since sometimes pollination 
may have taken place before the spraying, but this pollination may be 
so light that without the additional hormone the pistil would not develop 
into a fruit. 

Recently two papers appeared adding another economic plant that 
profits by hormone spray. Blondeau and Crane (i) and Stewart and 
Condit (21) have reported that Calimyrna figs, which are usually pro- 
duced only as a result of pollination by the fig wasp, produced partheno- 
carpic fruits of normal size and color when sprayed with 2,4-dichlor- 
phenoxyacetic, 2,4,5-trichlorphenoxyacetic, or indolebutyric acids. 
These authors state that if the spray method proves to be adaptable to 
commercial groves it will be of inestimable value to the California 
fruitgrowers, as there will be no further need of growing the pollen- 
producing caprifig trees. 

Figuies I to 5 illustrate typical parthenocarpic fruits produced by the 
writer. Figure i of the John Baer tomato shows that externally there 
is no difference between the seeded and seedless fruits; and in Figure 
2 it is seen that internally the parthenocarpic fruit is like the normal 
fruit in that it is fleshy, possesses prominent locules, but the ovules have 


enlarged only slightly. In the crook-neck summer squash (Fig. 3) the 
ovary developed into a fruit of normal length without fertilization, but 
when no seeds were pioduced the locular region did not grow as ex- 
tensively as when seeds were produced. The result was that the partheno- 
carpic fruits were quite long and thin and showed none of the bulging 
of the seeded fruit. Figure 4, of buttercup squash, brings out the fact 
that sometimes the resulting fruit may lack entirely the locules and be 
composed only of solid flesh. This has also been found in the tomato. 
The tomatoes lacking locules are, as a rule, considerably smaller than 
the normal seeded fruits. The writer has also observed this in naturally 
occurring parthenocarpic avocados, where the stone is lacking. 

Parthenocarpy in nonfleshy fruits is illustrated by the Maryland 
Mammoth tobacco shown in Figure 5. In this plant the pedicel of the 
flower is quite stout, and it was possible to inject a solution into the 
ovary through the thickened pedicel. Ovaries injected with a solution 
of 0.2 per cent potassium indoleacetate grew more during the first five 
days after injection than the ovaries from pollinated flowers, and reached 
nearly the same final size as the seeded fruits. 

Janes (13,14) made a comparative chemical study of parthenocarpic 
and seeded fruits of the tomato and pepper. He found that in the tomato 
total sugar and starch was greater in the parthenocarpic fruits. During 
early stages of development the titratable acidity was the same in both 
types of fruits, but during the ripening period the seeded fruits had a 
higher concentration and this was especially noticeable in the locular 
region. In the ripe fruits the percentage of dry weight was a little greater 
than in the seeded fruits. The mature parthenocarpic peppers had a 
slightly higher per cent of dry weight, soluble soUds, sugars, and total 
nitrogen than the seeded fruits, but the difference was not great. 

Gardner and Kraus (4) found in their extensive anatomical study 
of the development of the parthenocarpic fruits of Ilex opaca that with 
the exception of the lack of the seed there was no difference between the 
seeded and seedless fruits. In tobacco ovaries injected with 0.2 per cent 
potassium indoleacetate the ovules grew to nearly one fourth the size 
of ripe seeds and there was some growth in the embryo sac, but no 
seeds with an embryo ever formed (6). Several investigators have re- 
ported that the ovules may grow into empty seeds of considerable size 

Dollfus (2) found that ovules supply all or most of the hormone 


necessary for the enlargement of the ovary into the ripe fruit. When he 
removed the ovules little growth took place; but if lanolin containing 
indoleacetic acid was placed in the locular cavity in place of the ovules 
nearly normal growth occurred. Gustafson (6) corroborated this finding 
with the crook-neck summer squash. He cut the ovary from unopened 
flower buds at different distances from the base. By this procedure the 
base was left without any ovules or with smaller or larger numbers 
depending upon the position of the cut. When no ovules were included 
in the ovary the growth was slight, and the amount of growth increased 
with the number of ovules left. Indolebutyric acid in lanoUn smeared 
on the cut surface caused the portion of the ovary without any ovules 
to grow as much as it would have grown in a normally fertilized ovary 
(Fig, 6). Both of these investigators emphasized the importance of 
ovules and seeds in the development of fruits. Meyer (i6) and also the 
writer (8) have shown that ovules, placentae, and seeds are much richer 
in growth hormones than other parts of the ovary or fruit. 

As a result of these investigations the theory was developed (7) that 
fruit growth is initiated by the growth hormone brought into the ovary 
by the pollen tubes carrying the sperm nuclei into the embryo sacs. In 
plants like the navel orange, lemon, and grape where seedless fruits 
are normally produced, the growth-hormone concentration in the ovary 
was found to be greater than in similar varieties, which required poHina- 
tion or fertilization for fruit production. Van Overbeek (24) has ques- 
tioned the suggestion that growth hormones are supplied to the ovary 
by the pollen tubes, and he suggested the alternative that pollen tubes 
might carry into the ovary enzymes, or more specifically, prosthetic 
groups which would form enzymes, that acted on bound growth hor- 
mones to release the active form. 


1. Blondeau, R. and Crane, J. C, Science, 108:719 (1948). 

2. DoLLFUS, H., Planta, 25:1 (1936). 

3. Gardner, F. E. and Marth, P. C, Botan. Gaz., 99:184 (1937). 

4. Gardner, F. E. and Kraus, E. J., ibid., 99:355 (i937)- 

5. Gustafson, F, G., Proc. Nat. Acad. Sci. U. S., 22:628 (1936). 

6. , Am. J. Botany, 25:237 (1938). 

7. , ibid., 26:135 (1939). 

8. , ibid., 26:189 (1939). 

9. , Proc. Am. Soc. Hort. Sci., 38:479 (1940). 


10. , Botan. Rev., 8:599 (^94^)- 

11. Hagemann, p., Gartenbauwiss., 11:144 (1937). 

12. HowLETT, F. S., Proc. Am. Soc. Hon. Sci., 39:217 (1941). 

13. Janes, B. E., Am. J. Botany, 28:639 (1941). 

14. , Proc. Am. Soc. Hort. Sci., 40:432 (1942). 

15. Laibach, F., Ber. dent. Botan. Ges., 50:383 (1932). 

16. Meyer, F., Dissertation, Johann Wolfgang Goethe Universitat z. Frank- 

furt am Mainz. 1936. 

17. Mitchell, J. W. and Hamner, C. G., Botan. Gaz., 105:474 (1944). 

18. Murxeek, a. E. WimvER, S. H., and Hemphill, D. D., Proc. Am. Soc. 

Hort. Sci., 45:371 (1944). 

19. Olmo, H. p., ibid., 34:402 (1936). 

20. Roberts, R. H. and Struckmeyer, B. E., ibid., 44:417 (1944). 

21. Stewart, Wm. S. and Condit, I. J., Am. J. Botany, 36:332 (1949) 

22. Strong, Miriam C., Mich. Agri. Exp. Sta. Quart. Bull., 24:56 (1941). 

23. Thimann, K. v.,/. Gen. Physiol., 18:23 (1934). 

24. VAN OvERBEEK, J., CoNKLiN, Marie E., and Blakeslee, a. F., Am. J. 

Botany, 28:647 (1941). 

25. Webber, H. J., Cal. Citrograph, 15:304 (1929-30). 

26. Witt\ver, S. H., Proc. Am. Soc. Hort. Sci., 51:371 (1948). 

27. Wong, Cheong-Yin, ibid., 37:158 (1939). 

28. Yasuda, S. Agr. and Hort., 9:647 (1934). 

The Growth Hormone Mechanism 
in Fruit Development 


THE studies of Gustafson (5,6) and others on parthenocarpy indicate 
that growth hormones control the development of fruit. The 
mechanism whereby this control is implemented in the normal process of 
pollination and fertiUzation is incompletely known, however. Relatively 
few investigations of this aspect of auxin physiology have been made and 
their results are not in complete agreement. Undoubtedly some variation 
exists among plant species as is indicated by the differences in degree 
of natural parthenocarpy; yet, iDcfore ascribing different mechanisms to 
the species investigated, the differences which have been reported must 
be evaluated on the basis of the techniques used in the assay of auxin. 
Since the normal stimulus for fruit development is fertilization subse- 
quent to pollination, it follows that the pollen may furnish directly 
the growth hormones which cause the enlargement of the ovary, or it 
may furnish a part of a system responsible for the production of the 
hormone in the ovary. Laibach (8) first identified auxin in extracts of 
pollen of several orchids and Hibiscus and later (9) reported that extracts 
of Cucurbita pollen were active in the Avena test but those of a number 
of other species were not. Extracts of pollen of Sequoia (18), Zea 
(10,12,13), and Helianthus (10) have been shown to contain auxin. 
Laibach and Meyer (10) report relatively large amounts of auxin in 
both unripe anthers and ripe pollen of corn, and Wittwer (21) found that 
ether extracts of mature corn pollen were five times as active as extracts 
of immature anthers. This sequence of ontogenetic changes in the hor- 
mone content of pollen is different from that reported by Hatcher (7) 
for rye in which water, phosphate buffer (/?H 10), and N/50 NaOH 


extracts of green, unripe anthers gave much higher auxin yields than 
similar extracts of yellow, ripe anthers, and extracts of mature pollen 
grains did not contain active hormones. By grinding the mature pollen 
grains with glass and acidifving the mLxture before extraction with 
chloroform, auxin is obtained from the pollen of Xicotiana tabacum, 
AmirThinum majtis. Cyclamen persicum, and Datura suaveolens (15). 
L sually more auxin is obtained if the pollen grains are germinated on 
I per cent agar containing sucrose before extraction, and if hydrolysis 
with i.o N NaOH precedes extraction of the ground pollen grains, the 
yields of auxin are uniformly high. The poUen of all plants probably 
contains auxin, but it may be present in variable amounts as free, bound, 
or precursor forms, and in some species several auxins may be present. 
The extracdon procedures used are responsible for the unsuccessful 
attempts to obtain auxin from pollen which have been reported. 

TTie gro\M:h hormones of the pollen are not the principal hormones 
involved in the enlargement of the ovar}' as is shown by a comparison 
of the amount of auxin present in the ovar\' after fertiHzation and 
the amount present in the pollen of a normal pollination. The as- 
say of auxin in the ovary can be accompUshed by placing the tissue 
on an agar block and allowing the auxin to diffuse into the agar for 2 or 
3 hours. Numerous determinations of diffusible or free auxin in pistils 
of Nicotiana tabacum have shown that relatively small quantities are 
present at full anthesis (14). Similar results are obtained with pistils of 
Aruirrhinum and corn. Following pollination in Nicotiana tabacum an 
increase in diffusible auxin is detected in the style accompanying the 
penetration of the pollen tubes and when fertilization occurs there is a 
marked increase in the amount of diffusible auxin in the ovary. The 
amount found in the style is 30 times the maximum amount obtained 
by extraction of the pollen and the amount in the ovary is 100 times as 
great (15). Thus the stimulus for fruit development provided by the 
pollen is something other than the growth hormones which it contains. 

TTie high level of diffusible auxin content is maintained in the ovary 
oi Nicotiana for at least 45 hours following fertilization although a down- 
ward movement from the ovary to the stem must occur since considerable 
amounts of auxin can be diffused from the pedicel alone (14). This auxin 
inhibits the development of the abscission layer (ii,i6j and thus one of 
the primary requirements for the development of the fruit is fulfilled. 


These results indicate that following pollination and fertilization an 
auxin production mechanism is established in the ovary which brings 
about the enlargement into the fruit. 

A similar sequence of changes in hormone content of the ovary occurs 
in Heliopsis laevis according to Soding (17). The amount of diffusible 
auxin in the flower head increases from the bud stage until the flowers 
begin to turn yellow then decreases until no detectable auxin is present 
when the flowers are fiilly open. After fertilization diffusible auxin 
again is present in the flower head but the amount decreases as the 
fruits mature. Soding inferred that the free, diffusible auxin of the young 
fruit is transformed into the immobile reserve auxin of the seed. 

According to Hatcher (7) no diffusible auxin is found in the rye grain 
until three weeks after pollination whereupon a marked increase occurs 
with a maximum at five weeks, and then a steady decline follows to 
maturity. He concludes that the growth rate of the rye grain is not 
related to the amount of auxin present in the grain since very little 
auxin is present during the first three weeks of its development and 
maximum amounts are present when its growth has ceased. However, the 
most important auxin-growth relationship in the development of the 
fruitof A7ro//a«j appears to be the initiation of the enlargement associated 
with the presence of relatively large amounts of diffusible, fi-ee auxin as 
shown in Table i . Enlargement of the pollinated pistil begins promptly 
when the pollen tubes reach the ovules (approximately 45 hours after 
pollination) at which time there is a large increase in diffusible auxin, and 
during the following 40 hours the ovary nearly doubles in size whereas 
the ovarv of the unpoUinated pistil enlarges very little. It should be 
realized that changes in the auxin content of a head of flowers or a 
capsule containing hundreds of ovules represent composite effects of 


Average length of ovaries of Nicotiana tabacum 

Hours after anthesis Pollinated L'npolxjnated 



- .0 mm. 

5 mm. 

5.2 mm. 

5. r mm. 

5.6 mm. 

7.5 mm. 

5 T mm. 

TO 3 mm. 

6.0 mm. 


fertilization and are measurable whereas similar changes in a fruit such 
as the caryopsis with a single ovule might not be measurable with the 
techniques available. 

Determinations of auxin content by extraction procedures have identi- 
fied bound forms and precursor forms of auxin in addition to the free 
auxin measured by the diffusion technique. The results of experiments 
employing extraction procedures are difficult to interpret because of the 
uncertainty as to the auxin forms involved. Some of the investigations, 
however, substantiate the results of the diffusion experiments and show 
the production of free auxin beginning in the fruit after fertiUzation. 
Laibach and Meyer (10) extracted auxin with alcohol finding small 
amounts in pistils of Helianthus anniius before fertilization and none in 
those of corn. After fertilization they found a sharp increase in the 
amount of auxin in the pistils of both species. These results have been 
confirmed for corn by Avery et al. (i) and Wittwer (21) who found 
that the auxin content increases for 2 or 3 weeks following fertilization 
and then decreases until maturity. Extracting the auxin from the rye 
grain with water, alkaline phosphate buffer, and N/50 NaOH, Hatcher 
(7) found little or no auxin until three weeks after anthesis which agreed 
with the determinations by the diffusion technique. 

Of particular concern here are the results of the determinations of 
auxin in the ovary prior to fertilization. In the investigations cited above 
Httle or no auxin in the free, combined, or precursor forms was found 
in rye or in corn grains with the exception of some assays of kernels of 
the Country Gentleman variety of corn (i). In some experiments per- 
formed by the writer the grains of a hybrid corn at the silk stage yielded 
only 2 or 3 degrees of curvature by the diffusion technique, but extraction 
of the lyophilized grains with ether containing 5 per cent water for 10 
hours at 23° C. gave curvatures of 30 to 40 degrees per grain. With the 
same extraction procedure high yields of auxin were obtained from both 
fertilized and unfertilized ovaries of Nicotiana tabaciim 75 hours after 
anthesis, the yield from the fertihzed ovaries being greater than that 
from the unfertilized ovaries (Table 2). It has been shown (20) that the 
yields of auxin by such an extraction are probably due to the conversion 
of tryptophan or a similar precursor to the auxin, indoleacetic acid. 
Prehminary experiments indicate that both fertilized and unfertihzed 
Nicotiana pistils contain an enzyme system which can convert tryptophan 
to auxin with remarkable facility. Thus the stimulus furnished by the 



Auxin yields from 20 mg. ovary tissue of Nicoiiana tabacum 75 hours after 


Ml. of Avena test Curvature 

AGAR curvature PER OVARY 

Pollinated 0.4 i6.6±i.5 184 

Unpollinated 0.4 ly.Srbi.y 198 

pollen for the production of auxin in the ovary is not part of the enzyme 
system concerned with the transformation of tryptophan to indoleacetic 
acid, although the possibility that it might be a part of a system re- 
sponsible for the formation of tryptophan is not precluded. 

Further information on the production of auxin in unfertilized ovary 
tissue of Nicotiana is obtained by incubation of the tissue at varying pW 
levels with and without an aqueous extract of pollen as shown in Table 
3. Small amounts of auxin are obtained from the ovary tissue incubated 
at /?H 5.9 but large amounts are obtained if the tissue is incubated at 
pW 8.0. This is in agreement with our knowledge of tryptophan convert- 
ing enzymes in plant tissues, for, as Wildman et al. (19) have shown, the 
optimal pH for the enzyme system in spinach cytoplasm is pY{ 7.5, and 
below pH 6.0 or above pW 8.5 the activity of the enzyme is greatly 
restricted. However, if a small amount of an aqueous extract of ground 
pollen (containing no detectable auxin) is added to the medium, con- 
siderable auxin is produced at pW 5.9. Apparently the auxin production 
under acid conditions is not one of tryptophan conversion but involves 

Auxin yields from Nicotiana tissue following incubation at 37° C. for 24 hours 

Avena test 


Buffer solution, pW 5 . 9 

20 mg. ovary tissue 5.5±i.2 

Extract of 10 mg. pollen 0.0 

Ovary tissue + pollen extract i5.odbi.3 

Buffer solution, pW 8.0 

20 mg. ovary tissue 34.6 ± 1.7 

Extract of 10 mg. pollen 0.0 

Ovary tissue + pollen extract 40.2 ± 3.2 



the transformation of other types of precursors (3,2). The existence of 
such precursors in the unfertiHzed ovary tissue of Nicotiana tabacum 
and Antirrhinum is indicated by the yields of auxin obtained following 
hydrolysis with o.i N HCl as shown in Table 4. Hydrolysis of Nicotiana 
tissue with i .0 N NaOH gives equal yields of auxin, but similar hydrolysis 
of Antirrhinum tissue does not. 

Hatcher (7) proposes that for rye the auxin system of the anther is 
different from that of the pistil, since in the former the maximum amount 
of auxin is found 2 to 3 weeks earlier than in the pistil and later disappears 
completely while the auxin of the mature pistil can be recovered by 

Auxin yields from 20 mg. ovary tissue following hydrolysis 

Type of 

Avena test 

Micrograms of 





0.1 NHCl 

lO.O ± 0.2 



12.4 ±0.8 



0.1 NHCl 

14.0 d= 0.8 



3-9± 0-7 


alkaline hydrolysis. He infers that auxin-a is the principal auxin of the 
embryo and indoleacetic acid is the principal auxin of the endosperm, 
the latter being the auxin concerned in the development of the pistil 
and anther. However, Zimmerman and Hitchcock (22) have found that 
negative results for indoles are obtained when active extracts of corn 
pollen are tested by the Winkler method, and they suggest that auxin-a 
may be the principal auxin of such extracts. A comparison of the yields 
of auxin from pollen and ovary tissue by hydrolysis with i.o N NaOH 
or 0.1 N HCl strongly suggests the existence of several types of auxin 
(Tables 4 and 5). The auxin of the pollen of Nicotiana and Datura 
is alkali-stable, acid-labile, and therefore indicated to be indoleacetic 
acid whereas the pollen of Antirrhinum contains indoleacetic acid and an 
acid-stable auxin or auxin-a. Similarly in the ovary tissue of Nicotiana 
and Antirrhinum there appears to be both acid-stable and alkaU-stable 
auxins. These are tentative interpretations, however, for as Bonner and 



Wildman (4) have pointed out, the acid-alkah destruction test is in- 
conclusive because of the interference by proteins and inhibitors. The 
possibihty that different auxin types occur in the pollen and ovary 
tissue merits further investigation. 

Remaining to be investigated are many other aspects of the mechanism 
of auxin production in the pistil following fertilization. The greatest 
need exists for the re-examination of the auxin content of the pollen 
and pistil of the plants previously studied, this time using uniform tech- 
niques of auxin analysis to obtain conclusive evidence as to the similarity 
or dissimilarity of the mechanism in different species. In the pistil of 

Auxin yields from 30 mg. pollen following hydrolysis 

Type of 

Avena test 

Micrograms of 





X 10 Ymg. 



30.5± 1.8 


0.1 NHCl 





19.7 ± I.O 


0.1 NHCl 

19.2 ± 1.0 




27. 6± 1.8 


0.1 NHCl 



Nicotiana tabacum, and perhaps other species also, following fertihzation, 
a system for the production of diffusible, free auxin is established. The 
auxin production parallels the enlargement of the fruit, greatly exceeding 
the amount of auxin present in the pollen. The auxin in the ovary may 
be formed from tryptophan or a similar precursor, the pollen furnishing 
a part of a system responsible for the production of tryptophan in the 
tissues; or the auxin may be formed from other precursors, perhaps 
protein in nature, a substance in the pollen bringing about the release of 
the auxin from its inactive combination. The activator or coenzyme 
nature of the effective substance in pollen remains to be established. 



1. Avery, G. S., Jr., Berger, J., and Shalucha, B., Am. J. Botany, 2g:j6^ 


2. Avery, G. S., Jr., Berger, J., and White, R. O., tbid., 32:188 (1945). 

3. Berger, J. and Avery, G. S., Jr., ibid., 31:199 (i944)- 

4. Bonner, J. and Wildman, S. G., Growth, 11:51 (1947). 

5. GusTAFSON, F. G., Proc. Nat. Acad. Sci. U. S., 22:628 (1936). 

6. , Am. J. Botany, 26:189 (1939). 

7. Hatcher, E. S. J., Ann. Botany, N.S., 9:235 (1945). 

8. Laibach, F., Ber. deut. botan. Ges., 50:383 (1932). 

9. , ibid., 51:336 (1933). 

10. , and Meyer, F., Senckenbergiana, 17:73 (1935)- 

11. La Rue, C. D., Proc. Nat. Acad. Sci. U. S., 22:254 (1936). 

12. Mitchell, J. F. and Whitehead, M. R., Botan. Gaz., 102:770 (1941). 

13. Moulton, J. E., ibid., 103:725 (1942). 

14. MuiR, R. M., Am. J. Botany, 29:716 (1942). 

15. , Proc. Nat. Acad. Sci. U. S., 33:303 (1947). 

16. Myers, R. M., Botan. Gaz., 102:323 (1940). 

17. Soding, H., Flora, 132:425 (1937). 

18. Thimann, K. v.,/. Gen Physiol., 18:23 (1934). 

19. Wildman, S. G., Ferri, M. G., and Bonner, J., Arch. Biochem., 13:131 


20. Wildman, S. G. and Muir, R. M., Plant Physiol, 24:84 (1949). 

21. WiTTvvER, S. H., Mo. Agr. Exp. Sta. Res. Bull., 371 (1943). 

22. Zimmerman, P. W. and Hitchcock, A. E., Ann. Rei/. Biochem., 17:601 


Growth Substances in Fruit Setting 


POLLINATION and subsequent fertilization are prerequisites for fruit 
set on most plants. In their absence the ovary will usually fail to 
enlarge, and abscission occurs shortly. Pollination, pollen germination,, 
pollen tube growth, gametic fusion (embryo), and triple fusion (endo- 
sperm) are, with few exceptions, all essential for seed and fruit produc- 
tion. Closely associated and directly coupled with some (perhaps all) 
of these processes are complicated hormonal mechanisms which largely 
control fruit setting and bring about its eventual maturation. 

Certain plant reproductive structures are loci of production and ac- 
cumulation of natural growth substances. This is especially true of pollen 
grains and the young fruit or fertilized ovary. Literature concerned with 
the proposition that the included growth substances in pollen may be 
causal in the induction of natural fruit setting has been reviewed by 
Skoog (41) and van Overbeek (47). The small actual amounts of auxin 
in the usually few pollen grains which function in fruit formation do not 
indicate that pollen is the source of growth substances necessary for fruit 
setting. Nevertheless, the facts remain that fruit setting has been induced 
by pollen extracts and the exact chemical nature of growth sub- 
stances in pollen continues to attract considerable interest (39,59). Per- 
haps of greater importance is the rapid accumulation of growth sub- 
stances in the ovary following fertilization. These hormonal relationships 
as they exist in the corn plant and their close association with the cyto- 
genetically important processes of synapsis and syngamy (53) are illus- 
trated in Figure i. Similar hormonal relationships during the ontogeny 
of reproductive structures in corn (3,24) and in other plants (16,23,26) 
have been reported. It is highly probable that normal growth and 
development in the fruit of most plants is initiated and continued by a 



series of hormonal stimulations beginning with the auxin released in the 
developing gametophytes and ending with its production and accumula- 
tion in the developing fruit. The effects of the release of these natural 
growth substances during various phases of reproduction are frequently 
confined not alone to stimulating fruit setting and growth but are 
extended to vegetative parts as well, exerting a profound effect upon 



pe 400 









, » ^ FLOWERS 
-i- ^ FLOWERS 

Synaptit / Allowed 


August 5 


September 10 

Figure i. Changes in the growth hormone content of the reproductive 
organs of the corn plant during their development. There is complete absence 
of growth substance in the male inflorescence prior to synapsis, and in the ovule 
before fertilization. Subsequent to chromosome conjugation in the tassel, and 
the union of gametes in the ear, growth hormones appear in considerable 
quantities in these structures. 

the growth and metabolism of the entire plant (53). Such mechanisms of 
auxin action involving intact higher plants are complicated, indeed, and 
can hardly be explained in terms used for describing auxin effects on 
excised tissues (2). 

From the number of independent observations reported recently, 
there can be little doubt that the immediate causal factors in fruit setting 
are hormonal, providing nutrition is adequate. Considerable confusion 

S. H. WITTWER 367 

exists, however, pertaining to results that can be expected by the use 
of growth substances on many economic crops which exhibit from time 
to time difficulties in natural fruit setting. Most issues could be reconciled 
by a proper consideration of the effects of differing environments. To 
date, an almost total disregard of the importance of prevailing weather 
in determining fruit setting response to hormone chemicals has left a 
confused and somewhat distorted picture, making it extremely difficult 
to interpret many of the contradictory reports. The most profound 
increases in yield and fruit setting on crops reported as responding to 
the apphcation of growth substances are obtained when the prevailing 
environment is not conducive to good fruit set. 

Fruit Set in Tomatoes. — In at least one crop, the tomato, naturally 
produced hormones responsible for fruit set can be completely replaced 
by a great number of synthetic chemicals (18,45,58). These materials 
may be applied externally to the floral parts by several methods. Yet, 
even in the tomato the responses to hormone treatment, as measured 
by yield and fruit size increases, have not been consistent. 

The significance of variety and season on fruit production In green- 
house tomatoes as they relate to the changing day-to-day pattern of 
solar radiation has been emphasized (54). The effects of these variables 
on yield and fruit size are presented in Table i. That yields in the 


Effects of fruit setting treatments on yield and fruit size of greenhouse 
tomatoes as conditioned by variety and season 

Yield of fruit Average weight of fruit 


in lbs. per plant in ounces 



Flowers Vibration+ Flowers Vibration + 

Control vibrated hormone* Control vibrated hormone 


9.5 12.0 14.0 3.5 4.7 4.8 

Bay State 

2-3 3-7 5-2 4-3 4-4 4-3 


II. 6 12.2 II. 5 2.4 3.0 3.2 
2.7 4.3 4.9 3.2 2.9 2.8 

*Flower clusters sprayed with a mixture consisting of 10 ppm of p-chloro- 
phenoxyacetic acid (CIPA) and 30 ppm. of P-naphthoxyacetic acid (NOA). 


spring crop exceeded by almost 3 times those of the fall crop may be 
partially explained in the fact that the average daily solar radiation in 
gram calories per square centimeter received in the spring crop was 
354.2 and 437.3 during the periods of fruit setting and fruit harvest, 
respectively, compared with 183.2 and 102.6, respectively, for the fall 
crop. Spartan Hybrid, an American Globe type of forcing tomato, 
responded favorably to supplementary fruit-setting treatments in both 
the spring and fall, while Improved Bay State, an English forcing tomato, 
showed no response in the spring but gave variations comparable to 
Spartan Hybrid in the fall. Differences in floral structure and quality 
of the pollen of the two types as affected by light intensities and photo- 
period have been offered as possible explanations of the varied results 
obtained (5,20). In fruit size both varieties showed a similar response 
in the two crops. 

The use of growth substances on outdoor tomatoes for improving 
yields and fruit set has resulted in varied and contradictory data. Both 
successes (27,29) and failures (31,34) have been reported. The usual 
controlling factor for early fruit setting in field tomatoes is night 
temperature. Exacting studies of Went and Cosper (49) under controlled 
environments, those of Smith and Cochran (42) on pollen germination 
and pollen tube growth, and our own (56) under field conditions have 
estabhshed that the optimal range of night temperature for fruit setting 
in tomatoes is 59° to 68°F. (15° to 20°C.). Temperatures below 55°F. 
will cause failure of fruit set, even on early varieties, and irrespective 
of the fact that the vines are making good vegetative growth and ap- 
parently flowering normally. Little viable pollen is produced and much 
of that appears incapable of normal germination and of producing tubes 
of sufficient strength to traverse the style. Limitations on tomato fruit 
set imposed by cold night temperatures (56,57) or extremely hot tem- 
peratures (29) can be overcome by using hormone chemicals. 

Rather striking results have been obtained. Typical comparative data 
on early yields, total yield, and fruit size of hormone-treated versus 
nontreated plants obtained in the summer of 1948 in East Lansing, 
Michigan, are given in Table 2. The explanation for the unusual results 
in early yield is found in the low night temperatures prevaiHng during 
the month of June (Fig. 3), which delayed fruit set by i to 3 weeks on 
the nontreated plants. 

The averaged effect of a hormone spray consisting of p-chlorophcnoxy- 



































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acetic acid (CIPA), at 30 parts per million (ppni.) applied to the flower 
clusters of 14 varieties compared with respective controls in altering the 
pattern of production of field tomatoes is illustrated in Figure 2. Such 
alterations in harvest intensities should help to avoid seasonal overloading 
of markets. Peaks of production are leveled and the harvest time ex- 
tended. The harvest pattern as illustrated for treated plants is more 
desirable not only for the grower of fresh market tomatoes but for 
processing as well. 


Figure 2. Comparative seasonal patterns of fruit production in hormone- 
treated and control tomato plants. The harvest period is extended and peaks 
of production are avoided when growth substances are used to overcome 
delayed fruit set induced by cold night temperatures. 

An interesting and practical aspect of the effect of night temperatures 
on tomato pioduction is the key it provides in enabling one to predict 
when during the harvest season the price will break on the fresh market. 
From the data that have been compiled, it appears that the time of the 
first flush of tomatoes on the fresh market is predetermined by an ex- 
tended period of night temperatures favorable for fruit setting in a 
given area. These intervals of optimal temperature precede fruit ripening 
by 45 to 50 days, the usual time necessary in Michigan for fruit to ripen 
after it is set. In Figure 3 are given the average night temperatures 
prevaihng during the month of June for the years ot 1947, 1948, and 



1949. In the three years temperatures became optimal for fruit set on 
the twenty-seventh, the twenty-first, and the tenth of June, respectively. 
Price data compiled from the combined reports of the Detroit and 
Benton Harbor Farmers' Markets revealed an abrupt drop of over 50 
per cent in the fresh market price of tomatoes on August 13 in 1947, 
August 9 in 1948, and July 26 in 1949. 







— — , 




~y ^ 




00" /^» 

f % 0)' . t" 

1 • J / ^ ' 

i\ I M 




1 1 

1 1 t 1 






Figure 3. Averaged day to day night temperatures during the month of 
June in East Lansing, Michigan. Fruit setting on field tomatoes was delayed 
until June 10, June 21, and June 27, for the years 1949, 1948, and 1947, 
respectively. (Optimum range for fruit setting 59-68°F.). 

Hormone spravs for greenhouse tomatoes have been used as supple- 
mentary (33) to other grower practices for improving fruit set, size, 
and yield. In the greenhouse temperatures are controlled so that some 
pollination and fertilization occurs even in very dark weather if flowers 
are sufficiently "vibrated." Resulting tomatoes usually contain seeds. 
The importance of time of application on the greenhouse crop has 
recently been emphasized (17,21), and present evidence indicates that 
pre-anthesis treatments with growth substances greatly inhibit flower 
bud development and are notorious for causing complete seedlessness 


and the well-known defects of puffiness, green pulp in the seed locules, 
and premature softening. The use of whole plant sprays as compared 
with flowei cluster sprays has at times reduced the set of fruit probably 
because of the inhibiting effects of growth substances upon the young 
flower buds (31,34). The application of growth substances to the soil (37) 
has been eff'ective in setting tomato fruit, but such a technique suffers 
from the same disadvantages as whole plant sprays in adversely affecting 
the development of young flower buds. 

On outdoor tomatoes when hormone chemicals are applied to the 
flower clusters early in the season, while night temperatures are still 
below 55°F., the normal reproductive processes of pollination and fer- 
tilization are totally nonfunctional. The growth substances provide a 
complete replacement rather than a supplement. Results as measured 
by early yield and size increases have been more phenomenal than any 
reported for greenhouse tomatoes. In the field excellent fruit set and 
quality is obtained, and the fruit is completely seedless. A change of 
weather to warmer nights, in turn, will result in normally seeded fruit, 
and httle or no response to hormone treatment except in fruit size. 

The use of whole plant sprays or dusts is desirable if growth substances 
are to be utilized for improving fruit set and yields on large commercial 
outdoor plantings. Success in their use on greenhouse crops has been 
reported if one avoids spraying the growing tips (33), an impractical 
precaution with field power equipment. Considerable work is in progress 
on the use of whole plant apphcations for canning tomatoes (57). 
Increases in early and total production have been obtained, but these 
are not always equal to those reaHzed when the chemical is confined to 
the flower clusters. As a flower cluster spray, the most effective chemical 
has consistently been CIPA at a dilution of 30 ppm. Considerable evi- 
dence supporting this has been accumulated not only in Michigan 
but elsewhere (27,29). Alpha-ortho-chlorophenoxypropionic acid (ClPP) 
at 75 ppm. has also been relatively effective as a flower cluster spray and 
gives no leaf distortions or formative effects. When ClPP is used at 
20 to 40 ppm. as a whole plant spray good results have been obtained, 
but an immediate response in fruit setting comparable to flower cluster 
spraying is not realized. 

The incidence of blossom-end rot associated with hormone treatment 
of tomatoes has attracted the attention of physiologists since water 
relations of the plant are involved, which, in turn, may be influenced 



by the auxin level (2). In Table 2 the number of fruit showing blossom- 
end rot is listed for both treated and control groups. It is somewhat 
surprising that with all varieties the incidence of the disorder was less 
as a result of treatment. This can hardly be ascribed to any direct effect 
of the applied growth substance but likely exists because nontreated 
plants grew more vegetatively (less correlative inhibition (30) from early 
fruit development), and in subsequent periods of drought their water 
requirement was less. 

Fruit Set in Beans. — Snap beans are frequently grown in environments 
not favorable for fruit set. Factors which have been reported as associated 
with blossom drop are hot dry weather, rapid fluctuations in temperature 
and moisture (4,8), and, more recently, insects (14). The most striking 
results obtained through the use of growth substances for improving 
pod set have been obtained in weather with daily maximum temperatures 
above 90°?. and no rainfall (14,32,36,43,55). Data obtained from spray 
treatments of CIPA to Stringless Greenpod beans grown under such 
conditions of high temperatures and no rainfall are presented in Table 
3. Results with growth substances on snap beans have not always been 
so extreme. In repeated tests under more normal temperatures (Table 3) 

Effects of hormone sprays on yields, pod size, and seed content of snap beans 



Midsummer crop 

(Mean maximum 

temperatures during 

flowering 98 ± 3° f.) 

Fall crop 

(Mean maximum 

temperatures during 

flowering 80 ± 7° f.) 

Yield of pods Averages for Total Averages for 


per 500 plants harvested in grams harvested 

Weight Seed per 500 Weight Seed 
Early Total (Grams) Number plants (Grams) Number 

I ppm. 









2 ppm. 









5 ppm. 









Tap water 








*p-chlorophenoxyacetic acid. 


and humidity in both Missouri and Michigan, hormone sprays and dusts 
of CIPA and CIPP have given 10-25 per cent yield increases. Most of 
the increase in yield has been accounted for by larger fruit size rather 
than an increased set. This observation is in agreement with the recent 
work of Randhawa and Thompson (36). Fisher et al. (14), however, 
reported an increase due to the production of more pods rather than 
larger ones. Unfortunately, in snap beans precise information is lacking 
as to the exact temperature and humidity requirement for optimal fruit 
set. From a practical standpoint growth substances could be profitably 
used on snap beans as a type of insurance against low yields induced by 
unpredictable adverse weather. Consistent increases in yield of a sig- 
nificant magnitude and a hastening of maturity even under weather 
conditions favorable to pod development should further the use of 
growth substances in the production of snap beans. 

Some of the more disappointing studies to date on the use of growth 
substances for improving fruit set have been with lima (7,50) and dry 
shell (11) beans. The hormonal stimulation of ovarian tissue adjacent 
to the seeds in tomatoes and snap beans resulting in increased fruit 
production has generally been observed to depress rather than to stimu- 
late seed formation. When growth substances are used in a manner similar 
to those employed for improving fruit set in tomatoes and snap beans, 
they are likely to be ineffective on crops wherein yields are measured 
in terms of seed production, such as peas, lima beans, dry shell beans, 
and so on. 

General Considerations. — A series of recent reports on the fig, with 
possible fascinating implications for other fruits, have been published 
(6,9,10,44). The possibility through growth substances of not only 
controlling fruit set but also its time of maturity as well in fruits other 
than the fig and, as has been reported previously, in the pineapple (48), 
should attract interest and stimulate research in this possible role of 
the plant growth hormones. 

Some interest has been focused on possible alterations of nutritional 
values in fruits (tomatoes and snap beans) when induced to set by the 
use of growth substances. Variations in nutritional values have not been 
great nor of sufficient magnitude either to encourage or discourage the 
use of growth substances as fruit-setting sprays on the basis of their 
improvement or impairment of nutritional or market quality of the 
resulting produce (19,22,35,36,38,55,57). Supplementary to these in- 

S. H. WITTWER 375 

vestigations, Mitchell et al. (28) have reported a decided improvement 
in the retention of vitamin C and moisture in snap beans during storage 
when CIPA at 400 ppm. was used not as a spray designed for improving 
fruit set, but as a spray treatment applied four days prior to harvest. 
Additional studies should be made on the effects of growth substances 
on postharvest quality and shelf-life of such perishables as beans, peas, 
sweet corn, and asparagus. 

The use of growth substances for improving fruit set and seed pro- 
duction as an aid in plant breeding has attracted an increasing interest 
on a great variety of crops. The work of Whitaker and Pryor (52) with 
melons, Schomer and Hamner (40) with berries, Emsweller and Stewart 
(12) with lilies, and Wester and Marth (51) with Hma beans indicates 
that growth substances may increase both the number of successful 
crosses and the number of seeds per cross, and that they may also help 
overcome certain incompatibilities and assist in special types of vegetative 
propagations. Why seed production with some crops is stimulated while 
in other crops, and in some instances the same crop (51,55), with similar 
treatments it is retarded is hard to reconcile. Time and method of 
application are undoubtedly factors, as well as the prevention of abscission 
of the young fruit. Perhaps stimulatory effects of the growth substances 
on pollen germination and tube growth, as suggested by Eyster (13), 
and the data of Addicot (i), offer a partial explanation. Using identical 
treatments of growth substances, our results with hormone spraying 
of snap beans show marked decreases in seed content on some plantings 
and in others significant increases. 

One of the most puzzling series of reports is the failure of the known 
growth substances in effecting fruit set on such tree fruits as the apple, 
pear, peach, plum, and cherry. Insofar as the author is aware, only one 
(46) of many who have investigated the possibilities in this field has 
reported positive results. Lewis (25) has summarized papers published 
on the stimulation of fruit development with chemicals by the statement 
that many-seeded fruits, such as tomatoes, cucurbits, and Oenothera 
are stimulated, whereas in few-seeded fruits, such as cherries, plums, 
pears, and apples, fruits are not formed. A possible exception to the rule 
is the snap bean pod, a few-seeded fruit, which is definitely stimulated. 

Plant reproductive organs, especially the pollen and young fruit, 
offer interesting possibilities as source material for the isolation of new 
plant growth substances. Growth hormones occur in these tissues in 


concentrations higher than those in any other plant tissues. Crude 
preparations of natural growth hormones specific for fruit setting have 
been prepared from the pollen and young fruit of several economic 
plants (15,26,45,53). Continuing efforts in the isolation of these natural 
growth substances should provide the key for the further elucidation of 
the many still baffling problems associated with the control of fruit 



1. Addicot, F. T., Plant Physiol., 18:270 (1943). 

2. AuDUS, L. J., Biol. Rev. Cambridge Phil. Sac., 24:51 (1949). 

3. Avery, G. S., Berger, J., and Shalucha, B., Am. J. Botany, 29:765 

(^942). . o , ^ 

4. BiNKLEY, A. M., Proc. Am. Sac. Hart. Sci., 29:489 (1932). 

5. BuRK, E. F., ibid., 26:239 (1929). 

6. Blondeau, R. and Crane, J. C, Science, 108:719 (1948). 

7. Clore, W. J., Proc. Am. Sac. Hort. Sci., 51:475 (1948)- 

8. Coroner, H. B., ibid., 30:571 (1933). 

9. Crane, J. C. and Blondeau, R., Plant Physiol., 24:44 (1949). 

10. , Proc. Am. Soc. Hort. Sci., 54:102 (1949). 

11. Dexter, S. T,, Michigan Agricultural Experiment Station Quarterly Bulletin, 

25 (1943). 

12. Emsweller, S. L. and Stuart, N. W., Proc. Am. Soc. Hort. Set., 51:581 


13. Eyster, W. H., Science, 94:144 (i94i)- 

14. Fisher, E. H., Riker, A. J., and Allen, T. C, Phytopathology, 36:504 


15. GusTAFSON, F. C, Am. J. Botany, 24:102 (1937). 

16. Hatcher, E. S. J., Ann. Botany, 9:235 (1945). 

17. Hemphill, D. D., Mo. Agr. Exp. Sta. Res. Bull., 434 (1949). 

18. Hoffman, O. L. and Smith, A. E., Science, 109:588 (1949). 

19. Holmes, A. D., Spellman, A. F., Kuzmeski, J. W., and Lachman, W. H., 

/. Am. Dietet. Assoc, 23:218 (1947), and Food Technology, 2:252 (1948). 

20. Howlett, F. S., /. Agr. Research, 58:79 (1939). 

21. , Proc. Am. Soc. Hort. Sci., 53:323 (1949). 

22. Janes, B. E., Afn. J. Botany, 28:639 (1944). 

23. JuDKiNS, W. P., ibid., 32:249 (1945). 

24. Laibach, F. and Meyer, F., Senc^enbergiana, 17:73 (1935)- 

25. Lewis, D., /. Pom. Hort. Sci., 22:175 (1946). 

26. Luckwill, L. C, ibid., 24:32 (1948). 

27. MiNGES, P. A. and Mann, L. K., Hilgardia, 19:10 (University of Califor- 

nia, Davis, 1949). 

28. Mitchell, J. W., Ezell, B. D., and Wilcox, M. S., Science, 109:202 

(1949)- ^ . _, 

29. MuLLisoN, W. R. and Mullison, E., Botan. Gaz., 109:501 (1948). 

30. Murneek, a. E., Plant Physiol, 1:3 (1926). 

31. , Proc. Am. Soc. Hort. Sci., 50: 254 (1947). 

S. H. WITTWER 377 

32. , WiTTWER, S. H., and Hemphill, D. D., ibid., 44:428 (1944). 

33. , ibid., 45:371 (1944). 

34. Paddock, E. F., ibid., 52:365 (1948). 

35. Palmer, C. O., Master's Thesis, Michigan State College, East Lansing 


36. Randhawa, G. S. and Thompson, H. C, Proc. Am. Soc. Hort. Sci., 52:448 


37. , Science, 108:718 (1948). 

38. , Proc. Am. Soc. Hort. Sci., 53:337 (1949). 

39. Redemann, C. T., Wittwer, S. H., and Sell, H. M,, Plant Physiol, 

25:356 (1950). 

40. Schomer, H. a. and Hamner, C. L., U. S. Patent 2,4^^,0^6 (1948). 

41. Skoog, p., Ann. Rev. Biochem., 16: 529 (1947). 

42. Smith, O. and Cochran, H. L., Cornell University Agricultural Experiment 

Station Memoir, 175 (1935). 

43. Stark, F. C, Ann. Report Vegetable Grotvers' Assoc, of Am., lyi, (1948). 

44. Stewart, W. F. and Condit, I. J., Am. J. Botany, 36:332 (1949). 

45. SwARBRicK, T., Nature, 156:300 (1945). 

46. , ibid., 156:591 (1945). 

47. van Overbeek, J., Ann. Rev. Biochem., 13:631 (1944). 

48. VAN Overbeek, J., Botan. Gaz., 108:64 (1946)- 

49. Went, F. W. and Cosper, L., Am. J. Botany, 32:643 (1945). 

50. Wester, R. E. and Marth, P. C, Proc. Am. Soc. Hort. Sci., 49:315 (1947). 

51- , i^id; 53:315 (1949)- 

52. Whitaker, T. W. and Pryor, D. E., ibid., 48:417 (1946). 

53. Wittwer, S. H., Missouri Research Bulletin, 371 (1943). 

54. , Proc. Am. Soc. Hort. Sci., 53:349 (1949). 

55. , and Murneek, A. E., ibid., 47:285 (1946). 

56. , Stallworth, H., and Howell, M. J., ibid., 51:371 (1948). 

57. , and Schmidt, W. A,, Unpublished Data (1949). 

58. Zimmerman, P. W. and Hitchcock, A. E., Proc. Am. Soc. Hort. Set., 

45:353 (1944)- 

59. , Ann. Rev. Biochem., 17:601 (1948). 

Growth Substances 
in Pathological Growth 

Experimental Induction and Inhibition 
of Overgrowths in Plants 


OVERGROWTHS Can be induced on plants by a variety of agents, 
including chemical substances, genetic factors, viruses, bacteria, 
fungi, nematodes, and insects. In this paper four different kinds of over- 
growths induced by a chemical substance, a genetic factor, a virus, and 
a bacterium will be described. An account will also be given of the 
experimental inhibition of the growth of one of these tumors. 

In 1936 and 1937 a series of papers (27,4,21) was published describing 
the effect on certain plant organs of indole-3-acetic acid (lAA). This 
substance was applied to plant organs in lanolin, the concentration of 
lAA varying from 1.5 to 3 per cent. The stems of both bean and tomato 
plants and the pods of the bean responded to this treatment with the 
production of overgrowths some of which had a diameter of as much as 
2 cm. The overgrowths were made up in part of parenchymatous tissue 
and in part of the primordia of adventitious roots. Any parenchymatous 
tissue of the bean stem, according to Hamner and Kraus, could be 
rendered meristematic by this treatment. 

These studies on the action of lAA on intact plants were supplemented 
by the investigations of Gautheret (18) on the response to this substance 
of plant tissue cultured in vitro. Gautheret showed that the response 
of a plant tissue to lAA depended largely on the concentration of this 
substance in the medium. For carrot tissue he defined its action as 
cambiogenic at a concentration of o.i mg. per liter and as rhizogenic 
at a concentration of i mg. per hter. At a concentration of between 10 
and 100 mg. per Hter he found that the substance ceases to be rhizogenic 
but induces instead a disorganized type of growth composed of hyper- 
trophied cells. 


Similar disorganized growth was observed by de Ropp (13) in frag- 
ments of sunflower stem tissue cultured on agar containing i mg. per 
liter of lAA. After a week on this medium the fragments lost their 
original structure and developed into shapeless, semitranslucent tissue 
masses. These tissue masses resembled in outward appearance the bac- 
teria-free crown -gall tumor tissue previously isolated from sunflower by 
White and Braun (45). Subsequent culture of these tissues on a medium 
devoid of lAA showed that the change in growth pattern was not 
permanent. After about six months on this medium the shapeless tissue 
masses differentiated into roots. White and Braun (45) published similar 
findings in relation to the action of indoleoxaloacetic acid on plant tissues. 

In the experiments quoted above the application of lAA to plant 
tissues did not result in a permanent change in their pattern of growth. 
Gautheret (18), however, has been able to isolate from carrot tissue, 
cultured for several years on a medium containing o.i mg. per liter of 
lAA, a strain of tissue capable of growing on a medium free of lAA 
without any reduction in its rate of growth. This strain of tissue was not 
only altered as regards its reaction to lAA, it was also changed in external 
appearance, having become friable and translucent instead of compact 
and opaque. Gautheret called the change which had taken place in this 
tissue "accoutomance" which can be translated as habituation. The 
tissue which had undergone this change he referred to as habituated 

The phenomenon of habituation was also studied by Morel (30). 
Tissue cultures of Virginia creeper which had been grown on a medium 
containing 0.3 mg. per liter of napthaleneacetic acid (NAA) were trans- 
ferred to a nutrient devoid of this substance. They ceased growing, but 
after a period of 14 months in one of the six cultures and on a limited 
portion of the tissue a callus developed which grew in the absence of 
NAA, although the rest of the culture remained unchanged. This callus 
on transfer continued to grow on a medium devoid of NAA. 

It is diflicult to explain this observation of Morel's by assuming 
that the NAA itself had a mutagenic action on the tissues; nor does it 
seem likely that anything in the nature of adaptation had occurred. The 
phenomenon can be more adequately accounted for by assuming that a 
somatic mutation occurred spontaneously in a small part of one of the 
tissue fragments used. This would explain why habituation occurs 
sporadically rather than regularly when tissues are grown in the presence 



of lAA or NAA. To place these compounds in the category of carcino- 
genic or mutagenic agents along with such substances as the nitrogen 
mustards or methylcholanthrene, seems hardly justifiable at present. 

The biological status of habituated tissue has been studied by Camus 
and Gautheret (10). Habituated tissue of scorzonera was grafted to 
unaltered root tissue of this plant, on which it gave rise to a voluminous 
neoplasm similar to that induced by crown-gall tumor tissue. These 
investigators favor the view that habituated tissue occupies an inter- 
mediate position between normal and crown-gall tumor tissue. 

We will next consider overgrowths of genetic origin. Such over- 
growths were experimentally induced by Kostoff (26) by hybridizing 
certain species o( Nicofiana, notably A^ langsdorfii W\l\\ N.glatica. On the 
stems, roots, and leaves of these hybrids tumors developed either spon- 
taneously or as a result of wounding. The overgrowths varied in appear- 
ance from fasciations such as are seen on plants affected with witches'- 
broom to outgrowths lacking any outwardly visible structure. Whitaker 
(42) attributed the formation of these overgrowths to a cytoplasmic 
disturbance occasioned by the introduction of the chromosome compli- 
ment of A^. langsdorfii (used as pollen parent) into the cytoplasma of 
N. glauca (used as seed parent). 

Tissue cultures of these overgrowths were made by White (44) who 
proved that the callus was capable of making indefinite growth on a 
medium containing 2 per cent sucrose, mineral salts, and yeast extract. 
Like habituated tissue and crown-gall tumor tissue this callus is able to 
grow without added auxin. Though it grew at first in the form of white, 
undifferentiated tissue masses, it was later shown by White (43) and 
Skoog (37) to be capable of differentiating roots, stems, and leaves under 
certain environmental conditions. White (46) also showed, by grafting 
fragments from cultures of this hybrid callus onto the stem of healthy 
A^. glauca plants, that the hybrid tissue fragments grew under these 
conditions as tumors, and apparently possessed the property of propagat- 
ing their tumorous nature indefinitely. 

The experimental induction of overgrowths by a virus was accom- 
plished by Black (i) in 1945 using the virus Aiireogemts magnivetja, 
the causal agent of wound tumor disease. This virus produces a systemic 
infection in a considerable number of unrelated plants into which it can 
be introduced either by agallian leaf hoppers or by grafting. Insects 
infected with the virus were found to become infectious only after an 


incubation period of several days (3). Though the virus generally in- 
vaded the whole plant the tumors were local in character. Histological 
studies of virus tumors on sweet clover revealed that they were initiated 
by tangential divisions in cells of the pericycle, opposite the primary 
phloem (25). Abnormal cell multiplication rather than cell enlargement 
was found to be responsible for tumor development. The tumors were 
composed of a central core of xylem elements consisting of short reticulate 
tracheids of various widths surrounded by a meristematic zone outside of 
which was a layer of phloem. Tumor cells were apparently unable to 
differentiate into the most specialized cell type found in the xylem, 
the vessel, or into its counterpart in the phloem, the sieve tube. Non- 
granular, nonvacuolated, smooth, spherical bodies staining intensely 
with safranine were found in the cytoplasm of some of the tumor cells. 
Tumor tissue isolated from sorrel roots was successfully grown on a 
synthetic medium without auxin (2) and found to double its volume 
about every three weeks. The tissues in culture did not become organized 
into stems, roots or leaves. When such tissues were grafted to healthy 
sorrel plants the stock plants developed the systemic tumor disease, 
showing that the virus was still present in the tissue cultures. The 
heredity of the host was found to play an important part in tumor reac- 
tion. Clones of sweet-clover plants were selected which, when infected 
with the virus, developed minute, scarcely visible tumors; other clones 
were selected on which a dense mass of tumors developed. In most 
instances it could be shown that the overgrowth had developed as the 
result of a wound. 

The most intensely studied of all plant overgrowths is that produced 
by the crown-gall organism, Agrobacteriwn tumefaciens. Erwin F. Smith 
who, together with Townsend, discovered the bacterial etiology of 
crown gall (38) emphasized the resemblance of this plant disease to 
animal cancer and went so far as to argue, by analogy, that some 
microorganism must also be associated with malignant tumors in animals. 
Careful study of animal cancer tissue failed to reveal any microorganisms 
associated causally with the cancerous condition, for which reason Smith's 
contention that crown gall is cancer (40,41) was rejected by most workers 
in the field of cancer research. 

The experimental production of overgrowths on plants by means 
of the crown-gall organism is influenced first by the strain of the 
bacteria, second by the nature of the plant, and third by the manner in 



which plant and microorganism are brought together. That strains of 
crown-gall bacteria differ in tumefacient power has been known since the 
time of Smith (39). Some strains exist which have lost their tumefacient 
power completely, others generate small galls on susceptible plants, and 
still others cause the formation of large rapidly growing galls. The work of 
Riker and others (33,28) has shown that the tumefacient power of the 
crown-gall organism can be eliminated by culturing it on a medium 
containing certain amino acids, notably glycine. Bacteria grown on a 
medium containing this substance lost their tumefacient power in from 
10 to 20 transfers. The effect was reversible. 

Crown-gall bacteria, however virulent, will produce no overgrowths 
unless brought into direct contact with wounded tissue. Riker (31) 
showed that the size of the gall produced on tomato stems was propor- 
tional to the area of waterlogged tissue resulting from the wound. He 
found that tissue ceased to respond with tumor production five days 
after it had been wounded. De Ropp (15) found that fragments of 
sunflower stem cultured iti vitro also lost their capacity to respond 
in about this time. E. M. Hildebrandt (22), using micrurgical methods, 
showed that single bacteria would induce gall formation on stems of 
tomato plants. The size of the gall was related to the depth of the wound 
rather than to the number of bacteria introduced. When injected into 
the living surface cells of the tomato stem no galls were initiated by the 
bacteria, which seemed unable to survive in the intracellular environ- 
ment. The work of Riker (31) and of Robinson and Walkden (36) sug- 
gests that the organisms are inter- rather than intracellular parisites. 

Even when tissues are wounded they may still be unsusceptible to the 
tumefacient effect of crown-gall organism. In a series of experiments 
using slices of carrot roots inoculated with crown-gall bacteria, the 
writer (r6) has shown that tumor formation occurs primarily along 
the line of the cambium. The secondary xylem which forms the core of 
the root is capable only of very bmited tumor production. The secondary 
phloem outside the xylem responds rather more readily. The periderm 
does not respond at all. It was also shown that, in any given batch of 
carrot roots, grown under the same conditions and belonging to the same 
variety, as many as 10 per cent are generally immune to the tumefacient 
action of the crown-gall organism and a higher proportion are only 
slightly susceptible. These variations in susceptibility may be due to 
hereditary factors. It would probably be possible by selection to develop 


highly tumorous and nontumorous strains of carrots, as has been done 
with mice. 

The temperature at which the tissues are held after inoculation with 
crown-gall bacteria has an effect on tumor formation. Riker (32) found 
that galls developed poorly on tomato plants kept at temperatures 
between 28° and 30°C. and that none developed above 32°C., a finding 
which was later confirmed by Braun (7). The tumefacient process 
appears to take place within 36 to 48 hours from the time of introduction 
of the bacteria (6). 

That plant hormones have an effect on the tumefacient process was 
demonstrated by Riker (34) whose observations were further extended 
by Braun and Laskaris (8). Tomato stems inoculated with an attenuated 
strain of crown-gall organisms were found to generate galls if treated 
at the same time with lAA in lanolin. This finding caused Braun and 
Laskaris to suggest that tumefaction by crown-gall bacteria takes place 
in two stages, the host cells being converted to tumor cells in the first 
stage and stimulated to continued multiplication by a growth substance 
in the second. 

Some of the most important studies on the physiology of crown-gall 
tumor tissue were initiated in 191 8 by C. O. Jensen (24), who showed 
that tumors from red beet could be transplanted to sugar beet or mangel 
and that, under these conditions, they continued to grow as tumors. This 
suggested a close analogy between the behavior of animal cancer tissue 
and crown-gall tissue and gave support to E. F. Smith's contention 
that crown gall is a plant cancer. The most significant part of Jensen's 
work was overlooked at the time. It consisted in the observation that 
crown-gall bacteria could not be isolated from tumors thus transplanted, 
from which he concluded that the cells of the beet tissue, under the 
influence of the bacteria, had developed abnormally increased pro- 
liferated power which persisted independently of continued stimulation. 
Smith had frequently observed that crown-gall bacteria were difficult 
or impossible to isolate from galls on composites, but it was not until 
1943 that Braun and White (5) proved that up to 96 per cent of the 
secondary galls which form on infected sunflowers are devoid of crown- 
gall bacteria. 

This discovery led to the isolation of bacteria-free crown-gall tumor 
tissue from secondary galls on sunflower (45). This crown-gall tissue 
grew indefinitely on a medium containing 2 per cent sucrose, mineral 



salts, and thiamin. On this medium normal stem tissue of sunflower grew 
slowly, was green in color, woody in texture, and tended to differentiate 
roots. The tumor tissue, on the other hand, grew rapidly as a white, 
rather friable mass and did not differentiate organs. Bacteria-free crown- 
gall tumor tissue was subsequently isolated from primary galls on sun- 
flower (36), from galls on Periwinkle that had been freed of bacteria 
by heat treatment (47), also from galls on marigold and Paris-daisy 
(23), scorzonera, Jerusalem artichoke (20,19), ^^^^^ vinifera, Opiintia, 
Carthammis, Abutilon, and Nicotiana (29). It seems, in fact, that these 
galls tend, as they grow older, to become free of the causal organism. 

The isolation of bacteria-free crown-gall tumor tissue made possible 
a closer study of the physiology of this tissue. The respiratory behavior 
of crown-gall tumor tissue was compared with that of healthy tissue by 
White (48), who found no significant change in the quahtative respira- 
tory picture of tumor tissue but detected a lowering of the respiratory 
level. The insensitivity of sunflower crown-gaU tissue to indoleacetic 
acid, napthaleneacetic acid, and indolebutyric acid was contrasted by 
de Ropp (13) with the extreme sensitivity of healthy sunflower stem- 
tissue to these substances. Gautheret (19) working with artichoke crown- 
gall tissue made similar observations. Riker, Hildebrandt, and coworkers 
(23,35) have carried out extensive studies on the nutrient requirements 
of bacteria-free crown-gall tissue showing that crown-gall tumor tissue 
of various plants can use dextrose, levulose, and sucrose as sources of 
carbon for growth but have little or no ability to use organic acids or 
alcohols. Nitrate and urea proved to be the best sources of nitrogen of 
the various compounds tested. Several of the amino acids tested proved 
to have an inhibiting action on growth. 

The influence of crown-gall tumor tissue on healthy tissue of the 
same plant was studied by de Ropp (12) using the technique oiin vitro 
grafting. Induced tumors having a characteristic anatomical structure 
developed on many of the stem fragments to which tumor tissue had 
been successfully grafted. These induced tumors, on isolation, proved 
to have the physiological properties of crown-gall tumor tissue although 
they had apparently arisen from the cambium of the stem fragment. 
It was concluded that a tumefacient factor exists in crown-gall tumor 
tissue capable of being transferred to normal tissue across a graft union. 
Attempts to transmit the tumefacient principle by other means were 
not successful (14). Induced tumors were subsequently obtained on 



healthy artichoke tissue by Camus and Gautheret (9) using the same 
technique. The possibility that crown gall is actually a virus disease 
transmitted by a bacterium was discussed by White and Braun (45), 
and the results of these grafting experiments lend some support to the 
hypothesis though they do not prove it. 

It remains necessary to say a few words about the experimental 
inhibition of the growth of vegetable tumors. One of the most important 
lines of cancer research consists of a quest for a chemotherapeutic agent 
which will differentially inhibit the growth of cancer cells. This pre- 
supposes that there is some fundamental difference in metabolism be- 
tween the cancer cell and the normal. In crown-gall tumor tissue we 
definitely know that such a difference exists. Crown-gall tumor tissue 
grows on a medium without added auxin probably because it has acquired 
the capacity to manufacture its own. It either has more efficient en- 
zymatic equipment than normal tissue possesses, or it has less exacting 
requirements and can thus make do with a simpler set of primary 

In the course of the past year the writer has studied the effect of many 
substances on the growth of crown-gall tumors on carrot. These sub- 
stances included so-called anti-auxins such as 2,4-dichloranisole, anti- 
biotic substances, sulfonamides, purine and pyrimidine derivatives, and 
analogues of some of the B vitamins. To test these substances standard 
fragments of carrot cambial tissue were first inoculated with crown-gall 
bacteria and four days later, when the tumors were just becoming visible, 
were treated with 0.05 cc. of a solution containing i part per 1,000 
of the substance to be tested. The fragments were incubated for two 
weeks then examined for tumors. 

The anti-auxins, antibiotic substances, and sulfanamides used were 
found unable to inhibit tumor growth once the tumor had been initiated. 
Streptomycin was capable of inhibiting tumor formation but this seemed 
due to its action on the bacteria rather than on the tumor tissue. Com- 
plete inhibition of tumor growth was obtained with certain analogues 
of folic acid. These compounds had already been tested on animal cancers 
and found to have an inhibiting effect on the growth of tumor tissue, 
though their high toxicity limited their therapeutic value. The sub- 
stances found most active in inhibiting the growth of crown-gall tumor 
tissue on carrot are issued by Lederle Laboratories under the names 
Aminopterin, A-methopterin, A-denopterin, A-ninopterin and A-terop- 



terin. A total dosage of as little as 0.05 ng. of these substances was suffi- 
cient materially to reduce tumor growth (17). At low dosage levels the in- 
hibition could be partially reversed by a one per cent solution of folic 
acid. It is not yet possible to tell whether this inhibiting action is dif- 
ferential, that is, whether it affects the tumor cells without interfering 
with the growth of healthy tissue. 


1. Black, L. M., Am. J. Botany, 32:408 (1945). 

2. , Nature, 158:56 (1946). 

3. , Sixth Growth Symposium, 79 (1947). 

4. BoRTHWicK, H. A., Hamner, K. C, and Parker, M. W., Botan. Gaz., 

98:491 (1937). 

5. Braun, a. C. and White, P. R., Phytopath., 33:85 (1943). 

6. Braun, A. C, Am. J. Botany, 30:674 (1943). 

7. , ibid., 34:234 (1947). 

8. Braun, A. C. and Laskaris, T., Proc. Nat. Acad. Sa., 28:468 (1942). 

9. Camus, G. and Gautheret, R. J., C. R. soc. bioL, 142:15 (1948). 

10. , C. R. acad. sci., 226:744 (i94^)* 

11. DE Ropp, R. S., Phytopath., 37:201 (1947). 

12. , Am. J. Botany, 34:248 (1947). 

13. , ibid., 34:53 (1947). 

14. , Bull. Tor. Bot. Club, 75:45 (1948). 

15. , Cancer Res., 8:519 (1948). 

16. , Am. J. Botany, 37:352 (1950). 

17. , Nature, 164:954 (1949). 

18. Gautheret, R. J., Bull. Soc. Chim. Biol, 24:13 (1942). 

19. , C. R. acad. sci., 224:1728 (1947). 

20. , ibid., 226:270 (1948), 

21. Hamner, K. C. and Kraus, E. J., Botan. Gaz., 98:735 (1937). 

22. Hildebrandt, E. M.,/. Agr. Res., 65:45 (1942). 

23. , and RiKER, A. J., Am. J. Botany, 36:74 (1949). 

24. Jensen, C. O., Den K. Veterinaer- og Landboh^Js{ole. Aarss\rift., 1918:91 


25. Kelly, S. M. and Black, L. M., Am. J. Botany, 36:65 (1949). 

26. KosTOFF, D., Zentralbl.f. Bakt. 11. , 81:244 (1930). 

27. Kraus, E. J., Brown, N. A., and Hamner, K. C, Botan. Gaz., 98:370 


28. LoNGLEY, B. J., Berge, T. O., VAN Lanen, J. M., and Baldwin, I. L., 

/. Bact., 33:29 (1937). 

29. Morel, G., Private communication. 

30. , C. R. soc. biol, 140:269 (1946). 

31. Riker, a. J.,/. Agr. Res., 25:119 (1923). 

32. , ibid., 32:83 (1926). 

33. , Chron. Bot., 6:392 (1941). 

34. , Growth, 4th Symposium, sup. to vol. 6:105 (^942)« 


35. RiKER, A. J. and Gutsche, A. E., Am. J. BoL, 35:227 (1948). 

36. Robinson, W. and Walkden, H., Am. Bot., 37:299 (1923). 

37. Skoog, F., Am. J. Botany, 31:19 (1944)- 

38. Smith, E. F. and Townsend, C. O., Science, 25:671 (1907). 

39. Smith, E. F., Brown, Nellie A., and McCulloch, L., U. S. Dept. Agr. 

Bull., 255:1 (1912). 

40. Smith, E. F., Science, 43:871 (19 16). 

41. , /. Cancer Res., 1:231 (1916). 

42. Whitaker, T. W., /. Arnold Arboretum, 15:144 (i934). 

43. White, P. R., Bull. Tor. Bot. Club, 66:507 (1939)- 

44. , Atn. J. Botany, 26:807 (1939). 

45. White, P. R. and Braun, A. C, Cancer Res., 2:597 (1942). 

46. White, P. R., ibid., ^•.'J()i (i944)- 

47. , Am. J. Botany, 32:237 (1945). 

48. , Cancer Res., 5:302 (1945). 

/;/ Vitro Experiments on Tissues of Pathological Origin 


THE role of growth-regulating substances in pathological growth has 
received wide attention. Much of this work has centered around the 
bacterial plant-gall disease incited by Agrobacterium tumefaciens (Smith 
and Townsend) Conn. The early Uterature on crown gall has been re- 
viewed by Riker and Berge (20) and by Riker, Spoerl, and Gutsche (21). 
Such gall formation may result from a balance of factors among which 
growth-regulating substances may be important (19, 10). 

Plant-tissue culture provides certain advantages over whole plants 
to study the fundamental aspects of normal and abnormal growth. 
For example, strains of callus cultures may be grown vegetatively in 
vitro for unlimited periods. The media for such cultures are easily 
reproducible since they initially contain only nutrients whose chemical 
formulae are known. This provides a fairly simple medium for closely 
controlled studies. Such cultures may be maintained in a relatively 
undifferentiated condition. In certain cases changes in the morphological 
character of the cultures were induced under controlled conditions. 
The cultures may be subjected over long or short periods to wide ranges 
of concentrations of supplements, including cell stimulating or inhibiting 
materials under otherwise constant conditions. Such cultures once es- 
tablished are no longer directly influenced by the association of other 
parts of the plant. These and other conditions seem to offer important 
opportunities for critical studies under closely controlled environments. 
Whole plants may respond in a variety of ways to the presence of 
growth-regulating substances. Such responses may include formation 
of adventitious roots, epinasty, stimulation of cambial activity, inhibi- 
tion of bud development, or delayed abscission of old leaves. A number 
of natural and synthetic growth-regulating compounds may induce 


various ones of these effects and may either stimulate or inhibit normal 
or abnormal cell proliferation (see reviews in 10,28,26,23). A wide 
variety of natural and synthetic growth-regulating compounds have 
been tested on many species of whole or decapitated plants (27,34,35). 

The influence of a limited number of growth-regulating substances 
on growth in vitro of cultures derived from normal tissue has been ex- 
amined (18,5,6). Gautheret (5), found that indole-3-acetic acid was 
important for unlimited growth of tissue cultures from a number of 
species and was indispensable in some cases. Indolebutyric acid and 
a-naphthaleneacetic acid could be substituted for indole-3-acetic acid 
when the latter compound was essential for tissue cultures. Gautheret 
(6,7) reviewed the growth-regulating substance requirements of his 
tissue cultures as follows: i) cultures obtained from normal carrot and 
endive tissue grew for limited periods in vitro without added indole-3- 
acetic acid but growth was strikingly improved by that substance; 
2) cultures from normal tissues of Jerusalem artichoke, black salsify, 
and turnip did not grow without indole-3-acetic acid; 3) cultures of 
normal tissue of black salsify having once been cultivated on media 
with added growth-regulating substance grew in the absence of the 
material (The compound was required to start the culture but thereafter 
was no longer required even as an external source. Gautheret has referred 
to such cultures as habituated); 4) cultures of normal carrot tissue 
incubated initially on media with indole-3-acetic acid after several 
months developed faster in the absence of the material than did normal 
tissue without added growth-regulating substance; 5) Jerusalem arti- 
choke tissue of crown-gall origin developed without added indoIe-3- 
acetic acid. Such tissues were similar to those which at first required the 
material but later grew in its absence. Thus, considerable information is 
available about the influence of added growth-regulating substances on 
growth and development of normal tissue. This is perhaps because 
generally such cultures required supplements of this kind to grow for 
unlimited periods, or at least they required an external source when 
originally isolated. This necessitated working out the proper concentra- 
tion requirements. 

The concentration of the growth-regulating substance is a critical 
item in modifying the shape and kind of cells and in determining the 
initiation or inhibition of root formation in the cultures of normal tissues 
from a number of species. For example, Gautheret (6) noted that tissue 


cultures from normal artichoke failed to grow without supplementary 
growth-regulating substance. A concentration of io~'^ of indole-3-acetic 
acid, indolebutyric acid or a-naphthaleneacetic acid stimulated cambium 
formation, and a large callus was formed on the part of the tissue in 
contact with the medium. At a concentration of io~^ the tissue con- 
tinued to form cambial layers, but root formation was induced in 
addition. At io~^ cell division and callus formation were no longer 
promoted, but a swelling of the cells was favored instead, and finally 
at io~* giant cells were produced which emphasized the action of the 
growth-regulating substance on the swelling of the cells. Gautheret (6) 
also indicated the bud-inhibiting power of the growth-regulating sub- 
stances. For example, a slice of chicory root incubated on a-naphthalene- 
acetic acid at a concentration of io~^ produced roots and a large callus, 
while a slice on media lacking the material produced mainly young 
shoots. Skoog and Tsui (24) showed similar morphological responses 
of normal tobacco tissue in vitro. They found that adenine induced bud 
formation in callus and stem internode tissues. Callus growth and root 
formation in stem segments were stimulated by a-naphthaleneacetic 
acid, but bud formation was inhibited. Combinations of adenine and 
a-naphthaleneacetic acid greatly stimulated cell proliferation and enlarge- 
ment of all tissues, especially the pith, but it did not result in organ 
formation. A few studies have dealt with the effects of these materials 
on tissues of pathological origin, and have compared the responses of 
such tissues with normal tissues as they grew in vitro. 

Tissues of pathological origin have been isolated from several species 
and studied in vitro. Tissues isolated from the hybrid, Nicotiana glauca 
Grab. 9 X M langsdorffii Weinm. cT, provided the first true callus 
cultures capable of unlimited growth (29). This and related hybrids 
have been of special interest because of the type of galls (evidently of 
genetic origin) that are commonly produced at points of injury (16). 
Such galls have the general appearance of crown galls produced on a 
wide variety of plants by the bacterium Agrobacterium tumefaciens. 
Tissue isolated from the apical stem of the hybrid tobacco forms un- 
differentiated callus when incubated on solid media, but may form leaves, 
buds, or stems when cultured in a liquid medium. Skoog (22) found that 
the differentiation in liquid media was completely inhibited by the 
addition of 0.2 to 10 mg. per liter of indole-3-acetic acid or a-naph- 
thaleneacetic acid. Concentrations which did not decrease the growth 


rate, nevertheless prevented differentiation under conditions otherwise 
favorable for it. The higher of these concentrations caused a marked 
inhibition of growth, but at the lower concentrations the fresh weights 
were as large and with optimal concentrations even larger than those 
of the controls. Skoog also found that the cultures produced consider- 
able quantities of auxin extractable with ether when grown either in a 
liquid or on a solid medium. 

Bacteria-free tissue cultures of crown-gall origin from a number of 
species have provided for study important material of a pathological 
nature. Tissue cultures from primary galls on sunflower (i). periwinkle 
(33), Paris-daisy (12), black salsify (9), and Jerusalem artichoke (8), and 
from secondary galls on sunflower (31) and marigold (12) have already 
received considerable attention. The growth of such tissue in vitro 
removed the complicating factors that occurred when the gall tissue 
was studied in the intact plant. A comparison of the influence of growth- 
regulating substance on tissue of crown-gall origin and of normal tissue 
indicated some fundamental differences in response to these materials. 
De Ropp (2), for example, in an interesting study placed fragments of 
tissue of normal and crown-gall origin from sunflower and periwinkle on 
media containing indole-3-acetic acid, indolebutyric acid, or a-naph- 
thaleneacetic acid at concentrations of o.oi and 10 mg. per liter. The low 
and high concentrations strikingly stimulated growth of normal sun- 
flower tissue. The low concentration resulted in decreased growth while 
the high concentration completely inhibited growth of the tissue of 
crown-gall origin. The high concentration was toxic to normal periwinkle 
tissues but the low concentration resulted in a slight stimulation. The 
periwinkle gall tissue was inhibited at the high concentration and un- 
changed by the low concentration. Furthermore, normal sunflower and 
periwinkle tissue produced abundant roots at the low concentration and 
the cambium proliferated at the high concentration. Exposure of the 
normal sunflower tissue to the high concentration of growth-regulating 
substance for one day was sufficient to induce root formation. These 
results with normal tissue were therefore similar to those described 
by Gautheret (5) for normal tissue of carrot and other species. The 
sunflower and periwinkle gall tissue showed no structural changes as 
a result of the growth-regulating substances. 

Gautheret (8) also compared the responses of normal tissue of crown- 
gall origin of artichoke, lndole-3-acetic acid resulted in progressively 


increased weight of the normal cultures with corresponding increases 
in concentrations from I0-^ lo"^ . . . iQ-^ The greatest weight appeared 
at a concentration of 10"^ Indole-3-acetic acid was not necessary for the 
culture of the crown-gall tissue, and the above concentrations had no 
effect on the wet weight of the cultures although a concentration of 
io~* resulted in a decreased wet weight as compared with controls lacking 
the supplement. Gautheret (9) similarly compared the responses of 
normal tissue, tissue of crown-gall origin, and of habituated tissues of 
black salsify to varying concentrations of indole-3-acetic acid. The 
tissues of crown-gall origin and the habituated tissues compared with 
the controls lacking indole-3-acetic acid were unaltered with respect 
to the increase in wet weight at concentrations from io~^, lo"'' . . . 
I0-^ but were inhibited at a concentration of 10-*. Both these tissues 
are capable of unUmited growth on media lacking growth-regulating 
substance. The normal tissue however was strikingly stimulated as evi- 
denced by the progressively increased wet weight through progressively 
increased concentrations of indole-3-acetic acid from lo""^, lo"'' . . .io~^ 
The results with the salsify normal and crown-gall tissues were therefore 
comparable to similar tissues of Jerusalem artichoke. 

Recently Kulescha and Gautheret (15) compared the amount oi 
growth-regulating substance elaborated by the three strains of tissue 
from black salsify. Ether extracts were prepared of the three tissues 
and Avena tests made in the usual manner. On a wet-weight basis 
normal root tissues extracted in January during the resting period con- 
tained activity corresponding to 0.5 x io~^ of indole-3-acetic acid. Similar 
tissue isolated and placed on a medium-lacking growth-regulating sub- 
stance had after seven days activity equal to o.i x lo"^ indole-3-acetic 
acid. Such tissue that produced buds after 10 days had an activity 
equivalent to 1.5 x I0~^ but if the buds were removed the concentra- 
tion decreased to from 0.2 to 0.3 x 10"^ Tissue of crown-gall origin 
had much greater activity indicating a concentration equivalent to 
5.3 to 5.5 X iQ-^ Similarly, the habituated salsify tissue had activity 
equivalent to 1.5 to 2.3 x iq-^ The concentrations of growth-regulating 
substance therefore in these latter two tissues corresponded to the con- 
centrations found earlier to be optimum for callus formation in tissues 
from a variety of species (5) and suggested why such tissues required 
no supplementary growth-regulating substance. Tissues of crown-gall 
origin and habituated tissues had similarities and differences as compared 


to normal tissue with respect to gall production when each, respectively, 
was grafted to healthy plants. 

Tissues of crown-gall origin from a number of species, after cultivation 
in vitro, when grafted back to healthy plants commonly continue to 
grow and produce a gall at the graft cite. This was first described by 
White and Braun (31) for sunflower tissue isolated from secondary 
crown galls. Such tissue was also grafted successfully to Jerusalem arti- 
choke plants. The gall tissue itself actually increased in size, the host 
plant providing a suitable medium and support. This same phenomenon 
of grafting was successfully demonstrated for the hybrid tobacco tissue 
(32), for periwinkle (33), and black salsify (9). De Ropp (3,4) carried 
this study further with some interesting results on the interaction of 
normal and gall tissue in in vitro grafts. This approach to the role of 
growth-regulating substances in normal and pathological growth offers 
some splendid opportunities and deserves further attention. 

The above information provides a general background to the field 
of plant tissue cultures with special attention directed toward the in- 
fluence of growth-regulating substances on such cultures. Most of the 
work has been concerned with cultures derived from normal tissue. 
A few comparisons were made of the influence of these materials on 
tissue of normal and pathological origin. Next to be summarized are 
some phases of the problem studied at Wisconsin. These were concerned 
with the influences of some representative natural and synthetic growth- 
regulating substances on tissue cultures from hybrid tobacco and from 
secondary crown gall on sunflower. The stimulating or inhibiting efl'ects 
on growth of difl"erent compounds, their critical concentrations and their 
action on different tissues were investigated. These items were of special 
interest since the stimulating and inhibiting effects on tissue cultures 
of sunflower and tobacco of a variety of plant tissue extracts could not 
be entirely explained on the basis of improved salt balances in the basal 
media (13,14). 

Influence of concentration of growth-regulatifig substance on iveight of 
cultures. — The general procedures for culturing plant tissue in vitro were 
observed. The hybrid tobacco tissue was supplied by P. R. White in 
December, 194 1. The secondary sunflower crown-gall tissue was isolated 
in December, 1941, and was free of the inciting bacteria. The basal 
media were modifications of White's (30) and were described by Hilde- 
brandt, Riker, and Duggar (14). 


The growth-regulating substances tested included cysteine hydro- 
chloride, indoIe-3-acetic acid, indolebutyric acid, /7-chlorophenoxyacetic 
acid, a-naphthaleneacetic acid, a-naphthaleneacetamide, /3-naphthoxy- 
acetic acid, 2,4-dichlorophenoxyacetic acid, sodium 2,4-dichlorophenoxy- 
acetate, and 2,4-dichlorophenoxybutyric acid. Each of the compounds, 
respectively, was added to the basal media in concentrations from 
I X io~\ I X io~^ ... I X io~^^ grams per liter except indoIe-3-acetic 
acid which was not tested at i x io~^ grams per liter. Controls without 
added growth-regulating substance were used. 

Four tissue pieces were incubated in each 125 ml. Erlenmeyer flask. 
Each concentration was "replicated" six or more times so that twenty- 
four or more tissue pieces were cultured on each concentration in each 
trial. In addition certain compounds were tested in trials made at 
different times of the year. The influence of the growth-regulating 
substance on growth was measured after six weeks incubation by remov- 
ing each piece from the flask and by weighing it. 

The average wet weights of cultures incubated on the varying concen- 
trations of some growth-regulating substances are indicated in Figure i. 
Only a few curves for some representative growth-regulating substances 
are presented to conserve space. The weights of the cultures on each of 
the materials tested were presented elsewhere (11). Statistical analyses 
of variance showed these differences between concentrations with any 
one compound to be highly significant. 

With sunflower tissue the differences in weights varied with the con- 
centration and with the types of growth-regulating substance. Inhibition 
of sunflower tissue occurred at extremely high dilutions of 2,4-dichloro- 
phenoxyacetic acid, sodium 2,4-dichlorophenoxyacetate, 2,4-dichloro- 
phenoxybutyric acid, a-naphthaleneacetic acid and of /J-dichlorophen- 
oxyacetic acid. An increase in wet weight resulted from high dilutions of 
a-naphthaleneacetic acid and indole-3-acetic acid. Strong to complete 
inhibition appeared with all supplements as their strength was increased. 
Some comparisons of the inhibiting concentrations are presented in 
Table i. 

With tobacco tissue none of the compounds tested resulted in any 
large increases in wet weights. All resulted in decreased wet weights at 
the higher concentrations. The comparative inhibiting concentrations 
are indicated in Table i. 

The dry weights of sunflower tissue cultures incubated on media 









'^r-S^ =;^-°"^J^>.<^ 






Oar— —- °v 




vi rr; 

..«»••<»• o»^«,. 















0— -^ '^ /- TOBACCO 

" " ^ " ° ^V 



^ o o- ^ 



IX 10 



I X 10'' 

IX 10" 

IX 10 


Figure I. Average wet weights in milligrams of sunflower and tobacco 
tissue cultures incubated in the dark for six weeks on basal, synthetic media 
supplemented with different concentrations, respectively, of growth-regulating 
substances as indicated. Zero concentration indicates the control cultures with 
no added growth-regulating substance. Each point represents the average 
wet weight of 36 to 108 tissue pieces from one to three experiments. 


with varying concentrations, respectively, of /j-chlorophenoxyacetic 
acid and a-naphthaleneacetic acid expressed as percentage of wet weights 
varied from 4.6 to 6.8. Similar experiments with a-naphthaleneacetic 
acid and indole-3-acetic acid, respectively, and tobacco tissue provided 
dry weights that ranged from 5.3 to 9.9 per cent of the wet weights. 
When the average wet and dry weights were plotted on a logarithmic 
scale the resulting curves had similar trends. The dry weight expressed 
as the per cent wet weight decreased as the average wet weight increased. 
(The lowest per cent dry weights were obtained with concentrations 

Comparative Inhibiting Concentrations of Some Growth-Regulating Sub- 
stances on Sunflower and Tobacco Tissue Growing in vitro 

Inhibiting dilution 
Growth-regulating substance Sunflower Tobacco 

tissue tissue 

2,4-dichlorophenoxyacetic acid 
2,4-dichlorophenoxy butyric acid 
sodium 2,4-clichlorophenoxyacetate 
/?-chlorophenoxyacetic acid 
indole-3-acetic acid 
/3-naphthoxyacetic acid 
a-naphthaleneacetic acid 
indolebutyric acid 
cysteine hydrochloride 

providing the greatest wet weight). This suggested that stimulation of the 
growth-regulating substances was associated with a swelling of the 

The inhibiting effect of the growth-regulating substances was more 
striking with sunflower tissue than with tobacco tissue. This was perhaps 
because the sunflower grew more rapidly. Sunflower tissue on control 
media increased in wet weight during six weeks to an average of 450 
mg. per tissue piece as compared to only 200 mg. for tobacco tissues. 
The sunflower tissue also had a greater water content. 

The sensitivity of these tissue cultures to extremely low concentrations 
of growth-regulating substances suggested some possibilities for assay 
ot those compounds that have been difficult or impossible to assay by 



I X I0~^^ 

X 10-^ 

I X ID"'' 

X 10"^ 

I X I0~^ 

X IQ-^ 

I X IQ-^ 

X I0~^ 

I X I0~^ 

X I0~^ 

I X I0~^ 

X IQ-^ 

I X I0~^ 

X I0~^ 

I X IQ-^ 

X I0~^ 

I X IQ-^ 

X I0~^ 

I X I0~^ 

X I0~^ 


other methods. For example, 2,4-dichlorophenoxyacetic acid and its 
sodium salt induced negative curvature on stems and leaves of sensitive 
plants at 1.5 x lo"- grams per liter and modified organs at 3 x io~^ 
grams per liter (34), while with sunflower tissue cultures 2,4-dichloro- 
phenoxyacetic acid inhibited growth progressively from the lowest con- 
centration of I X io~^^ grams per liter to the highest concentration. The 
sensitivity of this tissue to the other compounds was also greater than 
that observed with whole plants. The sensitivity of the cultures to some 
of the compounds compares with that of the Avena coleoptile. The 
exposure of the cultures to the materials over a six week period may 
account for the sensitivity to such minute amounts. 

No macroscopic evidence was observed with sunflower and tobacco 
tissue of differentiation of leaves, stems, or roots. Under certain con- 
ditions such changes did occur in tobacco tissue in a liquid medium 
(29,22). The stimulating or inhibiting effects of low and high concentra- 
tions of growth-regulating substances described here were also reported 
by Skoog (22) for tobacco hybrid tissue, and by de Ropp (2) for sun- 
flower gall tissue. The possibihty that histological differences occurred 
in such tissues with different concentrations of these materials was 
examined next. 

Histological effects of growth-regulating substances on sunflotver gall 
tissue. — The influence has been tested of the concentrations of some 
representative growth-regulating substances on the structure of sun- 
flower tissue of crown-gall origin (25). Cultures incubated on weak and 
strong concentrations of four compounds and on control media lacking 
supplements were studied. Cultures from weak concentrations of indole- 
3-acetic acid and a-naphthaleneacetic acid (i x io~i^ grams per liter), 
indolebutyric acid (i x io~^ grams per liter) and /7-chlorophenoxyacetic 
acid (i X 10"'^ grams per liter) were compared with cultures incubated 
on the strong concentration (i x io~^ grams per liter) and with the 
controls. The weak concentrations for the respective compounds were 
selected because they represented optimal concentrations. The strong 
concentrations were inhibiting (see Fig. i and Table i). 

The structure of the sunflower tissue was relatively simple. Such tissue 
consisted of hypertrophic, hyperplastic, and thick-walled scalariform 
cells. The control tissues contained all three kinds of cells. Very few 
mitotic divisions appeared perhaps because growth was at a minimum 
after the six-week incubation period. Cells with scalariform thickenings 



were most frequently scattered among the hyperplastic regions and often 
were not orientated in the same direction as those of adjacent tracheal 
elements. This suggested a lack of organization. 

Tissues grown at weak concentrations had larger hypertrophic cells 
than control pieces. The total number of cells per unit area was less, 
and there were many more scalariform vessels. To determine this rela- 
tionship actual counts were made of the number of cells in fifty fields 


Comparison of the Number of Meristematic Cells and of Tracheal Elements 

of Sunflower Tissue of Crown-Gall Origin Grown on Media Containing Weak 

or Strong Concentrations of Growth-Regulating Substance and on Control 

Media Lacking Added Growth-Regulating Substance 


substance and 

cell type 

Cells in 50 fields of sections of tissue 

cultured with indicated concentrations 

of growth-regulating substance* 

Strong Weak None L.S.D.f 

Indole-3-acetic acid 
Meristematic cells 
Tracheal elements 

Indolebutyric acid 
Meristematic cells 
Tracheal elements 

a-naphthaleneacetic acid 
Meristematic cells 
Tracheal elements 

/7-chlorophenoxyacetic acid 
Meristematic cells 
Tracheal elements 






























*Concen(xations indicated in text. fNumber of cells required between treat- 
ment totals for significance at the 5 per cent level. 

each about 1/4 sq. mm. in size in cultures incubated on weak or strong 
concentrations of the four growth-regulating substances and on the 
control media. The total numbers of cells in the tissue sections examined 
from these media are summarized in Table 2. These counts indicated 
that the weaker concentrations stimulated greater cell size and a greater 
proportion of scalariform vessels. 

Strong concentrations of growth-regulating substances resulted in a 
striking decrease in wet weight as compared with controls (Fig. i). 


Both hypertrophic and hyperplastic cells were larger than those of 
tissues incubated on weak concentrations or on control media. Accord- 
ingly the number of cells per unit area on each of the four compounds 
was fewer with the strong concentration. Also the number of tracheal 
elements was fewer. 

It appeared therefore that the strong concentration of growth-regulat- 
ing substance tested stimulated a swelling of the cells. This swelling was 
greater between the strong and weak concentrations than between the 
weak concentration and the control. The greatest number of scalariform 
vessels was found on media containing the weak concentrations. The 
greater differences in the number occurred between the strong and weak 
concentrations than between the weak concentrations and the control. 

These results were comparable to certain of those reported by de Ropp 
(2) for sunflower and periwinkle tissue of crown-gall origin. He reported 
that different concentrations of growth-regulating substances had no 
effect externally and internally except for differences in weight after a 
six-week culture period. The studies described here indicated however 
that internally the structure of sunflower tissue of crown-gall origin 
was considerably modified. The reasons for these differences are not clear. 

The influence of growth-regulating substances on the respiration of 
cultures of crown-gall origin may provide some of the answers, but this 
approach to the problem has hardly been touched because of certain 
technical difficulties. Mitchell, Burris, and Riker (17) found that 0.002 
M. indole-3-acetic acid inhibited respiration of sunflower gall tissue 
by sixty-eight per cent. This reduction in respiration with indole-3- 
acetic acid was comparable to that observed in stems, roots, petioles, and 
gall tissues from a number of sources. 

Summing up then, growth-regulating substances induced striking 
effects on plant tissue cultures just as they did on whole plants. At 
least three general types of responses were observed. Tissue cultures 
from normal plants were especially responsive to these materials. With 
tissues from a number of species these materials were indispensable. 
If the growth-regulating substance was not added to the medium the 
piece of tissue originally isolated from the plant failed to form callus, 
and therefore cultures capable of unlimited growth were not estabhshed. 
Furthermore, cultures of tissue from normal plants of certain species 
required added growth-regulating substance in the medium to maintain 
indefinite growth. Concentration of added growth-regulating substance 


was critical in determining whether the cultures continued to produce 
a callus mass or whether they differentiated roots, stems, and leaves. 
Weak concentrations of growth-regulating substance favored cambium 
development and cell division; a slightly stronger concentration was 
beneficial for cambial growth, but stimulated root formation in addition; 
while even stronger concentrations stopped cell division and favored 
cell enlargement or stopped growth completely. 

Cultures of tissue from normal plants of certain species only required 
added growth-regulating substance in the medium to support growth of 
the original isolate or of the first few transfers in vitro. These cultures 
following the original incubation period on a medium containing supple- 
mentary growth-regulating substance grew indefinitely when transferred 
to a basal medium lacking added growth-regulating substance. Such 
cultures were described as habituated and were isolated from several 
plant species. 

Tissue cultures of primary or secondary crown-gall origin from several 
species and tissues from the genetically unstable tobacco hybrid were 
capable of unhmited growth in vitro on media lacking any added growth- 
regulating substance. Different concentrations of supplementary growth- 
regulating substance induced macroscopic changes evident only as in- 
creased weight at optimal high dilutions or as inhibition or death at 
low dilutions. No leaves, stems, or roots were formed on cultures of 
crown-gall origin, but under certain conditions they were formed on the 
tobacco tissue. Different concentrations of supplementary growth-regu- 
lating substance influenced histologically the sunflower tissue of crown- 
gall origin. 

There appeared therefore certain similarities and differences between 
tissue cultures from normal plants and those of pathological origin. 
Some of these similarities and differences in the requirements of the 
respective types of tissue cultures for growth-regulating substance were 
indicated. The growth-regulating-substance activity of the extracts of 
the three types of cultures in the Avena test was also reported to vary 
with the three types. Thus, the importance of growth-regulating sub- 
stance for normal and pathological growth was suggested and has re- 
ceived considerable attention. However, the fundamental role of these 
materials in normal and pathological growth is still to be worked out. 
These and further studies with plant tissue in vitro may clarify the 
nature of the balances resulting in normal or pathological growth. 



1. DE Ropp, R. S., Phytopathology, 37:201 (1947). 

2. , Am. J. Botany, 34:53 (i947)- 

3. , ibid., 34:248 (1947). 

4. , ibid., 35:372 (1948)- 

5. Gautheret, R. J., La Culture destissus, Lagny-sur Marne, France (1945). 

6. , Grotvth (Supple. Sixth Growth Symposium), 11:21 (1947). 

7. , Compt. rend. soc. biol., 141:627 (1947). 

8. , Compt. rend. acad. sci., 224:1728 (1947). 

9. , Compt. rend. soc. biol., 142:774 (1948). 

10. Grieve, B. J., Proc. Roy. Soc. {Victoria), 55:109 (1943). 

11. HiLDEBRAXDT, A. C. and Riker, a. J., Am. J. Botany, 34:421 (1947). 
12. , ibid., 36:74 (1949). 

13. , and DuGGAR, B. M., Cancer Res., 6:368 (1946). 

14. , Am. J. Botany, 33:591 (1946). 

15. KuLESCHA, ZojA and Gautheret, R, J., Compt. rend. acad. sci., 227:292 


16. Levine, M., Am. J. Botany, 24:250 (1937). 

17. Mitchell, J. E., Burris, R. H., and Riker, A. J., ibid., 36:368 (1949). 

18. NoBECOURT, P., Reu. Sci. (Paris), 81:161 (1943). 

19. Riker, A. }., Growth (Supple. Fourth Growth Symposium), 6:105 (1942). 

20. , and Berge, T. O., Am. J. Cancer, 25:310 (1935). 

21. Riker, A. J., Spoerl, E., and Gutsche, A. E., Botan. Rev., \i'.y] (1946). 

22. Skogg, F., Am. J. Botany, 31:19 (1944). 
23. , Ann. Rev. Biochem., 16:529 (1947). 

24. , and Tsui, C., Am. J. Botany, 35:782 (1948). 

25. Struckmeyer, B. E., Hildebrandt, a. C., and Riker, A. J., ibid., 

36:491 (1949)- 

26. Thomson, B. F., Botan. Rev., 11:593 (1945)- 

27. Thompson, H. E., Swanson, C. P., and Norman, A. G., Botan. Gaz., 

107:476 (1946). 

28. VAN OvERBEEK, J., Ann. Rcv. Biochem., 13:631 (1944). 

29. White, P. R., Am. J. Botany, 26:59 (1939). 

30. , Ann. Rev. Biochem., 11:615 (1942). 

31. , and Braun, A. C., Cancer Res., 2:597 (1942). 

32. White, P. R., ibid., 4:791 (1944)- 

33. White, P. R., Am. J. Botany, 32:237 (1945). 

34. Zimmerman, P. W., Cold Spring Harbor Symposia Quant. Biol., 10:152 


35. , Ind. Eng. Chem., Ind. Ed., 35:596 (1943). 

The Interaction Between Causative Agents in 
Diseased Growth 


THE basic problems, what starts off diseased plant growth and what 
keeps it going, are among the most fundamental in biology. Galls 
may be incited by various agencies including physical and chemical 
factors, virus, bacteria, fungi, nematodes, and insects. However, much 
of the basic research has been done with crown gall caused by Agro- 
bacterium tumefaciens (Smith and Town.) Bergey et al. 

Many of the numerous working hypotheses given to explain gall 
formations have been centered on one or another chemical substance. 
Such ideas have developed naturally from work on other diseases where 
injury and death were caused by some single factor, such as too much 
heat, a toxic spray, or a single microorganism. Many of the earlier studies 
on pathological growth have been reviewed elsewhere, for example, by 
Riker and Berge (i6) and by Riker, Spoerl, and Gutsche (20). The 
activity of growth substances has an obvious bearing on diseased growth. 
The general subject has been covered in a previous paper (22) listing 
earlier reviews, and by various reports elsewhere in this symposium. 

While a single extraneous factor may cause death, the growth of cells 
obviously is more complex. For normal growth many physical and 
chemical factors seem to operate in suitable balance with one another. 

Our purpose in this discussion is to indicate how some of these factors 
may be thrown out of balance. Further we consider evidence bearing 
on the suggestion (15) that such a lack of balance may influence subse- 
quent abnormal growth. 

Histological picture. — Perhaps one of the easiest ways to visualize 
this problem is to consider what happens when crown-gall bacteria appear 


in the intercellular spaces between cells that normally never would divide 
again. From histological studies, Riker (14) showed in 1923 that within 
two days the cell walls increased in thickness near the bacteria, the 
entire cells became larger, the nuclei apparently took up a position 
near the bacteria. New cell walls were laid down within four days in a 
plane perpendicular to the location of the bacteria. This time interval 
parallels that determined by Braun (2) with heat treatments. Further 
cell divisions followed rapidly and in a disorganized manner until a gall 

Activity of bacteria in culture. — What happened above perhaps may 
be clarified by examining what the bacteria have done in culture media. 
There the details have been followed with considerable precision. At the 
same time we recognize the difference between a culture tube of glass 
and a capillary space in the plant. Among the early changes the bacteria 
induced in culture, after the lag period, was the lowering of the oxidation- 
reduction potential of the medium (21). Hydrogen-ion concentration 
of the cultures was changed little, if any, except in certain cases, when, 
for example, complex nitrogen compounds were used also as carbon 
sources (24). Osmotic pressures became less (19) as sugar was used 
and viscosity was lowered (i). 

Among the chemical products formed in culture in addition to carbon 
dioxide, the most abundant was a polysaccharide. This has been charac- 
terized through a series of investigations and shown to be toxic under 
certain conditions (7). Substances related in size have proved toxic in 
direct relation to their molecular size (8). In passing perhaps we should 
mention that toxic substances at sublethal concentrations frequently 
may be stimulating. Small amounts of various other substances have 
been isolated, including phosphatides (4), thiamin, riboflavin, biotin, 
pantothenic acid (13), and auxin. The last has received much special 

Groivth substances in cultures and galls. — The production of growth 
substances in cultures of virulent and attenuated bacteria has been 
approximately the same when peptone containing tryptophane was a 
part of the medium (10). However, in some recent unpublished work 
with Hodgson, Tsui, and Skoog, it appears that in a synthetic medium 
the virulent culture produces more growth substances than the attenu- 
ated culture. This is noteworthy when correlated with the different 
symptoms induced by these cultures on decapitated tomato plants (9). 


The virulent cultures induced large galls, and the attenuated cultures 
induced small galls covered with many little shoots. Recently Hodgson, 
Peterson, and Riker (unpublished) found that d-tryptophane inhibited 
the bacteria more than the attenuated culture. Schurr (unpublished) 
found by means of microbiological assays that the virulent culture 
produced more free tryptophane in a synthetic medium than the 
attenuated culture. Tryptophane can stimulate galls about attenuated 
bacteria. It is a precursor for other important substances. 

The presence of growth substance in crown gall has been shown by a 
number of workers. For example, growth-substance responses in plants 
with galls were described by Locke et al. in 1938 (9). A substance in 
tomato was found in 1939 by the same workers (11) to behave like 
indole-3-acetic acid in the presence of strong acid and alkali. In related 
studies, Riker et al. (18) detected no difference in the amount of growth 
substances in tomato plants grown at 27° C, where galls developed well, 
and 31° C, where the galls did not develop. 

Galls were shown (13) to contain more of tyrosinase, oxidase, peroxi- 
dase, and catalase than normal tissue. The question has appeared whether 
this excess of oxidizing enzymes had any connection with the lowering 
of oxidation-reduction potentials (21) by the bacteria and with the 
reduced oxygen uptake (12) induced by gall-stimulating growth sub- 

Indole-3-acetic acid has been reported by some workers to be the 
cause of crown gall. The subject is reviewed elsewhere (12). It was pointed 
out that thus far the technique employed had not been adequate to 
justify this conclusion. However, the association of such material with 
embryonic growth, including galls, has been well established. 

Chemical induction of galls. — Various chemicals have been employed 
to induce galls on different plants. Among these the growth substances 
are the most prominent. However, many other unrelated chemicals 
seem to be active (16). We (unpublished) have found that substances 
such as thiamin, ammonium carbonate, and 1,2,5,6-dibenzanthracene all 
will induce chemical galls under certain conditions. Thus the formation 
of galls seems to be a relatively nonspecific reaction. If this should be 
true, then the cause must involve rather broad biological phenomena. 

The possibility of stimulating growth of galls with chemicals after 
inoculation with attenuated strains arose from the observation by Locke 
et al. (9) that virulent bacterial galls a few inches above inocula- 


tions with attenuated bacteria induced large galls about the attenuated 
bacteria. Braun and Laskaris (3) and Riker (15) repoited similar results 
with growth substances. With this as a background, Thomas and Riker 
(23) employed some 56 chemicals on five different kinds of plants. Each 
plant was puncture-inoculated with attenuated bacteria, then decapi- 
tated several inches above. Each cut stem was treated with one or another 
of the chemicals used. Records were kept of chemically induced galls, 
the sizes of the attenuated bacterial galls, and of growth-substance 
effects which were induced. 

Of the 56 chemicals used 19 were found to stimulate, in varying 
degrees, the attenuated-bacterial galls. These substances represented 
a variety of different chemical compounds. No particular compound or 
type of compound was found exclusively to stimulate the attenuated- 
bacterial galls. The compounds most active in this respect were the 
growth substances a-naphthaleneacetamide, a-naphthaleneacetic acid, 
2,4-dichlorophenoxyacetic acid, 2,4-dichlorophenoxybutyric acid, and 
indolebutyric acid. 

Most of the effective gall-stimulating substances also induced other 
growth responses, such as tissue proliferation, root stimulation, bud 
suppression, epinasty, and formative effects. However, stimulation of 
galls about attenuated bacteria was not consistently associated with the 
production of any particular growth response. Neither did all of the 
active gall-stimulating substances induce all of the growth responses. 
Consequently, stimulation of galls about attenuated bacteria appeared to 
be a distinct type of growth response that could be induced by a variety 
of chemical compounds. This suggests that no one of the known growth 
substances used was, by itself, the cause of crown gall. 

Bacteria reisolated from the stimulated galls were still attenuated. 
This fact, and the wide variety of compounds capable of inducing gall 
stimulation, indicated that the effects of the chemicals used were 
probably on the host cells. 

In addition to the known chemicals, a water extract of virulent 
bacterial cells, which had been dried while frozen, had a slight gall- 
stimulating effect. Galls produced from attenuated bacteria, which de- 
veloped shoots in the absence of terminal and lateral shoots on the host 
plant, were also larger than normal. 

Unbalance offactors for diseased growth. — While continuing to examine 
individual factors, one of which might act as a trigger mechanism to set 


off a series of events, we may wisely consider the changes in growth that 
may arise if we have the factors necessary for normal growth in an 
unbalanced combination. 

As already mentioned a considerable number of physical, chemical, 
and biological factors may induce gall formation. The relatively non- 
specific nature of this phenomenon is striking. From the evidence avail- 
able, the bacteria in culture, bacteria in the tissue, and important growth 
substances all seem associated with lower oxidation-reduction potentials 
or reduced oxygen uptake. 

Other critical factors besides respiration may include changes in 
osmotic pressure and surface tension as well as altered amounts of growth 
substances, vitamins, enzymes, irritating substances, and food materials. 
Any living cell, even a resting cell, that would fail to react when the 
normal balance in such factors is disturbed would seem to be unresponsive 

With the development of plant-tissue-culture techniques we have 
improved means for determining the effects of known substances. A 
trigger mechanism might be touched oft' in various ways. Perhaps of 
more importance is the kind of a gun the trigger is on, what the source 
of energy may be, how much is present, and what inhibiting and direc- 
tional factors may opeiate. Tissue cultures help with such determinations. 

Much progress has already been made as discussed elsewhere in this 
symposium. The work with mineral salts (6), with sources of nitrogen 
(17), and with sources of carbon (5) all emphasize the importance not 
only of the particular substance but also of its concentration to make 
the proper balance in relation to other items. The encouragement or 
inhibition by certain amounts of particular substances has sometimes 
been quite conspicuous. 

Let us summarize. In normal growth a number of factors, including 
growth substances, apparently operate in suitable balance. However, 
if these factors are out of balance in one way we can expect pathological 
growth of a certain kind. If these factors are out of balance in another 
way we can expect diseased growth of a different kind. Whether it is 
right or wrong, this point of view suggests many interesting experiments. 


I. Berge, T. O., Riker, a. J., and Baldwin, I. L., Phytopathology, 26:86 


2. Braun, a. C, Am. J. Botany, 34:234 (1947). 

3. , and Laskaris, Thomas, Proc. Nat. Acad. Sci. U. S., 28:468 (1942). 

4. Geiger, W. B., Jr. and Anderson, R. J.,/. Biol. Chem., I29(2):5i9 (1939). 

5. HiLDEBRANDT, Albert C. and Riker, a. J., Am. J. Botany, 36:74 (1949) 

6. , and Duggar, B. M., ibid., 33:591 (1946). 

7. Hodgson, Roland, Peterson, W. H., and Riker, A. J., Phytopathology, 

39:47 (1949)- 

8. , ibid., 37:301 (1947). 

9. Locke, S. B., Riker, A. J., and Duggar, B. M.,/. Agr. Res., 57:21 (1938). 

10. , ibid., 59:519 (1939)- 

11. , ibid., 59:535 (1939). 

12. Mitchell, J. E., Burris, R. H., and Riker, A. J., Am. J. Botany, 36:368 


13. Nagy, R., Riker, A. J., and Peterson, W. H.,/. Agr. Res., 57:545 (1938). 

14. Riker, A. J., ibid., 26:425 (1923). 

15. , Growth (Sup. to vol. 6, 4 Sym. Devlpmt. and Growth), 105 


16. , and Berge, T. O., Am. J. Cancer, 25:310 (1935). 

17. Riker, A. J. and Gutsche, Alice E., Am. J. Botany, 35:227 (1948). 

18. Riker, A. J., Henry, B., and Duggar, B. M.,/. Agr. Res., 63:395 (1941). 

19. Riker, A. J., Lyneis, M. M., and Locke, S. B., Phytopathology, 31:964 


20. Riker, A. J,, Spoerl, E., and Gutsche, Alice E., Botan. Rev., 12:57 


21. Sagen, H. E., Riker, A. J., and Baldwin, L L., /. Bact,, 28:571 (1934). 

22. Skoog, F., Ann. Rev. Biochem., 16:529 (1947). 

23. Thomas, John E. and Riker, A. J., (Abstract) Phytopathology, 38:26 


24. Wilson, A. R., Phytopathology, 25:854 (1935). 

Deformities Caused by Insects 


THE hexapods gradually became adapted to a wide range of food 
hosts. They can thrive on inorganic as well as organic matter, but 
the greater proportion of them, however, feed upon plant life. In their 
habit of feeding, insects may distuib the physiology of the plant by 
causing a direct loss of tissue or its cell constituents by inoculating a 
transmissible toxic pathogen or by causing a pathological condition 
resulting from their feeding which is usually accompanied by toxic 
salivary secretions. 

The latter group of insect feeders have been referred to as toxico- 
genic insects, or those insects which cause a pathological disturbance of 
tissue not ascribable to mere mechanical injury nor fulfilling the criteria 
necessary to establish the presence of some microorganism. The greatest 
number of toxicogenic insects belong to the orders Hemiptera and 

Toxicogenic insects cause distinct deformative effects, and their ca- 
pacity to produce such effects is often inherent in a particular species. 
Most striking in this respect are the gall-formers. With them the 
deformation of tissue is so specific that the species of insect concerned 
can be identified by the type of gall formed. 

It is the opinion of many workers that gall formation is caused by the 
introduction of some insect-produced toxic substance. Darwin and 
writers before him frequently referred to chemical secretions injected 
by the "gall mother." Early thinking in regard to the possible causes 
of insect gall formation appeared long before our present knowledge of 
auxin behavior. During feeding it is possible that an insect could inject 
or withdraw active substances which would tend to increase or decrease 
the activity of plant responses to hormones. 


Although no evidence exists showing that insects can produce a 
natural auxin, it is significant to note that in 1940 Link reported 
curvature responses in Avena tests from extracts of certain aphid'. (15), 
and in addition, from extracts of plant tissue upon which insects had 
fed (27). More recently, Nysterakis (18,19) ^^^^ reported that certain 
ones do secrete auxin which affects plant growth. 

A review of investigations relating to this subject shows that there 
are certain similarities in growth changes in plants caused by hormones 
and the deformities in plants caused by insect feeding. In both cases 
enzyme activity appeals to be involved. An interesting case suggesting 
a relationship between plant hormones and the effects of insect feeding 
is the fact that bean plants treated with hormones have failed to show the 
expression of certain insect damage. 

Similarity in Growth Changes. — As has been previously mentioned 
perhaps the most significant evidence that disturbances in plants caused 
by insects and by hormones are similar is the change produced by the 
feeding of insect and mite gall-formers. It is the opinion of most workers 
interested in such insects that gall formation is caused by the introduction 
of substances into the plant tissue. In 1936 Felt (5) stated that stimula- 
tion by such substances was the fundamental principle in gall formation; 
and Martin (16) concluded in 1930 that since stem galls in sugar cane can 
be produced by injection of macerated leaflioppers, possibly auxins 
were involved. Brown and Gardner (2) state that the formation of galls 
caused by insects is a tissue response paralleling the response to growth- 
promoting substances of some plant tissues. 

Further similarities in changes in plants caused by insects and by 
plant hormones may be seen in the development of adventitious buds 
and reduction of length and width of internodes which may follow the 
application of hormones (9,25). Ripley (21) has reported that some 
mirids cause malformation in young trees and a witches'-broom rather 
than a single stem is produced from the terminal leaf bud. A disease 
associated with the feeding of a cercopid on sugar cane has been described 
as reducing the length and width of plant internodes with a development 
of adventitious buds (10). A rosetting and shortening of internodes of 
alfalfa and clover by the feeding of spittle insects has recently been 
recorded (6). Smolak (24) describes a witches'-broom of lilac caused by 
Eriophyes lowi (Nel). Psyllid yellows, which is associated with the feeding 
oi Paratrioza cockerelli (Sulc), possesses symptoms that include rosetting 

T. C. ALLEN 413 

of leaves in witches'-broom fashion at the internode. Many abnormal 
tissue responses have been pointed out (8,12,17) showing that the feeding 
of certain Empoasca species will cause stunting, rosetting, and prolifera- 
tion of dwarfed shoots. Carter (3) observes a close analogy between the 
development of adventitious buds and insect galls and suggests that a 
possible relationship may exist between gall, hosts, and hormones. The 
statement by Carter that "insects feed on a specific plant species only" 
would suggest that auxin relations may be involved between insects 
and plants. It was also his opinion that if symptoms following certain 
insect feeding are "much more rapid than that of fungus or bacterial 
infection, this appears to be the principal support for the numerous 
susgestions in the literature that the secretions are toxic." 

That the feeding of Lygus upon potato stems indicates systemic 
response or diffusion of a toxic principle in association with the feeding 
of the insect has been reported by Leach and Decker (13). Smith (23) 
concluded that when certain insects feed they inject toxic secretions 
into plants and that the saliva of Miridae is so toxic it may cause 
changes in the tissue much more rapidly than could be produced by a 
virus. This speed of action is similar to that of applied hormones, and 
the similarity is of considerable interest. 

Similarity in Enzymic Activity where it Appears hivolved. — Although 
the specific action of auxin may still remain to be demonstrated, there are 
good indications that auxin release and action are exerted in connection 
with enzymes. 

Numerous investigators have pointed out the presence of enzymic 
activity in connection with insect feeding upon plants. Cosens (4) in 
considering the physiology of gall formation concludes that a larva 
secretes an enzyme which converts starch to sugar. Typical lesions caused 
by cotton flea hoppers were obtained by Painter (20) following injection 
of diastase in plant tissue, and froghopper injury to sugar cane has been 
shown to increase the content of oxidizing enzymes (10). Herford has 
reported that certain leaf hoppers secrete diastase and invertase (11); 
and Andrew pointed out that the saliva of some mosquitoes contains 
an enzyme which produces a reaction in plant juices causing precipita- 
tion of its contents (i). 

Little information is available, however, on the effect of enzyme 
activity, insect feeding, or plant auxin on the growth and differentiation 
in plants. Studies by Wildman and Gordon (26) show that proteolytic 


enzymes, such as tryptic extracts, trypsin, or chymotrypsin, release 
active auxin from isolated plant protein preparations. An effect on 
the plant protein resulting from insect feeding has been found by Smith 
(22), who has shown that proteins and amino acids, as determined by 
color tests, make up a major portion of the sheath material after certain 
insect feeding. 

Suppression of Blossom and Pod Drop Caused by Lygus Species. — 
A similarity in the action of hormones and of insects upon plants was 
recently shown by the use of a-naphthaleneacetic acid to inhibit ab- 
scission of blossoms and bean pods caused by the feeding of Lygus 
oblineatus Say (7). Under field conditions the application of naphthalene- 
acetic acid has resulted in greater retention of number of small bean 
pods thereby improving the quality of the bean for market purposes. 

An explanation of the above behavior may be proposed as follows. It 
is known that auxin inhibits leaf abscission (14). Possibly, therefore, 
the insect feeding stops the normal auxin supply to the base of the petiole 
thus permitting the abscission layer to form. The appUcation of a-naph- 
thaleneacetic acid would then restore the normal supply of hormone and 
prevent the abscission. The insect therefore may produce an auxin inac- 
tivator or interfere in some other way with the normal supply. 


1. Andrew, E. A., Proc. 4th Ent. Sac, Pusa (Calcutta), 56-59 (1921). 

2. Brown, N. A. and Gardner, F. E., Phytopathology, 26:708-713 (1936). 

3. Carter, Walter, Botan. Rev., 5:273-326 (1939). 

4. CosENs, H., Trans. Canad. Inst., 9:297-387 (1912). 

5. Felt, E. P., Ann. Ent. Sac. Amer., 29:694-700 (1936). 

6. Fisher, E. H. and Allen, T. C, /. Econ. Ent., 39(6):82i (1947). 

7. Fisher, E. H., Riker, A. J., and Allen, T. C, Phytopathology, 36(7) :504- 

523 (1946). 

8. Granovsky, a. a., XXX /. Econ. Ent., 21:261-266 (1928). 

9. Greenleaf, Walter H., /. Heredity, 29(12) :45 1-464 (1938). 

ID. Hardy, F., Min. & Proc. Frog. Invest. Comm., Trinidad & Tobago, 8:218- 

235 (1927)- 

11. Herford, G. V. B., Ann. Appl. Biol., 22:301-306 (1935). 

12. Hollowell, E. a., Monteith, J., and Flint, W. P., Phytopathology, 

17:399-404 (1927). 

13. Laibach, F., Deutschen Botamschen Gesellschaft Band Li, Heft, 8:336-340 


14. Leach, J, G. and Decker, P., Phytopathology, 28(1) :i3 (1938). 

15. Link, George K. K., Eggers, Virginia, and Noulton, James E., Botan. 

Gaz., ioi(4):928-939 (1940). 

T. C. ALLEN 415 

16. Martin, J. P., The Haw. Planters Record, 42:129-134 (1930). 

17. MoNTEiTH, J., Phytopathology (Abstr.), 18:137-138 (1928). 

18. Nysterakis, pRANgois, Compt. rend. soc. biol., 141:1218-1219 (1947). 

19. , Compt. rend., 226:1917-1919 (1948). Cf. ibid., 831-832. 

20. Painter, R. H., /. Agr. Res., 40:485-516 (1930). 

21. Ripley, L. B., Farm, in S. Africa, 1:423 (1927). 

22. Smith, F. ¥.,J. Agr. Res., 47:475-485 (1933). 

23. Smith, K. M., Ann. Appl. Biol., 7:40-55 (1920). 

24. Smolak, J., Shorn Csl. Akad. Zemed., 8A:39-5o (1933). 

25. Thompson, Betty F., Botan. Rev., 11:593-610 (1945). 

26. Wildman, S. G. and Gordon, S. A., Proc. Nat. Acad. Sec, 28:217 (1942). 

27. Went, F. W. and Thimann, K. V., Phytohormones (Macmillan, 1937), 

p. 283. 

Comparative Studies of Metabolism in Insect Galls 

and Normal Tissues 


THE striking abnormalities produced by plant tissues in response to 
the stimuli of gall insects are familiar to all of us. On the leaves 
of a single hickory tree there may be, for example, as many as eight 
or nine different types of galls caused by as many closely related species 
of gall midges of the family Cecidomyiidae, while four or five kinds of 
midge galls can be found on the upper surface of a single leaflet. These 
may be tubular, conical, flask-shaped, or globular, and covered with 
trichomes, waxy bloom, or sugary exudate. On oaks, there is an even 
larger and more remarkable series of growths induced by the larvae of 
gall wasps or Cynipids. 

The great diversity shown by galls in their tissue differentiation and 
arrangement, pigmentation, compartmentalization, and shape appears 
the more remarkable because it may be exhibited on a single plant organ 
by closely related species of insects ovipositing on the same surface at 
about the same time. Such facts have led some investigators to assume 
that each species of gall-forming insect must elaborate specific morpho- 
genetic hormones to which the plant tissue responds by producing a 
highly specific structure. Such a viewpoint is common in current litera- 
ture on galls. It must be emphasized, however, that no growth substances 
have been isolated and identified from the gall insects, not to mention 
morphogenetic compounds, and that attempts to induce gall formation 
by insect parts or extracts have met with Uttle success. Recent contribu- 
tions to this field have been made by several investigators whose work 
will be reviewed briefly. 

Beck (2), studying the gall-fly Eiirosta solidaginis, could not obtain 


gall formation in Solidago stems with extracts of gall tissues, maggot 
cultures, or maggots, although he induced some gall formation by 
injections of trypsin, mixtures of amino acids, and protein digests. Martin 
(io,ii), working with sugar cane, reported success in producing stem 
galls following inoculation of extracts of leaf hoppers and mealy-bugs 
producing galls on this plant normally. He also obtained galls with 
autoclaved extracts. 

Parr (i8) investigated gall formation on chestnut oak by the coccid, 
Asterolecanium variolosum, and found that 90 pef cent of the injections 
of salivary gland extracts into young stems resulted in gall formation. 
The galls had the same shape as those produced by the living insects, 
though they were slightly smaller. Since no gall formation was obtained 
by injection of salivary gland extracts heated to 6o°C., Parr concluded 
that enzymes or enzyme-like substances were the causative agents. 
Ptyalin and indole-3-acetic acid failed to produce galls. 

The successes of Martin and Parr were achieved with insects producing 
relatively simple galls which belong among Kiister's "kataplasmas." 
Kiister (7) defined kataplasmas as those galls which are less highly 
differentiated than the normal structures on which they are borne, and 
are inconstant and indefinite in size and form. Prosoplasmas, on the other 
hand, were defined as the more specialized galls characterized by definite 
size and form, which in tissue differentiation are different from but 
not below normal. The kataplasmas include such galls as the familiar 
enlargements on goldenrod stems, while the prosoplasmas include most 
of the galls produced by gall midges and gall wasps. 

Gall insects are known to secrete various enzymes, and some workers 
have attributed gall-forming properties to these. Kiistenmacher (6), 
Magnus (9), and others showed that the larvae of gall insects produce 
diastase and invertase. Cosens (4) identified diastase in the secretions or 
excretions of gall insects, and believed it played an important role in 
gall formation. Beck (2) identified amylase, invertase, and a protease 
in the excrement oiEurosta maggots, and concluded that the proteolytic 
enzymes were important factors in the production of galls. In the salivary 
glands of the coccid, Asterolecanium, Parr, in 1940, found amylase, in- 
vertase, a protease, and an oxidase, but he was not able to detect perox- 
idase or ccllulase (18). Nierenstein (15) found that tannase is produced 
by the larvae of the sawfly, Pontania proxima, which produces galls on 
Salix caprea. 

E. H. NEWCOMB 4^9 

Lewis and Walton (8), in a study of the formation of the cone gall of 
witch hazel leaves caused by an aphid, found that the stem mother injects 
into the young leaf cells minute drops of a substance "initiating, stimu- 
lating, and directing development and differentiation." Globules con- 
taming a crystalloid were injected by the proboscis into the cytoplasm, 
from which they entered the nuclei and then the nucleoli. There the 
crystalloids broke up into smaller bodies. For gall formation to continue 
normally, repeated injections of additional material were required. 

Boysen Jensen (3), in reporting on experiments with larvae of a midge 
forming galls on beech leaves, interpreted his results to indicate the lack 
of a special gall-forming substance. Larvae placed on lanolin smeared on 
a leaf caused cell division, although when lanolin on which larvae had 
been held was smeared on a leaf, cell elongation resulted. It was assumed 
that gall formation is caused by the larva, which is guided by instinct 
to secrete substances similar to growth substances in definite places and 
in different concentrations on the leaf, thus causing the latter to produce 
the characteristic gall structure. 

Due to the minuteness of the gall-insects and the extremely small 
amounts of growth substances which they could possess or inject, the 
isolation and chemical characterization of such substances would require 
enormous numbers of insects, and appears to be a formidable problem 
indeed. Also to be remembered is the great difficulty of rearing even a 
few of the frail and ephemeral gall-formers responsible for the highly 
organized prosoplasmas. 

There is another approach to the study of galls, however, which might 
yield information on the nature of the insect stimulus. This is the charac- 
terization of gall metabolism and its comparison with that of the normal 
tissues on which the gall occurs. It is not inconceivable that knowledge 
of the nature and extent of change may throw light on the nature of 
the stimulus. Such comparative data should also aid in the interpretation 
of the role of certain metabolic mechanisms of normal tissue. 

One such difference in metabolism results in the abnormal accumula- 
tion in galls of polyhydroxyphenols or their derivatives or condensation 
products. Galls may contain on a dry weight basis as much as 75 per cent 
tannin, and rarely contain less than 25 per cent (5). Such galls as the 
Aleppo and the Chinese nutgall served for centuries as a source of tannin 
for ink production. Both tannins and anthocyanins, as well as lignin, 
contain polyhydroxy aromatic nuclei. It was suggested by WisUcenus 



(20) that such phenohc groupings arise by the dehydration of fructose. 
For example, the 2,3-enol of fructose (I) would yield pyrogallol (II): 



Yl H j 

ri--'ri -" 


3 HjO 


The close chemical relationship which may exist between tannins and 
anthocyanins is well illustrated by the formulas for the tannin, gambir 
catechin (I), and for cyanidin (II), the aglycone of cherry fruits, cran- 
berries, and so on. 



As regards the precursors of anthocyanins, many workers have sub- 
stantiated the fact that there are more sugars and glycosides in leaves 
high in anthocyanins, and that high anthocyanin content is found 
especially in leaves where the transport of carbohydrate has been im- 
peded by damaged conducting systems. 

In galls such as that caused by the grape phylloxera (to be discussed 
later), the numbers of chloroplasts are greatly reduced so that local 
carbohydrate production must be small. Yet such galls are rich in 
starch grains in certain areas, and are high in polyphenolic compounds 
which presumably arise at the expense of carbohydrate. Possibly the 
developing galls are regions into which sugars move from the photo- 
syntheiically more active normal leaf tissue. Pertinent to this point of 
view is Molliard's (12) analysis of elm leaf galls produced by two species 
of plant lice. The galls were higher in reducing sugars than the normal 
leaf tissues, and were four times as rich in tannin. 

Another parallel between anthocyanins and tannins lies in the correla- 
tion of high anthocyanin or tannin content with high oxidase activity. 
In general, the distribution of oxidase activity in flowers coincides 
exactly with that of the anthocyanin pigments. According to Armstrong 

E. H. NEWCOMB 4-^ 

(i) the petals of certain flowers which have been investigated show the 
greatest epidermal oxidase activity in the most deeply colored varieties, 
less in the less deeply colored, and none at all in the white varieties. 

Higher oxidase activity in galls than in unaffected tissue has been re- 
ported by several workers. Using the benzidine reaction. Parr (i8) 
found much greater activity of oxidases in the gall tissue than in the 
normal oak stem tissue. The oxidases increased in activity in the gall dur- 
ing the growth period and then diminished. With decreasing oxidase 
activity in a region of the gall, the tannin content of the region increased. 
MoUiard (12) reported increased laccase and tyrosinase activity in two 
elm leaf galls. Quantitative data obtained by the writer, presented in 
detail below, show a several fold increase of tyrosinase activity in the 
grape phylloxera gall during its development. 

Increased oxidase activity has also been shown for bacterial galls. 
Nagy and Riker (13) found that the oxidase, catalase, and peroxidase 
activity of tomato crown gall tissue is 130, 160, and 120 per cent greater, 
respectively, than for the contiguous tissue, on a fresh weight basis. 

Nierenstein has discovered that the striking colorations of insect 
galls are due to glycosides of a derivative of gallic acid, itself a constituent 
of tannin. It had been assumed that the gall pigments are anthocyanins, 
but in 1919 Nierenstein (14) reported that the pigment of a Cynipid gall 
on oak leaves is a glycoside of purpurogallin. The latter, which had not 
been reported elsewhere in nature, has the following formula: 

Nierenstein and Swanton (16) have shown that the pigments of a large 
variety of galls, caused by such diverse organisms as insects, mites, 
roundworms, and fungi, are all purpurogallin glycosides. All glycosides 
investigated yielded two glucose residues on hydrolysis. Since four 
biglucoside isomers are possible by attachment to one or another of the 
four hydroxyl groups, and two glucose residues can occur on each of 
two different hydroxyl groups in six different combinations to give six 
additional isomers, there are ten possible isomers. Eight of these were 
isolated from galls, crystallized, and distinguished chiefly on the basis 
of differences in melting points. 


Nierenstein proved that Cynipid larvae secrete diastase, invertase and 
tannase, and postulated that the gallotannin of oak galls precipitates 
the first two enzymes while the tannase hydrolyzes tannin to gallic 
acid. Pyrogallol was presumed to arise from gallic acid by decarboxyla- 
tion, and to be oxidized to purpurogallin by enzymes secreted by the 
larvae, the purpurogallin then being deposited in the gall as the glycoside. 
Although it is highly improbable that the purpurogallin, distributed 
throughout the gall or frequently in the superficial layers of the rind, 
arises through the action of enzymes secreted by the localized parasite, 
its widespread occurrence in and confinement to galls and its close 
relationship to gallotannin emphasizes the metabolic derangement in 
galls resulting in the elaboration of polyphenolic compounds. In con- 
firmation of Nierenstein and Swanton's generalization as to the nature 
of gall pigments, the writer has isolated the purpurogallin glycoside, 
dryophantin, from galls produced by two species of Cynipids not ex- 
amined by these authors, the acorn plum gall on black oak caused by 
Amphibolips primus and the leaf gall on white oak produced by Xysto- 
teras poculmn. 

The respiration of two insect galls, the large oak apple produced by 
Amphibolips cotiflitens on leaves of red oak {Quercus borealis var. maxima), 
and the grape phylloxera gall produced by Phylloxera vitifoliae on Vitis 
vidpina, has been compared with that of the normal leaf tissue. 

On a dry weight basis the rate of respiration of oak apple rind tissue 
ranges from about three-fourths to one-half of that of normal oak leaf 
tissue. The respiratory quotients of gall and normal tissues are close to 
unity. In both young gall and leaf disks, the respiration is reversibly 
inhibited up to about 50 per cent by such heavy-metal poisons as 
cyanide and azide, but as both galls and leaves age, the respiratory rates 
decline and the respiration becomes insensitive to, or rather, is even 
slightly stimulated by these poisons. The gall respiration becomes in- 
sensitive to the poisons earlier than the normal. The galls die in early 

The grape phylloxera galls are initiated by young aphids on embryonic 
bud leaves. Within 24 hours a depression is produced with hairs at the 
periphery on the upper surface, and within 3 to 4 days the tissue below 
and around the aphid has proliferated greatly, producing a pouch pro- 
jecting below the lower leaf surface and opening onto the upper surface 
through an orifice surrounded by trichomes. The gall reaches its maxi- 

E. H. NEWCOMB 423 

mum size in 12 to 15 days. Its morphology and development have been 
described by Rosen (19). 

The walls and floor of the mature gall are many times thicker than the 
normal lamina, and the mesophyll consists of a disorganized mass of 
much enlarged, irregularly shaped cells. There are few stomata, and an 
almost complete lack of air spaces. The paucity of chloroplasts is 
especially noteworthy and becomes more pronounced as the galls age. 
Although the galls usually occur on leaf veins, their vascularization is 
poor due to the marked hyperplasia and the failure of small veins to 
develop. The expressed saps of both young and old gall and normal leaf 
tissues have pH values of from 3.0 to 3.2. 

The young galls selected for study, about 2 mm. in diameter, were on 
young leaves 2-3 cm. long. They were compared both with normal con- 
tiguous tissue and with that of normal leaves of approximately the same 
age and size. In the study of mature tissues, gall activity was compared 
with that of contiguous normal tissue, and leaves of approximately the 
same size and position on the branches were used. The results in Tables 
I and 2 represent averages of several experiments. 

The respiration of young and old phylloxera galls is compared with 
that of normal leaf tissue in Table i. It is noteworthy that both young 
and old galls have a markedly lower percentage dry weight than the 
normal tissue. Furthermore, as the galls mature they decrease still 
further in percentage dry weight, although the normal tissue increases 
slightly. Consequently, the respiratory rates of gall tissues are higher 
than those of normal tissues on a dry weight basis, although lower on a 
fresh weight basis. 

The respiratory quotients are close to unity, indicating respiration at 
the expense of carbohydrate. Both normal and gall cells ferment under 
nitrogen, the ratio of fermentative to respiratory carbon dioxide being 
0.84:1 and 0.66:1 for young leaves and galls, and 0.68:1 and 0.85:1 for 
old leaves and galls, respectively, on a dry weight basis. While intact 
young galls in air show the same R.Q. as the sUces, old galls, due to thick- 
ness and the impervious epidermis, show a high R.Q. indicating that 
oxygen is limiting the respiration, and that some fermentation is oc- 
curring (Table i). On slicing such galls, it is found that the oxygen 
consumption rises and the R.Q. drops to unity. 

Homogenates of gall and normal tissue were assayed for oxidase ac- 
tivity. Cytochrome oxidase activity could not be demonstrated in the 














CQ = 
< ?« 

















< o 









































>-ic/:)(y) ^ u:) (Tt k^mU 














E. H. NEWCOMB 4^5 

homogenates of either young or old tissue since, when either ascorbic 
acid or /7-phenylenediamine was used as a substrate, the addition of 
cytochrome c did not result in an increased oxygen consumption. On 
the other hand, ascorbic acid oxidase and tyrosinase activity were found 
in normal and gall material of all ages. The presence of ascorbic acid 
oxidase was shown by the vigorous oxygen consumption supported by 
ascorbic acid. While laccase can also oxidize ascorbic acid, the inactivity 
of the homogenates toward hydroquinone indicated its absence. Tyro- 
sinase activity was shown by the oxygen consumption on a variety of 
poly- and monophenols known to be attacked by this enzyme. Catechol- 
ase, while not attacking monophenols, can oxidize the polyphenols 
employed, but its presence in any considerable amount seems unlikely 
because of the agreement between trends in rates of mono- and poly- 
phenol oxidation for material of different ages (Table 2). 

When gall and normal leaf tissues are compared, the trends in levels of 
ascorbic acid oxidase and tyrosinase activity as the tissues mature are 
striking (Table 2). Ascorbic acid oxidase activity is greater on a dry 
weight basis in both young and old galls than in leaves. As both gall 
and normal tissues age the activity decreases considerably. 

Tyrosinase activity decreases greatly during the maturation of normal 
leaf tissue. But whereas tyrosinase activity in young galls is only about 
one-half that of young leaves, it is approximately doubled in old galls, 
and exceeds that of mature leaves several fold. The significance of this 
increase in the amount of an enzyme acting on polyphenolic compounds 
which takes place as the galls age is not yet clear. The fact may be 
recalled, however, that such compounds are capable of functioning as 
H-carriers with tyrosinase. Whether one or both of these oxidases par- 
ticipate as terminal oxidases in the respiration of these tissues is not 
certain. Both are copper-containing enzymes sensitive to such poisons as 
8-hydroxyquinoline and allylthiourea. Inhibition of leaf respiration by 
these poisons is only partial (about 30 per cent for 5 x iq-^ M allylthi- 
ourea for both young and old tissue), while the gall respiration appears to 
be insensitive to both poisons. 

The disparity in the trends which occur in the activity of the two 
oxidases as the gall and normal tissues mature suggests the possibility 
that the two enzymes are not associated in the same types of cytoplasmic 
particles and that there is a differential rate of multiplication of the latter. 
Comparison of the types and numbers of plastids and mitochondria and 








































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E. H. NEWCOMB 4^7 

of the distribution of activity of the two oxidases between such cellular 
entities in both normal leaf and gall cells is now in progress. By centrifu- 
gation of homogenates of normal leaf tissue, the ascorbic acid oxidase 
activity has been found associated entirely with the fraction consisting 
of insoluble cell debris and unfragmented chloroplasts and nuclei. No 
ascorbic acid oxidase activity was found associated with chloroplast 
fragments, mitochondria, or smaller particles or soluble enzymes. 

Such shifts in levels of enzyme activity may be due directly to the 
effects of substances injected by the insect, or to environmental modifi- 
cations resultant fiom early changes, such as the increase in tissue 
thickness resulting in reduced oxygen tension within the tissue, poor 
vascularization, and so on. No data have been obtained for that interest- 
ing stage when the first cells affected by the aphid are dividing and dif- 
ferentiating to produce a recognizable gall. The earliest stages studied 
were of galls already possessing typical form and anatomy. 


1. Armstrong, E. F., Chemistry in the Twentieth Century (E. Benn, Ltd., 

London, 1924). 

2. Beck, E. G., Am. J. Botany, 33(3) :14s (1946). 

3. BoYSEN Jensen, P., Physiologia Plantarum, 1:95 (i94^)' 

4. CosENs, A., Trans. Canad. Inst., 9:297 (1912). 

5. GoRTNER, R. A., Outlines of Biochemistry, (Wiley, 1949), 3d. ed. 

6. KiJsTENMACHER, M., Jahrb.f. Bot., 26:82 (1894). 

7. KusTER, E., Die Gallen der Pflanzen (Leipzig, 1911)- 

8. Lewis, L F. and Walton, L., Science, 106:419 (1947). 

9. Magnus, W. Die Entstehung der Pflanzengallen verursacht durch Hymenop- 

teren (Jena, 191 4). 

10. Martin, J. P., Hawaiian Planters' Rec, 42:129 (1938). 

11. , Science, 96:39 (1942). 

12. Molliard, M., Rev. Gen. Bot., 25:341 (1913). 

13. Nagy, R„ Riker, a. J., and Peterson, W. H., /. Agr. Res., 57:545 (1938)- 

14. Nierenstein, M., /. Chem. Soc, 115:1328 (1919). 

15. , Nature, 125:348 (1930). 

16. , and Swanton, A., Biochem. J., 38:373 (1944). 

17. Pantanelli, E., Staz. Sperim. Agr. Ital., 42:305 (1909). 

18. Parr, T. J., Yale U. School of For. Bull., 46:1 (i94o)- 

19. Rosen, H. R., Am. J. Botany, 3:337 (1916). 

20. WiSLiCENUS, H., Papier-Fabr. {Fest- und Auslandshefi), 31:65 (1933). 

Vitamins and Amino Acids 
as Growth Factors 

Growth Factors in Bacterial Nutrition 


THE term "growth factor" has been used in a variety of wavs. In 
the discussion to follow we shall use it to mean those organic factors, 
exclusive of the compound or compounds utilized as an energs' source, 
which are required lor growth of an organism in a given environment. 
This definition excludes, on purely arbitrary grounds, consideration of 
the inorganic nutrition of bacteria or of the variety of compounds which 
these microscopic plants may use to supply their requirements for 


Bacteria vary widely in their requirements tor growth factors. Thus 
certain of the photosynthetic autotrophs — for example, members of 
the Thiorhodaceae — are similar to the higher plants in that they grow 
well in appropriate mineral media with light as their energy* source and 
carbon dioxide as their sole source of carbon. Such organisms require 
no growth factors in the sense defined above. The chemosynthetic 
autotrophs, such as Thiobadlliis thiooxxdans, and the simpler hetero- 
trophs, such as Escherichia coli, hkewise require no growth factors in this 
sense of the term, althouijh thev oxidize either inorganic or orcranic 
materials to supply energy for synthetic purposes. A great many bacteria 
do, however, require organic materials other than an energy* source for 
growth, and many organisms which do not require a specific growth 
factor mav be greatlv stimulated bv it. 

\\'e may now examine the nature of the compounds which have been 
found to be essential growth factors for one or more species of bacteria. 
It will be seen from Table i that these represent almost all of the known 
water-soluble vitamins, most of the common amino acids, and a variety 
ot miscellaneous biologically important compounds. The striking thing 
about this hst is that with one or two exceptions to be considered later, 



Summary of compounds recognized as growth factors for various bacteria* 

Amino acids 

Vitamins " 

Purine and pyrimidine 


L-aspartic acid 
L-glutamic acid 







/7-Aminobenzoic acid 

Folic acid 

Nicotinic acid 

Pantothenic acid 






Vitamin Be (pyridoxal 

Vitamin B12 
Vitamin K 

Conjugated vitamins 

Coenzyme I or II 
Pyridoxamine phosphate 
Thiamin pyrophosphate 

Fatty acids 

Acetic acid 
Oleic acid (or other 
unsaturated acid) 






Nucleosides and 


(several, e.g. 




Putrescine (spermine 
or spermidine) 

Unidentified factors 

(See Table 3) 

*Appr()priate fragments or bound forms of these growth factors may nlso satisfy 
the nutritional requirements of certain bacteria. 


all of the compounds in it are of recognized importance for all forms of 
life so far examined. Each of them, so far as we now know, is contained 
in the protoplasm of every living organism, be it plant, animal, or 

Since these compounds seem to be present generally in all protoplasm, 
it is clear that those organisms which do not require them as nutrients 
must synthesize them, and that bacteria which require a preformed 
supply of one or more of them in the medium must, for some reason, 
be unable to synthesize those which they require. 

This idea that the growth-factor requirements of microorganisms arose 
through loss of the ability to synthesize substances of importance in the 
metabolism of all organisms was given formal expression independently 
by Lwoff (19,20) and by Knight (13) about fifteen years ago, while 
knowledge of bacterial nutrition was still fragmentary. These authors 
envisaged a physiological evolution from the primitive and self-sufficient 
state of autotrophism toward complete parasitism, brought about by 
successive losses in the synthetic capacities of microorganisms. Subse- 
quently Beadle and Tatum (2) showed that by irradiation of Neurospora 
with X-rays or ultraviolet light, mutants could be produced which 
showed added nutritional requirements (see also 1,3). The work of 
Lederberg and Tatum (17,18) and of others (16) showed that similar 
nutritional mutants could be produced in bacteria, and that the nutritive 
requirements of such mutants were for the same growth factors as had 
been previously shown to function in the nutrition of natural populations 
of bacteria. Through these developments, the theories of Lwoff and of 
Knight concerning the origin of nutritional requirements in micro- 
organisms have been given a sound experimental basis. We may now view 
the nutritional requirements of natural populations of bacteria as having 
arisen by successive mutations with cumulative effects to produce 
organisms having requirements of varying complexity. 

Since mutation is a random phenomenon it might be expected that a 
requirement for growth factors would be found at random among a 
great variety of bacterial species, and that the identity and number of 
the growth factors required would vary widely from one species ot 
bacteria to another, and even from one to another strain within a single 
species. Such is indeed the case. As one example, the requirements ol 
various species of the genus Clostridium may be cited. Clostridium 
butylicum requires only the single growth factor, biotin, for growth in 



a glucose-salts medium (37). The closely related Clostridium acetobuty- 
licum requires biotin and /7-aminobenzoic acid for growth under similar 
conditions (15), whereas Clostridium perfringens requires several different 
vitamins (pyridoxamine, biotin, riboflavin, and pantothenic acid), 
adenine and uracil, and thirteen different amino acids for growth (5). 
Somewhat similar variations, if not so marked, may be found in the 
requirements of different representatives of other groups of bacteria, 
for example, the lactic acid bacteria, some of which have nutritive 
requirements which surpass those of Clostridium perfringens in complexity 

(24.33) • 

It would be of little interest here to tabulate the growth-factor re- 
quirements of various bacteria which have so far been investigated. For 
each organism such a tabulation would show one, a few, or many of the 
compounds listed in Table i as being required. Summary articles which 
contain this valuable information have appeared (14,24,33). Instead, 
certain other aspects pertinent to this problem may be discussed. 

The nature of the response to an essential growth factor is shown in 
Figure i, which depicts the response of Lactobacillus casei to additions 
of riboflavin. Within hmits, the amount of growth is dependent only 
upon the concentration of the vitamin. If, at these growth-Hmiting 
concentrations, one measures the amount of the vitamin present in the 
medium and the cells after growth has ceased, it is found that all of the 
vitamin (within experimental error) has been absorbed from the medium 


0.05 0.1 

0.15 0.2 0.25 0.3 0.35 0.4 

0.45 0.5 055 


Figure i. Relationship between the concentration of riboflavin and growth 
of Lactobacillus casei. 


(35), and most of this can be lecovered from the cells. This behavior 
typifies that of other essential growth factors. They are essential com- 
pounds for formation of protoplasm and as such are absorbed from the 
medium with great efficiency and incorporated into the cell. Many 
other closely related lactic acid bacteria synthesize the riboflavin which 
they require (36); L. casei does not and must have it preformed for 

In the past the inability of an organism to synthesize a growth factor 
has been most frequently explained by assuming that the mutation which 
gave rise to the nutritional requirement eliminated one of the essential 


The comparative requirement of H. parainfluenzae for purine bases in the 
presence and absence of guanine (9) 

Additions to basal medium* Turbidity f 

None 70 

Guanine hydrochloride 94 

Hypoxanthine 73 

Guanine hydrochloride + hypoxanthine 74 

Guanine hydrochloride + adenine sulfate 70 

*ioo M^g. of the indicated compound to each 10 ml. of medium. 

tPer cent of incident light transmitted, uninoculated medium = 100. 

enzymes concerned in synthesis of the growth factor. Thus we might 
suppose that L. casei lacked one of the enzymes necessary for ribo- 
flavin synthesis. So far as is now known this is a sufficient explanation 
for this case. In many cases, however, this explanation does not suffice. 
Several examples are now known where a bacterium synthesizes a given 
growth factor in one medium but does not do so in another. For 
example (Table 2), if Hemophilus parainfluenzae 7901 is cultured in a 
defined medium which contains guanine, it must be supplied with either 
hypoxanthine or adenine to permit growth (9). The simplest explanation 
of this finding assumes that hypoxanthine (or adenine) cannot be syn- 
thesized under these conditions and consequently must be supplied to 
permit synthesis of nucleic acid, which is in turn necessary for growth. 
Yet if guanine is omitted from this medium the organism grows well 
in the absence of both hypoxanthine and adenine and must under these 
conditions synthesize its own purine bases. Failure to synthesize adenine 


(or hypoxanthine) in the medium which contains guanine apparently 
results not from absence of the appropriate enzyme but from inhibition 
of some step in the synthesis by guanine. Beerstecher and Shive (4) 
found, similarly, that tyrosine inhibited growth of a strain oi Escherichia 
coli under appropriate conditions and that this inhibition was alleviated 
by phenylalanine. Apparently tyrosine inhibited synthesis of phenyla- 
lanine by this organism, a synthesis which occurred without difficulty 
when tyrosine was omitted from the medium. 

A particularly clear-cut example of a nutritional requirement which 
results from an inhibition rather than from lack of the enzymes necessary 
to carry out a synthetic process is provided by the yeast, Saccharomyces 
carlsbergensis. Under ordinary cultural conditions this yeast requires 
vitamin Be for growth, and its growth response to additions of this vita- 
min has been the basis for a widely used method for determining vitamin 
Be- Yet if thiamin is omitted from the culture medium and the inoculum 
has been grown in a thiamin-low medium, the organism grows well in 
the complete absence of vitamin Be (Fig. 2), and analyses of the cells 
have shown that vitamin Be is synthesized under these conditions (25). 
Addition of rather small amounts of thiamin — a substance which is itself 
required in metabolism and is normally present in this yeast — inhibits 
growth, and under these conditions vitamin Be is required to permit 
growth (Fig. 2). How thiamin inhibits growth and how this inhibition 
is counteracted by vitamin Be are both unknown. The important point 
is, however, that a growth factor which under one set of conditions is 
synthesized in amounts sufficient for growth cannot be thus synthesized 
under another slightly different set of conditions, or is synthesized in 
amounts insufficient for growth. Thus the nutritional requirement for 
the growth factor results from an inhibition rather than from lack of the 
enzymes to accomplish a given synthesis. 

From these examples it is evident that the failure of an organism to 
synthesize an essential growth factor may result either from lack of the 
necessary enzymes to effect the synthesis, or from inhibition of one or 
more synthetic processes by metabolites normally present within the 
cell, or added with the medium. In general terms, if b is essential for 
synthesis of protoplasm and is normally formed from certain precursors, 
a, by an enzymatic process, then the synthesis: 

a > b 

may be eliminated either by lack of one of the necessary enzymes or by 



the presence of an inhibitor for the process, and in either case b will 
appear as a nutritive essential for growth, since it can no longer be 
formed within the cell or is formed in amounts insufficient for growth. 
The diagram represents only one mechanism through which an inhibitor 
may operate to make a previously nonessential growth factor become 
essential for growth. Several others can be readily visualized. 

0123456 8 10 12 


Figure 2. Inhibition of growth of Saccharornyces carlsbergensis 4228 by 
thiamin and its reversal by vitamin Be- Curves i, 2, 3, and 4 represent the 
growth response to pyridoxal in the presence of 0, o.i, i, or 107 of thiamin, 
respectively, per 6 ml. of medium (25). 

This latter explanation for the occurrence of certain nutritive re- 
quirements in bacteria has been insufficiently emphasized in the past 
apparently because of a disinclination to admit that normal cellular 
metabolites might act as inhibitors for certain metabolic reactions. Yet 
several such cases are now known as the few instances cited above 
illustrate. The concept is novel only in viewing the inhibitor as a sub- 
stance normally present within the cell. It has been recognized for several 


years that certain drugs act in this fashion. The sulfonamides, for 
example, competitively inhibit transformation of /^-aminobenzoic acid 
to one or more essential catalysts in those bacteria which they inhibit. 
Their effects can be overcome either by addition of /7-aminobenzoic 
acid, which acts competitively to overcome the effects of the drug, or by 
a variety of other substances (folic acid, thymine, purine bases, serine, 
methionine), which act noncompetitively (26,44). Each of these sub- 
stances might be considered a new growth factor required by the cells 
under conditions where a single essential reaction has been inhibited, 
in this case by an inhibitor (a sulfonamide) foreign to the cell. 

This concept of the origin of some nutritional requirements presents 
several interesting possibihties. Examples of "nutritional symbiosis" 
(possibly related to true symbiosis), in which each symbiont supplies by 
synthesis an essential nutrient for the other symbiont, are easily observed 
experimentally (29). Perhaps a new type of nutritional symbiosis, in 
which one symbiont metabolizes to its own advantage a product elabo- 
rated by the second symbiont and which would otherwise inhibit growth 
of the latter by preventing essential synthetic reactions, may be observed. 
Perhaps the toxicity of some types of organic matter for certain auto- 
trophs may be exerted in this fashion. This point of view should also 
make possible a different approach to the discovery of new growth 
factors. For if many nutritional requirements arise naturally through 
the inhibition of essential synthetic reactions, then one might purposely 
inhibit growth of bacteria with all types of organic compounds. Wherever 
these toxic effects could be reversed by addition of natural materials, 
the possibility would present itself that synthesis of some metabohcally 
essential compound, supplied by the natural material, was the process 
limiting growth in the presence of the inhibitor. Isolation of the com- 
pound or compounds effective in the reversal might then bring to light 
new compounds of importance in metabolism. It was in this way that 
/?-aminobenzoic acid was isolated; a naturally occurring substance which 
overcame the toxic action of sulfanilamide was observed (45), and on 
isolation, proved to be /^-aminobenzoic acid (27). More recently Shive 
and coworkers (30) isolated thymidine by this approach. This compound 
proved to be one of the naturally occurring substances which prevented 
the toxic action of 7-methylfolic acid for Escherichia coli. The approach 
has never been systematically exploited, however. 

We have seen that growth of Saccharomyces carUbergensis 4228 is 

















^ 40 











5 60 

















9 80< 




0.2 04 0.6 0.8 1.0 

I I I 1 I 

0.4 0.8 1.2 1.6 2.0 


Figure 3. Comparative growth promoting action of pyridoxal and neo- 
pyrithiamine for S. carlsbergensis 4228. 

inhibited by small amounts of thiamin and that this inhibition is over- 
come by small amounts of vitamin Be. In many other organisms the 
growth-promoting effects of thiamin can be counteracted by the anti- 
vitamins, pyrithiamine (46) or neopyrithiamine (43). These products are 
antivitamins which are so closely related in structure to thiamin (see 
the accompanying formulas) that although they cannot duplicate the 
physiological function of the latter they do interfere with these functions 
in a competitive manner. It was therefore of interest to determine what 
effect neopyrithiamine would have on an organism for which thiamin 
was a growth inhibitor. Here too, the product counteracted the effect 
of thiamin, that is, it promoted growth of S. carlsbergensis (Fig. 3) in 
the same way as did pyridoxal (25). 


N^ NH. 


CHf ^C— S 






I 3 / 2 2 


+ X=C, 


H H 


This result is of considerable interest, for it illustrates how a substance 
which is entirely foreign to metabolic systems may simulate the action 
of a true growth factor. Indeed, if these effects had been first observed 
in a natural medium, and if the effect of vitamin Be had not been known, 
neopyrithiamine might well have been considered as a true growth 
factor. It should not be considered as such despite its growth-promoting 
properties under these conditions, since it plays no role in the normal 
metabolism of this or other organisms. 

Other somewhat similar examples are known. Lactobacillus bulgaricus 
for example, requires oleic acid (or other unsaturated fatty acids) for 
growth, but these acids are highly inhibitory to growth at higher con- 
centrations (41). Thus in a medium which contains a considerable 
quantity of free fatty acid no growth occurs. Addition of a synthetic 
wetting agent and surface-tension depressant, Tween 40, will now permit 
growth (Fig. 4) simply because it eliminates the toxicity of oleic acid 
without eliminating its growth-promoting properties (41). Again, if the 
mechanism of the effect were not known Tween 40 might be considered 
a true growth factor, although it obviously should not be so considered. 
These examples are from natural populations. That entirely similar 
phenomena occur in artificially induced mutants is shown by the isola- 
tion of a mutant culture of Neurospora which appeared to require 
sulfonamides for growth. The mutant apparently synthesized /J-amino- 
benzoic acid in amounts which indirectly inhibited growth; and by 
counteracting this action of /J-aminobenzoic acid the sulfonamides pro- 
moted growth of this organism (47). Although a sulfonamide is thus 
required for growth of the organism, it can scarcely be considered as a 
true growth factor in the sense that the various compounds of Table 1 
are growth factors. Recent reports that certain streptomycin-resistant 
mutants of bacteria come to require streptomycin for growth may have 
a similar explanation. 

These examples of substances foreign to normal growth processes 
which come to behave as growth factors can be readily explained once 
the view is accepted that substances normally present in growing cells, 
and themselves essential in small amounts for growth, may under certain 
conditions act as inhibitors of other essential metabolic reactions. It then 
becomes understandable how such inhibitions can be alleviated either 
by appropriate normal metabolites (for example, the products of the 
inhibited reactions within the cell), or by appropriately fashioned anti- 



metabolites, which by competing with the toxic metabolite will prevent 
its toxic action within the cell and thus permit growth. 

We have seen that an organism may synthesize a growth factor in 
one medium which it requires preformed in another. It is also true that 


20 40 60 80 



Figure 4. The comparative growth-promoting properties of oleic acid for 
Lactobacillus bulgaricus in the presence (top curve) and absence (lower curve) 
of Tween 40 (cf. 41). 

the magnitude of the requirement for an essential growth factor may 
vary tremendously, depending upon the composition of the medium, 
and under conditions such that the variation in the requirement cannot 
be ascribed to variations in the amount of the growth factor synthesized. 
Such variations frequently reflect the physiological function of the 
vitamin, and though more experimental work should be done to estabhsh 



the detailed mechanism of such effects, an explanation which undoubt- 
edly holds in many instances may be summarized as follows. 

It may be supposed that a bacterium requires a vitamin to carry out 
a number of catalytic functions necessary for growth. For example, 
the vitamin may be necessary for synthesis of a number of different 
essential components (Pi, P2, P3, etc.) of protoplasm, from their respec- 
tive precursors (pi, p2, ps) as indicated in the accompanying diagram. 




It is logical to assume that if a medium supplies one or more of the 
products (Pi, P2, etc.) preformed, the metabolic requirement for the 
vitamin involved in synthesis of these products might be greatly reduced 
as compared with the requirement in a medium which did not supply 
these products. 

Such relationships apparently explain the data of Figure 5. It will be 


O tl +2 


Figure 5. The effects of serine, and of purine bases and thymine on the 
requirement of Streptococcus faecalis for folic acid. Curve i, serine, thymine, 
and purine bases present. Curve 2, purine bases and serine present, thymine 
omitted. Curve 3, purine bases present, thymine and serine omitted (6). 


seen that the foUc acid requirement of Streptococcus faecalis varies re- 
markably with the nature of the medium. In a medium which lacks serine 
the folic acid requirement is over ten times that observed in the same 
medium with added serine. This is interpreted to mean that folic acid 
is required in relatively large amounts for synthesis of serine, and if 
serine is supplied preformed, then substantially smaller amounts of 
folic acid suffice to fulfill its remaining functions (6,ii). If to the 
medium which contains serine, thymine is now added, the folic acid 
requirement completely disappears (6,38). Under the latter conditions 
no folic acid can be detected in the cells (58). Apparently the folic acid 
requirement observed in curve 2 represents that required for synthesis 
of thymine by these cells, and when thymine is supplied preformed the 
requirement for this folic acid no longer exists. Traces of the vitamin 
may still be required for other purposes, but if so, these traces can be 
synthesized by the cells and are insufficient in magnitude to be detected 
by present methods of assay- 
Several other instances of similar effects are known which will not be 
given in detail. The vitamin Be requirement of lactic acid bacteria, for 
example, is greatly increased by omitting certain nonessential amino 
acids from the medium (21) and can be eliminated entirely for some 
lactic acid bacteria bv the addition of D-alanine to a medium which 
contains a complete assortment of L-amino acids (32,10). Vitamin Be 
is apparently required by these organisms primarily to permit synthesis 
of amino acids (both l and d) which are essential for synthetic process 
within the cell, and when all of these amino acids are supplied preformed, 
the vitamin Be requirement is reduced to the point where it can no longer 
be detected (10). Here, too, analyses have shown that the cells do not 
synthesize increased amounts (if any) of the vitamin under conditions 
where they grow without it. 

In a wholly analogous fashion the biotin requirement of many lactic 
acid bacteria can be eliminated by addition of aspartic acid and oleic 
acid (39,41), and the vitamin B12 requirement by appropriate desoxy- 
ribosides or reducing agents (12,31). Similar explanations for these 
results may hold, although further detailed investigations are necessary 
to establish the mechanism of these effects. It will be apparent from the 
above examples, however, that a given bacterial species does not neces- 
sarily require a fixed and unchangeable assortment of growth factors, 
but that different combinations, both quantitatively and quaUtatively, 


may suffice to permit growth by supplying the same deficiencies through 
different mechanisms. 

In the above discussion the attempt has been made to give, at least 
in outline, the present status of research in bacterial nutrition. The 
valuable leads that a study of the interrelationships between these 
nutrients is providing the biochemist should be apparent. The utility of 
bacteria as test organisms in the microbiological determination of the 
substances which they require for growth is also well known. One of 
the most important of the gains to be derived from a study of bacterial 
nutrition is the recognition of new substances which function as essential 
growth factors, for such substances have, in the past, always proved to 
be substances of general importance in metabolism. For example, the 
recent discovery that D-alanine (10,32) and putrescine or the related 
compounds, spermine and spermidine (8), are essential for growth of 
some bacteria lends new importance to these long-known compounds. 
It had not been previously known whether any of the D-amino acids 
played essential roles in metabolism; it now appears most certain that in 
some bacteria, at least, D-alanine is essential for growth (10). Similarly, 
although putrescine had long been known as a decomposition product 
of arginine, it was not known to play any essential role in metabolism. 
Such a role now seems certain from the observation that the compound 
serves as an essential growth factor for Hemophilus parainjliienzae (8). 

Elucidation of the chemical nature of the several unidentified growth 
factors reported as essential for growth of various species of bacteria may 
similarly be expected to contribute materially to our knowledge of bio- 
chemistry and metabolism, for these unidentified substances, like the 
growth factors of Table i, are generally distributed in natural materials 
and undoubtedly have general metabolic significance. Several of the 
better defined unidentified growth factors are listed in Table 3. 

In summary, we have emphasized that the growth factors required 
by bacteria comprise those compounds, such as the amino acids and the 
water-soluble vitamins, which are of general importance as essential 
metabolites in all Jiving organisms. A nutritional requirement for one, 
several, or many of these growth factors may arise through cumulative, 
random mutations which result in the loss by the organism of the capacity 
to synthesize the growth factors which it requires under a given set of 
environmental conditions. This inability to synthesize a growth factor 
may result either from loss of one of the enzymes necessary for its 


Some bacteria which require unidentified growth-factors 

Organism Source Material Reference 

Leuconostoc citrovorum Liver extracts Sauberhch and Baumann (28) 

Lactobacillus bulgarictis Yeast WilUams, Hoff-Jorgensen, and 

Snell (42). 

Lactobacillus casei* Yeast, grass Guirard, Snell, and Williams (7) 

Streptococcus faecalis* Yeast, liver McNutt and Snell (22) 

Tetrahymena geleii* Liver, yeast Stokstad, et al. (40) 


*Cross-testing of concentrates of protogen and of the pyruvate oxidation factor 
of O'Kane and Gunsalus (23) on these various organisms indicate that these 
various factors are idendcal (34). 

synthesis, or from inhibition of the synthesis at one or another stage by 
other metaboUtes normally present within the cell or the medium. The 
latter occurrence is viewed as being relatively frequent and has been 
insufficiently emphasized in the past. It permits a ready explanation 
of the observation that substances such as neopyrithiamine and the 
sulfonamides, which ordinarily act as growth inhibitors, may with occa- 
sional organisms simulate the action of true growth factors. It also 
explains in many cases the fact that a given organism may synthesize a 
growth factor in one medium and require it preformed in another. 
The "sparing action" which certain growth factors may have on the 
requirement of bacteria for other growth factors has also been discussed. 
Such sparing actions are evidence for a metaboUc relationship between 
the growth factors involved and are occasionally of such magnitude that 
the requirement for a specific growth factor may apparently be com- 
pletely ehminated. Thus in some instances more than a single combina- 
tion of growth factors may suffice to permit growth of a given organism, 
although under appropriate conditions each component of each com- 
bination may be essential for growth. 


1. Beadle, G. W., Chem. Rev., 37:15 (1945)- 

2. , and Tatum, E. L., Proc. Natl. Acad. Sci., 27:499 (1941). 

3. , Am. J. Botany, 32:678 (1945)- 

4. Beerstecher, E., Ir. and SmvE, W., /. Biol. Chem., 167:527 (1947). 

5. Boyd, M. I., Logan, M. A., and Tytell, A. A., ibid., 174:1013 (1948) 

6. Broquist, H. p. and Snell, E. E., Unpublished data. 


7. GuiRARD, B. M., Snell, E. E., and Williams, R. J., Arch. Biochem., 

9:381 (1946). 

8. Herbst, E. J. and Snell, E. E., /. Biol. Chem., 176:989 (1948). 

9. , J. BacL, 58:379 (1949). 

10. Holden, }. T. and Snell, E. E., /. Biol. Chem., 178:799 (1949). 

11. Holland, B. R. and Meinke, W. W., il?id., 178:7 (1949). 

12. KiTAY, E., McNuTT, W. S., and Snell, E. E., ibid., 177:993 (1949). 

13. Knight, B. C. J. G., Bacterial Nutrition {Med. Research Council Special 

Report Series 210, London, 1936). 

14. , Vitamins and Hormones, 3:105 (1945). 

15. Lampen, J. O. and Peterson, W. H., Arch. Biochem., 2:443 (^943)- 

16. Lederberg, J., Heredity, 2:145 (1948). 

17. , and Tatum, E. L., Nature, 158:558 (1946). 

18. , /. Biol. Chem., 165:381 (1946). 

19. LwoFF, A., Ann. Fermentations, 2:419 (1936). 

20. , L' Evolution physiologique. Etude des pertes de fonctions chez les 

microorganisms (Herman et Cie, 1943). 

21. Lyman, C. M., Mosely, O., Wood, S., Butler, B., and Hale, P., 

/. Biol. Chem., 167:177 (1947). 

22. McNuTT, W. S., and Snell, E. E., Unpublished data. 

23. O'Kane, D. }. and Gunsalus, L C., /. Bact., 56:499 (1948). 

24. Peterson, W. H. and Peterson, M. S., Bact. Rev., 9:49 (1945). 

25. Rabinowitz, J. C. and Snell, E. E., Fed. Proc, 8:240 (1949) and un- 

published data. 

26. Rogers, L. L. and Shive, W., /. Biol. Chem., 172:751 (1948). 

27. Rubbo, S. D. and Gilespie, }. M., Nature, 146:838 (1940). 

28. Sauberlich, H. E. and Baumann, C. A.,/. Biol. Chem., 176:165 (1948). 

29. Schopfer, W. H., Ergeb. Biol., 16:1 (1939). 

30. Shive, W., Eakin, R. E., Harding, W. M., Ravel, J. M., and Suther- 

land, J. E.,/. Am. Chem. Soc, 70:2299 (1948). 

31. Shive, W., Ravel, }. M., and Eakin, R. E., ibid., 70:2614 (1948). 

32. Snell, E. E., /. Biol. Chem., 158:497 (1945). 

33. , Physiol. Rev., 28:255 (1948). 

34. , and Broquist, H. P., Arch. Biochem., 23:326 (1949). 

35. Snell, E. E., Guirard, B. M., and Williams, R. }., /. Biol. Chem., 

143:519 (1942)- 

36. Snell, E. E. and Strong, F. M., Enzymologia, 6:186 (1939). 

37. Snell, E. E. and Williams, R. J.,/. Am. Chem. Soc, 61:3594 (1939). 

38. Stokes, }. L.,/. Bact., 48:201 (1944). 

39. , Larsen, a., and Gunness, M.,/. Biol. Chem., 167:613 (1947). 

40. Stokstad, E. L. R., Hoffmann, C. E., Regan, M. A., Fordham, D., 

and Jukes, T. H., Arch. Biochem., 20:75 (1949). 

41. Williams, W. L., Broquist, H. P., and Snell, E. E., /. Biol. Chem., 

170:619 (1947). 

42. , Hoff-Jorgensen, E., and Snell, E. E., ibid., 177:933 (1949)- 

43. Wilson, A. N. and Harris, S. A., /. Am. Chem. Soc, 71:2231 (1949). 

44. Winkler, K. C. and de Haan, P. G., Arch. Biochem., 18:97 (1948)- 

45. Woods, D. D., Brit. J. Exp. Path., 21:74 (i94o)- 

46. WooLLEY, D. W. and White, A. G. C., /. Exp. Med., 78:489 (1943). 

47. Zalokar, M., Proc. Nat. Acad. Sci., 34:32 (1948). 

Genetic Aspects of Growth Responses in Fungi 


THE study of growth factors of fungi may be said to have been 
initiated by the pioneering work of Wildiers with yeast in 1901 
(89). Further investigations of the nutritional requirements of this 
nonfilamentous fungus have contributed outstandingly to the fields of 
nutrition of both microorganisms and higher organisms. These contribu- 
tions began with the identification by Eastcott (19) of meso-inositol as 
one of the bios constituents. The relationship of the nutrition of this 
microorganism to that of higher organisms first became apparent with 
the work of Williams (90), who showed that vitamin Bi was a required 
growth factor. With this information as a starting point, further in- 
vestigations with yeast to date have added two additional new vitamins 
to the list of those important to both microorganisms and higher forms. 
These are biotin (43) and pantothenic acid (91). Shortly after thiamin 
had been demonstrated as a growth factor for yeast this vitamin was 
reported by Schopfer as the first identified essential metabolite required 
by a filamentous fungus, Phycotnyces blakesleeanus (70). Investigations 
with other filamentous fungi initiated by Kogl and Fries (42) and ex- 
tended since then to a great variety of other fungi (25,67) have fully 
substantiated the concept that these organisms require essentially the 
same metabolites as do other forms of life. Strains of fungi are now known 
which as isolated from nature require the vitamins thiamin, biotin, 
pyridoxine, inositol, and nicotinic acid. Although not yet identified as 
a requirement of a filamentous fungus, /^-aminobenzoic acid is required 
by certain yeasts. These include Rhodotorula aurantiaca (68) as well as 
strains of Saccharomyces cerevisiae (63,64). The available information 
thus amply supports the generalization that microorganisms, bacteria 
as well as fungi, and higher organisms have similar requirements for 
most of the vitamins of the B complex. 



Perhaps of equal importance and interest, investigations with various 
fungi have led to the experimental verification of the genetic basis of the 
losses in synthetic capacity which in evolution may have led to require- 
ments for exogenous supplies of essential metabolites. Work in this field, 
initiated with the ascomycete Neurospora crassa (6), has been extended 
during the past several years to a considerable number of other fungi. 
Mutant strains of a number of fungi deficient in the synthesis of vitamins, 
amino acids (as listed in Table i), and nucleic acid constituents, have 
been produced by treatment with a variety of mutagenic agents in- 
cluding radiation, mustard gas, and other chemicals. With the exception 
of the imperfect fungus PeniciUium and the phycomycete Absidia glauca, 
convincing genetic evidence has been obtained that the growth factor 

Requirements of induced mutant strains of fungi 



Amino acid 

Mutagens used 

AND reference 

Neurospora all but B2, choline, all but alanine, X-ray (7, 45, 81) 

biotin* hydroxyproline Ultraviolet (7, 81) 

S-mustard (39) 
N-mustard (48, 52, 
Ophiostoma all but B2, choline, for argmine. X-ray (25) 

thiamin,* Be* lysine, methionine N-mustard (24) 

Caffeine (26) 
Glornerella for nicotinic. Be for tryptophan, 

Penicillimn all but pantothenic, all but alanine, 

Ultraviolet (51) 

X-ray, ultraviolet 


N-mustard (35) 


for pantothenic, 


for Bi, nicotinic 

B2 hydroxyproline, 

serine, glycine, 

threonine, tyrosine 

for histidine, 

lysine, tryptophan 

for arginine, 



for lysine, 
PAB, *pantothenic,*leucine, methionine. Ultraviolet (62) 
biotin* phenylalanine, histi- 

dine, tryptophan 
Coprinus for cystine, N-mustard (23) 


Saccharomyces for nicotinic, Bi, 

Ultraviolet (29) 
Ultraviolet (59) 

N-mustard (64) 

* Requirement of parental stock. 

E. L. TATUM 449 

deficiencies in all these organisms have resulted from mutation of single 

When the various amino acids and vitamins required by strains of 
fungi isolated from nature and of those isolated following treatment with 
mutagenic agents are compared, it Is seen that requirements found only 
in mutant strains of fungi include riboflavin, pantothenic acid, p-amino- 
benzoic acid, and choline. Nicotinic acid has recently been found to be 
required by Blastodadia pringsheimii (i6). Folic acid and vitamin B12 
are the only two B-vitamins which neither strains from nature nor 
mutant strains have yet been found to require. In regard to the amino 
acids, very few strains of fungi found in nature fail to grow on inorganic 
nitrogen or on relatively simple organic nitrogen sources such as aspara- 
gine (67,70). Blastodadia with a requirement for methionine seems an 
exception (17). In contrast, mutant strains of fungi have been obtained 
which have requirements for all of the known amino acids with the 
exception of alanine and hydroxyproline. Experimentally induced de- 
ficiencies for amino acids seem somewhat more frequent than for vita- 
mins. The evidence might be taken to suggest, if mutation in nature is 
qualitatively and quantitatively comparable to that Induced In the 
laboratory, that most fungi In nature find themselves In environments 
where strains with deficiencies for most amino acids and many vitamins 
would be eliminated from the population. This is In contrast to the 
occurrence and survival In nature of strains of bacteria which require all 
of the known vitamins and amino acids with the exception of Inositol, 
choline, alanine, and hydroxyproline. In general bacteria are found in 
more varied and specialized environments than fungi, and they tend to 
have lower quantitative requirements for vitamins and amino acids, so 
that survival of deficient strains would be more likely. 

At present comparisons with a given microorganism of the qualitative 
effects of different mutagenic agents can only be Incomplete. Some 
evidence is available, however, suggesting that with Neurospora similar 
types of mutations are produced by nitrogen mustards, by X-ray, and 
by ultraviolet light, using a number of different strains as well as a 
variety of Isolation techniques (81). This evidence Is consistent with the 
view that mutation of a given gene Is related to the specific lability of 
that gene rather than to the type of mutagenic treatment. According 
to this concept a mutagenic agent only accelerates the normal mutation 
frequency without affecting the quantitative relations between the dif- 


ferent gene mutations produced. The types of deficiency mutations found 
in different fungi under mutational treatment, although similar, have 
been found to vary somewhat from one organism to another. For 
example, in yeast the most frequent deficiencies yet reported are for 
adenine, methionine, leucine, and lysine (62,64). Deficiencies for adenine 
or hypoxanthine, thiamin, nicotinic acid, and reduced sulfur are most 
frequent in Ustilago maydis (59). Deficiencies for arginine, lysine, and 
methionine have been most frequently found in Penicillium (11). In 
Ophiostoma the most common requirements of mutant strains appear to 
be for arginine, purines, and pyrimidines (25). In Neurospora with the 
mutagenic agents, strains, and techniques so far used, the most frequent 
deficiencies have been for methionine, lysine, arginine, and adenine. 
With Absidia glauca the most frequent mutation seems to be for histidine 
(29). If the specific effects of the various treatments and techniques 
involved in the isolation of mutants in these fungi has not resulted in 
selection of particular types of deficiencies, these results would suggest 
that genes controlling different steps in growth-factor biosynthesis in a 
given fungus differ in their stability to mutation, and that those genes 
concerned in particular biosynthetic steps may have entirely different 
labilities to mutation in different organisms. 

Another tentative conclusion regarding the nature of gene control of 
growth-factor synthesis may well be made at this time. Any technique 
of production and isolation of mutant strains of a microorganism which 
does not require carrying the organism through a sexual stage before 
detection and isolation of a mutant stock, would in theory permit the 
detection of nutritional deficiencies controlled by extra-nuclear factors, 
such as those involved in Paramecium (77), and in yeast with adaptive 
sugar utilization (46) and with respiratory systems (20), This condition 
is met with the fungi listed above, including Neurospora in which mutant 
strains derived from asexual uninucleate microconidia (4) have been 
isolated (81), Genetic examination of mutants in the fungi so far obtained 
has failed to indicate the existence of extra-nuclear control of growth- 
factor or metabolite synthesis. Even in Saccharomyces cerevisiae, in which 
extra-nuclear control of sugar utilization has been suggested, recent 
work by Lindegren and Lindegren (47) and by Pomper (62) has shown 
that growth-factor deficiency characters are inherited as if they were 
characterized by single gene changes. We may, therefore, conclude that 
in the fungi so far examined, growth-factor deficiencies in mutant 

E. L. TATUM 451 

Strains, and conversely growth-factor syntheses by normal strains, are 
controlled by genes located on the chromosomes in the nucleus of the 
organism rather than by extra-nuclear factors. It might even be suggested 
that a microorganism which had developed an extra-nuclear system of 
control of growth-factor synthesis might not survive since such an 
extra-nuclear system might be more susceptible to environmental modi- 
fication and accidental loss or destruction. 

Although strains of fungi, either of natural origin or mutant strains, 
are known which require almost all of the B-vitamins which are required 
by bacteria or higher organisms, rather less is known of the exact function 
of these vitamins in fungi than in some other organisms. Although there is 
every reason to believe that the B-vitamins have the same coenzyme 
functions in fungi as they do in other organisms, direct demonstrations 
of this in the filamentous fungi are available in only a few instances. That 
thiamin in the form of cocarboxylase is involved in the respiration of 
fungi is indicated by the accumulation of pyruvic acid in thiamin- 
deficient cultures of Vhycomyces blahesleeanus (32) as well as by the 
stimulating effect of thiamin on the respiration of homogenized Sclero- 
tium delphinii mycelium (61). The situation presumably is similar in 
Nettrospora since carboxylase has been demonstrated in this organism 
(83). Although the involvement of vitamin B2 in the glucose oxidase 
system oi Penicillium has been established (8), and D-amino acid oxidase 
has been demonstrated in Neurospora (37), the participation of riboflavin 
in this enzyme in Neurospora has not yet been established. In Neurospora 
pyridoxal phosphate has been demonstrated to be the coenzyme of the 
tryptophane synthesizing system (86). Very little evidence is available 
in regard to /?-aminobenzoic acid, biotin, and pantothenic acid with the 
exception of an early report by Giese and Tatum (28) that they were 
involved in respiratory systems in Neurospora. Although biotin has 
been shown to be involved in sexual reproduction of certain fungi, 
such as Sordaria fijnicola (3), only two lines of evidence link the action 
of biotin in fungi with its functions in other organisms. These are reports 
that biotin may be spared by oleic acid for Neurospora (36), and by 
aspartic acid for several other fungi (60). 

Investigations with mutant strains of Neurospora may provide a clue 
to the nutritional function of inositol. The effect of inositol on this 
organism was early described by Beadle (5). Limiting amounts of this 
substance have been found to alter profoundly morphogenetic growth 


processes so that a characteristic morphologically limited growth habit 
is assumed. Although a specific antagonism of inositol action by hexa- 
chlorocyclohexane (gammexane) has been suggested by work with yeast 
(41) and with a-amylase (44), attempts to demonstrate such a relation- 
ship in Neurospora have been unsuccessful (80). Recent investigations 
with the pea root (71) and with a variety of bacteria (27) have likewise 
failed to indicate a specific interrelation between inositol and gammexane. 
The morphogenetic effect of inositol in Neurospora may conceivably 
be related in some way, at present unsuspected, with the effects of 
paramorphogenic substances such as tergitol, desoxycholate, and 1-sor- 
bose, which have been shown to alter drastically the morphological form 
of growth oi Neurospora in a nonhereditary manner (80). The antibiotic 
produced by Penicillium griseofulvwn has been shown by Brian to be 
the "curling factor" which has an effect on the growth of a number of 
fungi, including Neurospora (15), apparently similar to the effects of 
the paramorphogenic substances mentioned above. Whatever the func- 
tion of inositol in the growth and metabolism of fungi may be, it is at 
all events extremely specific for meso-inositol as has been shown recently 
with Neurospora by Schopfer (72). 

Considerably more is known in respect to the roles of the amino acids 
as growth factors in fungi than is true for the vitamins. Most of this 
information has been obtained from studies with amino-acid-requiring 
mutants of fungi. These studies in general have substantiated the con- 
cepts of comparative biochemistry in that the metabolism and synthesis 
of amino acids have been found to be similar in fungi, in other micro- 
organisms, and in higher organisms (see 79), For example, we may 
mention the similarities in the metaboHsm and synthesis of the sulphur- 
containing amino acids and of arginine. 

In addition these studies with biosynthetically deficient strains of fungi 
have raised several general points in regard to the significance of amino 
acids aside from their obvious roles as protein constituents. One of these 
points concerns the metabolic relationships between certain amino acids 
and certain vitamins of the B complex. The function of tryptophan as a 
precursor of nicotinic acid, first suggested by animal experimentation, 
has been elucidated by studies with mutant strains of Neurospora. The 
findings illustrated in Figure i have shown that at least one important 
route of nicotinic acid synthesis is from tryptophan through kynurenine, 
perhaps hydroxykynurcnine, hydroxyanthranilic acid (12,56), and quino- 










^ I T ^ I 







Figure I. Biosynthesis of tryptophan and nicotinic acid in Neurospora. 

Hnic acid (34). Several different mutant strains oi Neurospora are known 
which require either tryptophan or nicotinic acid for growth. Strain 
Y-31881 is unable to use tryptophan but can use hydroxyanthranihc 
acid which is accumulated by strain 4540. Recently strain 4540 has been 
shown to utilize quinolinic acid (34) which in turn is accumulated by 
strain 3416 (9,34)- The available evidence indicates that this entire 
sequence of reactions holds also in the rat (33,34,57)- 

The other known instance of a relationship between an amino acid and 
a vitamin is somewhat less direct than that just discussed. This is the 
relationship established between the aromatic amino acids and ^-amino- 
benzoic acid. As illustrated in Figure 2 mutant strains of Neurospora 
are known with single deficiencies for each of the aromatic amino acids 
and for /7-aminobenzoic acid. In addition, a single gene mutation has 
been found to result in the requirement for all four of these substances. 
This suggests that all four are derived from a common precursor. (See 
also 58.) Recent investigations have shown that this common precursor 




/r6'994 75001 

Figure 2. Biosynthesis of aromatic substances in Neurospora. 


is related to shikimic acid. Shikimic acid will replace all four of these 
substances for the multiple aromatic mutant of Neurospora, Y-7655. 
That the suggested relationship between these substances is of significance 
in other organisms is shown by the demonstration by Davis of a corres- 
ponding mutant in E. coli, which also responds to shikimic acid (18). 
Fischer has suggested that shikimic acid is derived in the plant directly 
from hexose sugars (22). In microorganisms the aromatic nuclei of these 
amino acids and of /7-aminobenzoic acid may thus be derived from hexoses 
through a common precursor similar to shikimic acid. Previous results 
(82) with the /'-aminobenzoicless mutant strain suggests that in the 
synthesis of this compound nitrogen is normally introduced into a non- 
aromatic structure. Since shikimic acid is active only for the multiple 
mutant, and not for the /7-aminobenzoicless strain, this structure would 
seem to be a substance closely related to shikimic acid. 

The other important contribution of investigations with micro- 
organisms is in regard to the roles of many amino acids as biosynthetic 
precursors of still other amino acids. The interconversion of glycine and 
serine first indicated in the rat (74) and recently further investigated by 
Sakami (69), seems also to be true for Neurospora and for certain bacteria 
(31,40). Evidence obtained with mutant strains of Acetohacter suggests 
that glycine is normally formed from serine, which may arise from a 
nonnitrogenous precursor, since one strain will grow on either glycine 
or serine and a second strain only on glycine (31). The interconversion 
of cysteine and methionine by way of homocysteine has been amply 
demonstrated in Neurospora as well as in bacteria (38,76). The role of 
aminoadipic acid as a precursor of lysine in Neurospora (54) represents 
another extremely interesting example of amino acid interconversions. 
The role of serine in tryptophan biosynthesis in Neurospora is now well 
known. There seems little question as to the reality of this conversion 
in view of enzymatic studies which have been carried out (55,86) and 
in view of the demonstrated incorporation of labeled nitrogen in the 
form of serine into the tryptophan synthesized by this system (75). 
The relationships of glutamic acid, proline, and ornithine suggested for 
the rat (74) have been shown to hold for Pejiicillium (11) and more 
recently for Neurospora (21). Fincham has shown that a-hydroxy- 
5-aminovaleric acid is an intermediate in the interconversions of glutamic 
acid, proline, and ornithine (21). Another extremely interesting series 
of interrelations is that between the aliphatic amino acids, threonine, 

E. L. TATUM 455 

homoserine, and isoleucine. Teas, Horowitz and Fling (85) and Teas (84) 
have shown that isoleucine and threonine are interconvertible in Neuro- 
spora and that threonine and homoserine are likewise interchangeable. 
These relationships and the demonstrated activity of a-aminobutyric 
acid as a precursor of threonine and homoserine (84) are intriguing 
relationships which remain to be elucidated. One promising lead in the 
investigation of these relationships may prove to be the examination 
of mutant strains oiNeurospora which require both isoleucine and vaHne. 
Continuing the work initiated by Bonner (10), a precursor of isoleucine 
accumulated by such a mutant strain has been identified as a:,/3-dihy- 
droxy/S-ethylbutyric acid (i). The results of studies using carboxyl- 
labeled acetate are consistent with the fairly direct conversion of acetate 
to the 18-ethyl side chain in the isoleucine precursor (2). These results 
strongly suggest that isoleucine and therefore probably threonine and 
homoserine are synthesized in Neurospora from a common four-carbon 

Of fundamental importance in studies in biochemical genetics in 
fungi as well as in other organisms is the concept that gene mutation is 
specifically related to biochemical reaction. Implicit in this concept is 
the hypothesis that each gene controls a specific biochemical reaction 
through the action of a specific enzyme (13). The gene and the enzyme 
are pictured as being specifically related in such a way that gene muta- 
tion with a change in the spatial or configurational specificity of the 
gene results in corresponding changes in enzyme specificity. Mutation 
of a given gene could then result in failure of the biochemical reaction 
either if no enzyme at all is produced, or if an enzyme is formed with 
altered properties such that it can no longer carry out the specific 
enzymatic function. The examination of particular mutant strains of 
Neurospora for the presence of specific enzymes is of considerable im- 
portance in experimentally testing the general concept. In this examina- 
tion it is, of course, vital to test for differences in the presence or activity 
of the specific enzyme between the wild-type and the mutant strain. 

One of the first examinations of an enzyme system from this point 
of view was carried out by McElroy and Mitchell (49) in their studies 
of the adenine-deaminase system of a temperature-sensitive adenme- 
requiring strain of Neurospora. Since this strain could synthesize adenine 
at temperatures under 28°C. but could not do so at higher temperatures, 
and since hypoxanthine was inactive for the mutant at higher tempera- 


tures at which adenine was required, it was expected that the adenine- 
deaminase in the mutant might have a different temperature sensitivity 
from that in the wild-type. McElroy and Mitchell (49) found, however, 
no demonstrable differences in the enzyme from the wild-type and 
mutant strains. It was pointed out by these authors that this instance 
might not represent a critical test of the general concept since adenine 
synthesis might normally proceed by a reaction other than a reversal of 
the deaminating system. Another instance which has been examined in 
Neurospora is that of the enzyme involved in pantothenic acid synthesis 
from pantoic acid and |S-alanine. Wagner and Guirard (88) first reported 
the enzyme which brings about this condensation to be missing in the 
pantothenicless mutant, and to be present in the wild-type strain. 
However, Wagner has been able to demonstrate that the enzyme is 
actually present in the mutant strain (87). A third instance recently 
investigated is that of the enzyme bringing about the synthesis of 
tryptophan by condensation of serine with indole, first demonstrated 
by Umbreit, Wood, and Gunsalus (86). In an investigation of a mutant 
strain of Neurospora which is unable to grow on indole but requires 
intact tryptophan, Mitchell and Lein (55) reported that this enzyme 
was not present in the mutant. This example apparently provides ex- 
cellent evidence in support of the general concept discussed above. 

In two examples in Neurospora, which may prove to be pertinent, 
carboxylase has been demonstrated in a strain which requires acetate 
or ethanol for growth, and asparaginase has been found in a strain which 
has a specific requirement for asparagine (83). Although generahzations 
from relatively few specific examples are always dangerous, the instances 
just discussed might indicate that gene mutation may affect metabolism 
in some microorganisms by modifying intracellular conditions so that 
the enzyme is normally inoperative; or alternatively by leading to the 
production of a specific enzyme inhibitor, and thus indirectly to the 
production of an enzyme inactive in vivo. Gene mutation, therefore, 
in at least some instances, may modify a biochemical reaction in the cell 
not by direct modification or elimination of a particular enzyme, but 
rather by controlling its //; vivo activity in some manner not as yet 

Some additional suggestions as to the nature of the gene-enzyme 
relation may come from examination of the three general types of 
behavior of biochemically deficient strains of fungi which have been 

E. L. TATUM 457 

described. In mutant strains of Neurospora one type of behavior typical 
of what has been termed an "absolute deficiency" is characterized by 
the failure of the mutant strain to grow in the absence of the required 
supplement, or by its being capable of a very slight amount of growth 
which does not continue after the stored material in the inoculum has 
been exhausted. Most of the mutant strains used in biochemical studies 
appear to belong in this group. A second type of behavior is that shown 
by strains with so-called "partial deficiencies" which are capable of 
growing on minimal medium at a more or less constant rate which is 
less than that of the wild-type or of the mutant on supplemented 
medium. The behavior of these mutants has been attributed to a modi- 
fication of the enzyme systems such that the limiting reaction can proceed 
to a certain extent but at a rate insufficient to permit optimal growth 
(53). A third type of behavior is typified by a strain which on inoculation 
into minimal medium grows very slowly for a considerable period of 
time. Finally, after a lag period of variable duration, its growth begins 
to improve and may approach or even reach the wild-type rate. In some 
cases this growth in minimal has been shown to be due to gene reversion, 
as in the inositoless mutants investigated by Giles and Zimmer (30). 
In other cases the phenomenon seems not to be due to gene reversion 
and has been termed "adaptation" (14). In these instances the ability 
to grow in the absence of the specific supplement is lost on passing 
through the asexual spores, that is the conidia, or through the sexual 
spores, the ascospores. Reasonable interpretations of this phenomenon 
of adaptation are those of the development of an alternative route of 
synthesis by-passing the genetically blocked reaction, or of the recon- 
struction of the genetically blocked reaction through the production of 
an alternative adaptative enzyme system. Similar behavior patterns 
have been described in other fungi, for example in pleomorphic cultures 
of the imperfect fungus Trichophyton (66), in which the genetic basis 
of the phenomenon cannot be directly examined. 

The phenomenon of adaptation in Neurospora has been further ex- 
amined recently in a few strains in which gene reversion has been 
rigorously excluded by genetic methods. Regnery (65) has examined the 
adaptive behavior of leucineless 47313, and Tatum and Garnjobst (83), 
the adaptive behavior of tyrosineless Y-6994. With these mutants it 
has been found that mycelium which has reached a wild-type rate of 
growth, either after a prolonged lag period on minimal medium or even 


in response to optimal concentrations of the required substance, is 
completely independent of an exogenous supply of the growth factor 
since it can grow indefinitely at maximal rate on minimal medium from 
myceUal transfers. The adaptive behavior of these mutants is not carried 
through the conidia, and the cultures must again pass through a lag 
phase on minimal medium. Although a differential storage in the conidia 
of the required factor may differentiate the mutant and wild-type, this 
does not seem likely. In contrast to wild-type conidia or ascospores 
which germinate and grow immediately on minimal medium, ascospores 
of the leucineless mutant send out germ tubes but fail to grow further 
on minimal medium. Ascospores of the tyrosineless strain initially grow 
slowly even on supplemented medium. Although much additional in- 
formation is needed before a final conclusion can be reached, existing 
information suggests that as the result of mutation the enzyme systems 
involved in biosynthesis of these factors are lost in these mutants during 
the return of the cytoplasm to a resting state in conidiation or ascospore 
production, and that the reconstitution of these systems depends upon 
and always accompanies growth. We may therefore have in these adapting 
strains examples of gene control of a biochemical reaction by altering 
the efficiency of initiation of the synthesis of an enzyme rather than 
by affecting enzyme production either quantitatively or qualitatively 
as suggested in the cases of the complete or partial blocks as discussed 
above. Strains which behave in this manner may represent the closest 
approximation to an extra-nuclear control of enzyme production yet 
found in Neurospora, since the maintenance of the enzyme activity in 
the cytoplasm would seem to be to a certain extent independent of the 

Studies of the nutrition of fungi have gone through two general 
phases. First, the identification of specific growth requirements leading 
to culture of the organisms on media of known chemical constitution, 
with the substantiation of the tenets of comparative biochemistry that 
fungi require the same factors, amino acids and vitamins, as do other 
organisms. Second, the experimental demonstration of the gene control 
of biosynthesis of these factors by means of experimental gene mutation 
with consequent biosynthetic deficiencies. Detailed biochemical study 
of strains with such induced deficiencies has added significantly to 
biochemical knowledge of vitamin and amino acid syntheses and inter- 
relations. It may be predicted that future investigations will continue 

E. L. TATUM 459 

to contribute valuable Information in these fields. Even more important, 
however, may prove future contributions of research with fungi along 
a number of lines discussed in this paper. It seems probable that these 
may lead to a better understanding of the nuclear and cytoplasmic 
factors involved in enzyme production, specificity, and activity, a 
problem of fundamental importance in all fields of biology. 


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29. Giles, N. H., Jr., Am. J. Botany, 33:218, (1946). 

30. , and Zimmer, E., ibid., 35:150 (1948). 

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37. Horowitz, N. H., ibid., 154:141 (1944). 

38. , ibid., 171:255 (1947). 

39. , HouLAHAN, M. B., HuNGATE, M. G., and Wright, B., Science, 

104:233 (1946). 

40. HuNGATE, F. P., Stanford Univ. thesis (1946). 

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43. KoGL, F., and ToNNis, B., ibid., 242:43 (1936). 

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Physiol, 30:331 (1947). 

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51. Markert, C. L., Personal communication. 

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59. Perkins, D., Genetics (in press). 

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61. , ibid., 35:360 (1948). 

62. Pomper, S., Yale Univ. thesis (1949). 

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64. Reaume, S. E. and Tatum, E. L., Arch. Biochem., 22:331 (1949). 

65. Regnery, D., Personal communication. 

66. RoBBiNS, W. J., Ann. N. Y. Acad. Sci., 49:75 (1947). 

67. , and Kavanagh, V., Botan. Rev., 8:441 (1942). 

68. RoBBiNS, W. J. and Ma, R., Science, 96:406 (1944). 

69. Sakami, W., /. Biol. Chem., 176:999 (1948). 

70. Schopfer, W. H., Ber. dtsch. bot. Ges., 52:308 (1934). 

71. , and Bein, M. L., Experientia, 4:147 (1948). 

72. Schopfer, W. H., Posternak, T., and Boss, M. L., Rev. internal. 

vitaminologie, 20:121 (1948). 

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74. , and RiTTENBERG, D., ibid., 158:71 (1945). 

75. Shemin, D. and Tatum, E. L., Unpublished results. 

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77. SoNNEBORN, T. M., Adv. Gcnctics, 1:263 (1947)- 

78. Tatum, E. L., Unpublished results. 

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E. L, TATUM 461 

80. , Barratt, R. W., and Cutter, V. M., Jr., Science, 109:509 (1949). 

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Botany (in press). 

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83. Tatum, E. L. and Garnjobst, L., Unpublished results. 

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86. Umbreit, W. W., Wood, W. A., and Gunsalus, I. C., ibid., 165:731 


87. Wagner, R. P., Proc. Nat. Acad. Sci. U. S., 35:185 (1949). 

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Vitamin and Amino Acid Requirements for the 
Growth of Higher Plants 



ONE of the difficulties in discussing the vitamin requirements of 
higher plants is to define the term vitamin, and to dehmit the 
substances to be considered. 

In the authorized English translation of the second German edition 
of his book on vitamins, Funk (15) approved Willaman's (62) definition 
which is as follows: "\^itamins constitute a class of substances, the 
individuals of which are necessary for the normal metabolism of certain 
living organisms but which do not contribute to the mineral, nitrogen 
or energy factors of the nutrition of these organisms." This definition 
might be interpreted to include any organic substance which has the 
following characteristics: it is an essential metabolite, it functions in 
some other way than as a source of energy or as a major constituent of 
the plant or animal body, and some individual organism must be supplied 
with it from without for normal function and development. 

Although by usage the word vitamin has special connotations which 
limit its application to a more or less well-defined group of substances, 
the distinction between vitamins, hormones, auxins, growth substances, 
and cofactors, has tended to disappear. With some exceptions the dis- 
tinction between the vitamins required by animals and those required 
by plants has also become less marked. In fact, it is preferable to consider 
growth requirements from the standpoint of essential metabolites rather 
than to limit consideration to vitamins, amino acids, purine and pyri- 
midine bases, or any other particular group of substances. Students of 
the growth requirements of any group of plants find it advantageous 


to be acquainted with those of other groups, both plants and animals, 
and to inckide in their investigations not only vitamins as such but 
amino acids, hormones, and other substances which have been demon- 
strated to play a significant role in plant and animal metabolism. 

Intact Plants. — Whether we consider vitamins in the narrower sense, 
that is, the B vitamins, vitamin A, C, D, E, and so on, or in the broader 
sense, intact higher plants have no vitamin requirements. This is demon- 
strated by their ability to grow in a mineral medium with no organic 
supplements. The evidence at hand indicates also that with some possible 
exceptions and under special circumstances, higher plants have no partial 
deficiencies for vitamins, that is, their growth is not improved by the 
addition of vitamins to the medium. They appear to synthesize adequate 
quantities of all the vitamins they need. Bonner and Bonner (10) in a 
recent review of this subject say that thiamin is not a limiting factor 
In the growth of most species of higher plants and can become a limiting 
factor only for a few species or under particular environmental circum- 
stances. The same conclusion appears to hold for the effects of niacin, 
pyridoxine, adenine, pantothenic acid, riboflavin, and other similar sub- 
stances for which growth increases have been reported by one investigator 
or another. 

Excised Parts. — Although the intact higher plant is self-sufficient as 
far as vitamins are concerned, isolated parts are not. They may evidence 
heterotrophism for various vitamins. 

Excised roots of a number of species have been demonstrated to have 
complete or partial, deficiencies for thiamin, pyridoxine, and niacin. 
Stem tissues and the tissues of storage organs require indoleacetic acid 
(lAA) or its equivalent, and in some instances (willow and hawthorne) 
pantothenic acid and biotin. Thiamin and cysteine are not essential but 
may improve growth (18), The contrast between the relation of intact 
plants to vitamins and that of isolated organs or tissues emphasizes the 
interdependence of the parts of a higher plant and has led to the thesis 
that the B-vitamins may be considered to be plant hormones (10). 

Excised Roots. — We know more perhaps about the vitamin relations of 
excised tomato roots than about those of the roots of any other plant. 
White (58) first succeeded in obtaining unlimited growth of tomato roots 
in a solution of mineral salts, sugar, and dried yeast. Robbins and Bartley 
(43) demonstrated that excised tomato roots would not grow unless the 
yeast extract was included in the sugar-mineral salt solution. They found 


thiamin to be the essential factor in the dried yeast (47). This confirmed 
the earher suggestion by Robbins (40,41) that the failure of an excised 
root to continue growth when repeated transfers of the root tip are 
made is because the seedling root contains some materials derived from 
the seed other than water, mineral salts, sugar, and free oxygen, which 
are necessary for continued growth and cannot be synthesized in solution 
cultures from the materials supphed. 

Later Robbins and Schmidt (48) demonstrated that tomato roots 
have a partial deficiency for pyridoxine, and Bonner (5) found some 
clones to have complete deficiencies for thiamin and for pyridoxine 
and partial deficiencies for niacin. All three vitamins show a high degree 
of specificity. 

The roots of a substantial number of species have been cultivated in 
excised condition, and for many of them potentially unlimited growth 
has been obtained. Among these are Acacia melanoxylon\ alfalfa {Medi- 
cago sativa) ; aster {Callistephus chinensis) ; buckwheat {Fagopyrum escu- 
lentum)\ carrot {Daiicus carota); celery {Apium graveolens)\ chicory 
{Cichorium sp.); red clover {Trifolium pratense); white clover {Trifolium 
repens); sweet clover {Melilotus alba); cotton {Gossypium hirsutum)', 
Crepis rubra; Jimson-weed {Datura stramoniuni); mustard {Brassica 
nigra); pea {Visum sativum); Petunia violacea; radish {Raphanus sativus); 
soybean {Glycine soja); tobacco {Nicotiana Tabacum, N. langsdorjii); 
tomato {Lycopersicon sp.); and vetch {Vicia sp.) (6,10,60). 

A number of the above have been cultivated only in a sugar-mineral 
salt medium supplemented with yeast extract and their exact vitamin 
requirements have not been defined. For the others, thiamin, pyridoxine, 
and niacin are the only vitamins which have been demonstrated to be 
of importance. The excised roots of one species, flax, make limited growth 
through an indefinite number of transfers in the absence of any added 
growth substance. The addition of thiamin, however, increases the 
growth substantially. White clover is able to grow indefinitely in the 
absence of thiamin but requires niacin. Of 12 additional species, all 
require thiamin. Four species require thiamin and pyridoxine, but their 
growth is increased by the further addition of niacin. The results to 
date emphasize the importance of thiamin as a vitamin requirement for 
the growth of excised roots, but, depending upon the clone or species 
used, all possible combinations of complete and partial deficiencies for 
the three vitamins occur. 


There is, however, a substantial number of species for which the 
conditions for unUmited growth of excised roots are unknown. Among 
these are included: Bauhinia purpurea; beet {Beta vulgaris)', broccoH 
{Brassica oleracea); Bryophyllum calydnum; cabbage {Brassica oleraced)\ 
corn [Zea Mays); cucumber {Cucumis sativus); eggplant {Solanum 
Melongena); grape {Viiis sp.); grapefruit {Citrus maxima); kohlrabi 
{Brassica oleracea); lemon {Citrus Limonia); lettuce {Lactuca scariola); 
lupine {Lupinus sp.); orange {Citrus sinensis); Parthenium argentatum, 
Poa sp.; Poinciana gilesii; potato {Solanum tuberosum); rice {Oryza 
sativci); Simmondsia californica; Sterculia diversifolia; Thuja orientalis; 
3 species of tobacco {Nicotiana glutinosa, N. rustica, N. sylvestris); and 
Wisteria sinensis (6,10,60). Potentially unlimited growth has not been 
obtained for the roots of any monocot or cucurbit, and the roots of 
many woody plants have proved to be refractory. 

Other conditions than vitamin requirements may be important in 
determining unhmited growth of excised roots. This is suggested by the 
peculiar results obtained by Robbins and Maneval (45) with lupine, 
and by Bonner (6) with the roots of Stercidia diversifolia, Bauhinia 
purpurea, and Wisteria sinensis. In these cases out of many roots the 
majority failed to grow under excised conditions, but a single individual 
might show very substantial growth. McClary (31) has reported that he 
could obtain unlimited growth for the excised roots of a hybrid corn 
on an agar medium containing sugar and mineral salts and has suggested 
that physical factors are determinative rather than vitamin supplements 
or other growth substances. Bonner was unable to confirm McClary 's 
observations on the unlimited growth of corn roots, and we have not 
been successful in obtaining unlimited growth. 

Excised Stem Tips. — Efforts to cultivate excised stem tips are compli- 
cated because, as a rule, the stem tips develop roots and the investigator 
is then dealing with an entire plant. Dodder and asparagus stem tips are 
relatively free from this complication. Loo (29) found that seedlings 
of dodder grew well during the first week but ceased to grow in the third 
week in a sucrose-mineral salt solution in diffuse light. Stem tips of 
dodder weie kept alive in diffuse light in a sucrose-yeast extract medium 
for 10 months through a series of transfers. The stem tips developed 
considerable chlorophyll. In the dark, however, they failed to maintain 
their growth even in the sucrose-yeast extract medium. Loo (27,28) 
also cultivated isolated stem tips of asparagus in diffused light on nutrient 


media containing mineral salts and sugar. On this medium the stems 
grew actively through repeated transfers extending for 22 months. In 
the dark, however, growth approached zero after a few transfers. The 
growth requirements for excised stem tips cultivated in the dark are 
still obscure. The slow absorption of sugar and other materials by the 
excised stem may be an important factor. 

Excised Tissues of Stems or Storage Organs. — Nobecourt, Gautheret, 
and others have obtained unlimited growth of portions of stems, storage 
organs, or callus of a considerable number of species of plants. These 
include among others: carrot {Daucus carota)\ chicory [Cichorium 
Intybus); Cissus discolor; grape {Vitis incisa, V. vinifera var. Aramon., 
V. Coignetiae, V. Davidii); hawthorne {Crataegus monogyna); Jerusalem 
artichoke {Helianthus tuberosus)\ Parthenocissus tricuspidata; P. Hederae- 
folia; Rubus fructicosus; Scorzonera Hispanica; snapdragon {Antirrhinum 
majus); and turnip {Brassica campestris) (18). 

Indoleacetic acid or its equivalent is of primary importance for the 
growth of these tissues. It represents an essential factor for many of them. 
Some (carrot, grape, Jerusalem artichoke, salsify {Tragopogon), Scorzo- 
nera, and turnip) grow slowly without the addition of lAA to the medium 
(17,18), but their growth is much more rapid when the medium is 
supplemented with this substance. Apparently they synthesize a small 
amount of lAA but insufficient for maximum growth. Cysteine and 
thiamin improve the growth of some of these tissues; the callus of 
Salix caprea and the stem tissues of hawthorne {Crataegus monogynd) 
are reported to require pantothenic acid and biotin in addition to lAA 

The relation of lAA to the growth of excised tissues of stems and 
storage organs suggests that this substance or its equivalent acts as an 
essential metabolite which is not synthesized in adequate quantities by 
the isolated tissues of stems and storage organs. Its relation to these tissues 
appears to be of much the same order as the relation of thiamin, pyri- 
doxine, or niacin to the growth of excised roots. 

Plant Embryos. — Considerable attention has been devoted to the 
culture of plant embryos by Blakeslee (2), Hannig (20), LaRue (26), 
Tukey (55), van Overbeek (56), and others. Hannig more than 40 
years ago cultivated embryos of some of the Cruciferae. Immature em- 
bryos evidenced little development after removal from the seed; older 
ones were grown to maturity. In general, attention has been devoted 


to the conditions necessary for the development of immature embryos 
and of those which fail to mature in the seed. The most successful efforts 
in defining the growth requirements for immature embryos are those of 
Sanders and Burkholder (51) discussed later in this paper. Unfortunately 
their investigations do not establish the vitamin requirements of the 
embryos, if any. From scattered observations we know a little of the 
vitamin requirements of the embryos of higher plants. Kogl and Haagen- 
Smit (23) found that biotin and thiamin increased the growth of pea 
embryos freed of their cotyledons; Bonner et al. reported that panto- 
thenic acid (9), ascorbic acid (3), or niacin (4) benefited pea embryos; 
Noggle and Wynd (37) state that niacin induced good germination and 
excellent development of Cattleya. 

Tumor Tissue. — Plant tumors and "accustomized" tissues in contrast 
to normal tissues excised from stems or storage organs grow in a sugar- 
mineral salt medium with no supplements. They do not require an 
external supply of lAA; their growth may be improved by thiamin. 
Judging from the evidence presented by Gautheret and his colleagues 
(24,34), "accustomized" tissue and at least some kinds of plant tumor 
tissues have an enhanced power to synthesize lAA.* 

An obvious explanation for the difference in growth requirements of 
normal tissue as compared to "accustomized" tissue or tumor tissue is 
that in becoming tumerous or in becoming "accustomized"! the original 
tissue has mutated (14) and developed a strain with greater ability to 
synthesize lAA. It is impossible to say whether the mutation is nuclear 
or cytoplasmic. 

Somatic mutations (or saltations) resulting in increased power of 
synthesis are known elsewhere in the plant kingdom and there is no 
a priori reason why such mutations, spontaneous or induced, should not 
occur in higher plants. We have studied a strain of Fusarium avenaceum 
which evidenced a complete deficiency for biotin. A spontaneous mutant 
isolated from this strain was able to synthesize its own biotin (44). Many 
species of fungi produce spontaneous somatic mutants with enhanced 
powers of growth. The greater vigor of mutants (pleomorphisms) of 
some of the dermatophytes as compared to the strains from which they 

*Riker, Henry, and Duggar (38) found no more auxin in crown-gall tissue 
than in normal tissue. 
fSee the observations of Morel (33) . 


are derived appears to be associated with an increase in the efficiency of 
nitrogen metaboHsm (46). 

Some Unsolved Problems. — Our knowledge of the relation of vitamins 
to higher plants is still far from complete. I have called attention to the 
substantial number of species for which the conditions necessary for 
unlimited growth of excised roots have not yet been defined. The same 
comment applies to excised stem tissues. 

The evidence for the effects of vitamins on intact higher plants is 
confused and in most instances unconvincing. However, the growth 
requirements of such parasites as dodder or saprophytes like Indian pipe 
have not been defined. The effects of the additions of natural organic 
supplements on the growth of the Lemnaceae (50), the relations of 
mycorhiza to some higher plants, the increased growth frequently 
associated with polyploidy, the phenomenon of hybrid vigor, genetical 
dwarfs, flowering and photoperiodism, all deserve further investigation 
from the standpoint of partial deficiencies of essential metabolites in- 
cluding vitamins. 

We know very little of the relations of higher plants to pteroylglutamic 
acid or to vitamin B12. One green plant, Eiiglena, is heterotrophic for 
vitamin B12 (22). The presence of this vitamin in higher plants has not 
been demonstrated, and no instance of its importance for higher plants 
has been reported. 

The difference in vitamin requirements of tissues derived from stems 
or storage organs and those of excised roots deserves further investiga- 
tion. For the former, an external supply of lAA is the critical factor; for 
the latter, thiamin, pyridoxine, or niacin include the essential supple- 
ments. This distinction applies even when the excised roots and the 
excised stem tissue are derived from the same species. It exists also when 
the requirements of tissue obtained from a storage root are compared 
with those of a seedling or fibrous root of the same species. Bonner (5) 
obtained unlimited growth of seedling carrot roots in a sugar-mineral 
salt solution supplemented with thiamin and pyridoxine; both vitamins 
were essential. Tissue from the storage root of carrot requires for con- 
tinuous and vigorous growth only lAA or its equivalent; other supple- 
ments are not necessary. Nobecourt (36) observed that fibrous roots 
which develop from the tissue isolated from the storage root do not 
grow in the sugar-mineral salt medium supplemented with lAA when 


detached from the tissue. They require thiamin.* This suggests that the 
growth requirements for cambial meristem are different from those of 
the apical root meristem. 

When we include a consideration of the vitamin requirements of the 
meristems of such tissues as plant tumors, it seems likely that the 
metabolism of various meristems in or from the same plant is not 
identical. This might be assumed from the differences in the cellular 
elements and tissues formed by the various types of meristem. However, 
the determination of the growth requirements of these meristems defines 
some of the specific differences in their metabolism. How these dif- 
ferences come about, what elements in the cells are responsible for them, 
their implications for differentiation or morphogenesis and for abnormal 
growth, are questions for further research. 

Methods of Investigation. — Methods of investigation in this field are 
exacting and the interpretation of results requires an appreciation of 
certain general concepts. Some considerations which should be borne 
in mind are as follows: 

(i) Species differ in their requirements. Clones of the same species 
and even meristems from the same plant may exhibit different responses. 

(2) Vitamin deficiencies may be complete or partial, single or multiple, 
absolute or conditioned, permanent or temporary. 

(3) Because of the relation of microorganisms to vitamins, experiments 
under nonsterile conditions must be reviewed with caution. When vita- 
mins are added to soil or sand cultures, effects noted on higher plants 
may be the result of their action on the microflora. 

(4) Reserves of vitamin in the seed or excised portion of a plant may 
compensate for a deficiency in the medium. Successive passages in a 
vitamin-free medium are advisable. White (60) has suggested ten passages 
as a criterion for determining potentially unlimited growth. 

(5) In experiments involving vitamins, the basal medium should be 
adequate in all other respects and approach as nearly as possible that 
most suitable for the organism in question. 

(6) Environmental conditions, including hydrion concentration, tem- 
perature, and salt concentration, may affect vitamin requirements. 

(7) Because of the minute amounts of vitamins which are effective 
and because of their wide distribution in products of natural origin, 

*The difference in the requirements of excised fibrous carrot roots as observed 
by Bonner and Nobecourt may be due to tlie use of different varieties of carrot. 


special attention must be given to cleanliness of glassware and other 
utensils and to purity of chemicals. Effective quantities of one or more 
vitamins may be present in the carbohydrates (maltose of adequate 
purity is especially difficult to obtain), in agar, in gelatin, in cotton, cheese 
cloth, asparagine, or any other product of natural origin. We have found 
some samples of the vitamin thiazole contaminated with the vitamin 

Amino Acids 

As far as is known higher plants have no amino acid requirements in 
the sense that they are heterotrophic for a particular amino acid or group 
of amino acids and will not grow unless suppHed from without with one 
or more of these metabolites. Higher plants are able to synthesize all 
the amino acids included in their cell substance from inorganic nitrogen, 
for example, nitrates or compounds of ammonia. In tact, it is probable 
that any cell of at least some species of higher plants can construct amino 
acids from inorganic compounds of nitrogen. This is suggested by the 
demonstration that some kinds of excised roots (42,7) and stem tissues 
(12,27,39) grow in media in which nitrates are the only source of 

With the possible exception of embryos (51) and perhaps some stem 
tissues (16), higher plants appear to synthesize amino acids from inorganic 
nitrogen as rapidly as they can be used in the plant's metabolism. The 
burden of evidence indicates that better growth is obtained with in- 
organic nitrogen than with any amino acid or mixture of amino acids. 
Furthermore, most investigations fail to demonstrate beneficial effects 
from the addition of one or more amino acids to a solution containing 
inorganic nitrogen if the plant is adequately supplied with carbohydrate 
through photosynthesis or otherwise. 

I say, as far as is known, higher plants have no amino acid requirements. 
Not all of them, of course, have been investigated, and it is possible that 
such parasites as dodder or saprophytes like Indian pipe, have amino 
acid deficiencies. This question cannot be answered until these plants 
are successfully grown on a medium of known composition. Neither can 
we assert that all roots grow as tomato roots do with nitrates as the only 
source of nitrogen. It is conceivable that some of the roots which have 
not been successfully cultivated in excised condition may have amino acid 
requirements. The same may be said for stem tissues. 


We can summarize briefly by saying that, with the exception of 
embryos, investigations generally indicate that higher plants have no 
complete or partial requirements for amino acids. 

This does not mean, however, that higher plants are unable to absorb 
amino acids as such and utihze them. Numerous investigations on the 
direct utilization of organic nitrogen by higher plants beginning with 
those of Lutz (30) in 1898 have demonstrated that some amino acids 
supplied under sterile conditions can be absorbed and utiHzed under some 
circumstances. There is disagreement, however, as to which amino acids 
can be assimilated by intact plants and the conditions under which 
they are utilized are ill-defined. Hutchinson and Miller (21) in summariz- 
ing the literature in 191 1 said that more or less satisfactory evidence of 
assimilation had been obtained for leucine, aspartic acid, asparagine, and 
tyrosine. The gains of nitrogen were, however, generally very small, 
and in many cases negative results were obtained. With some modifica- 
tion in the list of specific amino acids, the situation nearly 40 years later 
is about the same. 

For almost any amino acid for which utilization is claimed, other 
evidence showing that it is not utilized or is toxic can be cited. Aspartic 
acid, for example, is reported by Molliard (32) to be assimilated by 
radish; Beaumont et al. (i) obtained negative results with tobacco; 
Hutchinson and Miller (21) found it to be a fair source of nitrogen for 
peas; Tanaka (54) states that it is not utiHzed by Sisyrinchium; Virtanen 
and Linkola (57) found it to be assimilated by peas and clover but not by 
wheat and barley for which it was injurious. Brigham (11) states that 
asparagine is superior to nitrates for dent corn; Beaumont et al. (i) 
found asparagine to be a fair source of nitrogen for tobacco; Steinberg 
(53) reports it to be quite toxic for tobacco. White (59) in 1937 concluded 
that 9 amino acids were essential for the growth of excised tomato roots 
and called attention to the close correspondence between the amino 
acids essential for the growth of rats, for diphtheria bacilli, and for 
tomato roots. Two years later (61) he reported that the 9 essential 
amino acids could be replaced by glycine which was not included in 
the original group. Bonner (7) and Day (13) were unable to demonstrate 
beneficial effects of glycine. We have grown excised tomato roots through 
137 passages extending over almost 13 years in a medium in which the 
only organic constituents were sugar and thiamin and the only source 
of nitrogen was nitrate. 


The confused and unsatisfactory status of our information on the 
relation of amino acids to higher plants may be ascribed in part to a 
variety of causes among which are the following. 

Sterility. It is probably not necessary to emphasize the importance of main- 
taining sterile conditions. Any research with amino acids under nonsterile 
conditions must be viewed with suspicion if the purpose is to determine the 
effects of the amino acid rather than of its decomposition products. Un- 
fortunately, a good deal of the earlier work on the influence of amino acids 
was carried out under nonsterile conditions. 

Purity of Amino Acids. The purity of the sample of acid used is always a 
matter of concern. Proline, for example, materially influences the action of 
hydroxyproline on dermatophytes, and many of the so-called pure samples 
of the latter compound contain sufficient proline to affect the results materially 
(46). In addition to the purity of the individual amino acid, attention must 
be given to its optical form. The natural form may act differently from the 
unnatural or from mixtures of the two. 

Secondary Effects of Amino Acids. In evaluating the action of amino acids 
per se, consideration must be given also to their buffer action. We found, for 
example, the favorable effect of glutamic acid on the gametic reproduction of 
Phycomyces to be due to its buffer effects (49). Amino acids may combine 
with heavy metals (52) reducing the toxicity of a medium or lowering the 
amount of a minor essential element below the optimum. Nielsen and Johansen 
(35) found that asparagine materially reduced the toxicity of copper for 
Rhizobium radicicola probably by forming a copper complex. Amino acids 
may react during heat sterilization with other constituents of the medium, 
especially dextrose (63). Lankford (25) reports that the sterilization of glucose 
with amino acids produces effects on lactic acid bacteria quite different from 
those obtained when the amino acids and glucose were separately sterilized. 

Specificity of Action. Another complicating factor is that the effect of an 
amino acid may vary with the plant species. Hydroxyproline is quite toxic 
for a number of dermatophytes but is relatively harmless, even beneficial, for 
other fungi (46). Virtanen and Linkola (57) report that peas and clover use 
both optical forms of aspartic and glutamic acids well for their nitrogen nutri- 
tion, but that aspartic and glutamic acids do not function as nitrogen sources 
for wheat and barley. In fact, aspartic acid appears to interfere with the growth 
of wheat in media containing nitrate or ammonium salts. Legumes and grasses 
seem, therefore, to exhibit entirely different behavior toward aspartic or 
glutamic acid. 

Single Amino Acids versus Mixtures of Amino Acids. The results obtained 
with microorganisms and the excellent investigation by Sanders and Burk- 
holder (51) on Datura embryos emphasize that mixtures of amino acids may 
have substantially greater beneficial effects than any single amino acid. Further 
work on amino acid requirements of higher plants should include a considera- 
tion of the balance between amino acids. 

Embryos versus Adult Plants— Parts versus Whole Plants. Attention must be 
given to the stage of development of a plant and to the results obtained with 
root or stem tissue as compared to the intact plant. 

Carbon Content of Amino Acid. The possibility that an amino acid may be 


effective because it furnishes a carbon configuration rather than because of its 
nitrogen content is another factor to be considered. 

Miscellaneous Factors. It is hardly necessary to mention that temperature, 
hght intensity, the pW of the medium, and other important environmental 
factors may affect the action of amino acids. Riker and Gutsche (39) have 
emphasized the significance of the concentration of an amino acid in determin- 
ing its effects. 

Importance of Research on Amino Acids. — Interest in the relation of 
higher plants to amino acids has extended in various directions. There 
has been a substantial amount of research on the possibility that organic 
fertilizers owe their effects in part to the absorption of nitrogen in 
organic form including amino acids. The burden of