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The 
Physiology 

of 

Flowering 



Biology 



►►► 



Studies 






THE PHYSIOLOGY 
OF FLOWERING 




These Studies are designed to inform the mature 
student— the undergraduate upperclassman and the 
beginning graduate student— of the outstanding ad- 
vances made in various areas of modern biology. 
The books will not be treatises but rather will briefly 
summarize significant information in a given field 
and interpret it in terms of our current knowledge 
of the rapidly expanding research findings within 
the life sciences. Also it is hoped that the Studies 
will be of interest to teachers and research workers. 



BIOLOGY 
STUDIES 



Bell, Ultrasound in Biology 

Carlquist, Comparative Plant Anatomy 

Carpenter, Fossil Insects and Evolution 

Crafts, Translocation in Plants 

Deevey, Historical Ecology 

Delevoryas, Morphology and 
Evolution of Fossil Plants 

Hillman, The Physiology of Flowering 

Slobodkin, Growth and 
Regulation of Animal Populations 

Sutton, Genes, Enzymes, and 
Inherited Diseases 



CO* 



William S. Hillman 

Yale University 



THE PHYSIOLOGY 
OF FLOWERING 



Holt, Rinehart 
and Winston 

New York • Chicago • San Francisco 
Toronto • London 




4 5 6 7 8 9 

COPYRIGHT © 1 962 BY HOLT, RINEHART AND WINSTON, INC. 

LIBRARY OF CONGRESS CATALOG CARD NUMBER! 62 8419 

ALL RIGHTS RESERVED 

2109452 
PRINTED IN THE UNITED STATES OF AMERICA 



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preface t 



To the botanist flowering is of interest as the means of sexual 
reproduction in the higher plants, and because the processes leading 
to it provide experimental systems for the study of environmental 
and internal controls of development— problems of basic significance 
throughout biology. To the rest of mankind, which often has more 
pressing problems to consider, flowering is nevertheless of the great- 
est practical importance since agriculture is based on the control of 
flowering and its resultant fruits and seeds. Flowering has been 
studied with both attitudes for many centuries; only during the 
past few decades, however, has a large body of knowledge about 
flowering been accumulated. It is the purpose of this book to survey 
this knowledge. The major emphasis, which simply reflects the 
direction of most research, will be on processes affecting the initia- 
tion and early development of flowers rather than on associated or 
subsequent events. Historical details are omitted except when they 
are required to clarify current concepts. 

I have tried to write for several kinds of readers, from graduate 
students in botany and other branches of biology to laymen with 
some formal training in science. Inevitably, then, any given reade? 
will find some passages too elementary or others insufficiently ex- 
plained. As for the relatively small group of professional plant 
physiologists who specialize in the study of flowering, I hope this 
book will serve as a useful review for them. They should not expect 
to find much new in it, except perhaps another point of view, and 
there are as many of these as there are specialists. 

This question of point of view, particularly in presentation, has 

v 



vi ■ Preface 

not been an easy one to resolve. There is much to be said for the 
practice of sketching the broad lines of a topic with a few intel- 
lectually satisfying concepts and not burdening the student im- 
mediately with exceptions and difficulties. If I have avoided this 
procedure— and surely the bewildered reader of Chapter Five will 
agree that I have-it is because 1 am afraid it can be fundamentally 
misleading. My intention is to introduce the reader to the field and 
if possible to give him the "feel" of it, bringing him close to the 
position of the research workers themselves. Since in my opinion 
science progresses, like all endeavors, by fumbling, backing out of 
dead ends, and now and then taking a few steps forward, it is often 
easy to believe in a clear pattern of conceptually clean "break- 
throughs" after some time has passed, but it is harder to do so as 
the work becomes more recent. Or, at least, I doubt my own ability 
at this sort of judgment. The alternative, then, is to stress the phe- 
nomena, the empirical observations: these are not so likely to be 
subjectively distorted, and it is these that must be lived with, ex- 
amined, correlated, and finally understood. 

All this is of course no excuse for a mere random collection of 
'[acts," and the reader will find nothing of the kind. It is, however, 
the justification for bringing in exceptions almost simultaneously 
Avith the tentative rules, for employing an often deliberate vague- 
ness in terminology— since words used in a systematic, authorita- 
tive way can often conceal ignorance— and for stressing, above all, 
the kinds of experiments and results rather than merely the con- 
cepts they may or may not illustrate. I can think of no better way 
to convey the extreme openness of the subject, the way in which few 
if any principles are irrevocably established. It is all a question of 
how much confusion is necessary to provide a true picture of the 
present state of things; I have tried to avoid an excess, but not to 
exclude it entirely. 

A general outline of the way in which I have grouped various 
topics for consideration is provided by the table of contents, and 
requires no further comment here. However, some remarks on the 
bibliography and the manner in which papers are cited may be 
useful. 

The proportion of general reviews to original experimental 
articles cited is relatively high, and I have made no attempt to 
include all the revelant literature. Frequently a paper is considered 



Preface • vii 

not because it is the first or most important of its kind but simply 
because it provides a particularly good example of a problem under 
discussion. The great preponderance of English-language refer- 
ences is simply a concession to the convenience of both reader and 
writer, and does not reflect the frequency or importance of publi- 
cations in other languages. Fortunately for the English-speaking 
world most of the work from other countries is reviewed, and much 
is even reported, in English by the original workers themselves. 

I have adopted the following convention with regard to cita- 
tions in the text. If a statement is followed simply by author (s) 
and date, for example, Hamner (1940) , the paper cited has original 
data on the point in question. Directions to see a paper, on the other 
hand, indicate reviews or other discussions from which further 
references may be obtained. All plants are referred to for the first 
time by both common (if any) and scientific names. Thereafter, the 
practice adopted is arbitrary, but the index can always be used to 
establish one from the other. 

In summary, I have tried to treat the field in a manner not 
quite like that to be expected from a technical review or article, but 
in such a way that the previously uninformed reader will afterward 
be able to read any of these with understanding and enjoyment; and 
then, best of all, perhaps try his own hand at the game. 

W. S. H. 

New Haven, Connecticut 
September, 1961 






acknowledgments t 



During the time this book was written, the author was sup- 
ported entirely by research grants from the National Science 
Foundation. 

The patient cooperation of Violet Esdaile and Margaret Wark 
in typing successive stages of the manuscript has been of great value. 

Discussions and correspondence with numerous investigators 
have contributed greatly to this survey, but particular thanks are 
due Dr. Bruce A. Bonner and Dr. Ian M. Sussex for critically read- 
ing the manuscript. 



\ in 



contents 



► 



chapter one ► Background 



MORPHOLOGY OF FLOWERING 1 

NATURAL HISTORY OF FLOWERING 
THE MEASUREMENT OF FLOWERING 



chapter two 



► Photoperiodism: An Outline 10 

DEFINITIONS OF PHOTOPERIODISM 10 

HISTORICAL NOTE 11 

GIANT TOBACCO AND SEPTEMBER SOYBEANS 12 

KINDS OF PHOTOPERIODIC FLOWERING 

RESPONSES 13 

THE ROLE OF LEAVES IN PHOTOPERIODISM. 

PHOTOPERIODIC INDUCTION 17 

THE CENTRAL ROLE OF THE DARK PERIOD 18 

REQUIREMENTS FOR HIGH-INTENSITY LIGHT 20 

MUTUAL INTERACTIONS OF LIGHT AND DARK 

PERIOD LENGTHS 22 

INTERACTION OF DIFFERENT PHOTOPERIODIC 

cycles: FRACTIONAL INDUCTION IN LDP 

AND LONG-DAY INHIBITION IN SDP 24 

PHOTOPERIODISM AND TEMPERATURE 25 

PHOTOPERIODISM AND VEGETATIVE GROWTH 27 

LITERATURE 29 



chapter three ► Photoperiodism: Attempts at Analysis 

A. Photoperiodism and light quality 30 

ACTION SPECTRA FOR LIGHT-BREAKS 30 



30 



IX 



Contents 

the red, far-red reversible system 34 
the red, far-red system in 

photoperiodism 35 
nature and function of the red, far-red 

PIGMENT 39 

PROLONGED EXPOSURES TO LIGHT OF 
DIFFERENT COLORS 40 

B. Time relations and endogenous rhythms 
in photoperiodism 42 

ENDOGENOUS C1RCADIAN RHYTHMS IN 

PLANTS 44 

ENDOGENOUS CIRCADIAN RHYTHMS AS THE 

BASIS OF PHOTOPERIODISM 46 

CIRCADIAN RHYTHMS AND THE ACTION OF 

LIGHT-BREAKS 47 

FLOWERING IN LIGHT-DARK CYCLES OF 

DIFFERENT LENGTHS; TEMPERATURE 

INTERACTIONS 5 1 

ENDOGENOUS CIRCADIAN RHYTHMS AND 

THE RED, FAR-RED SYSTEM 52 

CONCLUDING REMARKS ON CIRCADIAN 

RHYTHMS AND PHOTOPERIODISM 52 



chapter four ► Temperature and Flowering 54 

vernalization: cold treatments and 
flowering 54 

Vernalization in winter rye Vernalization 

in other plants 

DEVERNALIZATION 59 

RELATIONS BETWEEN VERNALIZATION AND 

PHOTOPERIODISM 61 

THE SEMANTICS OF VERNALIZATION! 

FURTHER EFFECTS OF TEMPERATURE ON 

FLOWERING 62 

TEMPERATURE AND FLOWERING IN BULB 

PLANTS 64 



chapter five 



chapter six 



chapter seven 



chapter eight 



Contents • xi 

► Floral Hormones and the Induced State 67 

DEFINITIONS AND BACKGROUND! AUXINS AS 

PLANT HORMONES 67 

PRELIMINARY EVIDENCE FOR THE EXISTENCE 

OF FLOWERING HORMONES 69 

TRANSLOCATION OF FLOWERING HORMONES 72 

TRANSLOCATION RATE 77 

FLOWER PROMOTION OR FLOWER INHIBITION? 

THE SPECIFICITY OF FLOWERING STIMULI 78 

VERNALIN AND METAPLASIN 82 

PERMANENCE AND LOCATION OF THE 

INDUCED STATE 83 

THE BIOCHEMISTRY OF INDUCTION 88 

CONCLUDING REMARKS 94 

► Chemical Control of Flowering 99 

THE GIBBERELLINS 100 

AUXINS, AUXIN ANTAGONISTS, AND OTHER 

GROWTH REGULATORS 106 

PLANT EXTRACTS OF VARIOUS KINDS 109 

MINERAL NUTRITION; MAJOR ELEMENTS 1 1 1 

HEAVY METALS AND FLOWERING 113 

► Age and Flowering 

AGE AND FLOWERING IN HERBACEOUS 

PLANTS 117 

FLOWERING IN WOODY PLANTS 120 

► A Miscellany 127 

ANTHESIS 127 

THE SEX EXPRESSION OF FLOWERS 1 30 

GENETICS OF FLOWERING RESPONSES 134 

FLOWERING AND DEATH 136 

PROSPECTS 137 



116 



Bibliography 

Index of Plant Names 

Subject Index 



141 
159 
161 







L 




Extreme modification of development by photoperiodism in the common weed 
Chenopodium rubrum. Right, a plant germinated and grown under 8-hour photo- 
periods; much of the bulk of the 3-week-old seedling consists of flower parts. 
Left, a plant germinated and grown under 20-hour photoperiods; after more 
than 3 months, it remains completely vegetative. (Right-hand photograph from 
Cumming [1959], by permission of the editors of Nature; both photographs 
courtesy of Dr. B. G. Cumming of the Canada Department of Agriculture.) 



► 
► 
► 
► 



chapter one £ Background 



Experimental work is the main concern of this study, but some 
purely descriptive information on flowering should be helpful. This 
chapter considers, first, the structure and origin of flowers as dealt 
with by morphologists. The natural history of certain flowering 
habits will then be briefly described, and an outline of some of the 
methods used to "measure" or evaluate flowering concludes the 
chapter. 

MORPHOLOGY OF FLOWERING 

The word "flower" is commonly used for structures of the 
greatest variety, from those of the elm, simple and inconspicuous, 
to the showy, complex blossoms of orchids or sunflowers. Morphol- 
ogists use the term "flower" to mean a determinate sporogenous 
shoot bearing carpels. Determinate means of strictly limited 
growth; sporogenous, bearing the reproductive microspores (male) 
or megaspores (female). The key portion of this definition, how- 
ever, is the presence of carpels. 

The carpel, characteristic organ of the angiosperms, or "flower- 
ing" plants, is an organ bearing and enclosing the ovules; the 
ovules, in turn, contain the megaspores. Under this definition of a 
flower, the sporogenous axes of gymnosperms— pine cones, for ex- 
ample—cannot be considered flowers; the absence of true carpels 
is one of the major characteristics setting off the gymnosperms— 
conifers, cycads, and the like— from the angiosperms. Strict use of 
this definition of a flower of course also eliminates those structures, 

1 



2 • Background 

borne by many true angiosperms, which are commonly called "male 
flowers"— that is, structures containing only the pollen-bearing 
stamens and without even rudimentary carpels. In practice, the 
restriction to carpel-bearing structures need not apply here. Studies 
of flowering in gymnosperms such as pines have been conducted 
and are, for physiological purposes at any rate, analogous to studies 
on angiosperms. For these purposes, then, flowering can be taken 
to mean the production of sporogenous shoots by either angio- 
sperms or gymnosperms; the term flower in its common usage will 
not be misleading. 

The parts of "typical" flowers— such as those found in botany 
texts— are usually described as follows: the floral axis is more or 
less shortened as compared with that of a vegetative shoot, and 
bears successive whorls of parts arranged around it. The structure 
on which the flower parts are placed is the receptacle, and the 
stalk bearing the flower is the pedicel. The lowest or outermost 
parts are the sepals, commonly enclosing the bud; within and above 
are the petals. Sepals and petals are collectively the perianth. 
Within this are the stamens, each consisting of a filament bearing 
a pollen-producing anther. The upper or innermost flower parts 
are the carpels, which, either singly or united, give rise to one or 
more ovaries, containing the ovules, and to a pollen-receptive sur- 
face, the stigma. Stigma and ovary together, whether derived from 
one or more carpels, are called the pistil. 

Many individual flowers often occur on a single simple or 
complex axis as in the sunflower (Helianthus) or in grasses; such a 
group of flowers is an inflorescence. Flowers may also be solitary, 
each borne on a separate pedicel attached to the vegetative axis. 
Flowers or inflorescences may be terminal (at the ends of shoots) or 
lateral, or both, and may also be enclosed or accompanied by leafy 
or scaly bracts. 

There are great differences between various plants in the num- 
ber, arrangement, shape, size, color, degree of fusion, and even 
presence or absence of the various flower parts. In spite of this, 
there is a good area of agreement among botanists both on the 
phylogeny, or evolutionary origin of the flower, and on its ontogeny, 
or development from the vegetative axis in the individual plant. 

The definition of a flower as a particular kind of determinate 
shoot already implies an interpretation of both phylogeny and 



Morphology of Flowering • 3 

ontogeny. The evidence suggests that the various flower parts, from 
sepals to carpels, are homologous with ordinary foliage leaves. 
That is, they bear essentially the same anatomical and morpho- 
logical relation to the axes on which they are borne as do the 
leaves. This does not necessarily mean that the flower parts have 
been derived from foliage leaves, even though the flower parts of 
many plants, particularly those considered more primitive, may 
show distinctly leaflike characteristics. Probably the most widely 
accepted view is that both leaves and flower parts were evolu- 
tionarily derived from similar structures. These may have been 
fused branch systems, some of them entirely sterile and represented 
in our present leaves, some of them sporogenous and represented 
in modern carpels and anthers, and still others with functions 
accessory to the sporophylls and represented in modern sepals and 
petals. While the details of such questions remain speculative for 
the present since the ancestry of the angiosperms is not really 
known, the homology between leaves and flower parts is generally 
accepted and may be of some importance physiologically; it is at 
least implicitly challenged, however, by some of the work on 
flower ontogeny to be considered next. 

The flower, like the leaves and the shoot itself, is derived 
from the apical meristem. This is a region of relatively small, 
undifferentiated, more or less actively dividing cells located at the 
very apex of the shoot. Meristems in general are the sources of new 
growth in all higher plants, and this has given rise to the concept 
that plants, unlike animals, show a "continuing embryogeny." 
Relatively little is known about the mechanism of the formation 
of new organs by such embryonic, seemingly slightly organized 
groups of cells. The central problem of the physiology of flowering 
might be stated as the question of how various factors affecting 
flowering, be they environmental or genetic, are translated by the 
plant into physico-chemical "signals" to the meristem, and how 
these determine whether the meristem will produce flowers. The 
major morphological question on which there is disagreement is 
whether the meristematic activity that produces flower primordia— 
recognizably distinct structures that will develop into flowers under 
favorable conditions— is qualitatively different from that which 
produces leaf initials, which develop into leaves. 

According to the majority of recent workers there is no essen- 



4 • Background 

tial difference between the organization of a meristem producing 
only leaves and one producing flowers. Gross differences of course 
exist between floral and vegetative apices in a given plant. These 
differences appear to be correlated with the vegetative and in- 
florescence structures of the particular plant involved, and no 
generalizations true for all plants can be made. But the question 
of essential organization goes beyond this, which is largely a matter 
of shape and size. 

The organization of many vegetative shoot apices can be ex- 
pressed loosely in terms of the tunica, or outer layers of cells, and 
the corpus, or inner core of cells, the developmental functions of 
which may be somewhat different. Most recent investigators have 
observed that where this organization is present it continues with 
no sharp change into the floral meristems, which are thus not 
qualitatively different from the vegetative. See, for examples, Wet- 
more, Gifford, and Green (1959); Stein and Stein (1960); and 
Tucker (1960). However, according to a minority of investigators 
working chiefly in France, floral development is the exclusive 
function of a "waiting meristem" (meristeme d'attente) that re- 
mains inactive until the onset of flowering, whereas leaf production 
and purely vegetative growth are carried on by an "initial ring" 
(annean initial) surrounding it. This work is reviewed by Buvat 
(1955). In this view, then, reproductive and vegetative development 
are quite different, originating in different meristem regions, 
whereas the majority view is that there are not two sorts of develop- 
ment but merely a continuum with extremes. 

The view of no essential difference seems to be supported by 
experimental work, to be described later, showing that certain 
plants (Cosmos, Kalanchoe), given a treatment insufficient to in- 
duce flowering but having some effect in that direction, may re- 
spond by producing a series of structures intermediate between 
normal inflorescences and leafy shoots (see Fig. 1-1). Although 
one can interpret such "vegetative flowering" as the interaction of 
two fairly distinct meristematic activities, the majority view appears 
to involve less difficulty. 

Descriptive morphology of the meristem has little more to tell 
the student of flowering physiology, although experimental (oper- 
ative) morphological studies may well do so in the future. The reader 
should bear in mind that, in general, experiments on the physiol- 



Morphology of Flowering • 5 

ogy of flowering have been more concerned with the conditions 
bringing about the production of flower primordia— with flower 
initiation, as it is called— than with subsequent flower development, 
although in practice both are studied. 




-■ — 





B 




D 



Fig. 1-1. Intermediate conditions between full flowering and vegetative habits 
in Kalanchoe blossfeldiana, from (A) normal, fully developed inflorescence through 
(B)-(D) increasingly vegetative forms, to (E) a fully "vegetative inflorescence" 
in which there are no flowers at all, but a branching habit still unlike that in the 
normal vegetative state. The sequence (A)-(E) reflects decreasing amounts of 
short-day treatment. (Photographs from Harder [1948], by permission of the 
company of Biologists, Ltd., and courtesy of Dr. R. Harder, University of 
Gottingen.) 



A concept occasionally found in the experimental literature 
is that of ripeness-to-flower. In the development of many plants 
from seed, there may be a stage before which flower initiation can- 
not occur, at least in response to conditions that would bring it 
about in older plants. A plant which has passed this stage is said 



6 • Background 

to be ripe-to-flower. This concept will be considered in connection 
with work requiring it, notably in Chapter Seven, but by itself it 
explains little about the physiological events taking place and 
seems not to reflect any basic morphological conditions common to 
all plants. 

For more detailed treatments of the topics discussed here, see 
Lawrence (1951), Esau (1953), and Foster and Gifford (1959). 



NATURAL HISTORY OF FLOWERING 

Most of what is known about flowering is based on work done 
either with plants native to the temperate zone or with cultivated 
plants. Flowering times and habits particularly have been studied 
more thoroughly in the higher latitudes than in the tropics. This 
limitation should be kept in mind in any discussion of flowering 
habits and physiological mechanisms. The general state of igno- 
rance on flowering in the tropics, and particularly its seasonal 
aspects, is well summarized by Richards (1957, pp. 199-204). 

Plants are often classified as annual, biennial, or perennial. 
Under these familiar terms a plant either germinates, flowers, and 
dies within a single season, germinates and develops during one 
season and flowers and dies in the next, or persists for many years 
flowering repeatedly. Such classifications are not always physiolog- 
ically meaningful, although, as will appear later, many biennials 
can be regarded as annuals in which a low-temperature treatment 
is required for flower initiation. But many plants commonly called 
annuals do not die after flowering and fruiting in all climates; 
they may be tropical perennials able to survive or cultivated as 
annuals in cooler regions. 

There might be more meaning, both ecological and physiolog- 
ical, to a classification into two groups— the first being perennials, 
defined as above, and the second, a group called monocarpic plants. 
Under this term can be classified true annuals, such as the edible 
pea (Pisum sativum), biennials, and certain others, all having in 
common the behavior of flowering only once, with fruiting fol- 
lowed by death. This group then would include plants such as the 
century plant (Agave) that may develop from five to twenty or 
more years before flowering, and many tropical bamboos, with life 
spans from two to perhaps over fifty years. Such plants clearly 



The Measurement of Flowering • 7 

differ somehow from typical perennials that flower and fruit over 
tens or even hundreds of years without evincing any ill effects. 

Many studies of flower initiation and development under nat- 
ural temperate-zone conditions have been made on individual 
species. A survey of a large number of species in Britain was re- 
ported by Grainger (1939). By determining the times of flower 
initiation, bud development, and subsequent anthesis (flower open- 
ing), Grainger distinguished three classes of temperate-zone plants. 
Direct-flowering plants are those in which development through 
anthesis follows on initiation without interruption; this is perhaps 
the commonest type of flowering behavior, found in both mono- 
carpic and perennial plants. Initiation and anthesis may occur 
either together with the maximum vegetative growth, as for ex- 
ample in bluebells (Campanula) and mint (Mentha), or at the 
period of minimum vegetative growth (winter or early spring) as 
in Saxifraga. A second class, indirect-flowering plants, contains 
those species in which a distinct period of rest intervenes at some 
stage between initiation and anthesis. Here again, initiation may 
coincide with the period of maximum vegetative growth, as in 
many fruit trees (Pyrus, Prunus) and in Anemone, or with the 
period of minimum vegetative growth, as in many bulb flowers 
(Tulipa, Narcissus) that initiate flower primordia in summer after 
the leaves wither. A third class, cumulative-flowering plants, form 
primordia over a long period of time, in regular succession, but 
anthesis of all occurs in a brief period. A number of weed species, 
notably dandelion (Taraxacum), are in this class. Grainger distin- 
guished a fourth class, climax-flowering plants, not found in the 
temperate zone but including long-lived monocarpic plants such as 
the bamboos mentioned above. 

Most experimental studies of flowering have been conducted 
on plants of Grainger's first class— direct-flowering plants initiating 
in the period of maximum vegetative growth. Other types have 
been studied, however, as will appear in the succeeding chapters. 
Unfortunately, but for obvious reasons, there has been little if any 
experimentation on long-lived monocarpic plants. 

THE MEASUREMENT OF FLOWERING 

The general structure of experiments on flowering is obvious- 
groups of plants given various treatments are kept under observa- 



8 • Background 

tion until the effects on flowering can be ascertained. The situations 
may be complicated by the fact that conditions bringing about 
initiation are not always the same as those favoring bud develop- 
ment, and these in turn may differ from those required for anthesis. 
As mentioned earlier, experimentalists have been most concerned 
with initiation; since, however, flower primordia in their earliest 
stages are detectable only by dissection and microscopy, the data 
in many studies have been based on the appearance of macroscopic 
buds or flowers. 

Within this general framework, methods of evaluating the 
results quantitatively are less obvious and vary considerably. The 
crudest method is simply to record the time required for the first 
appearance of the designated floral stage in the various treatments. 
This of course will vary between individuals given the same treat- 
ment, so averages are used. Alternative but related data are the 
percentage of plants in each treatment showing the designated 
stage at a given time after the start of the experiment. There are 
also plants, such as soybean (Glycine), in which flowering may 
occur at a number of nodes, and the effectiveness of treatments can 
be estimated by establishing the average number of nodes with 
flowers per plant after a given time. Still another related method 
is that of assigning arbitrary number values to various stages in 
the development of flower or inflorescence primordia. With a scale 
so established and an appropriate time for evaluation chosen, the 
plants in each treatment are dissected or examined and the result- 
ing values averaged. A danger of this method lies in the subjective 
judgments involved in assessing stages and assigning values to 
them. 

These procedures are all related in that the major independent 
variable, other than the nature of the treatments given, is time. 
That is, in a graph of results so obtained, each flowering value, 
however stated, is a function of time in or after treatment. A draw- 
back of such methods is that if the treatments differ in their effects 
on overall growth, and the times involved are (as is usual) a week 
or longer, differences in flowering values may simply reflect differ- 
ences in growth rate of the entire plant. For example, a 10° C 
increase in temperature might double the rate of vegetative growth 
and also that of the appearance of buds. But in such a case the 
rate of bud appearance relative to vegetative growth is unchanged, 
although time-based data would indicate more rapid flowering. 



The Measurement of Flowering • 9 

This sort of danger is widely recognized and it is usually avoided 
by careful workers. One way of doing this depends on the possi- 
bility, which, as will appear, is often present, of using treatments 
of short duration followed by a return of all the plants to the 
same conditions where the same rate of growth will be maintained. 
Or treatments may be found that have demonstrably little direct 
effect on vegetative growth rate. Another method, often combined 
with one of these, is to avoid the use of time as a variable. 

Instead of time, some index of the rate of vegetative growth 
can be used as an independent variable. The most common such 
index is simply the number of new leaves or nodes produced in or 
after treatment before the designated floral stage appears. The node 
or leaf index can be substituted for the time scale, and systems 
can be produced that are analogous to those using time. These 
matters of scale are not trivial. For instance, an experiment on a 
time scale might show that treatment A caused 45 percent flower- 
ing and treatment B 95 percent flowering after 20 days; the 
same results on a nodal scale (also after 20 days) might be: A, 
100 percent flowering by the third new node; and B, 10 percent 
flowering by the third new node. Results that "differ" as much as 
this are not uncommon and require care in interpretation. The 
reader may find it instructive to invent reasonable data from 
which such values could arise. 

Naturally, the choice of scale depends on the intention of the 
experimenter. For practical agricultural or horticultural purposes, 
emphasis is often placed on flowering time. Investigations on more 
fundamental questions however, such as the existence or non- 
existence of flower-inducing hormones, are bound to be concerned 
with flower initiation or development relative to vegetative growth. 
In the best practice, results are reported in sufficient detail so that 
the entire developmental situation can be assessed. Very few factors 
affect flowering exclusively, without modifying vegetative growth. 
Whether the changes are brought about indirectly, as a result of 
flowering, or directly, by the factors causing flowering, a plant 
which is flowering frequently differs from a vegetative one of the 
same age in height, branching, leaf shape, or pigmentation (to 
name only a few characteristics), and not simply in the production 
of flowers. Such changes may provide clues to the mechanisms 
underlying flower initiation, or they may be effects of flower 
development itself; in the cases studied so far, it is not clear which. 



chapter two 



Photoperiodism: 
An Outline 



For obvious reasons, flowering has been studied largely in 
plants in which it is controllable by environmental factors that 
in turn are easily controlled by the plant physiologist. Chief 
among such factors is the photoperiod, or daily length of illumina- 
tion. Whether or not it eventually turns out to be as significant 
for the flowering of most plants as it is for many that have been 
studied, the following three general statements can be made with 

certainty. 

The phenomenon to be defined as photoperiodism is observed 
not only among plants but in many animals as well, and is a wide- 
spread mechanism in the seasonal regulation of biological processes, 
particularly reproduction. Although it was first discovered through 
its connection with flowering, photoperiodism controls other plant 
processes also, even when it does not affect flowering. Finally, 
part of the basic mechanism involved in plant photoperiodism 
occurs in, and can modify the growth of, most higher plant cells 
and tissues. 

DEFINITIONS OF PHOTOPERIODISM 

Photoperiodism has been variously defined as a response to 
the daylength, photoperiod, or daily duration of illumination; as 
a response to the relative lengths of day and night, or light and 
darkness; or, in view of later information, as a response to the 

10 



Historical Note • II 

nightlength or daily duration of darkness. These definitions all 
convey the general idea, but they may be misleading. A more 
general definition is that photoperiodism is a response to the dura- 
tion and timing of the light and dark conditions. Total light 
quantity, even light intensity above a certain threshold level, is 
of secondary importance in photoperiodism, although it may be 
a modifying factor. The relative length, or ratio of the lengths of 
dark and light exposures, is also secondary. It is the time relations 
in which light and darkness succeed each other that appear to 
be crucial. 

Under natural conditions of a 24-hour day-night cycle, of 
course, the duration and timing of light exposure cannot be 
changed without a complementary change in the dark exposure, 
but cycle lengths totaling more or less than 24 hours have been 
used to study photoperiodism experimentally, as have brief light 
(or dark) interruptions of extended dark (or light) periods. Results 
from this sort of work have led to the definition given above. In 
nature, however, the lengths of day and night change seasonally 
except on the equator, and it is evident that photoperiodism might 
be expected to have some relation to the seasonal changes in 
biological events. In fact, it was observations on the relation 
between seasonal daylengths and flowering that led to the discovery 
of photoperiodism. 

HISTORICAL NOTE 

Like many important phenomena, photoperiodism was observed 
frequently before being finally "discovered." References to early 
observations by workers such as Tournois, Klebs, and others can 
be found in Murneek and Whyte (1948), a volume recommended 
to those interested in the history and early development of flower- 
ing physiology. Such observations suggested that flowering in 
plants such as hops (Humulus) or houseleek (Sempervivum) could 
be brought about by artificially shortening or lengthening their 
daily exposure to light. It remained, however, for Garner and 
Allard, plant physiologists in the U.S. Department of Agriculture, 
to show that such effects were not isolated curiosities. It was their 
early papers (1920, 1923) that attracted other workers to the field 



12 • Photoperiodism: An Outline 

and in which the term "photoperiodism" first appeared, although 
the definition favored above is not their original one. These papers 
are among the classics of plant physiology; not only do they outline 
many of the major problems still facing students of photoperiodism, 
but they are also models of the critical, at first almost reluctant, 
demonstration of what then seemed a revolutionary concept. 
Although there is no intention here to maintain a historical 
approach, a brief outline of two practical problems faced and 
explained by Garner and Allard will serve as a concrete introduc- 
tion to photoperiodism. 

GIANT TOBACCO AND SEPTEMBER SOYBEANS 

The preceding heading might well have been used by Garner 
and Allard to summarize the problems that led to their dis- 
covery. The tobacco, Nicotiana tabacum, was a mutant named 
Maryland Mammoth since it grew over 10 feet high in an experi- 
mental plot at Beltsville, Maryland. It nevertheless remained 
vegetative, thus frustrating its growers who wanted to use it in 
breeding experiments. Propagated by cuttings and grown in the 
greenhouse in the winter, however, the mammoth flowered and set 
seed when less than five feet high. Equally puzzling was the 
behavior of the Biloxi variety of soybean, Glycine (or Soja) max. 
When successive sowings were made at two-week intervals from 
early May through July, all of them showed their first flowers in 
September, so that the earliest planted had taken some 120 days 
to flower and the latest about 60. It was as if all were waiting for 
some signal at which to start flowering, irrespective of their age 
from germination— an improbable notion that turned out to be 
correct. 

After eliminating other factors such as temperature variations, 
nutrition, and light intensity, Garner and Allard concluded that 
the length of day was controlling flowering in both situations. 
Both Biloxi soybean and Maryland Mammoth tobacco are short- 
day plants, a term introduced by Garner and Allard. Neither will 
iflower unless the daylength is shorter than a certain critical number 
of hours (which happens to be different for the two plants). On 
sufficiently short days, flowering takes place. Thus Maryland 
Mammoth flowered in the greenhouse in winter under the naturally 



Kinds of Photoperiodic Flowering Responses • 13 

short days of that season, but merely vegetated and grew large in 
the field in summer and fall. Biloxi soybeans, no matter when 
they were planted, would not flower until the sufficiently short days 
of late summer. Garner and Allard were able to show all this 
experimentally both by artificially shortening the summer days 
(placing the plants in light-tight sheds or cabinets at various times) 
or artificially lengthening winter or fall days even with dim 
incandescent lights. They also examined the effects of various 
daylengths on other plants and discovered various kinds of flower- 
ing responses, as well as many other effects. Work on photoperiodism 
soon became world-wide and has remained so, with major contribu- 
tions coming from Britain, France, Germany, Italy, Japan, the 
Netherlands, Russia, the United States, and elsewhere. 

KINDS OF PHOTOPERIODIC FLOWERING RESPONSES 

The flowering responses of various plants to different day- 
lengths in a normal 24-hour cycle can be roughly grouped into 
the following classes, of which the first two are those commonly 
studied. 

1. Short-Day Plants: The abbreviation SDP will be adopted 
for these hereafter. Flower initiation in SDP is promoted by day- 
lengths shorter than a particular value, the so-called critical day- 
length, which differs widely from species to species. It is probably 
actually the nightlength that is the most critical factor in such 
plants; hence, they have been described as "long-night plants." 
Much more work has been done with SDP than with the other 
classes. Examples are Maryland Mammoth tobacco and Biloxi 
soybeans, discussed above, also the common cocklebur, Xanthium, 
and the succulent Kalanchoe blossfeldiana. See the illustration 
facing page 1 and Fig. 2-1 for two examples of SDP. 

2. Long-Day Plants: The abbreviation LDP will be used for 
these. Flower initiation is promoted by daylengths longer than a 
particular value, the critical daylength, which differs from species 
to species. Again, such plants have also been described as "short- 
night plants." Examples are the Black Henbane, Hyoscyamus niger, 
and some varieties of barley, Hordeum vulgare. 

3 and 4. Short-Long- and Long-Short-Day Plants: Flower 



14 



Photoperiodism: An Outline 



initiation in a relatively few plants appears to be promoted by 
successive exposures to the kinds of conditions promoting it in 
classes 1 and 2, in an order depending upon the particular species. 
Each requirement in a given species may have its own critical 
daylength. Such plants have been little studied but may be valuable 




Fig. 2-1. Short-day response in morning glory (Ipomoea hederacea var. Scarlett 
O'Hara). Plants are about 8 weeks old, all grown with 8 hours of sunlight per 
day. In addition, the plant to the right received a further 8 hours per day of dim 
(40 foot candles) incandescent light for a total photoperiod of 16 hours. (Photo- 
graph from Hendricks [1956], American Scientist, 44: 229-247, by permission of 
the board of editors of the American Scientist and courtesy of Drs. H. A. Borthwick 
and S. B. Hendricks, U. S. Department of Agriculture.) 



in analyzing the photoperiodic mechanism. Some varieties of wheat, 
Triticum vulgare, and rye, Secale cereale, may be short-long-day 
plants; some Bryophyllum species and the night-blooming jasmine, 
Cestrum nocturnurn, are long-short-day plants. 

5. Day-Neutral or Day length-lndifj event Plants: These simply 
flower after reaching a certain age or size and apparently irre- 
spective of daylength. Other processes, however, may be photo- 



Kinds of Photoperiodic Flowering Responses ■ 15 

periodically controlled. Flowering in such plants, which may 
constitute the majority, has been relatively little studied. Com- 
mon examples are tomato, Lycopersicon esculentum, and many 
varieties of peas, Pisum sativum. 

Note that in this classification the distinction between SDP 
and LDP is based not on the absolute values of the critical day- 
lengths (which may range from four to over 18 hours for LDP, 
for example); the distinction is whether flowering is promoted 
by photoperiods shorter or longer than the critical. The critical 
daylength for Xanthium, for example, is about \o l / 2 hours, and 
that for Hyoscyamus about 11 hours. Yet the former is properly 
classified as an SDP since it flowers on photoperiods shorter than 
its critical value, whereas the latter is an LDP, requiring photo- 
periods longer than its critical. It is necessary to belabor this 
distinction since it is possible to find textbooks that should know 
better implying that LDP flower with more hours of light per day 
than SDP. Such statements miss the point. Both Xanthium and 
Hyoscyamus flower with 14 hours of light per day. The daylength 
in which a plant flowers is no indication of its response class in 
the absence of further information. 

In addition to the classes of response described, the following 
considerations should be recognized before proceeding further. 
There are plants in which the appropriate photoperiodic treatment 
is an absolute requirement for flowering under all naturally 
occurring conditions. Neither Xanthium nor Hyoscyamus, for 
example, ever flowers unless exposed to the proper photoperiodic 
conditions. Such plants are referred to as having a qualitative 
photoperiodic response, or requirement. In other plants, differing 
photoperiodic conditions merely hasten or delay but do not abso- 
lutely determine flower initiation. Such plants have a quantitative 
response to photoperiod. There are also plants in which qualitative 
or quantitative photoperiodic responses are observed only under 
particular conditions of temperature or some other environmental 
factors; these would be conditional photoperiodic responses. Still 
other plants may require one photoperiodic condition for flower 
initiation but a markedly different one for flower development. 
Finally, there are many species in which the photoperiodic response 
may change with age; such changes are usually in the direction 



16 • Photoperiodism: An Outline 

of day-neutrality from an initial qualitative or quantitative long- or 
short-day response. 

A particularly clear example of the last sort of behavior is 
shown by a variety of sunflower, Helianthus annum, recently 
studied by Dyer et al. (1959). Seedlings raised under 12-hour 
daylengths all showed inflorescences after 40 days, while seed- 
lings raised under 16-hour daylengths showed no detectable flower 
primordia at the time. Over 90 percent flowering occurred on 
both 12- and 16-hour photo periods in experiments carried to 130 
days, however, and even 20-hour photoperiods gave over 70 percent 
flowering. In other words, young plants had a qualitative short-day 
response with a critical daylength between 12 and 16 hours, but 
older plants were either day-neutral or showed a weak quantitative 
short-day response. 

While this brief list by no means exhausts the ways in which 
photoperiodic responses may differ within the overall classification, 
and examples will appear frequently in what follows, there do 
appear to be limits on such variation. Although varieties of the 
same species often differ in critical daylength and frequently show 
a range from day-neutrality to a qualitative long- or short-day 
requirement, the writer knows of no species with both LDP and 
SDP varieties; it is even relatively unusual to find both types 
within a single genus. The range of variation that can be caused 
by age or environmental conditions is also apparently limited in 
the same way as that within a species; that is, no experimental 
treatment yet found will convert an LDP to an SDP, or vice versa. 
Such an effect would obviously be very valuable for studies of the 
mechanism involved. Aside from these generalizations, however, 
the responses of species and varieties within a given class are 
extremely various, and there is no evident correlation between 
photoperiodic response classes and any taxonomic or ecological 
category. Thus, although much of this discussion will proceed by 
considering some of the results from a few well-studied plants, let 
the reader beware: the country is large, and the map, so far, is 
small. For many variations and modifications in photoperiodic 
response that have not been studied systematically, see Chouard 
(1957). 



Leaves in Photoperiodism • 17 



THE ROLE OF LEAVES IN PHOTOPERIODISM. 
PHOTOPERIODIC INDUCTION 

Neither of these topics will be considered in detail until 
Chapter Five where the discussion is on the nature of the flower- 
ing stimulus, since both are more germane to that question than 
to photoperiodism proper. Brief summaries are given here simply 
to render the rest of this chapter intelligible. 

In almost every plant studied, it is the leaf blades that perceive 
the photoperiodic treatment. This has been shown in several ways. 
Photoperiodic treatments given to all, or in some cases one or a 
few, leaf blades on a plant will have the same effects as though 
the entire plant had been treated. Defoliated plants, with rare 
exceptions, are photoperiodically unresponsive. Photoperiodic 
treatment of the apices or other meristematic areas is usually in- 
effective, although the meristems are the actual sites of the change 
from vegetative to reproductive growth. One can conclude that 
the primary photoperiodic effect occurs in the leaves and that the 
leaves somehow communicate its results to the meristems. 

Certain plants require more or less constant exposure to 
appropriate photoperiodic cycles, at least until flower primordia 
can be easily detected, in order to flower successfully. In many 
others, however, exposure to only a few such cycles will cause 
flowering even when the plants are returned to unfavorable photo- 
periodic conditions. Such plants are said to be induced by the 
photoperiodic treatment; photoperiodic induction is an aftereffect 
of favorable photoperiods which will result in flowering or at least 
considerable primordium development, even on unfavorable photo- 
periods. An induced plant indicates clearly by this behavior that 
some change has taken place and persists, but no anatomical or 
morphological changes can usually be detected after the few induc- 
tive cycles required in such plants. Naturally, not only is induction 
of great theoretical interest but it is also experimentally useful. 
One of the major reasons for the widespread use of Xanthium 
in photoperiodic studies is that, under favorable conditions, a 
single short-day cycle (even given to a single leaf) will lead to 
flowering in plants kept the rest of the time on noninductive long 
days. This sensitivity to a single cycle is unusual, but is not unique 



18 • Photoperiodism: An Outline 

to Xanthium; it has been reported also in the Japanese morning 
glory, Phcrbitis (or Ipomoea) nil (Imamura and Takimoto, 1955a), 
a duckweed, Lemna perpusilla (Hillman, 1959a), and pigweeds, 
Chenopodium (Gumming, 1959), all SDP. Many other SDP also 
can be induced by 2 to 10 days of the appropriate photoperiodic 
treatment. Induction by a very few cycles is perhaps less common 
among LDP, although at least dill, Anetham graveolens (A. W. 
Naylor, 1941), and mature plants of the grass Lolium temulentum 
(Evans, 1960) are both inducible by one long-day cycle. 

THE CENTRAL ROLE OF THE DARK PERIOD 

While the terms "short-day" and "long-day" plant have been 
maintained by constant usage, probably the most important single 
difference between these two response classes is in their reactions 
to the nightlength, or dark period. In general, flowering in SDP 
is promoted by certain reactions taking place during the dark 
periods, and the "critical daylength" actually represents the maxi- 
mum daylength that will allow a dark period of sufficient length 
in a normal 24-hour cycle. In LDP, on the other hand, dark periods 
have an inhibitory effect on flower initiation, and the critical 
daylength is thus the minimum which in a 24-hour cycle will keep 
the dark period short enough to allow flowering. These generaliza- 
tions are supported by the fact that LDP usually flower best on 
continuous light, so that apparently the entire role of the dark 
period is inhibitory (A. W. Naylor, 1941; see Lang, 1952). Several 
SDP, on the contrary, flower in continuous darkness if they are 
given sucrose (see Doorenbos and Wellensiek, 1959; Hillman, 
1959a), suggesting that light is unnecessary if its photosynthetic 
function is replaced by another source of carbohydrate. However, 
at least one LDP, spinach, Spiiuicia oleracea, also flowers in total 
darkness when supplied with sucrose (GentschefT and Gustaffson, 
1940) so that reliance on this sort of evidence alone is undesirable. 

Hamner and Bonner (1938) were able to show that in 
Xanthium the critical time for an appropriate photoperiodic 
treatment lay in the dark period length. When 24-hour cycles of 
light and darkness were used, these plants flowered with dark 
periods of 8% hours or longer. Thus the critical daylength was 
15% hours. No flowering occurred on schedules of 16 hours light- 



The Central Role of the Dark Period • 19 

8 hours darkness. To determine whether it was actually the day- 
length or nightlength that was critical in this schedule, Hamner 
and Bonner performed several kinds of experiments. 

Using artificial light when necessary, they exposed some plants 
to schedules of 4 hours light-8 hours darkness. None of these 
flowered, although each light period was far shorter than the critical 
daylength of 15 y> hours. On the other hand, all plants flowered 
rapidly under cycles of 16 hours light-32 hours darkness, even 
though each light period was longer than the critical daylength. 
Two conclusions come from such data. First, it seems to be the 
length of the dark period, not that of the light period, that is 
important for Xanthium. Second, the relative length of day and 
night is clearly not the critical factor since the ratio of light to 
darkness was the same in both schedules used. 

Perhaps the best evidence concerning the role of the dark 
period in both LDP and SDP can be obtained by interrupting 
these dark periods with brief light exposures. Hamner and Bonner, 
for example, showed that the inductive effects of 9-hour dark periods 
could be completely annulled by interrupting each one in the 
middle with a minute of relatively dim (150 foot candles) incandes- 
cent light. This "light-break" effect is widespread among both 
response classes, and the general situation can be summarized as 
follows (see, for example, Borthwick, Hendricks, and Parker, 1956). 

In order to be photoperiodically effective in either SDP or 
LDP, a dark period of sufficient length has to be uninterrupted. 
Total light energies (100-1000 kiloergs/cm 2 ) that are very low 
compared to those of daylight, even given in a few minutes, are 
sufficient to constitute an effective interruption. In SDP such as 
Xanthium or Biloxi soybeans, light-breaks in otherwise inductive 
dark periods will completely inhibit flowering. In LDP such as 
Hyoscyamus or the Wintex variety of barley, Hordeum vulgare, 
light-breaks in otherwise noninductive periods (that is, in schedules 
with daylengths less than the critical) bring about flowering as 
though the plants had been on an adequate long-day schedule. 
As will become evident later on, light-break experiments have 
proved very useful for further studies on the mechanism of photo- 
periodism. At this juncture, however, they are simply presented as 
evidence for the role of the dark periods as the single most im- 
portant controlling factor in photoperiodism. Similarly, brief "dark- 



20 • Photoperiodism: An Outline 

breaks" during main light periods have essentially no effect on 
the process. 

The evidence reviewed above should make clear the reason lor 
emphasizing duration and timing of light (and darkness) rather 
than total energy in the definition of photoperiodism. It has 
also resulted in the term "critical nightlength" replacing "critical 
daylength" in some reviews and articles on the subject, in order 
to stress the relative importance of light and dark periods. However, 
as will be shown, light also plays a role, although perhaps less 
important, in the normal time requirements of photoperiodism, so 
that the second terminology is only slightly more accurate than the 
first. Either will be used, as occasion demands. 

Ancillary evidence for the more crucial role of the dark periods 
has also been derived from experiments in which temperature is 
varied, some of which will be considered elsewhere. 

REQUIREMENTS FOR HIGH-INTENSITY LIGHT 

The effects of brief or prolonged exposures to low-intensity 
light, nullifying dark periods, will be considered in detail in the 
next chapter. Meanwhile, after setting up generalizations that dark- 
ness plays the major role in photoperiodism and that the total 
light energy during a treatment or cycle is relatively unimportant, 
it is now necessary to consider what role, if any, is played by the 
high-intensity light periods which, at least in nature, normally 
alternate with dark periods. 

1. Short-Day Plants: Early work with SDP soon showed that 
in spite of the critical role of the dark periods, the main light 
periods also had to include at least a certain amount of high- 
intensity light for optimum (lowering to occur in many plants. 
An elegant demonstration of this was given by Hamner (1910). 
using Xantliium. 

It was obviousl) not reasonable to study the effect of a dark 
period preceded by a dark period, since the two together simply 
add up to a longer one. Hamner made use ol the light-break tech- 
nique, however, in the following manner. Xanthium plants can be 
kept vegetative on cycles of 3 minutes Light-3 hours darkness. 
After a lew such cycles, a single dark period of 12 hours, which 



Requirements for High-Intensity Light • 21 

would normally cause flowering if the plants were subsequently 
placed on long-day conditions, was entirely ineffective. Before such 
a dark period could be effective, the plants had to be exposed to 
at least a few hours of high-intensity light; within limits, the 
effectiveness of the dark period was then directly related to the light 
energy given before it. This "high-intensity light reaction" clearly 
differs from the low-intensity reaction sufficient to interrupt a dark 
period, since it requires light energies some 10,000 times higher for 
maximum effect. It has since been shown that C0 2 must be present 
for the high-intensity light to have its effect; in addition, feeding 
the leaves with carbohydrates or organic acids can at least partially 
replace the high-intensity light requirement (see Liverman, 1955). 
Such results suggest that this requirement is largely a requirement 
for products of photosynthesis. 

Another high-intensity light requirement has also been reported 
in Xanthium. To be maximally effective, an inductive dark period 
must be followed as well as preceded by a period of high-intensity 
light. Lockhart and Hamner (1954), for example, found that if 
only a brief light flash was given to end the inductive dark period 
and this was then followed by another dark period before the 
plants were replaced in long-day conditions, flowering was com- 
pletely or partially inhibited. A period of high-intensity light given 
before the second (inhibitory) dark period rendered it ineffective, 
but low-intensity light did not. Both auxin (see Chapter Six) and 
high temperature increased the effect of the second dark period. 
Subsequently, Carr (1957) found that sucrose given to the leaf 
during the second dark period almost nullified the inhibition, 
allowing flowering to take place. He thus suggested that the "second 
high-intensity light requirement," like the first, is a requirement 
for photosynthetic products. 

While experiments of this sort show that high-intensity light 
periods can have profound modifying effects on photoperiodic 
induction, these are probably due to effects of photosynthate as an 
energy source and on the translocation of the flowering stimulus 
(see Chapter Five) rather than on photoperiodism proper. Even 
Xanthium, on which the most detailed work of this kind has been 
done, can eventually initiate flowers in total darkness (Hamner, 
1940). Thus the primary role of the dark period in photoperiodism 
is not contradicted by these data. 



22 • Photoperiodism: An Outline 

The interpretation of high-intensity light requirements in SDP 
as basically photosynthetic is not entirely secure. Kalanchoe bloss- 
feldiana is an SDP incapable of flowering in continuous darkness. 
It will, however, initiate flowers if it receives one one-second flash 
of light in every 24 hours (see Harder, 1948; Schwabe, 1959). 
Although COo is indeed required during the light flash, it is not 
likely that a great deal of photosynthesis takes place during that 
time, so that a more specific requirement is at least suggested. 

Even the generalization that the photoperiodic responses of SDP 
are generally promoted by at least some exposure to high-intensity 
light does not hold for the widely studied Perilla. Using Perilla 
crispa, de Zeeuw (1953) found that the critical daylength becomes 
longer (dark requirement becomes shorter) as the main light period 
intensity is lowered; with sufficiently low light intensities, flower 
initiation occurs under continuous light. A set of experiments on the 
complex interactions of bright and dim light periods on Kalanchoe 
has been published by Krumwiede (1960), who also provides a 
thorough bibliography on the question. It seems clear that probably 
more factors than photosynthesis are involved in the effects of 
bright light. 

2. Long-day Plants: Since, in general, the longer the light 
period the better for flowering in LDP, analyses of the kind de- 
scribed above have attracted little interest. A number of LDP are 
nevertheless known to flower more rapidly in either continuous 
light or long photoperiods if at least part of each light exposure 
is at high intensity (see Bonner and Liverman, 1953). Much of the 
work on the main light periods of LDP, like some of that on SDP, 
has been on the effects of various light colors, and will be considered 
in the next chapter. 

MUTUAL INTERACTIONS OF LIGHT AND 
DARK PERIOD LENGTHS 

Extremely complex interactions between light and dark period 
lengths have been observed in both LDP and SDP, to the extent 
that the critical values of either light or dark periods are markedly 
aflected by the lengths of the complementary periods. 

Claes and Lang (1947) studied the effects of various light and 
dark schedules on the rapidity with which the LDP Hyoscya?nus 



Interactions of Light and Dark Period Lengths • 23 

niger would initiate flowers. As long as the light-dark cycles totaled 
24 hours, flowering occurred with at least 1 1 hours of light per 
cycle, and was most rapid with 15-16 hours. When cycles totaling 
48 hours were used, however, flowering occurred with as few as 
9 hours light per cycle, and reached its maximum rapidity with 
13 hours per cycle. Thus longer total cycle lengths actually reduced 
the "critical daylength" by at least two hours, in spite of the fact 
that the shorter daylength was active with a much longer dark 
period. 

Differing but equally complex results were obtained by 
Takimoto (1955) in experiments in which he exposed the LDP 
Silene armeria to 10-day treatments of cycles composed of various 
durations of light and darkness. Flower initiation was most rapid 
in continuous light. In cycles with light periods of 12 hours or 
shorter, initiation occurred only when the associated dark periods 
were shorter than 13 hours; in cycles with light periods of 14 or 16 
hours, however, even dark periods of 24 or 32 hours duration failed 
to prevent initiation. Some of the interactions between light and 
dark periods in the SDP Biloxi soybeans were studied by Blaney 
and Hamner (1957). Only a few of the results will be mentioned 
here, but this paper provides one of the best examples of the com- 
plexity of such interactions and resultant difficulty of reaching any 
general conclusions on the problem at present. The Biloxi soybean, 
like most SDP, requires several cycles of appropriate photoperiodic 
treatment to initiate flowers. When plants were given 7 cycles of 
8 hours fluorescent light and 16 hours darkness, then placed on 
long-day greenhouse conditions, high flowering values were ob- 
tained. Hence 8-hour light periods and 16-hour dark periods 
together constitute an inductive cycle. However, when each portion 
of such an inductive cycle was examined separately, the following 
results were obtained. Seven cycles of 8 hours light alternating with 
24-hour or 26-hour dark periods resulted in no induction at all. 
Seven cycles of 16-hour dark periods alternating with light periods 
either 4 hours or shorter, or longer than 12 hours, also resulted in no 
induction. For further results and tentative conclusions the original 
paper should be consulted. The concept of a minimum critical 
dark period requirement was still supported since induction was 
never brought about by any cycle with less than a 10-hour dark 
period, no matter what the associated light period; however, it 



24 • Photoperiodism: An Outline 

also did not occur on cycles containing 16-hour light periods, no 
matter what the dark period. 

The generalization that crucial events in photoperiodism take 
place during the dark period is evidently not annulled by results 
such as those presented in this section. The precise values of 
"critical nightlengths," however, arc markedly dependent upon the 
lengths of the associated light periods, and in a manner which 
conforms to no simple pattern. 

INTERACTION OF DIFFERENT PHOTOPERIODIC CYCLES: 

FRACTIONAL INDUCTION IN LDP AND 

LONG-DAY INHIBITION IN SDP 

In all the experiments so far considered, not more than one 
particular kind of light-dark cycle was used for each experimental 
treatment, although such cycles might be repeated several times. 
It is desirable to examine some results of using more than one kind 
of cycle in a given treatment. Most such experiments have been 
concerned with the effects of intercalating noninductive between 
inductive cycles, and have naturally been conducted largely with 
plants requiring more than one cycle for induction. The responses 
of LDP and SDP to such treatments differ fairly consistently from 
each other, but show considerable regularity within each class. 

Most LDP studied are susceptible to "fractional induction." 
This is best illustrated by an example reported by Snyder (1948). 
Plants of the plantain Plantago lanceolata showed 100 percent 
inflorescences after exposure to 25 long-day cycles (18 hours light- 
6 hours darkness). Exposure to only 10 such cycles resulted in no 
flowering when followed by exposure to short-day cycles (8 hours 
light- 16 hours darkness). However, if 10 long-day cycles were given 
and followed by 20 short-day cycles, only 15 more long-day cycles 
were required for 100 percent inflorescence formation. Thus the 
effect of the first 10 inductive cycles, though insufficient to cause 
flowering by itself, persisted throughout the short-day treatment 
so that only 15 more long-day cycles gave the effective total of 25. 
This remarkably accurate "memory" is apparently not unusual in 
fractional induction. It implies that, in such LDP at least, non- 
inductive cycles play a merely passive role and do not oppose the 
effects of inductive cycles. 



4 

Photoperiodism and Temperature ■ 25 

In several SDP, on the other hand, noninductive cycles have 
a clearly inhibiting action on induction. Schwabe (1959) has shown 
that for Perilla ocymoides, Chenopodium amaranticolor, and Biloxi 
soybean, noninductive cycles intercalated between inductive cycles 
positively inhibit the effects of the latter. Each long-day cycle, in 
fact, appears capable of counteracting the effect of two short-day 
cycles. A long-day cycle probably acts by annulling the effectiveness 
of the short days immediately following it, rather than by destroy- 
ing the effect of the short days preceding it. Such a conclusion agrees 
with the results of Harder and Biinsow (1954) who had found that 
the number of flowers formed by Kalanchoe blossjeldiana after a 
given number of short-day cycles was inversely related to the 
daylength used in the noninductive cycles on which the plants were 
kept previous to short-day treatment. However, Carr (1955) obtained 
fractional induction in a number of SDP, including some of the 
same plants used by Schwabe, above. Carr also cites other results 
that oppose the generalization that only LDP exhibit the phe- 
nomenon, holding instead that it shows no particular correlation 
with response type but rather is an individual species characteristic. 

Possibly the ability of Xanthium and a few other SDP to 
flower in response to one short-day cycle is due to the lack, or 
weaker operation, of inhibitory long-day effects. Even in Xanthium, 
of course, flowering intensity increases proportionately to the 
number of short-day cycles over a considerable range (see Chapter 
Five) so that the phenomenon may be quite general. 

PHOTOPERIODISM AND TEMPERATURE 

Temperature enters into the physiology of flowering in numerous 
ways, many of which will be considered later. A few interactions of 
temperature with photoperiodism will be mentioned now, but with 
the cautionary note that the results of such studies tend to defy 
generalization more completely than any other aspect of the field. 
For a major treatment of the effects of temperature on plant 
growth, see Went (1957). 

Temperatures differing slightly from one another may strongly 
modify the effects of daylength on flower initiation. For example, 
Roberts and Struckmeyer (1938) found that both Maryland Mam- 
moth tobacco and Jimson weed, Datura stramonium, were SDP only 



26 • Photoperiodism: An Outline 

at 24° C or higher, but tended toward day-neutrality at about 
13° C. Strawberry, Fragaria virginiana x chiloensis, shows a virtually 
identical response (Went, 1957, Chap. 11). The requirement of 
at least a flash of bright light for induction of Kalanchoe, men- 
tioned previously, has been confirmed by Oltmanns (1960) at 20° 
or 25°, but apparently is no longer present at 15° C, since Kalanchoe 
will initiate flowers in total darkness at that temperature. 

The critical daylength for certain LDP is reduced at low 
temperatures. Hyoscyamus niger grown at 28.5° C requires at least 
1 1 Vo hours of light per day to flower, whereas at 15.5° the critical 
daylength is reduced to 8 ]/ 2 hours (see Melchers and Lang, 1948). 
However, the LDP Rudbeckia bicolor will flower at relatively high 
temperatures (about 32° C) under photoperiods too short to permit 
flowering under cool conditions; Rudbeckia speciosa, a similar 
species, remains a true LDP under both conditions (Murneek, 
1940). 

Most effects of this kind have been ascribed primarily to dark 
period rather than light period temperatures (see Lang, 1952), 
but unusual temperatures can modify both light and dark period 
processes. Two of the early papers on Xanthium illustrate this 
point. 

Long (1939) found that Xanthium required at least six cycles 
of 9 hours light- 15 hours darkness for induction if the dark period 
temperature was 5° C, even though the light periods were given 
at 21° C. Further experiments showed that when plants were 
grown at 21° light temperature and 5° dark temperature, the 
( ritical nightlength was increased to about 1 1 hours compared with 
8% hours for plants held constantly at 21°. Long concluded that 
"variations in temperature greatly affect the length of the critical 
dark period," although his work has been cited, in a context to be 
discussed later, as showing a "relatively temperature-independent 
time measurement of nightlength" (Pittendrigh and Bruce, 1959). 

The light period processes in Xanthium also are temperature- 
sensitive, at least when they are made relatively limiting (Mann, 
1940). At least four hours of bright light (over 2000 foot candles) 
are required for the optimum action of a subsequent dark period 
if the light is given at 10° C, but only about one half hour of light 
is required at 30° for the same effect. 

The sensitivity of light or dark periods to temperature changes 



Photoperiodism and Vegetative Growth • 27 

has been studied extensively in connection with the possible rhyth- 
mic components of photoperiodism (see Chapter Three). The paper 
by Blaney and Hamner, previously cited, also contains data on the 
interactions of temperature with the various light-dark cycles used. 
A simpler example of such work is a paper by Schwemmle (1957) 
reporting the effects on the SDP Kalanchoe blossfeldiana of brief 
exposures to 30° C during various portions of 12-hour dark periods 
alternated with 12-hour light periods (inductive for Kalanchoe), 
the temperature otherwise being 20°. Such exposures promoted 
flowering significantly when given for the first three hours of each 
dark period, but inhibited it completely when given for the last 
three hours. Full 12-hour exposures to 30° during the night also 
inhibited completely. 

One of the most striking temperature effects reported recently 
deals again with Xanthium, which will apparently flower on a 
16 hours light-8 hours darkness schedule, completely noninductive 
at 23° C, if the first 8 hours of each light period are given at 4°. 
Low temperatures during the second half of each light period, or 
during the dark period itself, do not cause flowering, nor does 
flowering occur on continuous light with any alternation of tem- 
peratures used (Nitsch and Went, 1959); see Fig. 2-2. The SDP 
Pharbitis can be brought to flower even under continuous light 
by low-temperature treatments (Ogawa, 1960). 

On the basis of some experiments with Hyoscyamus and the 
SDP Chenopodium, as well as other results in the literature, 
Schwemmle (1960) has suggested in a brief paper that, in a physio- 
logical sense, high temperatures may be equivalent to light and 
low temperatures to darkness in their effects on photoperiodism. 
Whether this generalization will withstand critical examination 
remains to be seen. So far, all that can be said with certainty is 
that high or low temperatures can modify both dark and light 
processes in photoperiodism in a manner varying widely with the 
temperatures, species, specific cycle, and portion of light or dark 
period chosen. 

PHOTOPERIODISM AND VEGETATIVE GROWTH 

Structures and processes of all kinds can be affected by photo- 
periodism, and such results are widespread in the literature, 



28 • Photoperiodism: An Outline 




Fig. 2-2. Photoperiodic control of flowering in cocklebur {Xanthium pennsyl- 
vanicum) as modified by low temperature. Growing points of plants of the same 
age — with all except terminal leaves removed to show development — photo- 
graphed after 13 days of the following treatments: (A) 8-hour days at 23° C 
(flowering); (B) 16-hour days at 23° C (vegetative); (C) 16-hour days as in (B) 
but with 4° during first 8 hours of each light period; (D) 24-hour (continuous) 
days at 23° except 4° during 8 hours of each day. (Photographs from Nitsch and 
Went [1959], by permission of the American Association for the Advancement 
of Science and courtesy of Dr. J. P. Nitsch, Le Phytotron, Gif-sur-Yvette, France.) 

starting with Garner and Allard. Some of the characteristics 
frequently under photoperiodic control even when flowering is not 
are stem elongation, leaf shape and size, branching, pigmentation, 
tuberization, and pubescence (see, for example, Nay lor, 1953). 
Effects on these have been studied far less than the flowering 
responses, but the data at hand suggest that they are less likely 
to be inductive. That is, when the photoperiodic conditions are 
changed, the new vegetative growth quickly reflects the new con- 
ditions. This may even be true when the vegetative change would 
normally be associated with a truly inductive effect on flowering. 
In Murneek's work on Rudbeckia bicolor, for example, continuous 
treatment with long days (longer than 12 hours) caused both 
flowering and stem elongation. Exposure to only 25 long days still 
brought about flowering, both normal and abnormal, but the plants 
remained in a semirosette stage. 

Many papers on responses of all types make it difficult to decide 
whether they are truly photoperiodic or not. Paradoxically, this 



Literature • 29 

is more often true in very recent research, since air conditioning 
now makes it possible to grow plants entirely under high intensi- 
ties of artificial light. This frequently results in comparisons between 
plants grown, for example, in 8 and 16 hours of light per day, 
comparisons with the implicit or explicit assumption that the 
operative difference between treatments is in light duration, even 
though the total light energies also differ proportionately (see, 
for example, Galston and Kaur, 1961; also portions of Went, 1957). 
It would help clarify the literature if the term photoperiodic were 
properly restricted to effects that have been concurrently or pre- 
viously shown to be controlled by light and dark duration and 
timing, as indicated by light-breaks or low-intensity supplementary 
illumination. Any other use of the term only results in confounding 
photoperiodism with the effects of greatly increased or decreased 
photosynthesis, or other light actions. 

LITERATURE 

The literature on photoperiodism is vast. Some of the most use- 
ful reviews are by Lang (1952), Naylor (1953), Bonner and Liver- 
man (1953), Borthwick, Hendricks, and Parker (1956), and Door- 
enbos and Wellensiek (1959). A volume edited by the late R. B. 
VVithrow (1959) contains many valuable reviews and original re- 
ports on photoperiodism and related phenomena in both plants 
and animals. 



► 
► 



chapter three t Photoperiodism: 

Attempts at Analysis 



Faced with the various phenomena of the previous chapter, 
many investigators of photoperiodism have naturally tried to dis- 
cover characteristics common to the various response classes, and 
particularly to look for indications of whatever cellular and bio- 
chemical mechanisms might be involved. Two major lines of such 
research, by no means completely separate, are the subject of this 
chapter. 

A. PHOTOPERIODISM AND LIGHT QUALITY 

So far, photoperiodism has been considered simply in terms 
of white light versus darkness, but experiments with light quality— 
different colors or wavelengths of light— have proved very valuable. 
They have opened up photoperiodism itself to further manipula- 
tion and linked it to a biochemical system, still incompletely 
known, that is probably universal among plants except perhaps for 
the bacteria and fungi. The main point of departure for this work 
was the effectiveness of relatively brief, low-energy "light-breaks" 
in opposing the flower-promoting or flower-inhibiting (for LDP) 
effects of appropriate dark periods. 

ACTION SPECTRA FOR LIGHT-BREAKS 

In order to act on any process, light must first be absorbed. 
Compounds, called pigments, that absorb visible light are generally 

30 



Photoperiodism and Light Quality • 31 

complex organic compounds, although many inorganic salts are 
highly colored. The absorption spectrum of a given pigment, by 
which is meant a curve indicating the relative degree to which it 
absorbs various wavelengths of light, is characteristic of that com- 
pound alone, or at least of a small class of similar substances. Thus 




Fig. 3-1. Method of holding single leaves (these are soybean leaflets) in the 
image plane of a spectrograph for subsequent irradiation with various wave- 
lengths of light. (Photograph from Hendricks and Borthwick, Proc. First Int. 
Photobiol. Cong. [1954], courtesy of Dr. H. A. Borthwick, U. S. Department of 
Agriculture.) 



the action spectrum for any process affected by light— a curve 
indicating the relative effectiveness of different wavelengths on the 
process— may provide information as to the nature of the com- 
pound or compounds by which the light is absorbed. For example, 
part of the evidence for the role of chlorophyll in photosynthesis 
is the observation that the light most active in that process— blue, 



32 • Photoperiodism: Attempts at Analysis 

wavelengths 4000-4500 A (Angstrom units), and red, 6200-6800 
A— is also the light most strongly absorbed by chlorophyll solutions. 
That is, the action spectrum for photosynthesis resembles the ab- 
sorption spectrum of chlorophyll solutions. 

In principle, this seems simple enough; in fact, the accurate 
determination and evaluation of absorption and action spectra is a 
complex, still-developing branch of physics and chemistry, as well 
as biology; for some references, see articles in Hollaender (1956) 
and Withrow (1959). For present purposes, however, it should be 
evident that the action spectra for light-break elfects in various 
plants might indicate whether or not these effects are mediated by 
the same pigment and what that pigment might be. 

Much of the work on this question has been done by Garner 
and Allard's successors, a group at the U.S. Department of Agricul- 
ture, Beltsville, Maryland, and many reviews by the original 
workers are in the literature (see, for example, Borthwick, Hen- 
dricks, and Parker, 1956; Borthwick, 1959; Hendricks, 1958, 1959). 
Their procedures are basically simple, though not technically easy. 
Stating the situation more quantitatively than before, an action 
spectrum can be represented either as a graph of varying responses 
brought about by equal energies of light of given wavelengths, or 
as a graph of the energy which must be given at each wavelength 
to cause a particular degree of response. Thus it is necessary to 
measure the effect of each wavelength chosen at several energv 
levels, and on a considerable number of plants; this requires light 
of considerable intensities but in relatively pure wavelength bands 
spread out over considerable areas. For this purpose, the Beltsville 
group built a large spectrograph, in which high-intensity white 
light could be passed through a prism and projected as a spectrum. 
They then took advantage of the fact that in the plants chosen 
photoperiodic treatments need only be given to a single leaf if 
the other leaves were removed. The single leaf could be placed so 
as to receive light of a particular color and energy at the optimal 
time for dark period interruptions; after main such experiments, 
the relative effectiveness of the various colors can be calculated. 
(See Figs. 3-1 and 3-2.) 

From 1946 on, action spectra for light-break responses were 
obtained in both SDP and LDP, including Xanthium, Biloxi soy- 
bean, Hyoscya/nu.s. and Wintex barley. All these spectra seem 



Photoperiodism and Light Quality 



33 



substantially alike; the most effective wavelengths are in the 
orange-red range, 6000-6800, with a maximum at 6400-6600 and 
a steep drop beyond 6800 A. Blue light is much less effective and 
green is almost completely ineffective. Such results indicated that 




Fig. 3-2. Effects of various amounts of light given as dark-period interruptions 
on inflorescence primordium development in the LDP barley (Hordeum vulgare 
var. Wintex). Three-week-old plants were grown for 9 days with 12 H-hour 
dark periods interrupted in the middle with various energies of light, then 
allowed to grow for 19 days with uninterrupted dark periods. These dissections 
show the apices greatly magnified ; that at the far right was about 3 mm high. 
Relative energies used for the night interruptions ranged from none (extreme 
left) through 25 (middle) to 400 (extreme right) foot-candle minutes of white 
light. The study of similarly graded responses to various energies at various 
wavelengths indicated the effectiveness of the wavelengths tested. (Photograph 
from Borthwick, Hendricks, and Parker [1948], Bot. Gaz., 110: 103-1 18, courtesy 
of Dr. H. A. Borthwick, U. S. Department of Agriculture.) 



light-breaks inhibiting the flowering of SDP were probably ab- 
sorbed by the same pigment as those promoting flowering in LDP. 
The nature of the pigment remained a subject of speculation since 
no known pigment in higher plants had an absorption spectrum 
with a peak only in the red region. Further information came from 
outside photoperiodism proper, and it is therefore necessary to 
digress. 



34 • Photoperiodism: Attempts at Analysis 



THE RED, FAR-RED REVERSIBLE SYSTEM 

It had been known for a long time, in a general way, that the 
germination of many seeds was affected by light. Flint and Mc- 
Alister (1935, 1937) had found that the germination of lettuce, 
Lactuca sativa, was promoted by red light. If seeds previously ex- 
posed to enough red to cause subsequent germination were exposed 
to either blue or near-infrared (7000-8000 A) light, germination was 
inhibited. This work was taken up again by the Beltsville group 
(Borthwick et al., 1952a, 1954). They determined an action spec- 
trum for the promotion by red, which showed maximum activity 
at about 6500 A and resembled the light-break action spectra, and 
also an action spectrum for the infrared (now called far-red) inhibi- 
tion, which showed a maximum around 7350 A. More important, 
however, were observations leading them to postulate the existence 
of what is now known as the red, far-red reversible pigment system. 

Some data taken from the 1954 paper illustrate what is meant 
by red, far-red reversibility. Groups of lettuce seeds were allowed 
to imbibe water in darkness at 20° C for three hours, subjected to 
various brief light treatments, then kept in darkness at 20° C for 
two days, after which the number germinating in each lot was 
counted. The light treatments were either 1 minute of red (R) or 
4 minutes of far-red (F) at previously established intensities, or 
combinations of these in immediate succession. In typical results, 
treatment R alone caused 70 percent germination, and the treat- 
ment RF (red followed immediately by far-red) caused 7 percent, 
almost the same as germination in darkness. Such alternations 
could be carried much further: the treatment RF, RF, RF, R gave 
81 percent, and the treatment RF, RF, RF, RF, 7 percent again. 
The germination depended simply on whether R or F was given 
last, as if a switch were thrown one way or the other by the 
different radiations. Any red effect was reversed by far-red given 
immediately after, and vice versa. Similar results could be obtained 
even when the seeds were chilled to 6° C during the period of light 
treatments. This temperature-independence and a number of other 
observations led to the suggestion that the two opposed light 
effects might be mediated by the same pigment. The basic assump- 
tion is that the pigment can exist in two forms, a red-absorbing 



Photoperiodism and Light Quality • 35 

form (or form with greater red than far-red absorption) and a 
far-red-absorbing form. These two forms, call them P R and P F , 
would be photochemically interconvertible, thus: 

red light 

Pr - v — I p» 

far-red light 

and the final physiological result would then depend on whatever 
form remained after the last illumination, or on the ratio of the 
two. 

THE RED, FAR-RED SYSTEM IN PHOTOPERIODISM 

Evidence for the red, far-red reversibility of photoperiodic 
light-breaks was presented first by Borthwick et al. (1952b), using 
Xanthium. Following this, Downs (1956) showed that the effects of 
light-breaks were also far-red reversible in the LDP Hyoscyamus 
niger and Wintex barley and the SDP Amarantlnis caudatus and 
Biloxi soybean, and was able to demonstrate repeated reversibility, 
like that in lettuce seeds, in both Xanthium and soybeans. A more 
concrete account of some of these results may be illustrative at this 
point. 

By this time, simpler light sources than the spectrograph had 
been developed. The red source was simply white fluorescent light 
(about 1000 foot candles at plant level) with an interposed filter of 
two sheets of red cellophane. Far-red was obtained by filtering 
either sunlight (8000 foot candles) or incandescent light (800 foot 
candles)— both rich in far-red compared to fluorescent light- 
through two layers each of red and blue cellophane. These cut out 
almost all radiation of wavelengths shorter than 7000 A but allow 
far-red to pass. Using these sources, Downs then conducted a more 
detailed investigation of the time and energy relations of these 
effects on Xanthium. Groups of plants were given various experi- 
mental treatments for three 24-hour cycles. They were all then 
placed under noninductive long-day conditions and allowed to 
develop for some days, after which the flowering response was 
scored as an inflorescence-stage index from (vegetative) to 7 
(maximum response). 

The effect of red light in the middle of each dark period of 
three successive 12 hours light-] 2 hours dark cycles was propor- 
tional to the duration of exposure, that is, to total energy given. 



36 • Photoperiodism: Attempts at Analysis 

Uninterrupted controls had a mean flowering stage of 6.0; 10 
seconds red gave a value of about 4.8, 20 seconds brought it to 
about 2.5, and 30 seconds, to 0. One minute of sun-source far-red 
was sufficient to reverse the effects of two minutes of red if given 
immediately after, returning the value to 6, but twelve minutes of 
far-red brought it down again to nearly 4; such "overreversals," in 
which long exposures to far-red act more like red, occur in other 
plants as well, and will be discussed later. 

Downs next studied the effect of interposing a brief period be- 
tween the red and far-red treatments. In one experiment, far-red 
immediately after red gave a value of 6.5 compared with the un- 
interrupted controls of 7.0. With a 20-minute dark period before 
the same far-red treatment, the value was only 3.8, and with a 
40-minute dark period, 0.5. Thus the far-red treatment had to be 
given soon after the red to be effective; the simplest explanation is 
that when most of the pigment is in the far-red-absorbing form 
(after the red), a series of reactions inhibitory to induction is started 
and reaches such a stage after 40 minutes that even changing the 
pigment will no longer change the result. If the plants are held at 
5° C during the intervening dark period, this "escape from photo- 
chemical control" occurs much more slowly. With a 40-minute 
dark period, for example, the red effect was still almost completely 
reversible at this temperature, precisely as would be expected under 
the explanation given. The escape from photochemical control also 
explains w T hy, under ordinary conditions, repeated reversals cannot 
be carried on indefinitely and the red effect eventually predomi- 
nates. 

Downs's results typify the kind of control exerted by the red, 
far-red system in photoperiodism, but by no means exhaust the 
subject. Evidence was obtained, first in lettuce seed (Borthwick 
et al., 1952a) and later elsewhere, that the conversion from the 
far-red-absorbing to the red-absorbing form takes place not only on 
exposure to far-red but also, more slowly, in darkness by some 
thermal (temperature-dependent) process. This revises the relation 
previously written to: 

red 




far-red P F . 

^dark, thermal"" 



Photoperiodism and Light Quality • 37 

Certain data on flowering further suggested that this dark conver- 
sion might determine the length of the critical dark period. Borth- 
wick et al. (1952b) reported that if Xanthium plants were given a 
brief far-red exposure at the beginning of a dark period (end of 
the high-intensity white light), less than 7 hours of darkness were 
required for induction. If they received a brief red treatment 
instead, 9 hours of darkness were required, compared with the 
Sy 2 sufficient with no treatment after the white light. Downs (1959) 
has also shown that the quantitative SDP millet, Setaria italica, 
which flowers rapidly with 12-hour nights but very slowly with 
8-hour nights, will also flower rapidly with 8-hour nights if a brief 
far-red treatment is given at the beginning of each. This far-red 
promotion of flowering is reversed by red, and red alone has no 
effect at the start of the dark periods. (See Fig. 3-3.) 

At this point one may well wish for the solace of a theory 
unifying all these data. Such a theory exists (see Borthwick, Hen- 
dricks, and Parker, 1956) and can be briefly summarized. At the 
end of a long white-light period, the pigment is almost completely 
in the far-red-absorbing form; evidence for this is that red given 
then has little or no effect, and far-red a much larger one. It is 
this far-red-absorbing form that brings about the inhibition of 
induction in SDP and the promotion of induction in LDP. Thus 
SDP require a dark period long enough to allow thermal conver- 
sion of the far-red-absorbing form and its continued absence for 
some time, whereas LDP are inhibited by too long a dark period 
since this conversion and absence are unfavorable. Hence red (or 
white) light-breaks inhibit SDP induction and promote LDP 
induction by returning the pigment to the far-red-absorbing form. 
This theory takes into account all the data so far presented, and 
even fits the observation (Chapter Two) that the dark period for 
Xanthium has to be longer if the temperature is lowered, since 
thermal conversion to the red-absorbing form will be slowed. The 
only difficulty is that it does not fit the equally valid data to be 
considered next. 

According to the theory, far-red given to LDP at the start of a 
dark period barely short enough to allow induction should inhibit 
induction. Yet in at least two LDP, Hyoscyamus and dill, it pro- 
moted induction. Also, flowering in the SDP Chrysanthemum 
morifolium is not promoted by far-red at the start of the dark 



38 



Photoperiodism: Attempts at Analysis 



period, as it is in Xanthium and millet (see Borthwick, 1959). Still 
more complicated, yet confirmed now by the Beltsville group whose 
theory it confounds, is the response of the Japanese morning glory, 
Pharbitis nil. 




Fig. 3-3. Effect of far-red supplement at the end of the light period on the SDP 
millet (Setaria italica). All plants were grown with 16 hours of light; at the end 
of each light period the following treatments were given, represented by the 
plants from left to right: no further radiation; five minutes of far-red; five 
minutes of far-red followed by five minutes of red. (Photograph from Downs 
[1959], by permission of the American Association for the Advancement of 
Science, and courtesy of Drs. R. J. Downs and H. A. Borthwick, U. S. Depart- 
ment of Agriculture.) 



Pharbitis seedlings grown at about 26° C can be induced to 
flower by one or more 16-hour dark periods, and red light-breaks 
(perceived by the cotyledons) 8 or 10 hours after the start of the 
dark period completely inhibit induction. This is a typical SDP 
response. But the effects of red light are not reversed by far-red; 
far-red itself inhibits flowering when given during the dark period. 
Far-red even inhibits when given at the start of the dark period, 
and this effect is reversed by red. Thus the red, far-red reversible 
system is present and active, but in a way unlike that suggested by 



Photoperiodism and Light Quality • 39 

the theory (Nakayama, 1958). However, all this is true only when 
the cotyledons are the light-responsive organs. Older plants, in 
which the true leaves perceive the light, respond in the same way 
as Xanthium (Nakayama, Borthwick, and Hendricks, 1960). These 
observations provide an opportunity for studying the precise ways 
in which the red, far-red system may be linked to flowering, if the 
operative differences between the cotyledons and the true leaves 
can be discovered. 

A point requiring further comment is that white light acts 
more or less like red. This is not surprising for fluorescent light 
sources since their far-red emission is very low, but both incandes- 
cent light and sunlight have a high proportion of far-red. Their 
action as red light is probably due in part to the proportion of red 
to far-red, in part to the relative sensitivities of the two forms, and 
also to the fact, mentioned previously, that prolonged exposures to 
far-red may have an action more like red than short exposures. The 
latter has been explained (see Borthwick, 1959) as being due to the 
maintenance of a small amount of the far-red-absorbing form in 
equilibrium with the red-absorbing form during far-red radiation, 
since the absorption spectra of the two forms must overlap. Thus 
darkness following the far-red treatment is needed to allow the 
conversion to the red-absorbing form to be completed by the 
thermal process. It is, however, not strictly true that all white light 
sources are equivalent for photoperiodism. Fluorescent and in- 
candescent light differ considerably in their effects on both flower- 
ing and vegetative growth when used to lengthen light periods, and 
the differences can be ascribed to the different far-red emissions 
of the two sources (Downs, 1959; Downs et al., 1959). 

NATURE AND FUNCTION OF THE 
RED, FAR-RED PIGMENT 

Many effects of low-intensity red light on plants are now known 
to be reversible by far-red, but a discussion of the red, far-red 
control of vegetative growth— so-called photomorphogenesis— would 
occupy too much space here. References to the abundant literature 
on it are to be found in most reviews on photoperiodism; a 
particularly good introduction is Withrow's own article in Withrow 
(1959). Much speculation and calculation has in the past been 



40 • Photoperiodism: Attempts at Analysis 

devoted to the possible nature and metabolic function of such a 
reversible pigment system, on the assumption, of course, that it 
existed and was not a misinterpretation of two separate light 
effects. The assumption has since been justified, and the specula- 
tions may soon give way to data. Workers at Beltsville (Butler 
et al., 1959), using relatively sophisticated spectrophotometric 
techniques, have shown that intact tissues and properly prepared 
extracts of etiolated (dark-grown) seedlings of various species, such 
as corn, Zea mays, contain a pigment with the predicted reversible 
changes in absorption characteristics in the red and far-red. The 
pigment is present in very low concentrations— the etiolated tissue 
in which it was observed was nearly white— and is either a protein 
or closely bound to a protein. The development of better extrac- 
tion and purification techniques should soon make it possible to 
characterize the pigment further and aid in establishing its imme- 
diate biochemical function. The rapid developments which should 
ensue may make further discussion on these points obsolete when 
printed. 

Even discovery of the immediate biochemical function of the 
pigment, no easy matter in itself, will not completely clarify its 
role in photoperiodism. Much more physiological work is still 
required on this question. The only generalization that will hold 
at present is that the red, far-red system mediates the low-intensity 
light effects and may also be involved in the critical time-require- 
ments. There is no clear evidence, however, as to the precise way 
in which the pigment is linked to subsequent events in the induc- 
tion process, and the relation may well differ from species to species 
even within a given response class. 

The pigment has been dubbed "phytochrome" by its dis- 
coverers (see Borthwick and Hendricks, 1960). Though the name 
is unfortunate both because it is general (Greek for "plant" plus 
"color" or "pigment") and because it is liable to be confused when 
spoken with the cytochromes, so significant in the biochemistry of 
respiration, it will undoubtedly be perpetuated. 

PROLONGED EXPOSURES TO LIGHT OF 
DIFFERENT COLORS 

In the 1930's and 1940's, Funke (see Funke, 1948) used sunlight 
filtered through white, red, or blue glass to lengthen photoperiods 



Photoperiodism and Light Quality • 41 

for both LDP and SDP. Red and white were the only effective 
photoperiod-lengthening conditions for many, with blue equivalent 
to darkness. For a second large class, both red and blue were effec- 
tive, as well as white. For a third very small class, only white was 
effective, but neither red nor blue. Funke's "Class IV" has attracted 
the most interest; these were all of the Cruciferae (Mustard family) 
and almost all LDP, in which the blue and white, but not the red, 
were effective in lengthening photoperiod. 

Since Funke, there has been a great deal of work, most of it in 
the Netherlands, on the vegetative development and flowering of 
plants grown with relatively high energies (high intensities, long 
exposures, or both) of various colors of light. For reviews of this 
work, see Wassink and Stolwijk (1956), Wassink et al. (1959), Meijer 
(1959), and Van der Veen and Meijer (1959). Although many inter- 
esting phenomena have been observed, such work is, almost without 
exception, extremely difficult to evaluate for at least two reasons. 
First is the immense technical difficulty of obtaining high energies 
of light in pure spectral bands and over large enough areas to grow 
groups of whole plants. Often the sources have been more or less 
impure, as Funke's must have been, so that what appear to be 
high-energy effects of the main wavelength region may include 
low-energy effects of other wavelengths. Such contaminations have 
been gradually reduced (see Wassink et al., 1959) but may still be 
present. The second problem is, if anything, worse. Consider, for 
example, the effects of long exposure to high-intensity blue light, 
no matter how pure. The light may be affecting at least three sys- 
tems simultaneously. The red, far-red system itself and photosyn- 
thesis are already obvious, but one must also consider whatever 
pigments mediate phototropism— the orientation of plant parts with 
respect to the direction of light— since blue light is the most effec- 
tive in this process. In addition, fluorescence of chlorophyll and 
other compounds caused by the blue may expose the cells internally 
to longer-wave radiations. The difficulties of disentangling such 
effects and reaching satisfactory interpretations can hardly be over- 
estimated. Nevertheless, some of this work is of considerable 
interest. 

The unexpected promotion of Hyoscyamus flowering by far- 
red at the start of the dark period, mentioned above, was first 
reported by Stolwijk and Zeevaart (1955) who also observed that this 
LDP entirely failed to flower when grown in continuous red light, 



42 • Photoperiodism: Attempts at Analysis 

although it flowers rapidly in continuous white light. However, 
small amounts of far-red given with the continuous red brought 
about flowering, as did also blue light. Nine hours of blue once 
every third day would permit flowering under otherwise continuous 
red light. There is some question as to whether the slight far-red 
contamination in the blue might be responsible for the original 
effect reported, but it has since been repeated with much purer 
sources (Wassink et al, 1959). Thus, in Hyoscyamus, blue and 
far-red may be physiologically equivalent for flower initiation. 

Meijer (1959) has reported a number of complex experiments 
on flower initiation in the SDP Salvia occidentalis. One of the 
most interesting results is that a standard 15-minute red light- 
break during an inductive dark period does not inhibit flowering 
if the main (8-hour) light period is of red or green light. It does 
inhibit, however, if the main light period is of blue (all main 
light periods being of the same energy) or if the red or green 
periods are supplemented with far-red. It should also be noted 
that Salvia occidentalis, like Perilla crispa (Chapter Two) will 
flower even in continuous white light of sufficiently low intensities; 
at higher or even lower intensities, it again behaves like a proper 
SDP by failing to flower. Even more complex work on Hyoscyamus 
has been recently reported by De Lint (1960), to whose extensive 
work the reader should go for further details. 

Work of this kind has certainly indicated that light quality 
and intensity have more effects on flower initiation and other 
aspects of development than can readily be explained through 
what is known of the red, far-red system at present. Unfortunately, 
even the effects of blue on this particular system are not under- 
stood; there is evidence that, in various organisms, blue (at high 
energies) may act like either red or far-red. Whether this is a 
direct action on the red, far-red reversible pigment itself or an 
indirect one, through other pigments or metabolic systems, is un- 
certain. Due to the difficulties, already mentioned, of interpreting 
such studies, the only suggestions at present are purely speculative. 

B. TIME RELATIONS AND ENDOGENOUS 
RHYTHMS IN PHOTOPERIODISM 

The characteristic defining aspect of photoperiodism is the 
importance of the time relations of light and dark conditions. The 



Time Relations and Endogenous Rhythms • 43 

response to this timing is sometimes surprisingly precise; Xanthium 
can distinguish clearly between a dark period of 8 hours (non- 
inductive) and one of 8 hours, 40 minutes (inductive) (Long, 1939). 
On the reasonable assumption that the main survival value of 
photoperiodism in an organism is in the seasonal timing of devel- 
opment that it affords, Withrow (1959) has calculated that to be 
accurate, the timing mechanism must detect daylength differences 
of 14 to 44 minutes within a week in temperate latitudes. In addi- 
tion, it should be relatively insensitive to random changes in light 
intensity and temperature brought about by local weather. Insensi- 
tivity to intensity changes is provided by the fact that low intensi- 
ties are sufficient to bring about most photoperiodic responses, but 
insensitivity to temperature is more difficult to understand. 
Although both the accuracy and the temperature-insensitivity (see 
Chapter Two) of the photoperiodic control of flowering are, in the 
writer's opinion, often exaggerated, it is true that certain aspects 
of photoperiodism are less temperature-sensitive than most plant 
processes. 

The effects of low temperature in lengthening the critical dark 
period in Xanthium, discussed earlier, indicate that a drop of 
about 16° C increased the dark period required by only about 3 
hours, or less than 40 percent (Long, 1939). This contrasts with the 
general observation that the rates of most ordinary chemical re- 
actions, and thus of growth or other processes in most biological 
systems, are at least doubled by a 10° C rise in temperature within 
a fairly wide range. If the series of events constituting the dark 
period "timing mechanism" in Xanthium responded in this fashion, 
one would expect the 16° drop in temperature to bring about at 
least a 20- or 24-hour dark requirement, but it does not. This and 
similar evidence, although there is not a great deal of it, suggest 
that the photoperodic timing mechanism is not a simple linear 
series of ordinary reactions, but may be more complex. 

Neither timing nor temperature-insensitivity are peculiar to 
photoperiodism. In mammals and birds, of course, a self-regulated 
temperature could obviously permit the accurate timing of re- 
sponses and metabolic events by simple chemical means alone, but 
it is now well established that probably all plants and animals- 
even unicells, excluding perhaps the bacteria— have accurate timing 
mechanisms that are temperature-insensitive, more so, in fact, than 
most photoperiodic phenomena. Several groups of workers have 



44 • Photoperiodism: Attempts at Analysis 

thus suggested that photoperiodism, in both plants and animals, 
is merely a special case of a general rhythmic mechanism by which 
all organisms can register the passage of time. 

ENDOGENOUS CIRCADIAN RHYTHMS IN PLANTS 

Most of the recent data on rhythmic processes in higher plants 
have come either from Erwin Biinning and his co-workers in Ger- 
many or from work done elsewhere to test their hypotheses. Biin- 
ning's concepts (see Biinning, 1956, 1959) have developed from a 
number of basic observations, some antedating his own work. 

Most plant processes exhibit a diurnal rhythm in phase with 
the daily alternations of light and darkness. This rhythm is not 
simply a passive response to external conditions since as expressed 
in various processes— the nocturnal "sleep" movements of legume 
leaves, for example— it persists for at least a few days after the 
plants are placed in a constant-temperature dark room. In fact, 
periodic light-dark alternations are not necessary to initiate such a 
rhythm. The classic example is the behavior of bean, Phaseolus, 
seedlings germinated and grown in constant-temperature darkness. 
The movements of the young leaves, which can be recorded with a 
suitable apparatus, are small, more or less random, and unsynchro- 
nized among the population of seedlings. After a single flash of 
light the movements become larger, synchronized among all the 
seedlings, and exhibit a marked periodicity, with the leaves return- 
ing to the same position about once every 24 hours. The move- 
ments become weaker after several days and finally die out, but 
maintain their periodicity until they do. In Biinning's view, such 
results provide evidence of "endogenous daily rhythms" in plants. 

By "endogenous" Biinning means that the period, or length 
of a complete oscillation in such rhythms, is determined by the 
plant and not imposed by external conditions. There are at least 
three kinds of evidence for this in experiments with the leaf move- 
ments of bean seedlings. First, of course, the movements are evoked 
by a single exposure to light, not by a repeated light-dark schedule. 
Second, the phase of the rhythm— as indicated by the position of a 
leaf at any given time— is not affected by the solar time of day, but 
depends only on the time at which the light flash was given. A 
group of plants given a flash 12 hours before a second group will 



Time Relations and Endogenous Rhythms • 45 

show movements 12 hours out of phase with the second group. 
Finally, and perhaps most important, the rhythm of such move- 
ments is not exactly daily, not precisely 24 hours long. It may be 
from 20 to 30 hours; different varieties have rhythms with char- 
acteristic period-lengths, so that this is a genetically controlled and 
thus endogenous property. The term "circadian" (Latin: circa, 
about, and dies, day) has been coined for such rhythms with period- 
lengths of close to 24 hours. 

The relation of the bean circadian rhythm to temperature is 
shown by data from Biinning (1959a). In darkness (after a light 
flash) the period is 28.3 hours at constant 15° C and 28.0 hours at 
constant 25° C. Thus a 10° difference in ambient temperature has 
no effect. However, a change in temperature does have an effect. 
Seedlings moved from 20° to 15° had a period of 29.7 hours, and 
those moved from 20° to 25° had a period of 23.7 hours, for the 
first day or so after a change. Later, compensation occurred and 
the periods in the two temperatures became similar. Thus it is not 
strictly true to call such circadian rhythms temperature-insensitive, 
but they are clearly temperature-compensated and arrive at the 
same period in different constant temperatures. 

In general, the phase and amplitude of circadian rhythms in 
various organisms are greatly affected by the environment but the 
basic period-length can only be changed within narrow limits. An 
organism with a rhythm of 20 or 30 hours will adapt its period to 
a normal 24-hour day, but may either revert to its endogenous 
rhythm or exhibit highly disorganized activity under light-dark 
cycles totaling 12 hours in length. Not only light flashes but transi- 
tions from light to darkness and abrupt temperature shocks as well 
can reset the phase or initiate circadian rhythms, but it seems clear 
that they do not cause them. 

Many processes in an organism generally exhibit the same cir- 
cadian rhythm, probably manifesting the activity of a single "clock" 
mechanism. This "clock" may be a. basic property of the organiza- 
tion of most cells or a particular unknown process, but there is no 
general agreement even as to its possible nature. A major investi- 
gator (Brown, 1959) has recently abandoned the hypothesis of a 
completely endogenous origin, and suggests that organisms may 
register the passage of time by perceiving certain unknown geo- 
physical periodicities, although the way in which such an exogenous 



46 • Photoperiodism: Attempts at Analysis 

clock may be used would still vary greatly from organism to organ- 
ism. Most other workers, however, consider the clock truly endog- 
enous. For summaries of the state of this field with particular 
reference to animals and microorganisms, see Pittendrigh and 
Bruce (1959) and Brown (1959); a recent symposium also covers the 
field in great detail (Biological Clocks, 1960). Only experiments 
directly concerned with photoperiodism and flowering will be con- 
sidered below. 

ENDOGENOUS CIRCADIAN RHYTHMS 
AS THE BASIS OF PHOTOPERIODISM 

In the view of Bunning and co-workers, the endogenous 
circadian rhythm of plants passes through two phases of more or 
less opposite sensitivity to light: a "photophile" (light-liking) phase 
in which development is favored by light and a "scotophile" (dark- 
liking) phase in which light is unfavorable. These phases are said 
to be distinguishable by leaf movements as well as by differences 
in rates of respiration, photosynthesis, cell division, and other 
processes. As phases of a circadian rhythm they are affected but not 
caused by light-dark alternations; they are the means by which 
the plant can ''time" the light or dark exposures it receives. A 
particular version of this view, now considerably modified by 
Bunning (1948, 1959b), has provided the stimulus for much of the 
work on the problem. It relates SDP and LDP specifically by pro- 
posing that in both types each phase of the rhythm is about 12 
hours long, but whereas in SDP the photophile normally starts 
soon after illumination, in LDP it starts only some 8 to 12 hours 
after the start of light. Thus long photoperiods give the SDP 
excessive light in its scotophile, whereas short photoperiods give 
LDP most of the light in the scotophile and little in the photophile. 

An example of the kind of evidence supporting this proposal 
is from Bunning and Kemmler (1954). They found that flowering 
in the LDP dill occurred on a daily schedule of 17% hours light- 
6*4 hours darkness, but was more rapid if a 2-hour dark period 
was given 3 hours after the start of each main light period (making 
the schedule 3 hours light-2 hours dark-12% hours light-6 1 /. hours 
dark). This observation is consistent with the idea that dill has a 
scotophile phase that occurs shortly after the start of the main 



Time Relations and Endogenous Rhythms • 47 

light period, and thus darkness during this time promotes flower- 
ing. However, the effect was not detected in the LDP Plantago and 
spinach. 

Evidence has also been obtained from leaf movements, a 
particularly impressive case being that of Madia elegans. This 
desert composite was first studied by Lewis and Went (1945) who 
found that it flowered rapidly with 8, 18, or 24 hours of light per 
day, but slowly with 12 or 14 hours of light. This unusual bimodal 
sensitivity, with intermediate daylengths less effective than long or 
short, is apparently reflected in the leaf movements. Bunning (1951) 
was able to show that these movements corresponded to what his 
hypothesis would predict for a plant with two photophile phases 
within each circadian period, and he explained the peculiar photo- 
periodic response on this basis. Indeed, leaf movements have 
generally been used as the chief indication of the postulated phase 
changes. Those in various soybeans, for example, can indicate 
whether a given variety will show SDP or daylength-indifferent 
flowering responses (Bunning, 1955). Although leaf movements in 
Kalanchoe are difficult to detect, Schwemmle (1957) has found that 
the effects of high temperature given at various times during in- 
ductive dark periods (see Chapter Two) are well correlated with the 
effects of similar treatments on the rhythmic movements of the 
petals of plants in flower. Not all the leaf-movement work is so 
favorable, however; there is apparently no significant difference 
between the rhythmic leaf movements of the qualitative SDP 
Coleus frederici and Coleus frederici x blumei and those of the 
quantitative LDP Coleus blumei (Kribben, 1955). At best, of course, 
correlatory evidence is merely circumstantial, whether favorable or 
unfavorable. 

CIRCADIAN RHYTHMS AND THE ACTION 
OF LIGHT-BREAKS 

The most widely used tool in assessing the relation of circadian 
rhythms to photoperiodism, as in the study of low-intensity light 
processes, has been the light-break. Here, instead of quality and 
intensity, the timing of the light-breaks and the length of the dark 
periods are the factors varied. It was tacitly assumed during the 
preceding sections that light-breaks are most effective when given 



48 • Photoperiodism: Attempts at Analysis 

in the middle of the dark period. This is very approximately true 
in ordinary 24-hour cycles, but rarely so under other conditions, as 
such work has made evident. Under the rhythm hypothesis, light- 
breaks act not by merely breaking each long dark period into two 
short ones, but by supplying light in the scotophile (for SDP) or 
photophile (for LDP) phases. This has been tested extensively. 

When Claes and Lang (1947) examined the effects of 48-hour 
cycles on Hyoscyamus (Chapter Two), they found that cycles of 7 
hours light-41 hours darkness were noninductive. A 2-hour light- 
break would promote flowering if given not long after the start or 
before the end of each long dark period, but was ineffective in the 
middle. The times of maximum effectiveness were about 16 and 
40 hours, respectively, after the start of each main light period. 
These results were consistent with the idea that the photophile- 
scotophile alternation continued through the dark period with the 
first photophile maximum (typical of LDP) 16 hours after the start 
of the main light period and the second about 24 hours after the 
first. Yet there was an equally reasonable alternative explanation 
not depending on rhythms. Suppose that the light-break could act 
together with the main light period nearest it (either before or 
after) to constitute a long light period interrupted (without effect) 
by darkness. On this alternative the light-break was ineffective in 
the middle of the long dark period not because it fell in the scoto- 
phile, as in the rhythm explanation, but because it was too far from 
a main light period. Claes and Lang favored the second view. 

An experiment designed to avoid this ambiguity was reported 
by Carr (1952), who used the SDP Kalanchoe grown in 72-hour 
cycles of 12 hours light-60 hours darkness. On the Biinning theory, 
light-breaks during the dark period should show three times of 
maximum effectiveness in inhibiting flowering and causing the 
correlated changes in vegetative growth, whereas on the Claes and 
Lang alternative there should be only two, close to either end of 
the dark period. Carr's results indeed showed three maxima, about 
24 hours apart, although the middle one was not as well defined 
as one might wish. Carr concluded that "the theory of Biinning 
. . . must therefore be regarded as finally and decisively proved," 
thereby illustrating the partisan vigor that at least enlivens if not 
clarifies the question. 

Schwabe (1955a) repeated Carr's results but noted that the 



Time Relations and Endogenous Rhythms • 49 

crucial differences (evidence for the second, or middle, maximum) 
were very small, and reached opposite conclusions on other grounds 
(see below); but very clear data confirming Carr's results were later 
published by Melchers (1956). Meanwhile, Hussey (1954) had shown 
that the LDP Anagallis arvensis grown in 72-hour cycles with long 
dark periods showed only two maxima for the promotion of flower- 
ing by light-breaks instead of the three that would correspond to 
Carr's results. With Hyoscyamus, however, Clauss and Rau (1956) 
were able to show three optima in similar experiments, thus sup- 
porting Carr and Biinning. The quantitative LDP Arabidopsis 
thaliana was studied twice, with ambiguous results each time (Hussey, 
1954; Clauss and Rau, 1956). The SDP Coleus blumei x frederici 
disagreed with all others, since the time for maximum light-break 
inhibition (72-hour cycle) was in the middle of the long dark period, 
with no sign of three or even two optima (Kribben, 1955). 

Other work besides that on 72-hour cycles suggests Carr's 
quoted conclusion may have been hasty. Wareing (1954) voiced 
strong opposition to the idea that endogenous alternation of photo- 
phile and scotophile phases determines the action of light-breaks. 
He presented experiments with Biloxi soybeans grown on 9 hours 
light-39 hours darkness (48-hour cycles), or on 9 hours light-51 
hours darkness (60-hour cycles), testing the effects of light-breaks 
at various times during the long dark periods. In both cycles light- 
breaks about 6 to 8 hours before or after the main light periods 
were maximally inhibitory, whereas they promoted flowering in 
the middle of the dark periods. Since the dark periods used in the 
two cycles differed by 12 hours, one would not expect these results 
if the inhibitory action of light-breaks was due to a more or less 
unchanged circadian rhythm. One would expect them, however, if 
light-breaks interact with the main photoperiod when it is close 
enough, thus providing a total photoperiod that exceeds the "limit- 
ing value" for soybean flowering (see Chapter Two). Further evi- 
dence for this view was that in cycles totaling 48 hours, light-breaks 
given either 3 or 6 hours before the main light period were inhibi- 
tory when the latter was 9 hours long, whereas only a light-break 
6 hours before was effective with a 6-hour main photoperiod. 

Wareing also reported experiments with Xanthium in which 
light-breaks toward the end of a long dark period were not inhibi- 
tory. Since this plant, unlike soybeans, has no "limiting photo- 



50 • Photoperiodism: Attempts at Analysis 

period," these results were consistent with the explanation pro- 
posed. The inhibition of Xanthium induction by light-breaks given 
early in long dark periods was explained as due to a direct nul- 
lification of dark processes leading to flowering plus the fact that, 
after the light-break, the high-intensity light process (Chapter Two) 
is left unsatisfied. The induction of Xanthium by a critical dark 
period, regardless of length of the preceding photoperiod, was 
also cited by Wareing against Bunning's theory, since the latter 
appeared to hold that the phase of the rhythm was regulated by 
the start of each main light period. Thus the effect of a dark period 
should depend on how long the light continued. 

Biinning responded to all this in considerable detail. As to the 
Xanthium results, leaf-movement studies (Biinning, 1955) indicated 
that in this plant the phase of the circadian rhythm is indeed regu- 
lated by the light-to-dark rather than the dark-to-light transition, 
thus refuting Wareing's evidence based on the opposite assumption. 
A light-break given early in the dark period falls in the scotophile 
induced by the transition to darkness and thus inhibits, but a light- 
break late in a long dark period falls in the photophile that endog- 
enously follows and thus does not inhibit. The results with soy- 
beans may also be clarified, according to Biinning (1954), by 
attention to the actual course of the circadian rhythm as shown by 
leaf movements. These indicate that the rhythm continues for 
about 30 hours in darkness, after which a period of "dark rigor" 
(Dunkelstarr) sets in. A light-break during dark rigor brings about 
a new photophile phase which is then followed endogenously by a 
scotophile. Wareing's observation that the effect of a light-break 
toward the end of a long dark period depended not on the length 
of the dark period but on the light-break's relation to the following 
main light period is then due to the fact that the main light period 
and the scotophile phase of the newly reinitiated rhythm now over- 
lap, with resultant inhibition. In addition, Biinning pointed out 
that his observations on leaf movements would also predict the 
existence and optimum times for the light-break promotions of 
flowering observed by Wareing. To Wareing's position that light- 
break effects are due to interaction with nearby light periods, 
Biinning thus retorted: "Yes, that is so— because of the endogenous 
daily rhythm." 1 

i "Ja, das ist so, und es beruht auf der endogenen Tagesrhythmik." 



Time Relations and Endogenous Rhythms • 51 



FLOWERING IN LIGHT-DARK CYCLES OF DIFFERENT 
LENGTHS; TEMPERATURE INTERACTIONS 

If a circadian rhythm regulates photoperiodic responses, 
normal flowering should depend upon light-dark alternations of 
about 24 hours. Schmitz (1951) using Kalanchoe and Schwabe 
(1955a) using Kalanchoe, Xanthium, and an SDP variety of Im- 
patiens balsamina, concluded against Bunning's theory on the 
grounds that cycles with total lengths ranging from 15 to 50 hours 
proved inductive, with any failures to flower attributable to the 
length of either the dark or light periods but not to the periodicity 
of the cycles. Schwabe also criticized the extensive use of leaf- 
movements as indicators of the endogenous rhythm, since the 
photoperiodic response is often insensitive to conditions which 
may completely obscure the leaf movements. Calling attention to 
the remarkable plasticity of both the endogenous rhythm and 
Bunning's theory based on it, Schwabe questioned the value of the 
latter in explaining photoperiodism and asked Bunning to "define 
the sort of experimental result which he would regard as in- 
compatible with it." 

In contrast to the results of Schmitz and Schwabe, cycle-length 
experiments show clear quantitative effects on the flowering of 
soybeans (Blaney and Hamner, 1957; Nanda and Hamner, 1958, 
1959). Cycles totaling 24, 48, or 72 hours in length are far more 
favorable to flowering than, for example, 36- or 60-hour cycles, 
although neither of these most unfavorable cycles are completely 
inhibitory. This certainly supports the concept of a circadian 
rhythm in sensitivity to light and darkness. Finn and Hamner 
(1960) have also published a group of experiments with Hyoscya- 
mus in which the total length of the light-dark cycle appears to be 
a major controlling factor. For example, with a 10-hour light 
period, flowering was most rapid with a total cycle length of 18 
hours (with an 8-hour dark period), slowest or absent with a total 
cycle length of 24-30 hours (14- or 20-hour dark period), and faster 
again with a 42-hour cycle length (32-hour dark period). Such 
results may also be used to support a rhythm-based theory of 
photoperiodism. 

Further experiments with soybeans (Blaney and Hamner, 1957) 



52 • Photoperiodism: Attempts at Analysis 

indicate that the phase of the rhythm can be shifted by low 
temperatures during part of the cycles used. A recent paper by 
Oltmanns (1960) suggests that the interactions between tempera- 
ture, light, and rhythmic phemonena in the flowering of Kalanchoe, 
and by implication in the flowering of any other plant, are not yet 
sufficiently understood to be described by any simple hypothesis. 

ENDOGENOUS CIRCADIAN RHYTHMS 
AND THE RED, FAR-RED SYSTEM 

There appears to be a relationship between the red, far-red 
system, unquestionably involved in photoperiodism, and endog- 
enous circadian rhythms in plants. Red is the most effective light 
in initiating the movements of etiolated bean seedlings, previously 
discussed, and this effect is far-red reversible (see Bunning, 1959a). 
More directly related to photoperiodism is the observation by 
Konitz (1958) that far-red given as an interruption of the main 
light period of Chenopodium amaranticolor (SDP) inhibits the 
effectiveness of inductive cycles, just as does red given in the dark 
period. Since rhythms in plants demonstrably affect many processes 
under certain circumstances, the particular closeness of their con- 
nection with the red, far-red system is hard to judge, even from 
these results. Engelmann (1960) has found that when red light is 
given to Kalanchoe at various times during a 62-hour dark period, 
it inhibits induction in what would be predicted to be the scoto- 
phile phases and promotes it in the photophile phases. Far-red, 
however, does not show an inverse pattern, but simply inhibits 
during the first half (30 hours) of each dark period and inhibits 
less during the second half. 

CONCLUDING REMARKS ON CIRCADIAN RHYTHMS 

AND PHOTOPERIODISM 

If the reader is now confused, he is in good company; no aspect 
of flowering physiology has given rise to more complex experi- 
ments, tenuous interpretations, and heated controversy. The contro- 
versy is not over the existence of rhythms in plants, which is not 
seriously questioned, but over their usefulness and relevance in 
understanding photoperiodism. In this situation, even more obvi- 



Time Relations and Endogenous Rhythms • 53 

ously than in most, appeals to expert opinion are useless, since 
there are accomplished and respected investigators on both sides. 
The writer is frankly of two minds on the subject. On the one hand, 
the existence of rhythms and their influence in many processes 
recommend them as the underlying mechanism of the more particu- 
lar time-dependent response, photoperiodism. Yet hypotheses on 
the precise relationship tend to seem vague, or easily disproved, 
or ad hoc elaborations full of special exceptions. It has understand- 
ably been argued that they simply confuse the issue, explaining the 
relatively simple response of photoperiodism in terms of an equally 
unexplained set of more complex phenomena. Yet, if photoperiod- 
ism is indeed a special case of a basic biological process, it would be 
a pity not to recognize it as such. So far, the evidence on both sides 
consists largely of correlations or the lack of correlations, and these 
differ from plant to plant. Certainly endogenous circadian rhythms 
are at least modifying factors in photoperiodism; whether they are 
more than that, time will undoubtedly tell. 



chapter four 



Temperature 
and Flowering 



Temperature affects all plant processes, and some temperature 
interactions with photoperiodism have already been mentioned. 
There are many plants in which flowering is either qualitatively or 
quantitatively dependent upon exposure to near-freezing tempera- 
tures, and it is largely with these that this chapter will deal. A few 
other less well-defined relationships between temperature and 
flowering will also be considered. 

VERNALIZATION: COLD TREATMENTS 
AND FLOWERING 

It is evident from Chapter Two that photoperiodism provides 
not only a convenient method lor controlling and studying flower- 
ing in many plants, but also a basis for the explanation of many 
seasonal phenomena. The same is true of low-temperature effects, 
which play an important role in the life cycles of many temperate- 
zone plants. Among the monocarpic plants, both biennials and 
winter annuals are forms in which a cold treatment is required 
before flowering can take place with optimum rapidity; in winter 
annuals it can be given during germination to very young seedlings, 
whereas biennials must first have made substantial growth. Many 
perennials also, both woody and herbaceous, require cold treat- 
ments each season to continue flowering. The ecological and adap- 

54 



Vernalization: Cold Treatments and Flowering • 55 

tive significance of such behavior in regions with a period of winter 
cold, itself unfavorable to growth, need not be belabored. 

The cold treatment of germinating seeds in order to hasten 
subsequent flowering has come to be known as vernalization. This 
is a translation of the Russian yarovizatsya, and both words com- 
bine the term for "spring" (Russian, yarov; Latin, ver) with a 
suffix implying "to make" or "become," reflecting the ability of 
such cold treatments to convert "winter" strains of cereals to the 
"spring" habit by satisfying their cold requirement. Winter cereals 
must normally be planted in late fall or winter in order to flower 
and produce a crop in the subsequent year, whereas spring varieties 
may be planted in the spring of the year in which the crop is 
expected. The terms vernalization or yarovizatsya both actually 
postdate the first observations of such effects by many years, but it 
was Russian attention to the possible practical values of the process, 
particularly in the 1930's, that brought it generally to world-wide 
notice. For the history of early work on vernalization, see McKinney 
(1940) and Whyte (1948). 

Vernalization is probably the only aspect of plant physiology 
that ever became involved in political ideology. The agronomic 
use of vernalization in the Soviet Union was popularized by T. D. 
Lysenko, who viewed the effect as an actual inheritable conversion 
from winter to spring habit; later he even claimed the conversion of 
one species of wheat into another. Lysenko's theory eventually led 
to the establishment of a Marxist form of Lamarckism-an old 
and thoroughly discredited view, which holds that changes pro- 
duced by the environment are directly inherited by the offspring of 
the changed organism— as the Soviet dogma in biology. The adopt- 
ing of this view by the Soviets was probably partly due to simple 
opportunism on Lysenko's part, as he was its chief interpreter. Some 
of the finest biologists in the U.S.S.R. refused to support the official 
line and, as a result, simply disappeared or were demoted. This 
unfortunate episode in the history., of science has been recounted 
and analyzed by Huxley (1949) and Zirkle (1949) but does not 
appear to have run its course even yet, so that Soviet biology 
still labors under a disadvantage. Ironically, vernalization has not 
proved to be of great agronomic importance, since the breeding 
of varieties suitable for particular climates and uses has been far 
more successful. At present, the chief practical applications of an 



56 • Temperature and Flowering 

understanding of such low-temperature effects are in relatively 
small-scale horticultural and floricultural practices. 

Vernalization in winter rye 

Although accounts of the effects of chilling seeds and seedlings 
abound in the literature, there have been relatively few extensive 
studies of vernalization. The work of F. G. Gregory, O. N. Purvis, 
and their collaborators in England since about 1931, on the effects 
of vernalization and photoperiodism on flower initiation, develop- 
ment, and vegetative growth of spring and winter strains of the 
Petkus variety of rye, Secale cereale, is by far the most thorough. 

The spring strain is a typical quantitative LDP. Under 
sufficiently long days, flower initiation begins after approximately 
seven leaves have differentiated, whereas under short days (10 
hours light) it occurs only after at least 22 leaves have been pro- 
duced. The winter strain, when germinated at relatively high tem- 
peratures (for example, 18° C) , is not an LDP, but flowers equally 
slowly— again after about 22 leaves— under both long and short 
days. However, if the germinating winter strain is vernalized by 
holding it at 1° C for several weeks before planting, it subsequently 
responds to long days in the same way as does the spring strain 
(Purvis, 1934). The effect of vernalization is thus to render the 
seedling sensitive to long days; early flower initiation does not take 
place as a result of vernalization alone, or vernalization followed 
by short days. 

The effect of vernalization is proportional, within limits, to the 
duration of the cold treatment. Four days' exposure is sufficient to 
increase the subsequent relative growth rate of the stem apex, but 
has no effect on either the number of days from planting to full 
anthesis or the number of leaves preceding flower initiation. Both 
values are reduced to a minimum (under subsequent long days) by 
increasing the length of the cold treatment up to 14 weeks (Purvis 
and Gregory, 1937). 

To determine what portion of the germinating seed perceives 
the cold treatment, Gregory and Purvis (1938a) and Purvis (1940) 
studied the effects of low temperature on excised intact embryos 
and parts of embryos. Not only the intact embryo itself, separated 
from the rest of the seed, but even its isolated apex alone are 
susceptible to vernalization, giving rise to plants responding op- 



Vernalization: Cold Treatments and Flowering • 57 

timally to long days. Thus the site of vernalization is in the meri- 
stem itself, and the results of vernalization are somehow maintained 
throughout the development of the plant derived from the few 
cells originally exposed. The technique of vernalizing isolated 
embryos also made it possible to show that vernalization requires 
a carbohydrate source, presumably as an energy supply for the 
process involved. Rapid flowering takes place only if the embryos 
are cold-treated on a medium containing sucrose, although sub- 
sequent vegetative growth is excellent even if the medium consists 
of mineral salts alone (Gregory and DeRopp, 1938). 

Oxygen is also required during vernalization, confirming the 
suggestion that the process requires a considerable amount of 
energy. For example, Gregory and Purvis (1938b) found that germi- 
nating seeds held at 1° C for 9 weeks would eventually produce 
inflorescences after the eighth leaf if the cold treatment was given 
in air, but only after the twenty-third, as in the unvernalized 
controls, if the treatment was in nitrogen. As little as 1/500 of the 
normal air concentration of oxygen allowed some vernalization to 
take place, but not the maximum effect. 

Before proceeding further, one should bear in mind that 
confusion occasionally arises between vernalization and the favor- 
able effects of chilling on seed germination in many species. The 
former has relatively specific effects, inductive in the sense that they 
lead to subsequent changes in the flowering response of the plants. 
Mere cold treatment to hasten germination is not necessarily ver- 
nalization. It may indeed result in earlier flowering, but the use of 
developmental criteria (number of leaves before the inflorescence, 
for example) can usually indicate whether a genuine hastening of 
flowering relative to vegetative growth has occurred. 

Vernalization in other plants 

The flowering not only of winter cereal strains, but of many 
other plants, can be hastened by vernalization. Certain varieties of 
peas, Pisum sativum, can be made to produce their first flower at 
an earlier node. In the variety Zelka, the eighteenth or nineteenth 
nodes are the first to bear flowers if germination and growth take 
place at about 20° C, but if the germinating seeds are kept at 7° 
for 30 days before planting, flowers occur beginning with the 
fourteenth or fifteenth nodes. The physiological stage susceptible 



58 • Temperature and Flowering 

to vernalization appears to be very brief. If the germinating seeds 
are kept at 20° for 3 clays or at 26° for 1 or 2 days, they can 
no longer be vernalized, even though no new nodes have developed 
during the short time involved (Highkin, 1956). 

The term vernalization has been extended to cover similar 
effects of low temperature given not to germinating seeds but to 
already developed plants. Such effects are typically found in bien- 
nials and many perennials, and are at least formally similar to 
those obtained with the very young plants used in "true" vernal- 
ization. One plant frequently studied is the biennial strain of 
Hyoscyamus niger, previously introduced as an LDP. The strain 
discussed in Chapters Two and Three was the annual, from which 
the biennial appears to differ only in having a cold (vernalization) 
requirement. After this requirement is satisfied, it responds to 
davlength in the same way as the annual strain, but it cannot 
flower otherwise. It thus shows a qualitative vernalization require- 
ment, unlike the plants so far discussed. 

Some of Lang's (1951) results with biennial Hyoscyamus 
illustrate how vernalization depends on both the temperature and 
duration of exposure. Plants were exposed to temperatures from 
3° to 17° C under 8-hour day conditions for varying periods of 
time, after which they were placed in 16-hour days at 23° C. The 
vernalizing effectiveness of the various temperature treatments was 
then expressed by the time required under long days before flower 
initiation was detectable; the shorter the time, the more effective 
the vernalization. With a vernalizing time of 105 days, all tempera- 
tures from 3° to 14° were highly effective: flower initiation was 
detected after 8 days under the long-day conditions. With only 
15 days of vernalization, 10° was the most effective temperature, 
giving 23 days to initiation as compared to the 35 days given by 3° 
and the 28 days given by 14°. With an intermediate vernalizing 
time of 42 days, both 3° and 6° allowed initiation alter 10 long 
days; 17° gave initiation after 20, and the values for the other 
temperatures lay in between these. Thus die temperature optimum 
for vernalization shifts considerably depending on the length of 
exposure (10° for 15 days, 3 to 6° for 42 days), but ceases to exist 
if the exposure is long enough. 

As in the rye embryos, cold given to the apex alone is sufficient 
to vernalize Hyoscyamus and many other biennials. The gcrminat- 



Devernaijzation • 59 

ing seeds, however, are not vernalizable; this distinction between 
biennials and winter annuals is not always clear-cut, but in 
Hyoscyamus at least it is clear that seedlings are not sensitive to 
vernalization before 10 days of age, and not maximally sensitive 
until they are 30 days old (Sarkar, 1958). Work on the vernalization 
of Hyoscyamus has been reviewed by the original workers, Melchers 
and Lang (1948) and Lang (1952). Evidence for the existence of a 
translocatable product of vernalization has also been put forward 
and will be discussed in Chapter Five. 

An exception to the observations that vernalization is per- 
ceived by the stem apex is found in Streptocarpus wendlandii 
(Oehlkers, 1956), in which the leaf appears to be the receptive 
region and neither embryo nor stem apex can be vernalized at all. 

Several varieties of ornamental Chrysanthemum (Chrysanthe- 
mum morifolium) require vernalization. Here again the apex is the 
site of vernalization, and all the laterals subsequently derived from 
it over a long period of time show the vernalized condition 
(Schwabe, 1954). While most of the vernalizable plants studied 
require the treatment in order to respond as LDP, or are daylength- 
indifferent, vernalized Chrysanthemum is a quantitative SDP for 
both flower initiation and development. Three or four weeks at 
4 to 5° C has an optimum vernalizing effect. Low temperature is 
effective even if given discontinuously, and a particular total 
number of hours given during each dark period is more effective 
than the same number of hours given only during light periods, 
at least under short-day conditions. Chrysanthemum is a perennial, 
and yet requires renewed vernalization each year (Schwabe, 1950), 
a situation probably characteristic of many such plants. This brings 
up the general topic of "devernalization," which has been observed 
in a number of plants. 

DEVERNALIZATION 

Vernalized seeds of Petkus winter rye can be devernalized 
simply by drying them and holding them in the dry condition for 
several weeks. However, only the effects of vernalization on the 
subsequent flowering response (to long days) are so reversed; the 
effects on vegetative growth are more complex. This is well illus- 
trated by some data from Gregory and Purvis (1938a). Their unver- 



60 • Temperature and Flowering 

nalized controls in this experiment produced about 4.7 tillers 
(lateral branches from the base) per plant, and a flowering "score" 
of 19. The "score" is an arbitrary scale adopted to indicate the 
intensity and earliness of flowering. Vernalized seed held dry for 
one day only (which has essentially no effect) gave a score of 51 
and about 2.7 tillers per plant— vernalization typically decreases the 
number of tillers. Seed devernalized by being dry for 20 weeks, 
however, gave a score of 20 and about 13.7 tillers per plant; the 
promotion of flowering was completely reversed, but the number 
of tillers was much higher than in either vernalized or unvernalized 
plants. Thus devernalization here is not a simple reversal of vernal- 
ization but a conversion of its effects to a different physiological 
expression. Like vernalization itself, it is proportional, within limits, 
to the duration of exposure to the condition bringing it about. 

Even spring Petkus rye, which may be regarded as already 
genetically vernalized, can be devernalized to some extent. The 
leaf number preceding flowering (in long days) is increased from 
an average of 6.8 to 8.3 by a three-week germination period under 
anaerobic conditions, and this effect is removed by a subsequent 
three-week vernalization treatment (Gregory and Purvis, 1938b). 

The devernalization of vernalized biennial Hyoscyamus is 
brought about by relatively high temperatures. Vernalized plants 
may be kept under short-day conditions for at least several weeks 
at about 23° C and still retain their capacity to respond as LDP. 
Ten days at about 38° will completely remove this capacity, if 
started immediately after the vernalization treatment; if even a 
lew days of moderate temperature intervene between vernalization 
and the high temperature, however, the vernalized condition 
becomes stabilized and can no longer be removed (Lang and 
Melchers, 1947). In general, studies of various plants indicate that 
the more complete the original vernalization and the greater the 
length of the treatment, the more difficult devernalization becomes. 
Revernalization after devernalization is also possible in certain 
plants. 

As the only perennial studied in any detail, Chrysanthemum 
again appears unusual in that devernalization is not brought about 
by high temperatures alone, but requires several weeks of low light 
intensity (or darkness) as well as temperatures of 23° to 28° C. 
The mechanism of this effect is unknown. It is not due simply to 



Vernalization and Photoperiodism • 61 

starvation for carbohydrates since defoliation of the plants does 
not have the same effect, nor does sucrose feeding during treatment 
reduce devernalization (Schwabe, 1955b, 1957). Whether the de- 
vernalization that occurs in the natural yearly cycle is actually due 
to high temperatures and low light intensities (at the underground 
growing points) is still uncertain. 

RELATIONS BETWEEN VERNALIZATION AND 

PHOTOPERIODISM 

Many of the plants studied, and also work with the gibberellins 
(Chapter Six), may be used to support the idea of a close relation- 
ship between vernalization and long-day requirements, but the 
situation is probably more complex than this, varying greatly from 
plant to plant. 

Petkus winter rye and biennial Hyoscyamus niger are "typical" 
vernalizable plants in which the cold treatment brings about 
quantitative or qualitative LDP responses. In other plants, vernal- 
ization can even substitute partially or completely for a long-day 
requirement. Vernalization of spinach seeds, for example, reduces 
the critical daylength for flowering from 14 to about 8 hours 
(Vlitos and Meudt, 1955), whereas cold treatments given to seed- 
lings of certain strains of subterranean clover, Trifolium subter- 
raneum, can completely remove any marked dependence on day- 
length (Evans, 1959). 

Floral induction and development in several grasses depend 
upon both photoperiod and vernalization. Plants of orchard grass, 
Dactylis glomerata, studied by Gardner and Loomis (1953) require 
low temperatures and short days (less than 13 hours light) for 
floral induction, followed by higher temperatures and long days 
for optimum flower development. The short-day and vernalization 
requirements for induction can be satisfied separately but only in 
that order, not in the reverse. In a sense, then, Dactylis glomerata 
is one of the short-long-day plants (SLDP) mentioned in Chapter 
Two, except that a period of low temperature must occur between 
the two photoperiodic treatments or together with the first. 

In some plants, short-day treatments can substitute partially or 
completely for vernalization, making them SLDP. Petkus winter 
rye itself shows a response of this kind, although the situation is 



62 • Temperature and Flowering 

complicated by the fact that both short days and continuous light 
favor flower initiation more than do long days in unvernalized 
plants (Gott et al, 1955). A more clear-cut example of a vernalizable 
SLDP is Campanula medium (see Doorenbos and Wellensiek, 
1959), which has a qualitative requirement for either low tempera- 
ture or short days before it can respond to long days. 

Although even in the above plants, vernalization generally has 
to be followed by exposure to long days, CJirysanthemiim is not 
the only plant in which it promotes a response to short days. 
Junges (1958) found that short days following the vernalization of 
a strain of Kohlrabi, Brassica oleracea var. gongyloides, a biennial, 
promoted the subsequent flowering in long days and high tempera- 
tures. Such results make it unwise to regard vernalization require- 
ments as necessarily linked to any other environmental response. 



THE SEMANTICS OF VERNALIZATION: 

FURTHER EFFECTS OF TEMPERATURE 

ON FLOWERING 

A restricted definition of vernalization was given earlier, but 
it is now time to acknowledge its fluidity. For one thing, the term 
is so often misapplied to the breaking of bud or embryo dormancy 
by low temperatures that it has become a mere jargon substitute 
for "cold treatment"; this is deplorable, but perhaps too late to 
mend. Even if one restricts its usage to effects on flowering, how- 
ever, difficulties arise. It is clear enough how certain effects of near- 
freezing temperatures on biennials and perennials are similar to 
those on germinating winter annual seeds, and why the term 
vernalization may well be used for both. As long as one is dealing 
with an obviously inductive action on flowering of temperatures 
low enough to prevent growth, the phenomena seem relatively 
clear-cut. But when the same or very similar effects occur at 
temperatures high enough to allow rapid growth, or are not induc- 
tive, or interact with the conditions of light and darkness during 
exposure, are they still vernalization? This is not simply a matter 
of semantics; the point is that the influences of temperature on all 
aspects of development are so manifold that "typical" vernali/a- 
tion, as in rye or Hyoscyumus, probably is an extreme case of a 



The Semantics of Vernalization • 63 

very general situation. If so, then perhaps the erosion of the word 
vernalization is fortunate. 

The plasticity of some vernalization requirements is illustrated 
by celery, Apium graveolens var. dulce. If the plants are kept at 
usual vernalizing temperatures (about 7° C) for a month, they will 
flower rapidly when transferred to cool (10-16°) or moderate 
(16-21°) but not warm (about 24°) conditions. The initial vernaliza- 
tion is not absolutely necessary for flowering, which will also take 
place eventually under constant cool conditions, or under the 
moderate conditions after two weeks under cool conditions. No 
temperature pretreatment of any kind will permit flower initiation 
under the warm conditions (Thompson, 1953). In short, vernaliza- 
tion is only weakly inductive and can take place at temperatures 
high enough to allow growth. The latter of course is true to a lesser 
extent even of Hyoscyamus, and one can still see in celery the 
occurrence of vernalization and devernalization in the Hyoscyamus 
sense, but the effective temperatures are considerably closer 
together. 

The flowering response of stocks, Matthiola incana, as sum- 
marized by Kohl (1958), represents a situation in which it is uncer- 
tain whether the term vernalization can be applied or not. Neither 
germinating seeds nor seedlings can be induced by low tempera- 
tures, but maturing plants require at least three weeks at 10 to 
16° C for flower initiation. If the temperature rises above 19° for 
as little as 6 hours per day, initiation is completely inhibited; the 
plants must remain at the favorably low temperatures until full 
differentiation of floral primordia has occurred. After this, however, 
they remain induced and produce new flower primordia even at the 
higher temperatures. This behavior can of course be regarded as 
vernalization with a very low degree of induction and a small 
difference between vernalizing and devernalizing temperatures, but 
speaking simply of optimum and maximum temperatures for 
flower initiation seems to be as accurate. Many plants probably 
respond in a similar fashion, with optima and maxima varying 
widely depending on the species. 

Also relevant here is another temperature effect on plants, 
thermoperiodism. This term indicates the responses of plants to 
differing day and night temperatures— growth and development in 
mo6t of those tested are favored by night temperatures markedly 



64 • Temperature and Flowering 

lower than those optimal during the light period (see Went, 1957). 
Work on this question will not be dealt with here, since relatively 
little of it directly concerns flower initiation. In addition, the 
interactions of temperature changes with high-intensity light 
periods of different lengths are extremely complex and have not 
been carefully analyzed. Many of the data do suggest, however, 
that "typical" vernalization, the effects of moderately low tempera- 
tures, the effects of varying day and night temperatures, and the 
interactions of temperature with photoperiod (Chapter Two) all 
intergrade. 

Recall in this connection the observation of Schwabe (1955b, 
1957) that discontinuous vernalizing cold treatments were more 
effective on Chrysanthemum when given during each night rather 
than in the day. This sounds very much like thermoperiodism. 
Note also that the tomato, Lycopersicon esculentum, in which 
major effects of temperature have been studied as thermoperiodism, 
is quantitatively vernalizable; exposure of the seedlings to tempera- 
tures near 10° C soon after cotyledon expansion significantly de- 
creases the number of leaves formed before the first inflorescence 
and increases the number of flowers in that inflorescence (Wittwer 
and Teubner, 1956). Since one effect of low night temperatures is 
also to increase the number of flowers per inflorescence (Went, 
1957, Chap. 6), vernalization in the tomato, as in Chrysanthemum, 
is perhaps not completely distinguishable from thermoperiodism. 

A further expansion of the phenomena that need to be con- 
sidered in connection with vernalization is suggested by some 
work of Guttridge (1958). By the definition previously given, 
vernalization results in the promotion of flowering. However, a 
cold treatment affects certain varieties of strawberry (Fragaria) in 
the opposite fashion, inductively bringing about a condition in 
which flower initiation is delayed and runner production promoted 
when the plants are subsequently transferred to conditions that 
would otherwise make for continued flowering and low vegetative 
growth. This effect is certainly formally similar to vernalization, 
though inverse in result. 

TEMPERATURE AND FLOWERING IN BULB PLANTS 

Among the most detailed studies yet done on temperature 
and flowering are those of Blaauw, Hartsema, Luyten, and their 



Temperature and Flowering in Bulb Plants • 65 

collaborators in the Netherlands, particularly in the period 1920- 
1935, on the initiation and development of flowers in bulb plants. 
This work is largely recorded in Dutch but has been reviewed by 
Went (1948), from whom this account is taken. The basic pro- 
cedure was to store bulbs at different temperatures for different 
lengths of time and determine, by anatomical studies, the optimum 
temperature for the various developmental events taking place 
within them. 

After the current year's foliage has died, the next year's apical 
meristem within the tulip (Tulipa) bulb already has several leaf 
primordia. Flower initiation, including differentiation of all the 
flower parts, then takes about three weeks at 20° C, the optimal 
temperature for this process. If further flower development is to 
take place (still entirely within the bulb), the temperature must 
now drop and remain at about 9° C for 13 to 14 weeks. After this 
low-temperature period the optimal temperatures for leaf and stalk 
elongation are successively higher, reaching 20° and above for com- 
plete anthesis. This increase in optimal temperature for the final 
stages of flowering is more or less gradual, but it appears to be 
characteristic of tulip and certain other plants that flower initiation, 
favored by relatively high temperatures, must be followed quite 
abruptly by low temperatures for the best subsequent development. 
In the hyacinth (Hyacinthus) bulb, on the other hand, the changes 
in temperature optima are not as abrupt as in the tulip, though they 
are similar, and all the values lie somewhat higher. 

Such studies have since been conducted, in the Netherlands 
and elsewhere, on many plants having bulbs, rhizomes, or other 
fleshy organs that can be stored for a considerable part of the year. 
The detailed results of course differ from plant to plant, but are 
usually of great practical value since they make it possible to 
control development or arrest it at desired stages to suit almost any 
shipping and planting schedule. Tulips and hyacinths, for example, 
can be held completely dormant without injury for weeks by 
storage at 35° C. As soon as further development is required, the 
temperature can again be lowered to the optimal level for the stage 
previously attained. Recent references to this sort of work can 
be found in journals and textbooks on horticulture. 

It needs to be stressed that this sort of temperature response is 
not characteristic of all bulb plants, but merely of those adapted to 
temperate climates with a well-defined winter. The tropical bulb 



66 • Temperature and Flowering 

Hippeastrum, for example, also studied by Blaauw (see Went, 
1948), has no such requirement for a long period of low tempera- 
ture, and flowers several times a year at high or moderate 
temperatures. The similarity between the cold requirement in a 
plant such as the tulip and typical vernalization should also be 
noted. Here of course the effect is on flower development, not 
induction or initiation, but the conditions involved and the final 
results are the same, although the underlying physiological con- 
ditions are unknown in any case. 

Unlike light or certain chemical factors, temperature cannot be 
given or withheld but only changed, and it ailects essentially all bio- 
chemical processes. This makes it at once the most important single 
factor in development and the most difficult to study in any de- 
limited way. Hence it is not surprising that terms such as vernali- 
zation are almost meaningless except to indicate a particular kind ol 
manipulation, and may not designate any single specific physio- 
logical process. The brevity of this discussion relative to those on 
other factors affecting flowering should be taken to reflect not a 
lesser importance of its problems, but only how little is known 
about them in any fundamental sense. See Went (1953, 1957) for 
a much more thorough treatment of the effects of temperature on 
all aspects of plant growth; a review by Chouard (1960) emphasizes 
the complexity of vernalization and related low-temperature effects. 



► 
► 



chapter five t Floral Hormones 

and the Induced State 



Even before the effects of light and temperature— the major 
natural environmental influences on flowering— were known, the 
question of what internal changes lead to flowering was of obvious 
importance; photoperiodism and, to a lesser extent, vernalization 
made experimental approaches to it more feasible. The next three 
chapters are largely concerned with this question in one way or 
another; the present will examine the nature and origin of sub- 
stances controlling flowering and transmissible from one part of a 
plant to another or from plant to plant by grafting. 

DEFINITIONS AND BACKGROUND: AUXINS AS 

PLANT HORMONES 

Hormones can be defined as substances produced in one part 
of an organism and acting in another, and active in very low con- 
centrations. Action at a distance from the site of production is the 
most crucial characteristic of a hormone; activity in low concen- 
trations simply serves to distinguish it from substances furnishing 
energy or structural materials and used in large quantities. Sugars, 
for example, are produced in aerial parts of the plant and used 
in the roots (as well as elsewhere) but cannot be considered hor- 
mones. 

67 



68 • Floral Hormones and the Induced State 

The idea that the formation of flowers, and of other organs as 
well, is controlled by hormones specific for each type of organ- 
" organ-forming substances"-was favored in the nineteenth century 
by Julius Sachs, the so-called "father of plant physiology." Evidence 
at the time was almost nonexistent; more recent evidence, at least 
for flowering hormones, will be considered below. First, however, 
it is useful to describe briefly a different and better known class of 
plant hormones, the auxins. Research on these substances, starting 
in the 1920's, has had a strong influence on the less successful 
investigations on possible flowering hormones; in addition, auxins 
may play at least a minor role in the control of flowering. 

If the tip of a growing shoot is removed, the elongation of the 
remaining stump generally ceases rapidly. If the tip is replaced, the 
stump may resume and continue elongating for some time, although 
not necessarily as fast as in the intact plant. This effect of the tip 
may even occur if it is separated from the stump by a thin layer of 
agar or gelatin. In such cases, elongation can be brought about 
simply by placing on the stump a piece of gelatin or agar on which 
the cut surface of the tip, or several similar tips, have rested for 
some time. Such results indicate that a substance or substances that 
can move out of the tip and into or through gelatin are required for 
the continued elongation of the tissue below. Such substances are 
termed auxins. It is now known that low concentrations of many 
substances, both natural and synthetic, can promote the elongation 
of shoot tissue deprived of its natural auxin sources. Most of them 
are relatively simple organic compounds, such as indole-3-acetic 
acid; those occurring naturally are clearly plant hormones since 
they are produced in shoot tips (or other young, actively growing 
regions) and affect tissues elsewhere. The action of auxins is not 
confined to causing the elongation of shoot cells, however; depend- 
ing on the concentration, they may either promote or inhibit 
many plant processes, including root initiation, leaf abscission, and 
cell division. Space forbids further discussion of auxins as such, 
but they will figure in a number of the topics to be considered. 
For additional information on the general topic of auxin physi- 
ology, which has a voluminous literature, see Audus (1959), 
Leopold (1955), or the recent volume, Plant Growth Regulation 
(1961). 



Evidence for Flowering Hormones • 69 



PRELIMINARY EVIDENCE FOR THE EXISTENCE 
OF FLOWERING HORMONES 

The clearest early investigations indicating the existence of 
floral hormones were by Chailakhyan in Russia. One of his major 
experiments (1936a) showed that if the upper portion of the SDP 
Chrysanthemum indicum were defoliated, it would initiate flowers 
if the lower (leafy) portion received short days, even if the de- 
foliated part were kept on long days. With the conditions reversed 
—if the upper defoliated part were kept on short days and the 
lower leaves on long days— no flowering occurred. He interpreted 
these results as indicating that under the proper photoperiodic 
conditions the leaves could form a hormone that moved to the 
apex and brought about flowering. From subsequent work he 
concluded also that this hormone, which he named "florigen" 
(flower-maker), could move either up or down the stem and could 
be transferred from one plant to another through grafts (Chail- 
akhyan, 1936b, 1936c, 1937). 

Several investigators at first obtained data suggesting that 
florigen, like the auxins, could pass through a nonliving connec- 
tion, but these proved to be illusory. Moshkov (1939), for example, 
soon reported his inability to repeat his own earlier experiment in 
which the Chrysanthemum floral stimulus had apparently passed, 
through a thin film of water, and he concluded that such move- 
ment could take place only through living tissue. A similar en- 
couraging but false start was made by Hamner and Bonner (1938). 
They showed that a photoperiodically induced Xanthium plant 
grafted to a noninduced plant could bring about flowering in the 
latter. They further observed that interposition of a piece of fine 
lens paper between the stock and scion would still permit this 
effect. This suggested that florigen could move from the induced 
plant (the donor) to the noninduced plant (the receptor) without, 
direct tissue contact. When this work was repeated by Withrow and 
Withrow (1943), using various kinds of membranes including lens 
paper between the cut surfaces of donor and receptor, it appeared 
that the original interpretation was mistaken. Anatomical studies 
showed that tissue union could occur by the growth of cells through 
the lens paper; the transmission of florigen took place only when 



70 • Floral Hormones and the Induced State 

there was such union, and all membranes that would prevent 
actual "taking" of the graft also prevented transmission. 

Chailakhyan (1937) had already concluded from experiments 
with Perilla and Chrysanthemum, and the Withrows (1943) con- 
firmed with Xanthium, that florigen movement occurred only 
through the "bark"— the phloem and cortical tissue. If this was 
removed in ringing or girdling experiments, no movement of the 
floral stimulus across the girdle was observed, although water con- 
tinued to pass through the xylem (wood) and the shoots remained 
healthy. Presumably the major route of transport is the phloem 
itself, in which most organic substances are transported; but we 
will return to this question later. 

Questions obvious from the start of this kind of research are 
whether the florigen of one kind of plant is effective on another 
and, more particularly, whether that of an SDP will act on an LDP 
and vice versa. Auxins are not species-specific, but such questions 
are more difficult to answer with respect to flowering hormones, 
transmissible from plant to plant only by grafting. Successful grafts 
are generally possible only between closely related plants so that no 
completely general answer can be given. Within these limitations, 
however, the floral stimulus produced by one species is often 
effective on other, closely related species. 

Maryland Mammoth tobacco and annual Hyoscyamus niger 
are members of the same family (Solanaceae) and can be success- 
fully grafted. In such a graft partnership, the LDP Hyoscyamus 
will flower under short-day conditions if the SDP tobacco is also 
kept under short days, but not if the tobacco is exposed to long 
days. That is, under short days the tobacco is itself induced and 
serves as the donor of stimulus of florigen to Hyoscyamus. Con- 
versely, the tobacco can be made to flower under long-day condi- 
tions if the Hyoscyamus is induced by also being kept under long 
days, but not if the Hyoscyamus receives short days. Here Hyoscy- 
amus becomes the donor and Maryland Mammoth the receptor 
(Lang and Melchers, 1947; see Lang, 1952). The simplest conclu- 
sion is of course that the florigens produced by Hyoscyamus in 
long days and by Maryland Mammoth tobacco in short days are 
physiologically equivalent if not identical. 

There are many similar experiments in the literature. The 
SDP Xanthium, lor example, can be made to (lower on long days 



Evidence for Flowering Hormones • 71 

when grafted to any of several LDP members of its family, the 
composites, such as species of Erigeron or Rudbeckia (Okuda, 
1953). Using members of the family Crassulaceae, Zeevaart (1958) 
found that the LDP Sedum ellacombianum or Sedum spectabile 




Fig. 5-1. Transfer of flowering stimulus between LDP and SDP by grafting, 
showing role of leaves. (/I) Induction of flowering in an LDP (Sedum spectabile) in 
short days by grafting onto an SDP (Kalanchoe' blossfeldiana) . In the graft to the right, 
the Kalanchoe (below) was kept defoliated. Photograph made 96 days after grafting. 
(B) Induction of flowering in an SDP (Kalanchoe) in long days by grafting onto an 
LDP (Sedum) — the reciprocal of the experiment in (.4). Again, in the graft to the 
right, the Sedum was kept defoliated. Photograph made 130 days after grafting. 
(Photographs from Zeevaart [1958], courtesy of Dr. J. A. D. Zeevaart, Agricultural 
Institute, Wageningen.) 



could flower under short days when grafted onto the SDP Kalan- 
choe blossfeldiana, whereas the latter would flower under long days 
when grafted to the Sedians (see^ Fig. 5-1). Such effects can be 
turned to practical use. Many varieties of the cultivated sweet 
potato, Ipomoea batatas, flower irregularly if at all, no matter 
what the environmental conditions, which is a distinct hindrance 
to breeding programs. This recalcitrance can be overcome by 
grafting shoots to any of several free-flowering (SDP) genera of the 



72 • Floral Hormones and the Induced State 

same family (Convolvulaceae— morning glories) and then inducing 
the latter (Lam et al., 1959). 

The occurrence of transmissible flowering stimuli is not con- 
fined to photoperiodic plants. This is of course implicit in the 
fact, noted earlier, that many plants are only quantitatively photo- 
periodic, or are photoperiodic only under certain conditions, 
whereas some are completely daylength-indifferent; the processes 
leading to flowering may or may not be under photoperiodic 
control and still have the same end result. Lang (1952) has reviewed 
work in which daylength-indifferent plants can serve as donors of 
a flowering stimulus to closely related LDP or SDP. 

Not all results on the transmission of flowering stimuli have 
been straightforward, and before proceeding further it is well to 
keep the fundamental difficulty in mind. Auxins can be obtained 
from plants either by diffusion from cut tissues, as previously 
described, or by extraction. They can then be reapplied and will 
cause growth in responsive tissue. This makes possible not only the 
identification and quantitative assay of naturally occurring auxins 
but also the study of the biochemistry of their origin and function. 
Not so for the hypothetical florigen— which remains hypothetical 
for the very reason that, with one possible exception, no work to 
date has successfully isolated it from the living plant; attempts to 
do so will be discussed in the following chapter. Thus it has not 
been possible to study flowering hormones chemically, and all the 
evidence is necessarily circumstantial. Hence the use in this chapter 
of all sorts of terms-florigen, floral hormones, flowering stimuli, and 
so on— to avoid implying a precision that does not exist. We must 
now pay closer attention to the experimental systems involved in 
such work— the plants themselves— following which we can return 
more critically to the question of whether floral hormones actually 
exist. 

TRANSLOCATION OF FLOWERING HORMONES 

The conclusion that florigen moves only through living tissues 
is based on observations besides those already presented. Borthwick, 
Parker, and Heinze (1941) showed that a soybean plant defoliated 
to only a single leaf could flower in short days, but not if the petiole 
was chilled to 3 C C. This was true even if another leaf was left on 



Translocation of Flowering Hormones • 73 

the plant, below the first, and exposed to long days without any 
other treatment. Hence the inhibition of flowering was not due 
simply to lack of carbohydrate transport through the chilled petiole, 
since carbohydrates were still supplied by the long-day leaf, but to 
the inhibition of the transport of the stimulus specifically from the 
short-day leaf. These and similar results indicate that transport is 
the result of cellular activity. Further circumstantial evidence im- 
plicates the phloem in florigen transport by indicating that the 
latter is associated with the movement of carbohydrates in the 
plant. This evidence is not unequivocal, and is based largely on 
experiments dealing with the effects of noninduced leaves on the 
flowering response. 

Note that in Chailakhyan's experiment with Chrysanthemum, 
discussed earlier, the upper portion of the plant was defoliated in 
order to demonstrate the movement of a flowering stimulus from 
the lower leaves on short days. Many observations, including those 
of Chailakhyan, indicate that in some plants translocation can only 
be demonstrated in this manner. A technique often used to study 
this sort of question involves the use of two-branched plants, 
produced by removing the apical portion of seedlings and allowing 
two approximately equal lateral branches to develop. One branch 
can then be exposed to inductive conditions, making it the donor 
of flowering stimulus, and the other, on noninductive conditions, 
is used as the receptor. When Biloxi soybeans are used in this way, 
the receptor (long-day) branches flower only if they are defoliated 
but not if the leaves are left in place, even though the donor 
branch flowers well whether or not the receptor has leaves (Borth- 
wick and Parker, 1938b). Similar results have been obtained in 
other plants but are by no means universal. In the SDP Amaranthus 
caudatus, defoliation of the receptor (long-day) branches greatly 
inhibits, rather than promotes, their flowering, which is otherwise 
almost as rapid as that of the donor branch itself (Fuller, 1949). 

Noninduced leaves can be kept in total darkness, rather than 
removed, in order to avoid their inhibiting transmission. This 
observation was actually first made by Garner and Allard in 1925; 
the only reason they are not generally credited with the discovery 
of the translocatable effects of photoperiodism is that they them- 
selves stressed the localization of such effects in Cosmos, the SDP 
they chose for work on this question. In this as in many other 



74 • Floral Hormones and thf. Induced State 

plants, flowering in normal, intact individuals is confined to the 
area exposed to induction. A portion of the plant kept in total 
darkness, however, will exhibit a flowering response, provided an 
adjacent portion is kept on inducing (short-day) conditions. An 
elegant experiment by Stout (1945) illustrates the same situation 
for an LDP, the sugar beet (Beta vulgaris). Plants with three shoots 
were made by root grafts. If one of these shoots was exposed to 
long days, it flowered and also brought about flowering in a second 
shoot kept in darkness. The third shoot, however, maintained on 
short days, remained vegetative. 

As suggested earlier, most experiments of this kind can be 
interpreted as indicating that florigen moves in the prevailing 
direction of carbohydrate movement. In this view, darkening or 
removing leaves from a noninduced part of the plant results in a 
lower carbohydrate production in that part, so that carbohydrate 
(and florigen) movement in its direction is increased. There are 
alternative explanations, however, as will become evident later. In 
some cases, darkened leaves may inhibit translocation; this has 
been interpreted as a "diversion" of the movement into such leaves 
(see Lang, 1952). 

Interesting evidence on the translocation of floral hormones 
and the effects of noninduced leaves comes from work on the SDP 
Kalanchoe blossfeldiana reported by Harder (1948). With a mini- 
mal short-day treatment, development of the complex, branching 
inflorescence is slow and "vegetative"; that is, the flowers are small 
or abortive and the bracts among them overdeveloped and leaflike. 
If only a single leaf receives short-da y treatment, inflorescence 
development may be normal, provided the treatment continues 
long enough; but commonly it is notably asymmetrical, being more 
normal on the side directly above the induced leaf and vegetative 
on the side away from it (see Fig. 5-2). Examination of the vascular 
svstem shows that this is consistent with the idea that florigen 
simply moves in the phloem: the lateral connections in Kalanchoe 
are relatively minor, so that little lateral movement of the effect 
would be expected. 

Experiments on the effects of noninduced leaves in Kalanchoe 
depend on its decussate leaf arrangement; that is, each pair of 
opposite leaves is at right angles to the pair above or below it. 
Thus looking down on the plant one sees four ranks of leaves at 



Translocation of Flowering Hormones • 75 

right angles to each other. If all but two leaves (of different pairs) 
are removed from the plant, and the lower is given short days and 
the upper long days, several different results can be obtained. I! 
the long-day leaf is in the same rank with (directly above) the 
long-day leaf, flowering is prevented. If the long-day leaf is in the 
rank opposite that of the short-day leaf, flowering is the same as if 



Fig. 5-2. Localization of flower- 
ing stimulus in Kalanchoe. A single 
leaf situated on the left-hand side 
was repeatedly exposed to short 
days whereas the rest of the plant 
received long days. (Photograph 
from Harder [1948], by permis- 
sion of the company of Biologists, 
Ltd., and courtesy of Dr. R. 
Harder, University of Gottingen.) 




the long-day leaf were absent. Finally, if the long-day leaf is in 
either of the two ranks at right angles to that of the short-day leaf, 
some inhibition of flowering is evident. In this sort of experiment, 
the transport of florigen is evidently upward from leaf to growing 
point, but appropriately trimmed plants can be used for similar 
studies on the transport downward from a short-day leaf to an 
axillary shoot. Here again, a long-day leaf between the short-day 
leaf and the shoot inhibits most effectively if it is in the same rank, 
and least effectively if it is in the rank opposite. In short, whether 
movement is up or down, the inhibition only occurs if the non- 
induced leaf lies effectively between the induced leaf and the 
growing point in question. This is apparently true for many plants 
besides Kalanchoe and is again consistent with the postulated 
movement of florigen with the carbohydrate stream. In addition, 



76 • Floral Hormones and the Induced State 

however, it is also consistent with the idea that the flowering 
hormone might be taken up by the noninduced tissue and de- 
stroyed by it. 

The latter interpretation is also suggested by analogous experi- 
ments in which parts of a single leaf are subjected to long-day or 
short-day treatments. If the basal part of the leaf is given short 
days and the apical long days, flowering occurs, but if the situation 
is reversed, the flowering is weak or absent. This is not due to the 
inability of the apical portion to respond to short days and lead 
to flowering, since it does so if the entire basal part is trimmed off 
as long as the vascular connection to the stem is left intact. Here 
again, noninduced tissue evidently inhibits flowering when it is 
situated between induced tissue and the growing point, and possibly 
does so by destroying the floral stimulus. Earlier experiments by 
Chailakhyan with Perilla leaves also lead to the same conclusion 
(seeNaylor, 1953). 

The most thorough recent studies of the interactions of various 
parts of the plant on the effectiveness of localized inducing treat- 
ments are those of Lincoln, Raven, and Hamner (1956, 1958), using 
Xanthium. The first paper bears most directly on translocation. 
With two-branched plants, the intensity of flowering in the re- 
ceptor branch (long days) is inversely proportional to the amount 
of mature tissue left on it. If, however, a carbohydrate deficiency 
is produced in the receptor by heavy shade, the inhibition by the 
long-day leaves is greatly reduced. Conversely, shading the donor 
(short-day) branch, which would produce a carbohydrate deficit in 
it, reduces flowering in the receptor. So also does removing the 
receptor's young leaves, which are responsible for a great portion 
of its carbohydrate uptake. Although these results are consistent 
with the carbohydrate-flow hypothesis, several others suggest a 
more complex situation. The inhibiting effect of mature leaves on 
the receptor is not simply proportional to the amount of light they 
receive but depends on its timing; that is, the effect is photo- 
periodic. For example, the inhibition caused by leaves given 12 
hours light-12 hours dark cycles is much greater if each night is 
interrupted by three evenly spaced 10-minute light-breaks than if 
interrupted only once, in the middle, by a 30-minute light-break. 
If only carbohydrate production were involved in the inhibition, 
such results would not be expected. 



Translocation Rate • 77 

In certain plants, such as the SDP Piqueria trinervia (stevia), 
the effect of inductive treatment remains relatively localized no 
matter what manipulations are performed (Zimmerman and Kjen- 
nerud, 1950). Thus the only summary statement that can be made 
about the movement or apparent movement of flowering hormones 
is that it takes place in living tissue, probably through the phloem; 
that it can be either acropetal (base to apex) or basipetal; that it 
may be localized or systemic depending on the plant, the structure 
of its vascular system, and the condition of noninduced portions. 
There is evidence that noninduced leaves act in an inhibitory 
fashion primarily but not exclusively by affecting the predominant 
direction of carbohydrate movement, with which the florigen may 
be carried. 

TRANSLOCATION RATE 

There are very few studies on the rate of movement of floral 
stimuli, again because of the difficulty that only the final response, 
not the postulated hormone, can be measured. Early work by 
Chailakhyan suggested values of about 2 cm in 24 hours in Perilla, 
but it is doubtful whether conditions were optimal (see Lang, 
1952). Some ingenious experiments by Imamura and Takimoto 
(1955b) provide the best data so far available. 

Plants of the SDP Pharbitis nil (Japanese morning glory) can 
be reduced to a stem with a single leaf, and then decapitated so 
that the bud in the axil of the leaf will start to grow. The position 
of the first flower on the axillary shoot will then depend on the 
time between the start of growth (decapitation) and the start of a 
single 16-hour inductive dark period given to the leaf. In one 
experiment, for example, if the dark period was started imme- 
diately after decapitation, the average position of the first flower 
on the axillary shoot was at node 2.8 (that is, node 2 in some 
plants, node 3 in most). If 24 hours elapsed between decapitation 
and the dark period, the average position was node 3.5, and so on. 
Such differences are developmental expressions of the amount of 
time during which the axillary bud (shoot) was growing before the 
flowering stimulus reached it. Parallel experiments can be done at 
the same time with plants in which the distance between the single 
leaf and the receptor bud is greater-for example, by having the 



78 • Floral Hormones and the Induced State 

latter not in the axil of the leaf but on the opposite branch of an 
otherwise debudded two-branched plant. The rate of stimulus 
translocation can then be calculated by the difference in the first- 
llowering-node values of the shoots in the plants with the receptor 
buds close and the receptor buds far from the induced leaf. An 
example from one experiment may make this clear. In the "close" 
series, in which the average distance from leaf to bud (mainly 
through petiole tissue) was 90 mm, the mean first flowering node 
was 3.4 if the dark treatment was given 24 hours alter decapitation. 

4.5 with it given 48 hours after, and 5.3 with it given 72 hours 
after. In the "far" series, the distance between leaf and bud was 
about 235 mm, through both branch and petiole tissue. Here, 
inductive treatment started immediately after decapitation gave a 
first-flowering-node average of 4.6. By interpolation from the pre- 
ceding figures, it is as if the inductive treatment for the "close" 
series had been delayed some 55 hours. Since the difference be- 
tween "far" and "close" is about 145 mm, this difference of 55 
hours represents the movement of the stimulus at 145/55, or about 

2.6 mm per hour. 

Such experiments of course give an average value for the 
transport through a petiole, then both down and up a branch: 
other experiments suggested that upward transport may be faster 
than downward. Also, the transport rate in plants so mutilated 
may well differ from that in intact plants. In any case, all experi- 
ments with Pharbitis gave values of the order of 3 mm per hour. 
This represents a considerably slower movement than that observed 
for carbohydrates in phloem tissue (often exceeding 200 mm per 
hour), but rates of virus transport in the phloem sometimes fall 
in this low range (see Esati et ai, 1957). 



FLOWER PROMOTION OR FLOWER INHIBITION? 
THE SPECIFICITY OF FLOWERING STIMULI 

The florigen hypothesis in its simplest form postulates a single 
substance, common at least to many plants, uniquely responsible 
lor flower initiation. Much of the evidence so far presented is 
consistent with this hypothesis, but some investigators, on the con- 
trary, have concluded that flowering is controlled by an inhibitory 



Flower Promotion or Flower Inhibition? • 79 

substance or substances that prevent initiation until they are 
removed by the proper conditions. 

It may be surprising that most of the very evidence presented 
in the preceding section for movement of a florigen can be reinter- 
preted as indicating simply deinhibition (see von Denffer, 1950). 
Under this interpretation, noninduced leaves constantly produce a 
flowering inhibitor that moves to the growing point along with the 
products of photosynthesis; induced leaves no longer produce this 
inhibitor. Hence the removal or darkening of noninduced leaves 
often promotes flowering not, as under the florigen hypothesis, by 
preventing interference with the carbohydrate stream in which the 
florigen moves, but by reducing still further the sources of the 
inhibitor; flowering thus occurs simply as a result of sufficient 
quantities of inhibitor-free assimilates. It has been suggested, on 
the basis of work to be discussed later, that the inhibitor in 
question might be an auxin, and the general form of this hypothesis 
fits some of the experimental data well enough. At least, it often 
fits no worse than the other hypothesis, as a brief reconsideration 
will show. 

Stout's (1945) work with "three-headed" beet plants indicated 
that the presence of a shoot on short-day conditions did not inhibit 
the response of a darkened receptor shoot to the long-day donor 
shoot; thus if the noninduced (short-day) shoot produces an inhib- 
itor, it is not detectable. This does not help the inhibitor hypoth- 
esis. On the other hand, the further result that even 4 hours of 
light per day (compared with 17 hours for the donor) prevents a 
shoot from being an effective receptor also does not help the simple 
fiorigen-movement-with-carbohydrates hypothesis, since it is un- 
likely that the predominant direction of carbohydrate movement 
would be reversed under these conditions. Another ambiguous 
situation is of course that the inhibitory effect of noninduced 
Xanthium leaves appears to be a photoperiodic phenomenon in its 
own right, not simply a matter^ of affecting carbohydrate (and 
florigen) flow. 

The florigen hypothesis can be saved from many difficulties, 
including these, by the suggestion that noninduced leaves act not 
by producing an inhibitor but by destroying florigen. On balance, 
the simple inhibitor hypothesis is probably less satisfactory; the 
strongest argument against it is the effectiveness of small amounts 



80 • Floral Hormones and the Induced State 

of induced leaf tissue, of which there are many examples in the 
literature. Xanthium is striking in this regard. Several double- 
branched plants grafted together in series can all be brought to 
flower by short-day treatment of a single leaf on one of them (see 
Naylor, 1953). Khudairi and Hamner (1954a) found that a total 
leaf area of less than one square centimeter was enough to bring 
about flowering from a single 16-hour dark period. Xanthium may 
be more extreme in this regard than most species, but the idea 
that induced leaves simply supply an inhibitor-free stream of 
assimilates is hard to reconcile with such results. However, some 
form of inhibitor hypothesis is still favored by certain investiga- 
tions, of which a few should be considered. 

In annual Hyoscyamns (LDP), removal of all the leaves brings 
about flower formation, which then takes place at the same rate 
irrespective of light or dark conditions. Presumably, then, the 
effect of long days on an intact plant is to prevent an inhibition 
of flowering exerted by the leaves under short-day conditions. Since 
clearly in the defoliated plant the floral stimulus is present or can 
be formed in the stem or roots, leaves on short days apparently not 
only fail to produce it themselves, but also destroy it, or inhibit its 
production, or produce an inhibitor of flowering. The latter hypoth- 
esis can be avoided either by adopting the first or by suggesting 
a mechanism for the second— for example, that the short-day leaves 
remove some substance that could otherwise act as a precursor for 
production of the stimulus. So far, there is no clear evidence in any 
direction (see Lang, 1952) . Whatever the explanation, such effects 
may be responsible for some of the ambiguous results obtained 
from grafting experiments, as in the following example taken from 
Zeevaart (1958). 

Defoliated scions of the LDP Nicotiana sylvestris grafted on 
stocks of the SDP Maryland Mammoth will flower on short days, 
suggesting florigen transfer from the induced stock. However, such 
scions also flower on long days, noninductive for Maryland Mam- 
moth, although similarly defoliated but ungrafted Nicotiana sylves- 
tris fails to flower on long days. Does Maryland Mammoth then 
produce florigen under, for it, noninductive conditions? The ex- 
planation may be that defoliated Nicotiana sylvestris, like Hyoscya- 
mus, has the capacity to flower if sufficient assimilates are present. 
In Hyoscyamus these come from the large storage root, whereas in 



Flower Promotion or Flower Inhibition? • 81 

the Nicotiana sylvestris experiment they are supplied by Maryland 
Mammoth whether on long or short days. 

Flower initiation in strawberries, Fragaria, requires short days, 
at least under certain conditions. Hartmann (1947) showed that 
daughter plants would initiate flowers in long days if the adult 
plant, to which they were still connected by runners, was exposed 
to short days; he interpreted these results in the conventional 
"florigen" manner. Guttridge (1959) has since performed experi- 
ments suggesting the opposite— that flowering occurs when the 
level of a flowering inhibitor, which also promotes vegetative 
growth, is sufficiently reduced. This postulated substance would be 
produced in long but not in short days, and might even be 
destroyed in the latter. The evidence is analogous to that on the 
translocation of flowering hormones. 

Plants kept on long photoperiods (using light-breaks) promote 
vegetative growth and inhibit flowering in runner-attached plants 
under short photoperiods. This is favored by earlier daily illumina- 
tion of the plants on long days, although earlier illumination itself, 
without light-breaks to create an effective long photoperiod, has no 
effect. These results of course again suggest translocation of the 
substance in question— this time the flower-inhibiting, growth- 
promoting substance— in the predominant direction of carbohydrate 
movement. Experiments with radioactive phosphorus as a tracer 
confirmed the postulated direction of assimilate movement.. 
Guttridge's results are thus more consistent with the "simple 
inhibitor" hypothesis than with "florigen"; here the "donor" is 
vegetative, the "receptor" potentially flowering. 

The earliness of flowering in certain pea varieties— by which 
is meant whether the first flower appears at a lower or higher node 
—can be influenced in several ways other than (in some varieties) 
photoperiod or cold treatment. These include removing the 
cotyledons, making cuttings from the young seedlings, grafting of 
early onto late varieties or vice versa, or even grafting stock and 
scion of the same variety. The situation is complicated by the fact 
that certain treatments, which can be broadly described as in- 
hibitory, may inhibit vegetative growth more than flowering so 
that the latter actually occurs at an earlier node, though no sooner 
in time. Haupt (1958) has concluded on the basis of his own 
experiments and those of others that transmissible flower-promoting 



82 • Floral Hormones and the Induced State 

and flower-inhibiting substances both play a part in these effects, 
but their nature is unknown. 

Resentle (1959) also supports the concept that flowering 
generally depends on a change in a complex balance rather than 
on either simple flower-promoting or flower-inhibiting substances, 
since his experiments with the Crassulaceae (Bryophyllum, 
Kalanchoe, Bryokalanchoe species) have indicated all degrees of 
transfer of the "floral state" or "vegetative state" from one plant to 
another by grafting. Further discussion on the merits of various 
hypotheses will be deferred until the concluding section of the 
chapter. 

VERXALIN AND METAPLASIN 

In addition to florigen and flowering inhibitors, the participa- 
tion of other transmissible substances in flowering or processes 
related to it has been suggested. With regard to vernalization, 
Melchers (see Melchers and Lang, 1948; Lang, 1952) has assumed 
the existence of a substance called "vernalin" on the basis of 
experiments with biennial Hyoscyamus. If two of these Hyoscyamus, 
one previously vernalized and one unvernalized, are grafted 
together, both will flower in response to long days, although an 
unvernalized plant alone will not. This might indeed be due to 
transfer of vernalin from the vernalized to the unvernalized plant, 
but it can be equally interpreted as a movement of floral stimulus 
from the vernalized, long-day treated plant to the other that, 
unvernalized, cannot respond to long days. The "vernalin" inter- 
pretation is based on the additional observation that unvernalized 
biennial Hyoscyamus grafted to Maryland Mammoth tobacco will 
flower in long days, in which the tobacco itself is not induced. 
The tobacco is visualized as a donor of vernalin— produced without 
vernalization in a non-cold-requiring plant— enabling the unver- 
nalized biennial to respond to long days. In this view, vernalin 
is either a direct biochemical precursor of florigen or makes its 
synthesis possible. 

The difficulties of interpreting grafting experiments with 
tobacco (Nicotiana) species, some of which were mentioned 
earlier, make this evidence less than completely convincing. To 
the writer's knowledge, there has never been any clear demonstra- 



Permanence and Location of the Induced State • 83 

tion of the transmission, by grafting or otherwise, of a stimulus 
resulting from vernalization alone rather than vernalization fol- 
lowed by long days; such a demonstration would be necessary to 
establish the existence of vernalin. 

In the course of work on Kalanchoe, Harder (1948) concluded 
that short-day treatment caused the production not only of flower- 
ing hormones but also of "metaplasin," a substance responsible for 
the large and easily measured changes in vegetative habit (par- 
ticularly leaf succulence) accompanying flowering. Studies on its 
transport, analogous to those on the floral hormones in Kalanchoe, 
did not permit any separation of one from the other. The entire 
evidence for the existence of metaplasin as a separate entity is this: 
subjecting the upper portion of a plant on short days to a prolonged 
chloroform treatment that will strongly inhibit flowering has no 
influence on the vegetative effects of the photoperiod. This is 
hardly unequivocal proof that short days result in the production 
of two different substances, one specific for flowering and one for 
the vegetative changes. It is equally reasonable to assume that the 
processes leading to flowering are in some way different and more 
sensitive to this inhibition than those controlling vegetative growth, 
but it does not follow that the initiating conditions or substance 
brought about by photoperiodic treatment is necessarily multiple. 

If the conclusion at present must be that vernalin and meta- 
plasin may be myths, they nevertheless serve a purpose here. They 
remind us, to whom these particular errors may seem obvious, that 
the difficulties of analyzing the responses of complex organisms, 
coupled with the desire to achieve simple interpretations, may lead 
even some foremost investigators astray. 

PERMANENCE AND LOCATION OF THE 
INDUCED STATE 

As indicated in the preceding chapters, the effect of a par- 
ticular treatment, temperature or photoperiodic, may persist and 
be expressed in flowering response later, even though no anatomical 
changes are evident when the treatment is stopped. Induction, as 
this aftereffect is called, is widespread though not universal, and 
differs considerably in both permanence and location within the 
plant. Confining this discussion first to the photoperiodically in- 



84 • Floral Hormones and the Induced State 

duced state, we find that it is transient in certain plants— that is, 
they may require almost continuous exposure to the appropriate 
photoperiod in order to flower— and remarkably long-lived in others 
(see, for example, Doorenbos and Wellensiek, 1959; Chouard, 1957). 
Probably most plants are at neither extreme but, like Biloxi soy- 
bean, revert to vegetative growth after flowering over a period pro- 
portional to the previous photoperiodic treatment (Borthwick and 
Parker, 1938a; Hamner, 1940). For obvious reasons, however, the 
induced state has been studied chiefly in a few plants in which it 
is relatively permanent, notably in two SDP, Xanthium and 
Per ilia. 

The induced state in Xanthium is both persistent and trans- 
missible from plant to plant. The transfer of a florigen from a single 
leaf on short days through several grafted plants has already been 
mentioned, but it is possible to separate the final receptor from 
the short-day donor in time as well. If a plant induced by short 
days is grafted to a receptor plant in long days, the latter will 
flower. If the first graft is broken and the first receptor then grafted 
to another vegetative plant, that plant will also flower on long 
days, and so on (see Bonner, 1959a). Thus the induced state, by 
which is meant here the capacity to continue producing florigen, 
appears to be transferable from plant to plant along with the 
florigen itself; this might be called "indirect" induction, in con- 
trast to direct induction by short days. 

If all the actively growing buds of a single-leaved Xanthium 
plant are removed before and for a few days after a single short-day 
cycle, the plant remains vegetative. A given leaf can produce the 
flowering stimulus, but not over a long period of time; the young 
leaves and buds can apparently be indirectly induced by older 
leaves, however, and can themselves either store or continue to 
produce the stimulus in quantity. The experiments indicating this 
interaction are too complex to describe here (Salisbury, 1955; 
Lincoln, Raven, and Hamner, 1958), but suggest that in Xanthium 
the induced state is not permanently localized but depends on the 
renewed indirect induction of the younger portions of the plant. 

The situation obtaining in Perilla, as reported by both Lona 
(1959) and Zeevaart (1958), is quite different. A photoperiodically 
induced leaf continues to produce florigen throughout its life. It 
can be grafted onto a plant on long days, bringing it to flower, 



Permanence and Location of the Induced State • 85 

then removed and grafted onto another plant, with the same result; 
this can be repeated as long as the leaf remains healthy, which may 
be for several months (see Fig. 5-3). There is no evidence that any 
other part of the plant has a role in the maintenance of the 
induced state; detached leaves are easily induced by the appropriate 
photoperiod, as can be demonstrated by subsequently grafting them 
onto plants on long days. Experiments of this kind are rarely 
successful with Xanthium. The clearest difference between Perilla 
and Xanthium lies in the lack of any indirect induction in the 
former. When Perilla in long days is brought to flower by grafting 
an induced leaf to it, the leaves it subsequently produces remain 
noninduced, incapable of causing flowering in another plant on 
long days. 

On the basis of these observations, the relationship between 
florigen and the induced state in Perilla and Xanthium. appears to 
differ considerably. In the former, the induced state is localized 
in the leaf, produced only by photoperiodic treatment and obviously 
separable from the transmitted florigen. In Xanthium, indirect in- 
duction of the developing leaves goes on continually, either as a 
result of the transmission of florigen itself— in which case the pro- 
duction of floral stimulus in Xanthium is autocatalytic— or brought 
about by a second unknown substance moving with it. Without 
further evidence, the first possibility clearly requires the fewest 
assumptions, although it raises problems which will be considered 
later. 

As the induced states in Xanthium and Perilla are maintained 
in different ways, their permanence also differs. Implicit in much 
of the Xanthium literature is the idea that, once induced, a plant 
remains induced throughout its lifetime. In a sense this is not true, 
since Lam and Leopold (1960) showed that reversion can be brought 
about by constantly removing the flowering shoots and forcing new 
ones to grow out, until finally vegetative shoots appear. Several 
interpretations of these results have been suggested, none preferable 
to others on the basis of available evidence; but it is nevertheless 
clear that without such drastic treatment, Xanthium seldom or 
never reverts even after induction by a single short-day cycle. The 
Perilla plant, unlike Xanthium, reverts easily to the vegetative 
state under long days, since the induced older leaves die and there- 
is no indirect induction to reinduce the younger. It is thus some- 



86 • Floral Hormones and the Induced Statf 




1 


■ -« 


A 
__Au 


i 








^^r 




^T 


*k 


m 




Ejifj 


jk& 


^^^^S 


■ 


I 






il 


\^dtL 


n9 


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B 



Fig. 5-3. Experiments with grafting of single leaves in 
Perilla. (A) Technique. Left, donor leaf in polyethylene bag. 
Right, bag removed; in this case the leaf has been trimmed 
to give a standard surface area. (B) Induction of flowering 
in long days by a grafted leaf previously exposed to 36 short 
days. Photograph made 41 days after grafting. (Photographs 
from Zecvaart [1958], courtesy of Dr. J. A. D. Zeevaart, 
Agricultural Institute, Wageningen.) 



Permanence and Location of the Induced State • 87 

what paradoxical that the induced state in Perilla leaves themselves 
appears indestructible. Attempts by Zeevaart (1958) to remove it 
were completely unsuccessful, for after various treatments the 
capacity to bring about flowering was retained. The treatments 
included exposure of the detached leaves to continuous light of 
low or high intensity, solutions of a synthetic auxin (naphthalene- 
acetic acid), high temperatures (up to 5 hours at 42° C), and the 
respiratory inhibitors dinitrophenol and sodium azide. As long as 
a leaf survived, so did its induced state. However, Lam and Leopold 
(1961) have recently obtained results indicating that, under certain 
circumstances, the induced state in a Perilla leaf may be gradually 

lost. 

One of the most curious properties of the induced state in 
Xanthium is its quantitative nature. This is not to be confused 
with the phenomenon previously mentioned (for example, in 
Biloxi soybean) in which eventual reversion to the vegetative stale 
is preceded by an "amount" of flowering proportional to the in- 
ductive treatment. In Xantfiium, too, the intensity of flowering is 
quantitatively related to the inductive treatment (for example, 
Salisbury, 1955), but since intact plants do not revert, they merely 
continue flower development at a very slow rate if the initial 
induction treatment is minimal. F. L. Naylor (1941) compared the 
development of plants under repeated short days with that of others 
given only a single short day and then placed in long-day con-, 
ditions. In the former, inflorescences with all flower parts complete 
were evident after 13 days, and the seeds were almost mature within 
a month. The second group did not show complete flower develop- 
ment until over two months from the single short day, but the slow 
progress toward fruiting gave no sign of stopping before the experi- 
ment was discontinued, shortly thereafter. This kind of observation 
seems much more difficult to explain than a mere reversion to 
vegetative growth. The latter could be due to exhaustion of florigen 
or, as in Perilla, of the capacity to produce it, but maintenance of 
a long-lived but low "steady state" of flowering cannot be visualized 
on this basis. In a sense it is analogous to the fractional induction 
described in Chapter Three, except that in fractional induction 
there is no morphological or anatomical change after the first, 
subminimal, treatment. 

There is little to be added here to the description of the state 



88 • Floral Hormones and the Induced State 

induced by vernalization covered in the preceding chapter. Perhaps 
j is most remarkable property, :ikin to the way in which a small Leaf 
area brings about flowering in a large plant, is the way in which only 
.1 small portion (the meristem) need be vernalized. Present evidence, 
however, does not point to the existence <>i a transmissible stimulus, 

.ind the vernalized State probably occurs only in tissues actually 

derived from the cells originally treated. lake photoperiodic in- 
duction, the effect <>l cold treatment is quantitative and "fra< tional" 
undei < ertain < onditions. 

THE BIOCHEMISTRY ()!• INDUCTION 

Whit oi the cellular and biochemical changes involved in 
induction and the final (lowering response? These changes must 

be understood il knowledge ol (he physiology <>l flowering is to be 
more than supei Ik ial, but up to the present time very little evidence 
sufficient to answer the question has been un<o\eied. The subject 
cannot be dismissed so briefly, however, il only because many 
investigators have tried to remedy the situation and one should be 
awaie ol their attempts. 

As indicated in Chapters Two and Three, photoperiodic 

induction is a highly complex process. In SDP, at least, it is often 
regarded as comprising several steps, or "partial processes"-— the 

lust high intensity light pioeess, the dark process, the low intensity 
light process by which the dark process can be inhibited, and the 
second high intensity light process. To these can also be added 

Horigen synthesis (marking the attainment of the induced state), 

followed by Horigen translocation, and then the changes in the 
iiiciistcin (see, lor example. Bonner, 1959a; Bonner and Liverman, 

1953; Liverman, l!). r >r>). This analysis is more appropriate lor some 

plants than lot others, and none has been studied enough to 
disclose- the iiatuie ol ,mv ol the partial pioc esses, except perhaps 

the two involving high light-intensity. These may be photosyntheti< . 

.is we have seen in Chapter Two. and thus supply both energy lot 
the othci changes .ind c .u bohvclrales with which the Horigen moves. 

LDP have been less amenable t<> such an analysis, particularly with 
the evidence ol both promoting and inhibiting actions clue to the 

leaves .ind both ol which may be- affected by light and darkness. 
One ol the lew consistent observations is that the dark (and low 



The Biochemistry of Induction • 89 

light-intensity) processes in most plants studied appear at least to 
have the red, far-red reversible system in common, but its bio- 
chemical function is unknown. Again, the role of endogenous 
rhythms is uncertain. 

Many specific mechanisms have been proposed for various 
processes in induction, mostly involving transformations and inter- 
actions of hypothetical substances. As Lang (1952) has pointed out, 
they are often little more than generalized restatements of particu- 
lar data. Since expositions of these hypotheses abound in the 
reviews and papers cited, no attempt will be made to represent 
them here. Instead we will briefly consider some of the general areas 
of investigation involved. 

One of the earliest and still most favored ideas is that auxin 
plays a major part in photoperiodic induction and flower initiation. 
The possibility that induction might be caused by a change in 
auxin content was tested by Chailakhyan and Zhdanova (1938); 
they concluded that this was unlikely since auxin content in a 
number of plants was greater on long than on short days, irre- 
spective of whether they were LDP or SDP. More recent work of 
the kind has confirmed their general conclusions (see Hillman and 
Galston, 1961; Doorenbos and Wellensiek, 1959), but a major prob- 
lem is the multiplicity of auxins as well as other growth-promoting 
and growth-inhibiting substances in plants; it is difficult to be sure 
that all the relevant compounds have been assayed in a given 
investigation. Thus changes in one or another identified or un- 
identified substance may or may not be correlated either with a 
change from one photoperiod to another or with flowering response, 
but are not easily interpretable as the cause of flowering (Cooke, 
1954; Vlitos and Meudt, 1954). 

A study by Harada and Nitsch (1959a), in which paper 
chromatography was used to separate and help identify various 
compounds, illustrates the complexity of the situation. They fol- 
lowed changes in the amounts of growth substances extractable 
from an LDP, an SDP, and a vernalizable plant at various times 
during or after induction. In each plant there was a number (3 to 
6, perhaps more) of active substances; the levels of some changed 
in such a way as to suggest that they might be the cause of the 
developmental changes rather than being merely correlated with 
them. These results are only suggestive at present, but intensive 



90 • Floral Hormones and the Induced State 

pursuit of this kind of work may eventually clarify the relation 
of auxins and similar substances to flower initiation. 

Another approach is shown in the work of Konishi (1956). His 
studies of auxin level in several LDP (Sileiie, Rudbeckia, Spinacia) 
were based entirely on biological assays without previous separation 
of possible multiple substances, but he also considered enzyme 
systems that might be involved in the synthesis and destruction of 
the known auxin, indoleacetic acid. Increased activity of the former 
and reduced activity of the latter were associated with the "bolting" 
—rapid stem elongation— characteristic of flowering in many LDP; 
evidence is lading, however, that these changes actually cause 
bolting and flowering. 

Some indirect evidence of a role for auxin in flowering has 
been obtained with radiations believed to affect auxin concentra- 
tion, including both ultraviolet (UV) and x-rays. As early as 1887, 
Julius Sachs concluded that UV promoted flowering, since both 
Tropaeolum (nasturtium) and Lepidium flowered readily in sun- 
light filtered through water but not through a colorless solution of 
cjuinine, which absorbs UV. The flowering of Linum usitatissimum 
(flax) and Statice bonduelli is greatly hastened by exposure to a 
minute or two of intense UV each day, according to von DenfTer 
and Schlitt (1951). Supporting von Denfter's (1950) idea that auxin 
is a major inhibitor of flowering, they concluded that this effect of 
UV was due to an inactivation of auxin within the plants, and 
believe it explains the rapid flowering occasionally encountered at 
high altitudes where more UV readies the vegetation. Many other 
plants tested, however, did not respond in this way. An example of 
the promotion of flowering by low x-ray doses, known to reduce 
;iuxin synthesis, is reported by Leopold and Thimann (1919); 
flowering in Wintex barley was increased by over 20 percent after 
three weekly treatments with 25 roentgens. 

Further indirect evidence comes from the eflects of gravity. 
Cieotropic stimulation is known to cause a changed pattern of 
auxin distribution in plants, although the mechanism is unknown 
(see Audits, 1959; Leopold, 1955); it can also hasten flowering. The 
Cabezona variety of pineapple (Ananas comosus) can be brought 
to flower at any time by bending the stem into a horizontal position 
and keeping it bent for as few as three days; assays confirm the 
assumption that this treatment results in auxin redistribution (van 



The Biochemistry of Induction • 91 

Overbeek and Cruzado, 1948). In certain soybean varieties also, 
keeping the stem apex bent over causes earlier flowering, which 
Fisher (1957) again attributes to auxin redistribution, presumably 
a lower level at the older nodes resulting from an accumulation at 
the apex. 

Hypotheses on the role of auxin in flowering have been based 
largely on the effects of externally applied auxins and related 
compounds, to be considered in the next chapter, rather than on 
the kind of work described above. Neither type of evidence has 
lent itself to any simple interpretation. In addition to hypotheses 
in which auxin simply inhibits or promotes flowering, one of the 
most elaborate schemes suggested relates its action directly to the 
red, far-red reversible system (see Liverman, 1955). The evidence 
is derived largely from work with processes other than flowering, 
and the "morphogenetic photocycle," as the scheme has been called, 
has not been widely accepted, at least in its original form (see Lang, 
1959; Hillman, 1959c). 

The gibberellins, a class of compounds to be discussed in the 
next chapter, can cause flowering in many LDP when applied 
externally. So far there is little information on whether the control 
of the level of these substances by photoperiod or temperature may 
explain certain flowering responses. Some of the Harada and Nitsch 
(1959) results are suggestive of a change in gibberellin levels follow- 
ing induction, but the bioassay used was relatively unspecific. A- 
more specific assay was used by Lang (1960), whose preliminary 
results show a higher gibberellin level in induced than in non- 
induced annual Hyoscyamus. That this may be a cause of flowering 
rather than simply correlated with it is indicated by the fact that 
the increase shows up soon after induction and is less pronounced 
after flowering is well under way. This sort of work is now develop- 
ing rapidly; and, as mentioned earlier about research on the 
red, far-red pigment, what is reported here may well be obsolete 
by publication. 

The role of respiratory systems has also been studied. Elliott 
and Leopold (1952), for example, following oxygen uptake in leaf 
tissues of certain SDP and LDP, concluded that respiration rate 
increased in the former and decreased in the latter with photo- 
induction, whereas rates in two daylength-indiflerent plants were 
dependent on the total light given. Whether such correlations are 



92 ■ Floral Hormones and the Induced State 

general, and what their significance might be, is unknown. The fact 
that various well-known respiratory poisons, including cyanide, 
azide, and fluoride, may inhibit the dark period induction (Naka- 
yama, 1958, on Pharbitis nil) does not afford any special insight 
into the processes involved, but indicates simply that normal 
respiration is required to support them. This is true also of ver- 
nalization, at least on the basis of the oxygen-level and sugar-feeding 
experiments mentioned in the previous chapter. 

There has been a series of investigations on the fixation of 
carbon dioxide in darkness, particularly by Kalanchoe, since photo- 
period influences its time-course and intensity in a manner sug- 
gestive of the effect on flowering. In addition other work has shown 
that exclusion of CO, during dark periods can reduce the induction 
of several SDP. These results are reviewed by Kunitake et al. (1957), 
who concluded from their own experiments with radioactive tracer 
techniques that short-day induction of Kalanchoe affected not the 
proportion of COo fixed in various compounds but only the total 
amount. This conclusion, together with the fact that even this 
change occurs relatively late in induction, affords no support for 
the suggestion of a specific significance for dark C0 2 fixation in 
the inductive process. 

The induced state in many plants has some of the character- 
istics of infection with a virus, or some other self-replicating entity. 
This is true both of photoperiodically induced Xanthium, in which 
llorigen production appears to be autocatalytic, and, in a different 
way, of vernalization in those plants in which the vernalized state 
is maintained in all cells descended from those originally treated. 
Unfortunately this stimulating hypothesis of flowering as a virus 
disease has as yet no direct evidence in its favor. Changes in the 
levels of both ribonucleic and desoxyribonucleic acids during and 
following photoinduction have been observed (Gulich, 1960, and 
bibliography therein), but all attempts to show qualitative dif- 
ferences between the nucleic acids or proteins of induced and non- 
induced plants have been unsuccessful (see Bonner and Liverman, 
1953; Bonner, 1959b). However, some indirect evidence has been 
obtained by the use of compounds believed to inhibit nucleic acid 
synthesis. Hess (1959) found that 2-thiouracil given during the 
vernalization of Streptocarpus could reduce or abolish flower 
initiation without affecting vegetative growth; 5-fluorouracil is 



The Biochemistry of Induction • 93 

reported to inhibit photoperiodic induction in Xanthium in a 
manner possibly suggestive of an effect on the synthesis or effective- 
ness of the flowering hormone (Salisbury and Bonner, 1960). But 
2-thiouracil also causes a strong inhibition of induction in another 
SDP, hemp (Cannabis sativa); careful histological observations sug- 
gest that this action and, by inference, those above are due to a 
general effect on the differentiation capacities of the meristem 
rather than to a specific effect on flowering (Heslop-Harrison, 
1960). 

A question of fundamental importance concerning photo- 
periodic induction was recently raised by R. M. Sachs on the basis 
of his and other work with LSDP (see Sachs, 1959). It has been 
widely assumed that the basic induction process in both LDP and 
SDP is alike, there being at least two grounds for this assumption. 
One is the participation of the red, far-red system in both types 
and the other is the apparent equivalence of florigen in both types, 
at least among many closely related plants. But Sachs points out 
that in the LSDP Cestrum nocturnum (night-blooming jasmine) 
long- and short-day induction appear to differ considerably. The 
product of long-day induction ■ is not translocated from the treated 
leaves; short-day induction following long-day induction, however, 
gives rise to a translocatable flowering hormone. Further, the se- 
quence of long- and short-day induction is not reversible for any 
plants requiring both— in LSDP the former must precede the latter, 
whereas in SLDP the reverse is true. Thus if one assumes that long- 
day induction in both LSDP and SLDP (as well as in simple LDP) 
controls the same step in a series of reactions, one then suspects 
that the short-day induction step in LSDP is not equivalent to that 
in SLDP. Similarly, assuming that short-day induction in both types 
(as well as in SDP) is the same, then the long-day induction in the 
two types must differ. In addition to indicating that short- and long- 
day induction may affect different processes, Sachs suggests that "we 
should be wary of the assumption that LD induction affects the 
same stage of synthesis of the floral stimulus in every LDP (the 
same doubt exists with regard to SD induction in all SDP)." The 
question will be finally answered only by a complete understanding 
of the biochemistry involved, which may take many years. The logic 
of Sachs's analysis warns that the answer will not be simple, and 
may also be different for different plants. 



94 • Floral Hormones and the Induced State 



CONCLUDING REMARKS 

An attempt at some sort of evaluation is desirable here, if only 
to avoid ending on a note of complete confusion. Some of the views 
to be expressed differ greatly from those held by other writers, who 
also differ among themselves; anyone seriously concerned with 
theoretical interpretations should consult various reviews cited 
earlier. 

The "all-or-none," qualitative character of both floral initia- 
tion and photoperiodic induction has been widely stressed (for 
example, Lang, 1952). In the writer's opinion, it is a questionable 
concept. Admittedly, there are situations in which one either sees 
or does not see a floral primordium, so that the final judgment is 
either "flowering" or "vegetative." The same could be said, how- 
ever, about the growth or nongrowth of a piece of tissue; at the 
lower limit of the technique used, one either detects growth or 
does not, yet there is no general opinion that growth is an all-or- 
none phenomenon. Bonner (1959a), accepting the photoperiodic 
response as in a sense quantitative, nevertheless goes on, "each 
bud and each plant is either reproductive or vegetative." Logically, 
this is true enough. But in developmental, morphological terms, 
one has only to consider work like that of Harder (1948) on 
Kalanctwe to reali/e that there can be a continuum between obvi- 
ously vegetative and obviously reproductive growth. 

One origin of the all-or-none view may be an overemphasis 
on flower initiation (although such studies usually involve some 
degree of development) with too little attention to the fact that 
optimum flower development often requires a continuation of the 
inducing conditions. A good illustration of this common situation 
was recently given by Zabka (1961) working with Amaranth us 
raudatits. At a certain age this is a very sensitive SDP; when older, 
it initiates flowers even under long days. Under any circumstances, 
however, inflorescence development and fruiting are strongly 
favored by short days, no matter how initiation came about. 

Another major support of the all-or-none view has been the 
fact that, in SDP lor example, flowering does not occur at day- 
lengths above the critical but does occur at lower values. This thus 
n( emed to represent a sharp, qualitative cut-oil in the curve of 



Concluding Remarks • 95 

response versus daylength, but only on the assumption that day- 
lengths above the critical had no other effect than to be noninduc- 
tive. Work mentioned in Chapter Three, however, indicates now 
that such daylengths are often positively antiinductive, not merely 
ineffective, and that this antagonistic effect is quantitatively related 
to the amount by which the noninductive daylength exceeds the 
critical. While no generalization is likely to hold for all plants, 
it is possible that the processes involved in induction proceed con- 
tinuously, and that only the ratio of the rates of, say, two or more 
of them differs under different daylengths. The critical daylength 
would then be that value at which the ratio neither promotes nor 
inhibits the train of events finally leading to flowering. 

Many of the subjects touched on in the preceding chapters, 
including the question of the degree of difference between the 
structure of vegetative and floral meristems, bear on this sort of 
problem, but cannot be enlarged upon now. The relevance of such 
theoretical considerations to more concrete questions is largely 
in the suggestion that flowering does not represent a sudden change, 
some sort of developmental "quantum-jump," but is probably under 
controls similar to those affecting vegetative growth, to the small 
degree that these are understood. 

Consider, for instance, the nature of floral stimuli. That some- 
thing moves between induced and noninduced parts of a plant, or 
between grafted plants, cannot be doubted. Movement of active 
substances from vegetative to reproductive tissue is also highly 
probable. In physiological terms, then, both florigen and anti- 
florigen appear to be valid concepts, but in the absence of extracted 
samples one can only speculate as to their nature and whether they 
are the same in all plants. In the light of the considerations above, 
it appears extremely unlikely to the writer that florigens, whether 
simple substances or as complex as a virus, are likely to be specific 
floral hormones in the sense that they are involved only in the 
processes of floral initiation and development but no others. Julius 
Sachs's concept of specific organ-forming substances has not stood 
the test of experimentation, since most vegetative systems studied 
indicate that particular aspects of development can be controlled 
by the concentrations and interactions of substances that affect many 
other processes as well. A few examples will be helpful here. 

The use of the auxin indoleacetic acid in rooting cuttings is 



96 • Floral Hormones and the Induced State 

well known; in addition, much of the rooting behavior of cuttings 
can be explained in terms of their auxin content and sensitivity. 
Yet it is also known that the same compound plays a major role 
in other developmental processes having nothing to do with root 
initiation, so that it would be grossly misleading to call it 
"rhizogen" (root-maker). That development is controlled by the 
balance of various substances common to many processes is strik- 
ingly illustrated by the work of Skoog and Tsui (1948) and Miller 
and Skoog (1953). Tobacco stem segments grown in aseptic culture 
produce roots if supplied with a particular level of auxin and 
shoots if supplied with another substance, adenine. Both com- 
pounds together cause the production of more or less disorganized 
callus tissue; but increasing the adenine again leads to shoot forma- 
tion, whereas increasing the auxin leads to root formation. Thus 
the balance of auxin and adenine controls the production of roots 
or shoots in this system. Adenine, as a component of the nucleic 
acids and many respiratory co-enzymes, is probably present in every 
living cell; the many roles of auxin have already been mentioned 
(see Audus, 1959). 

A simpler example of control by an unspecific substance was 
found by Wetmore (1953), who studied the development of young 
fern apices in aseptic culture. The first few leaves produced by 
ferns, as by many other plants, may differ considerably from the 
later ones, being characteristically "juvenile" in some way; the 
ferns in question (Todea, Adiantum) have juvenile leaves with 
few or no divisions, whereas the older leaves are deeply lobed. In 
culture, mere variation of the sucrose content of the medium 
suffices to bring about almost any degree of "juvenility" or 
"maturity" in leaf shape, with the lowest sucrose level giving the 
least lobed leaves. Thus the normal leaf progression, regarded as 
a fundamental developmental property of the meristem and one 
of considerable evolutionary significance, is susceptible to regulation 
by a substance that presumably serves merely as a general energy 
source. This result may have more than illustrative value here. 
If, as Philipson (1949) suggests, the reproductive apex simply 
represents a normal later stage in the ontogeny of the shoot, as 
does the transition from juvenile to mature foliage, then perhaps 
a local increase in carbohydrates may play a central role in 
flowering itself. 



Concluding Remarks • 97 

One further study on vegetative growth should be considered 
since it bears comparison with the quantitative yet long-lived 
induced state which seems so puzzling in Xanthium. The reader 
whose sensibilities were disturbed by "flowering as a virus disease" 
will have to make the best of another similar analogy, this time to 
the plant disease crown-gall. In many ways resembling cancer in 
animals, crown-gall is brought about by a bacterium; following 
infection, the tissues become tumorous, growing rapidly in a 
disorganized fashion, and continue to do so even when the bacteria 
are no longer present. Pieces of such bacteria-free tissue grow 
rapidly in culture on a simple mineral medium with sucrose and 
a few vitamins, whereas normal callus tissue from the same plant 
fails to grow under the same conditions. Braun (1958) has been 
able to make a whole series of tissue clones intermediate between 
typical crown-gall and typical normal tissues in their growth rate 
on the basic medium. This was done by letting the bacterial infec- 
tion proceed for different lengths of time before a heat treatment 
that stops it without harming the tissue. In order to make normal 
tissue grow as fast as fully tumorous crown-gall tissue in culture, 
one must add to the basic medium 6-furfuryl amino purine, 
guanylic and cytidylic acids, asparagine, glutamine, inositol, and 
naphthaleneacetic acid. If the tissue has been exposed to infection 
for a short time, the first compound may be omitted; if it has been 
exposed for a longer time, the first four may be omitted, without 
reducing the rate below that of the fully tumorous tissue. 

Each strain of tissue maintains its particular nutritional re- 
quirements in culture and does not revert to normal. Braun con- 
cludes that "a series of quite distinct, but well-defined, growth- 
substance-synthesizing systems becomes progressively activated" 
during the crown-gall induction. In short, a quantitative gradation 
exists as a result of several qualitative changes in metabolism. 
Perhaps photoperiodic induction in some plants is a process of this 
kind, with many intermediate stages, and not a unitary process 
at all. 

With such work as background one might envision florigen 
as either a single substance, or a combination of substances, 
normally occurring in many plant cells, but frequently present 
in insufficient quantities or improper balance for the meristem 
to proceed to reproductive development. If production in another 



98 • Floral Hormones and the Induced State 

part of the plant, the leaf, is susceptible to modification by day- 
length, there will be evidence of photoperiodically induced, trans- 
locatable floral stimuli or inhibitors. When such production is not 
under photoperiodic control, the stimuli or inhibitors may still 
be demonstrable. There is no a priori reason to assume that these 
are the same for all plants simply because they appear to be so 
in certain closely related forms. (They do not appear to be so in 
all: see Zeevaart, 1958.) On the other hand, work with the gib- 
berellins indicates that the same compound can cause flowering 
in many unrelated LDP, although gibberellins themselves cannot 
be florigen, as will be indicated in the next chapter. 

The fact that floral stimuli to the present have proved non- 
extractable, and are transferable only by grafting, has been used 
as supporting evidence for the "virus" concept (see Bonner, 1959b) 
in spite of the fact that many viruses are easily extracted and 
transmitted by other means. It is at least as likely that the com- 
pounds involved are simply unstable under most extraction tech- 
niques. Still another possibility is precisely that florigen activity 
is either due to a particular balance of substances or, as suggested 
by Went (1959), is the reflection "of rhythmic concentration 
changes" of one or more substances. In either case, extraction of 
the right combination would prove extremely difficult, and move- 
ment through a nonliving gap might disrupt the relationships 
involved even though the substances themselves were stable. 

The reader may well protest that the intent of this section, 
"to avoid ending on a note of complete confusion," has been badly 
betrayed. In answer, the entire point here is that there is no con- 
fusion, only ignorance. There are undoubtedly many growth- 
regulating substances and systems of which we know nothing as yet, 
and which will change present attitudes as much as work with the 
red, far-red system or the gibberellins is changing those of the past 
decades. Therefore a comprehensive statement on the subject of 
this chapter is not only impossible but undesirable, since it would 
have to assume that all parts of the puzzle are now in hand and 
simply need putting together. All of the concepts in the literature 
are valuable to the extent that they are useful as working 
hypotheses, but they should not be mistaken lor anything else. 
What we need is more of the missing pieces, wherever or however 
thev mav be found. 



► 
► 
► 



chapter six t chemical Control 

of Flowering 



Attempts to bring about or prevent flowering by the applica- 
tion of chemicals are carried on for both practical and theoretical 
reasons. The former are self-evident, the latter hardly less so. As 
already indicated, studies on the mechanism of induction have 
included work with various metabolic inhibitors, which will not 
be considered further here. More attention has been paid to the 
effects of naturally occurring compounds and of other substances 
that modify plant growth; variations in the supply of various 
minerals have also been studied with respect to flowering. 

A major motive of this kind of work has been the hope of 
discovering compounds, either naturally occurring or synthetic, 
with florigen activity. Although there have been reports of success 
from time to time, none of these has as yet proved valid. Either 
the work has been unrepeatable or the substance in question has 
not fulfilled the criteria for florigen. Drawing on the previous 
chapter, the minimal requirement for such activity is the ability to 
bring about flowering both in LDP^ under short days and in SDP 
under long days, as well as in cold-requiring but un vernalized 
plants. In addition, if the substance is to be considered a true 
(naturally occurring) florigen, it should of course be produced 
only under inductive conditions. It is well to keep these criteria 
in mind, since the effects of the first class of compounds to be con- 
sidered are dramatic enough to be misleading in this regard. 

99 



100 • Chemical Control of Flowering 



THE GIBBERELLINS 



The single most striking property of the gibberellins, besides 
the effects on flowering to be discussed, is their ability to cause 
greatly accelerated growth in intact plants. This is evident mainly 
in the stem, but occurs also in other parts and is especially obvious 
in certain "dwarf" varieties. No other group of compounds, includ- 
ing the auxins, is known to have such effects on a wide variety of 
intact plants. Gibberellins also act on many of the same phenomena 
affected by red and far-red light. Such action is not consistently 
in one direction— in some cases, such as seed germination, gibberel- 
lins appear to mimic the effect of red, but in others (for example, 
stem elongation) they act in the same direction as far-red. It has 
thus been suggested that gibberellins may be involved in the action 
of the red, far-red system, but none of the specific hypotheses pro- 
posed is as yet sufficiently grounded to be considered here. 

Several gibberellins have been isolated from higher plants, but 
the group was originally discovered as products of a fungus 
(Gibberella fujikuroi) causing a rice disease characterized by 
excessive stem elongation. They are complex compounds that can 
be regarded as derivatives of the hydrocarbon fluorene with lactone, 
hydroxyl, and other substituents. The detailed structures of some 
of them, notably gibberellin A 3 (gibberellic acid), are fairly well 
established. Much of the work to be discussed has been done with 
gibberellic acid, but other gibberellins have been studied as well, 
and the general term "gibberellin" will often be used. Research on 
the gibberellins has been pursued for several decades in Japan, 
but became known outside that country only relatively recently. 
The first generally available review, by Stowe and Yamaki in 1957, 
has since been followed by others, and all should be consulted for 
a thorough knowledge of this rapidly developing topic (Brian, 
1959; Phinney and West, 1960; Stowe and Yamaki, 1960; Wittwer 
and Bukovac, 1958). For an excellent discussion of gibberellin and 
flowering, see Lang and Reinhard (1961). 

The first thorough publication on gibberellin and flowering 
was that of Lang (1957), showing that a few drops of a dilute solu- 
tion (chiefly gibberellic acid) given repeatedly to the growing point 
or leaves brought about flowering of unvernalized biennial 



The Gibberellins • 101 

Hyoscyamus, carrot (Daucus carota), and several other biennials, 
all under long-day conditions (see Fig. 6-1). Several LDP kept on 
short days, including annual Hyoscyamus, Samolus pannftorus, and 
Silene armeria, also flowered in response to such treatment. No 
promotion of flowering occurred in the SDP Xanthium and Biloxi 
soybeans kept on long days. These experiments were conducted 
with gibberellins of fungal origin. Similar results on both Samolus 
and biennial Hyoscyamus were later obtained with extracts of 
wild-cucumber (Echinocystis) seeds, known to be rich higher-plant 
sources of gibberellins (Lang et ah, 1957). Evidently, then, gibberel- 
lin can substitute for the cold requirement of certain vernalizable 
plants and for the long-day requirement of certain LDP, but not 
for the short-day requirement of SDP. This general conclusion still 
appears valid, but requires expansion. 

Vernalization or long-day requirements have not been suc- 
cessfully replaced by gibberellin in all plants tested. One reason 
for this may be the known difference in activity, for a given plant, 
among the various gibberellins themselves (see Phinney and West, 
1960) well illustrated by Fig. 6-2. Possibly plants that have not 
responded so far will do so when other gibberellins are tried. In 
the "classical" experimental objects for vernalization studies, the 
winter cereals, gibberellic acid can hasten flowering in unvernalized 
seedlings, but only when applied at a particular stage; in addition 
to flowering, which is often abnormal or abortive, other changes in 
meristem development occur (Caso et al., 1960; Koller et al., 1960; 
Purvis, 1960). Further lack of exact correspondence between gib- 
berellin effects and vernalization is found in the work of Sarkar 
(1958), discussed in the next chapter, showing that optimum 
sensitivity to gibberellin or to cold treatment need not occur at 
the same stage of development. Moore and Bonde (1958) have 
observed that gibberellic acid actually devernalizes or prevents 
vernalization in a variety of Pisum, depending on whether it is 
applied after or before the cold treatment. 

It is important to realize that, at least so far, all the LDP in 
which gibberellin does replace long days are those in which flower- 
ing is associated with "bolting"— the rapid elongation of the axis 
from the almost stemless "rosette" of leaves characteristic of the 
vegetative condition. In caulescent LDP, having elongated stems 
even when vegetative, gibberellin apparently cannot bring about 



102 



Chemical Control of Flowering 




Fig. 6-1. Substitution of gibberellic acid (GA) for cold treatment in the flower- 
ing of the biennial, carrot (Daucus carota). Left to right: controls on long days only; 
long days plus GA, no cold treatment ; long days plus previous cold treatment, 
no GA. (Photograph from Lang [1957], courtesy of Dr. A. Lang, California 
Institute of Technology.) 



The Gibberellins 



103 




Fig. 6-2. Effects of various gibberellins on flowering of the LDP lettuce 
(Lactuca sativa var. Grand Rapids) on short days. From left to right: controls 
(vegetative), and gibberellins A x (flowering), A 2 , A 3 , and A 4 . Plants were 
treated with a total of 4 applications of 10 microliters of 10~ 3 M solutions at 
weekly intervals starting when 6 to 8 true leaves were present. (Photograph 
courtesy of Dr. M. J. Bukovac, Michigan State University.) 



flowering. Examples of such plants are Roman nettle (Urtica 
pilulifera) and enchanter's nightshade (Circaea lutetiana) (Lona, 
1956). Since most of the widely studied LDP are rosette plants, 
the notion that gibberellin promotes flowering in all LDP has been 
current but is probably untrue. Not even all rosette plants tested 
have proved responsive. 



104 • Chemical Control of Flowering 

Most of the other situations in which gibberellin substitutes 
for long days involve stem elongation. It causes flowering in the 
LSDP Bryophyllum crenatum grown under short days, thus satis- 
fying the long-day requirement; this again is a matter of bringing 
about bolting (Biinsow et al., 1958). Another example is its action 
on strawberry plants, in which it causes runner initiation, petiole 
elongation, and flowering inhibition. These effects are all similar 
to those of long days, and the postulated flower-inhibiting, growth- 
promoting substance produced on long days may be related to gib- 
berellin. (Thompson and Guttridge, 1959; see also Chapter Five 
in this volume.) 

The action of gibberellin on stem development may well be 
primary, with the promotion of flowering in rosette plants— both 
LDP and biennials— an indirect result. Lang (1957), for example, 
noted that although flower initiation in the rosette plants studied 
occurred with the start of bolting under normal conditions— long 
days, or vernalization followed by long days— bolting in gibberellin- 
treated plants generally preceded flower initiation. In some rosette 
plants, gibberellin causes bolting only, without flowering (Lona, 
1956; see Wittwer and Bukovac, 1958). In many rosette plants, 
normal flowering occurs only if the environmental requirements 
are partially satisfied (see Brian, 1959; Chouard, 1960). Anatomical 
investigations by Sachs, Lang, and collaborators (Sachs et al., 1959, 
1960) show that the early effect of gibberellin treatment on several 
rosette plants is the activation of the "subapical meristem," some- 
what below the growing apex. The increased cell divisions in this 
area are largely transverse; this, plus the subsequent cell elongation, 
results in rapid stem growth. Gibberellin can also completely 
reverse the effects of the complex growth-regulating compound 
Amo-1618, which causes a dwarfed or rosette habit in normally 
caulescent plants such as Chrysanthemum by inhibiting the 
activity of trie subapical meristem. While such work bears no direct 
relationship to flowering, it strengthens the view that gibberellin 
may indirectly remove some inhibition on flowering through its 
direct effect on stem growth. 

Gibberellin may either promote or inhibit later flower develop- 
ment in SDP, but is entirely unable to bring about initiation under 
noninductive conditions. In addition to the work already men- 
tioned, a striking example of its ineffectiveness occurs with the 



The Gibberellins • 105 

species Chrysanthemum morifolium. In those varieties requiring 
only cold treatment to flower, irrespective of daylength, gibberellic 
acid can cause flowering. In those that are SDP, however, it does 
not (Harada and Nitsch, 1959b). In Kalancho'e, gibberellin reduces 
the flowering of plants kept on short days, although it promotes 
vegetative growth. In spite of this, the effect is not identical with 
that of long days since it makes no difference whether or not the 
gibberellin-treated leaf lies between the short-day (induced) leaf 
and the growing point (Harder and Biinsow, 1956, 1957). 

At least two detailed studies on Xanthium have appeared. 
Both agree that gibberellic acid cannot cause flowering under long- 
day conditions; it can, however, increase the flowering response to 
a limited number of short-day cycles. Greulach and Haesloop 
(1958) obtained such results with intact plants; Lincoln and 
Hamner (1958), on the other hand, found this effect only in de- 
budded plants, and concluded that the compound acted by 
increasing the capacity of the young leaves to store the flowering 
stimulus. 

Flowering in a strain of the duckweed Lemna perpusilla may 
take place under any daylength or may require short days, depend- 
ing upon factors to be discussed later. In both situations, however, 
gibberellin can completely abolish flowering at levels that promote 
vegetative growth, although other associated morphogenetic effects 
prevent this from being considered a specific inhibition of flower^ 
ing (Hillman, 1960). 

In summary, the gibberellins have already contributed greatly 
to the study of flowering: they are the first compounds discovered 
with which many kinds of plants can be brought to flower almost 
at will. Further understanding of the way in which they fully or 
partially satisfy requirements for long-day or cold treatments, at 
least in rosette plants, will be of great value. The closeness of their 
relation to flowering, as compared with other developmental 
processes such as stem elongation, is still in doubt, and the results 
with SDP indicate that no gibberellin so far tested can be con- 
sidered a florigen. However, there is good preliminary evidence 
that native gibberellin levels in certain plants increase as a result 
of treatments leading to flowering, and such changes may be part 
of the normal mechanism involved (Chapter Five). 



106 • Chemical Control of Flowering 



AUXINS. AUXIN ANTAGONISTS, AND OTHER 
GROWTH REGULATORS 

Since auxins were widely known long before the gibberellins, 
there has been more work on their effects on flowering. In addition 
to auxins, one must consider also the effects of auxin antagonists. 
This broad term is used here to cover any substances believed to 
act in a manner opposed to that of auxin. Such action may be 
exerted through a molecular structure sufficiently similar to that 
of an auxin to interact with the same biochemical site, yet not 
sufficiently similar to participate further in whatever system auxin 
normally acts. Such an auxin antagonist, competitive with auxin 
molecules, would be a true "antiauxin." Other auxin antagonists 
may act by interfering with native auxin synthesis, by blocking the 
transport of auxin from the site of action, or by interfering with 
the effectiveness of auxin in some other way. Finally, many other 
organic compounds effective as growth regulators— capable of modi- 
fying development in various ways— have also been tested on 
flowering. All of these topics will be considered briefly. None of 
the results so far has provided much clear information on flower- 
ing, since most of the evidence suggests that the effects obtained 
are extremely indirect. 

As noted in the preceding chapter, studies on the changes in 
native auxin levels associated with flower induction are incon- 
clusive. In considering the effects of applied auxins, one should 
bear in mind that these frequently cause all kinds of abnormalities 
in growth, depending upon the concentrations (see, for example, 
Audus, 1959; Leopold, 1955). With respect to auxin effects on 
flowering, comparison of earlier reviews (for example, Lang, 1952; 
Bonner and Liverman, 1953) with more recent ones such as 
Leopold's (1958) or the excellent critical article by Lang (1959) 
indicates a marked decline in the certainty with which any general 
statement can be made. 

There have been indications that auxin treatment promotes 
flowering in LDP and inhibits in SDP. The results of some of the 
papers on this question should illustrate the general uncertainty. 

In experiments by Liverman and Lang (1956) flower initiation 
in annual Hyoscyamns and Silene was promoted by the auxin 



Auxins, Growth Regulators ■ 107 

indoleacetic acid (IAA) under conditions in which the controls 
remained vegetative. These, however, were "threshold" conditions- 
supplementary light of intensities not quite sufficient to cause 
flowering by itself was used to extend the photoperiod beyond its 
critical value. No auxin promotions were observed under strict 
short-day conditions. Promotion of flowering in another LDP, 
Wintex barley, has been observed by Leopold and Thimann (1949). 
This effect was obtained under inductive conditions and appears to 
be simply a promotion of later inflorescence development. Note 
that in the same experiments (see Chapter Five) x-irradiation, 
which may reduce the auxin level, also increased flowering. 

In the SDP Xanthium, Bonner and Thurlow (1949) reported 
that application of the auxins IAA, naphthaleneacetic acid, or 2,4- 
dichlorophenoxyacetic acid (2,4-D) to cuttings or to leaves of intact 
plants prevented the flowering response to short days. This effect 
was opposed by the auxin antagonists 2,4-dichloroanisole and 
2,3,5-triiodobenzoic acid (TIBA). The antagonists themselves, under 
threshold conditions— night interruptions barely sufficient to keep 
the controls vegetative— caused the initiation of "flowerlike buds," 
which, however, did not develop into flowers (Bonner, 1949). 

Auxin inhibitions of flowering in Xanthium have been studied 
further by Lockhart and Hamner (1954) who showed that IAA 
increased both the magnitude and consistency of the inhibition 
caused by a second dark period following the inductive night 
(Chapter Two). Additional data on auxin inhibition in both 
Xanthium and Biloxi soybean are provided in Hamner and Nanda 
(1956). Salisbury (1955), again with Xanthium, found that auxin 
inhibited flowering only if applied before translocation of the 
"flowering stimulus" appeared to be completed— that is, before the 
end of the period during which removal of the induced leaves 
could reduce the flowering response. If applied later, it promoted 
flower development, particularly under reduced light intensities 
or in the absence of actively growing buds. Inhibitions by IAA 
applied before and during the inductive dark period have also 
been reported in the SDP Pharbitis (Nakayama, 1958), although 
earlier work showed promotions under similar conditions (Naka- 
yama and Kikuchi, 1956). 

One of the few plants in which auxins have a major effect on 
flowering is the pineapple {Ananas comosus). As noted in Chapter 



108 • Chemical Control of Flowering 

Five, one variety flowers in response to geotropic stimulation, an 
effect that has been ascribed to a change in native auxin distribu- 
tion. In addition, a number of varieties can be made to flower by 
applications of synthetic auxins such as naphthaleneacetic acid 
(NAA) or 2,4-D. IAA appears to be a native auxin in pineapple, 
and, paradoxically, it has been suggested that NAA may act in this 
situation as an auxin antagonist— an antiauxin, in fact, competing 
with the native IAA— and that flowering may result from a lowering 
of the effective level of the native auxin (Bonner and Liverman, 
1953; Gowing, 1956). Whatever the explanation, the phenomenon 
itself is easily repeatable and of considerable economic importance; 
sprays of synthetic auxins are used to schedule flowering, and hence 
fruiting, in commercial plantations (see van Overbeek, 1952; 
Leopold, 1958). 

Flowering in pineapple can be brought about also by several 
(< unpounds structurally unrelated to auxins, including /?-hydroxy- 
ethylhydrazine, acetylene, and ethylene (see Leopold, 1958). Indeed, 
pineapple is not the only plant in which ethylene can cause 
flowering. Khudairi and Hamner (1954b) found that ethylene 
chlorohydrin vapor caused flower initiation in Xantliiinn plants 
under 16-hour photoperiods. As with the auxin-antagonist results 
mentioned previously, the experiments were carried out under 
threshold conditions, with supplementary light of low intensities. 

The mechanism of ethylene action on flowering or any other 
plant process is unknown, but there is some evidence that it acts 
as an auxin antagonist, possibly reducing the native auxin content. 
If this is so, then its effects on both Xanthium and pineapple are 
in accord with the hypothesis that synthetic auxins act as anti- 
auxins for the pineapple, and the whole set of observations can be 
used to support the hypothesis that, at least under certain condi- 
tions, flowering may occur after the lowering of a superoptimal 
auxin level. However, with the bits of evidence discussed in Chapter 
Five, this hypothesis remains highly speculative. 

Auxin antagonists have provided another major difficulty in 
analyzing the auxin relationships to flower initiation. Certain com- 
pounds believed to be true antiauxins (such as 2.1-dichloro- 
phenoxyisobutyric acid or 2,4-6-trichlorophenoxyacetic acid) and 
others that may rather inhibit auxin transport (such as 2,3,5- 
triiodobenzoic acid) promote flowering in annual Hyoscyamus under 



Plant Extracts of Various Kinds • 109 

threshold conditions just as do several auxins. No convincing 
hypothesis about such results has yet been stated (see Lang, 1959). 

Many growth regulators can speed or delay flowering some- 
what under particular circumstances. These effects are usually 
minor and are also associated with equal or greater effects on 
vegetative growth. Occasionally, dramatic and at present inexpli- 
cable effects of particular compounds on particular plants are discov- 
ered, of which two examples will be cited. For further information, 
see Audus (1959) and Leopold (1958). 

Furfuryl alcohol, a compound not previously known to have 
growth-regulating activity for higher plants and not obviously 
related to known growth regulators, promotes flowering and bolting 
in the LDP Rudbeckia speciosa under short days in the same way 
as does gibberellin (Nitsch and Harada, 1958). In one of the two 
experiments reported, some of the control plants flowered as well, 
so the conditions may have been close to threshold. Effects on other 
plants are unknown. 

The compound N-metatolylphthalamic acid is one of a group 
of growth regulators that profoundly affects flowering as well as 
other processes in a number of plants. It is particularly effective in 
increasing flowering in the tomato {Ly coper sicon esculentiim), a 
daylength-indifferent plant, chiefly by increasing the number of 
flowers in each cluster. High doses may even cause the development 
of a large inflorescence at the apex, causing further vegetative 
growth to stop. Such promotions of inflorescence development 
appear to be due to temporary or permanent suppression of the 
branch that would otherwise arise beneath an inflorescence and 
compete with it, and are almost certainly not direct effects on 
flower initiation (Cordner and Hedges, 1959). 

PLANT EXTRACTS OF VARIOUS KINDS 

Many naturally occurring substances have been tested for 
possible flower-promoting activity, often as extracts of uncertain 
composition. No such work, other than that with gibberellins, has 
as yet been conspicuously successful, but it is well to consider some 
representative efforts. 

An extract of the young inflorescence of a palm, Washing- 
tonia robusta, apparently brought about flowering in Xanthinm 



110 • Chemical Control of Flowering 

under long days in experiments by Bonner and Bonner (1948). 
Unfortunately their attempts to repeat this work, with inflorescence 
extracts from the same and other species of palm, were completely 
unsuccessful, so the result remains unexplained. 

In 1951, Roberts also reported the extraction of a substance that 
induced flowering in Xanthium under long days. It appeared to be 
of a lipide nature and obtainable only from flowering, not vegeta- 
tive, individuals of a number of species including Xanthium itself. 
Although attempts in several other laboratories have failed to con- 
firm Roberts's results, a long-chain keto-alcohol with activity as an 
auxin antagonist can be prepared from certain plants by the pro- 
cedures used (see Struckmeyer and Roberts, 1955). Its florigenic 
properties, however, remain as doubtful as those of the palm extract. 
An extract with weak but significant flower-promoting activity for 
Xanthium plants in long days has recently been prepared by 
careful lyophilization of Xanthium inflorescences. Only future 
work will decide whether this result will go the way of the others 
cited, but the initial report is very encouraging (Lincoln et a\., 
1961). 

In an extensive investigation on the development of a straw- 
berry (Fragaria) variety, Sironval (1957) has reported that unsaponi- 
fiable lipide fractions from flowering plants promote flowering of 
those in the vegetative condition. In only a few experiments, how- 
ever, are the untreated controls completely vegetative, and often 
the differences between control and treated series are discouragingly 
small. The active substances in the extracts may include Vitamin E, 
which is itself active in the strawberry-plant test, and certain uni- 
dentified sterols. 

Flowering in at least one vernalizable variety of pea (Pisum 
sativum) can be promoted by first allowing the seeds to imbibe 
"diffusate" prepared from other pea seeds (Highkin, 1955). Like 
vernalization, such treatment results in flowering at a lower node 
than in the controls; in the data published, the node number to 
the first flower was about 20 in the controls to about 18 in the 
treated, but was highly significant statistically. By a "diffusate" is 
meant an extract prepared not by grinding seeds in water but 
simply by soaking them, intact, under sterile conditions for varying 
periods of time during which active substances diffuse out into the 
water. Such diffusates probably contain many metabolically impor- 



Mineral Nutrition; Major Elements • 111 

tant compounds. In the investigation cited, the effect on flowering 
was about the same whether the diffusate was made by soaking 
the seeds at 23° or at 4° C; since only the latter temperature would 
vernalize, the activity cannot be considered to represent a vernalin 
(Chapter Five). 

MINERAL NUTRITION; MAJOR ELEMENTS 

The question of the relationship between mineral nutrition 
and flowering is embodied more in practical lore, and less in experi- 
mental data, than almost any other aspect of flowering physiology. 
Because of this, relatively little can be said here. Not that such 
lore is necessarily incorrect, but it is usually uncertain and often 
extremely local. One reason is that distinctions between relatively 
specific effects on (lowering and those simply associated with changes 
in vegetative growth are usually not made, as indeed they do not 
need to be, for many practical purposes. Thus one frequently finds 
that nutritional conditions that simply favor optimal growth will 
be recommended to increase flowering and fruiting. 

Interestingly enough, one of the commonest examples of such 
practical lore is the opposite belief, that flowering may result from 
conditions causing poor vegetative growth or restraining growth 
in some way. Although this may be simply an inverse recognition 
of the fact that in many plants flowering and fruiting are associated 
with and may cause a reduction in vegetative growth (see Leopold 
et al., 1959), there may be more to it. The clearest recent study 
on this question has nothing to do with mineral nutrition, but 
tends to confirm the view that, at least in certain plants, growth 
restraint can promote flowering. Kojima and Maeda (1958) studied 
a variety of radish (Raphanus) in which flowering is hastened by 
vernalization. In unvernalized seedlings, flowering and bolting 
were promoted by several treatments that greatly impeded the 
growth of the stem apex. The most effective was to imbed the 
upper part of the seedling for several days in gypsum; another was 
to immerse the seedlings in relatively concentrated sugar solutions, 
which inhibited growth osmotically. The mechanism by which a 
growth restraint might promote flowering is unknown, but the data 
seem clear and suggest that such notions are better tested than 
dismissed. 



112 • Chemical Control of Flowering 

The suggestion that nitrogen nutrition plays an important role 
in the control of flowering and fruiting in a manner related to the 
considerations above was strongly supported, although not origi- 
nated, by Kraus and Kraybill in 1918 (see Kraus, 1925). They 
concluded that fruitfulness in the tomato plant depended on the 
ratio of carbohydrate to nitrogen— the C/N ratio. Under a given 
light intensity (to supply the carbohydrates) and at a given tem- 
perature (which would govern the rate at which they are metab- 
olized), the C/N ratio can obviously be controlled by controlling 
the nitrogen supply. In Kraus and Kraybill's experiments, a 
moderate ratio was favorable to flowering and fruiting, whereas 
a low ratio (high nitrogen) favored luxuriant vegetative growth 
but little reproductive development. This conclusion in generalized 
form was for a while inflated out of all proportion to the data 
supporting it, which appear to have been valid largely for the 
particular conditions used. However, one should note in fairness 
that Kraus and Kraybill were chiefly interested in later flower 
development and fruiting, not in flower initiation. 

A more recent study by Wittwer and Teubner (1957), also on 
tomato, does not support the notion that high nitrogen favors 
vegetative growth at the expense of flowering. On the contrary, in 
solution culture the highest nitrogen level used gave the best 
flowering even under optimal temperature conditions. With respect 
to photoperiodic plants, El Hinnawy (1956) found that high nitro- 
gen promoted earlier flowering in Perilla and Kalancho'e (both 
SDP) under inductive conditions, slowed it in mustard (Brassica) 
and dill, and had no effect on spinach (all three LDP) under induc- 
tive conditions. It had no effect on the photoperiodic response as 
such, and he concluded that the effects of nitrogen and other major 
element changes were highly indirect. 

Eguchi et al. (1958) have studied the responses of some photo- 
periodic, vernalizable, or daylength-indifferent plants to levels of 
nitrogen and phosphate nutrition. They concluded that in the first 
two types the time of flowering, both chronologically and develop- 
mentally, was almost unaffected. In the daylength-indifferent plants, 
however, which included tomato, pepper (Capsicum), and eggplant 
(Solanum), there was a much greater effect. In a tomato variety, 
for example, flowering was earliest at the highest levels of nitrogen 
and phosphate used, with the first flower at node 8 or 9. Reducing 



Heavy Metals and Flowering • 113 

either nitrogen or phosphate to the lowest level used delayed 
flowering to node 12 or 13 at the earliest. The authors proposed 
the interesting generalization that flowering in many tropical 
daylength-indifferent plants is far more dependent upon nutrition 
than it is in photoperiodic or vernalizable plants in which the 
environmental requirements have been satisfied. In this connection, 
note that Gott et al. (1955) found that a low nitrogen level delayed 
flowering in unvernalized or partially vernalized winter rye but 
hardly affected vernalized plants. 

Although the literature on nutrition and flowering is more 
extensive than that presented here, these examples serve to indicate 
that, at least at present, there is no good evidence for a close rela- 
tionship between a particular major element and flower initiation 
in most plants. 

HEAVY METALS AND FLOWERING 

There is some indication that iron nutrition may be more 
critically involved in photoperiodic induction. In a preliminary 
survey to see whether any of a large number of different mineral 
deficiencies would reduce the capacity of Xanthium to respond to 
short-day treatment, Smith et al. (1957) noted that iron, and possi- 
bly boron and magnesium deficiencies, had some effect. In further 
experiments they found that plants suffering from iron-deficiency 
symptoms failed to flower or flowered abnormally even when trans- 
ferred to a high-iron medium after photoinduction. Such results 
are suggestive, although the inhibition of vegetative growth as well 
as the response to short-day leave them somewhat equivocal. Any 
special significance for iron in flower initiation has been questioned 
by Shibata (1959) in a brief investigation on rice (Oryza sativa). 

A more clear-cut result was obtained by the writer (Hillman, 
1961a), using a clone of the duckweed Lemna perpusilla growing 
in a well-chelated medium (see below). The plants were pretreated 
by growing them in media with various levels of iron for several 
days, given one (inductive) long night, and then all returned to a 
high-iron medium. Under these conditions, the flowering response 
to the single long night was essentially abolished by pretreatment 
with a level of iron not low enough to affect vegetative growth. 
In other words, the iron requirement for induction appeared to 



114 • Chemical Control of Flowering 

be higher than that for vegetative growth only. Whether this might 
be true also for other micronutrient elements in this plant, or 
whether it truly indicates a special role of iron in photoperiodic 
induction, is not yet clear. Yoshimura (1943) has reported promo- 




Fig. 6-3. Duckweeds (Lemna) as experimental organisms for the study of 
flowering under highly controlled conditions. (^1) An aseptic culture of L. 
perpusilla. (B) A group of L. gibba, showing anthers. (Photographs by Dr. J. H. 
Miller and Yale University Photographic Services.) 

tion of flowering in another duckweed, Spirodela, by molybdenum 
deficiency. For a review of other early reports on duckweed flower- 
ing, see Hillman (1961a). 

The writer has pursued evidence of important metal effects 
in photoperiodism originating in observations on the effects of 
chelating agents on the flowering of two species of Lemna (see 
Fig. 6-3). Chelating agents are compounds that form particularly 
stable complexes with many metal ions and thus affect their chem- 



Heavy Metals and Flowering ■ 115 

ical reactivity. Many compounds of biological importance (for 
example, amino acids) are chelating agents in addition to their 
other properties. Especially effective chelating agents, such as 
ethylenediaminetetraacetic acid (EDTA, "versene"), bring about 
considerable changes in plant metabolism, probably by affecting 
processes involving metals. 

When EDTA is added in sufficient quantity to a mineral 
medium supporting good growth, it profoundly modifies the photo- 
periodic responses of a clone of Lemna perpusilla and a clone of 
Lemna gibba. Lemna perpusilla, previously daylength-indifferent, 
now responds as a typical SDP; Lemna gibba, unable to flower 
under any photoperiod on the first medium, now flowers rapidly 
as an LDP in the medium with EDTA. The effects of EDTA on 
vegetative growth are quite minor and not related to photoperiod. 
It seems obvious that the major effect of EDTA here is not directly 
on flowering itself but on flowering through its sensitivity to photo- 
period, since in Lemna perpusilla EDTA permits a long-day inhibi- 
tion of flowering whereas in Lemna gibba it permits a long-day 
promotion. These effects are related to a report by Kandeler (1955) 
—the first in which the control of flowering in any duckweed was 
observed— that Lemna gibba flowered under long photoperiods 
given with fluorescent light only in "aged" medium, in which the 
plants had grown for some time. It now appears that EDTA substi- 
tutes for this "aged" medium effect and vice versa. Since, at least in. 
Lemna perpusilla, chelating agents other than EDTA are effective, 
the action is not specific to EDTA alone and is probably a conse- 
quence of chelation (Hillman, 1959a, 1959b, 1961a, 1961b). 

It has recently appeared that in more purified media, these 
two plants show their photoperiodic responses even in the absence 
of EDTA. Under these conditions, very low levels of cupric or 
mercuric ions promote Lemna perpusilla flowering in long days, 
have no effect in short days, and inhibit Lemna gibba flowering 
in long days. Thus these ions, by the reasoning above, appear to be 
relatively specific inhibitors of the response to long days; the action 
of the chelating agents observed earlier probably represents preven- 
tion of the effects of contaminants (undoubtedly copper) in the 
medium. Such results may provide new tools for the analysis of 
photoperiodism; however, much further work will be required to 
explore such a complex and sensitive experimental system (Hillman, 
1961c). 



► 



chapter seven t ^g e a nd Flowering 



In the growth of most plants from seed, an appreciable period 
elapses before flowers are initiated even under conditions that 
would cause rapid flowering in more mature individuals. This is 
often expressed by saying that in order to flower a plant must 
reach the stage of readiness or "ripeness-to-flower," the latter being 
a rendering of Klebs's (1918) term Bliihreife. Put so abstractly the 
concept seems merely circular, but it is not unique in this regard. 
Dormancy often seems to be defined as a state in which growth 
does not take place under conditions favorable in all respects— 
except for that condition required to break "dormancy." However, 
this merely illustrates the limitation of abstract statements since 
the questions involved in both dormancy and ripeness-to-flower are 

quite real. 

The relationship of age or developmental stage to the ability 
to flower is not well understood, and differs vastly from species to 
species. The requirement for a considerable amount of vegetative 
growth is particularly marked in woody plants; many trees do not 
flower until at least ten years of age, and some "juvenile" phases 
are characterized not only by inability to flower but also by growth 
habits and leaf shapes differing from those of the adult phase (see 
Sax, 1958a). In herbaceous species, similar events lasting a much 
shorter time are often observed. 

Since plants differ so greatly in the speed with which they 
become ripe-to-flower, and probably in the mechanism involved, 
the concept itself has little use except to call attention to a whole 
range of phenomena. In spite of this, an even more general concept, 

116 



Age and Flowering in Herbaceous Plants • 117 

that of "phasic development," has been associated with some studies. 
It views plant growth as a succession of recognizable phases, each 
requiring a specific set of environmental conditions for its fulfill- 
ment, and none of which can be bypassed (see Murneek and Whyte, 
1948). A concept as unspecific as this is hardly susceptible either to 
proof or disproof once it is admitted that the characteristics of 
the phases will not be the same in all plants. Hence, it will not be 
considered further. Instead, some relationships of age and flowering 
in some of the familiar herbaceous plants will be discussed first, 
and will be followed by a consideration of the problems posed by 
flowering in woody species. 

AGE AND FLOWERING IN HERBACEOUS PLANTS 

Certain plants produce a characteristic minimum leaf number 
before flower primordia are initiated. In the best-known examples, 
spring and vernalized winter rye, a minimum of seven leaves 
appear before the inflorescences no matter what the conditions 
used, at least in most of the older research with these plants. A 
partial explanation is that four leaf primordia are already present 
in the mature embryo, and so precede the inflorescence. However, 
three more are apparently differentiated during or after germina- 
tion. Although it is possible to reduce the "minimum leaf number" 
below 6 by the use of continuous light from germination, or by. 
starting with prematurely harvested embryos that have differen- 
tiated fewer leaf primordia, apparently at least one or two leaves 
in addition to those in the embryo still intervene before flower 
initiation (Gott et al., 1955). 

Holdsworth (1956) has considered the concept of minimum 
leaf number extensively, and questions its general usefulness. The 
number in Xanthium— 8— appears to be accounted for by those 
leaves present in the embryo plus those developing before induc- 
tion and the translocation of the floral stimulus have taken place. 
In certain other plants the number is higher than can be accounted 
for in such ways. However, both types of observation may depend 
on differences in the sensitivity of successive leaves to photoperiodic 
induction, which will be considered below. Other factors affecting 
minimum leaf number may be the movement of flower-inhibiting 
or promoting substances from the cotyledons, as observed, for 



118 • Age and Flowering 

example, in grafting experiments by Paton and Barber (1955) and 
Haupt (1958) on early and late (lowering in peas (see Chapter Five). 
There are also plants in which the flower primordia, following a 
certain number of leaves, are already present in the seed (see 
Naylor, 1958). 

One should attempt to distinguish between minimum leaf 
number, as in the case above, representing a condition in which 
a certain amount of development takes place before and during 
the treatments leading to flowering, and ripeness-to-flower under- 
stood as a condition before which a given treatment is completely 
ineffective in promoting flowering. In practice, such distinctions 
may be difficult to make. If the treatment in question is vernaliza- 
tion, however, it is clear that the difference between winter annuals 
and biennials (Chapter Five) simply reflects the fact that the latter 
are not responsive until they have attained a considerable size. In 
this sense, some winter annuals are ripe-to-flower as germinating 
seeds. The reason for the size requirement in biennials is not 
known, and has been ascribed to many factors, including the 
amount of food reserves. De Zeeuw and Leopold (1955) found that 
the age at which seedlings of Brussels sprouts, Brassica oleracea 
gemmifera, a biennial, could be vernalized was decreased if the 
synthetic auxin NAA was given together with the cold treatment; 
the effect was not great, so that evidence that the size requirement 
in biennials is related to auxin content is scanty. 

A series of experiments by Sarkar (1958) on a winter-annual 
strain of the crucifer Arabidopsis thaliana illustrates not only the 
complexity of possible relationships between development and 
receptivity to cold treatment, but also the fact that the cold treat- 
ment itself may have a multiple action, as evidenced by the ability 
of gibberellin to replace it at some stages but not at others. The 
strain of Arabidopsis in question is easily vernalizable in the seed, 
during germination, or in the mature rosette stage. Young rosettes 
are less easily vernalized. Gibberellic acid, however, is most effective 
on the young rosettes, less so on the older, and totally ineffective 
on seeds. 

Many studies bearing on ripeness-to-flower deal with respon- 
siveness to photoperiod. In certain plants, of course, previous 
vernalization is a major factor aflecting such responsiveness and 
thus also ripeness-to-llower in this sense. Since this relationship was 



Age and Flowering in Herbaceous Plants • 119 

discussed earlier, the discussion below will be concerned primarily 
with other prerequisites for the photoperiodic control of flowering. 

Klebs (1918) originated this field of inquiry by observing that 
Sempervivum funkii did not show a flowering response to long 
days until it had been growing for some time, and he concluded 
that the best conditions to bring about this Bliihreife state were 
those involving a high degree of carbon dioxide assimilation and 
a relatively meager mineral nutrition. This, as well as other obser- 
vations by Klebs, was in part the origin of investigations on the 
C/N ratio (Chapter Six). It seems clear now that for most photo- 
periodic plants, probably including Sempervivum, gross nutrition 
is less important than the morphological stage of development 
attained. 

Certain plants do not respond to an inductive photoperiod 
until they have produced true leaves, but there are some in which 
the cotyledons themselves are sensitive. These include the SDP 
Pharbitis (Nakayama, 1958) and Chenopodium rubrum, some 
strains of which may flower as tiny seedlings barely emerged from 
the seed coat (Cumming, 1959; see illustration facing page 1). The 
SDP Xanthium and Perilla, on the other hand, are of the former 
type. The development of at least one true leaf is necessary before 
Xanthium can respond to short days. Jennings and Zuck (1954), 
testing the possibility that this might be due to insufficient area of 
the expanded cotyledons, found that an area of true leaf consid : 
erably smaller than the total cotyledon area could induce flowering. 

In Perilla, the sensitivity to induction Of successive pairs of 
leaves increases from the second to at least the fifth pair, with the 
first and second being almost insensitive. This again does not appear 
to be a matter of leaf area or even of plant size, but represents 
a developmental difference in the leaves. For example, if equal areas 
(see Fig. 5-3, p. 86) are cut from second and fifth leaves, grafted 
onto other plants in long day, and then induced with short-day 
treatments so that they will function as donors, the tissue from the 
fifth leaves is by far more effectived However, the fact that intact 
older plants respond more quickly than younger plants is also due 
to greater total leaf area (Zeevaart, 1958). In the grass Lolium 
temulentum, the increasing sensitivity of the entire plant to photo- 
period is attributable entirely to the increasing sensitivity of suc- 
cessively produced leaves. When only several lower leaves are left 



120 • Age and Flowering 

on a mature plant, as many long days are required to induce as are 
required by a much younger plant. However, a small portion of 
the area of one later-produced leaf is sufficient for induction by 
one long day (Evans, 1960). 

The change in sensitivity of successive leaves, as in Perilla, 
may be a function of meristem aging. It is also possible that as the 
meristem itself ages, it becomes more sensitive to the floral stimulus 
from other parts of the plant; the general question of meristem 
aging and flowering may also be important for flowering in woody 
plants (see below) but little is known about it. 

At least in Xanthium , the photoperiodic sensitivity of each leaf 
varies during its development. Khudairi and Hamner (1954a) 
studied the flowering responses of plants in which single leaves of 
different ages and at different stages in expansion were present. 
Within a wide range of absolute sizes, leaves were most sensitive 
when they had expanded to about half their final size, being much 
less so either when very young or when mature. Undoubtedly 
similar relationships between individual leaf development and 
photoperiodic sensitivity obtain in other plants as well. 

It is not always true that photoperiodic sensitivity increases 
with plant age or development. The opposite situation has already 
been noted in sunflower (Chapter Two). It is an SDP when young 
but later becomes daylength-indifferent (Dyer et al., 1959); stated 
otherwise, long days inhibit flowering in the young plant but not 
in the older. On the other hand, this can still be regarded as an 
increased sensitivity in the sense that a shorter nightlength is induc- 
tive in older plants. The mechanism is unknown. 

FLOWERING IN WOODY PLANTS 

It is in the woody plants that the problem of ripeness-to-flower 
is most obvious. The two major environmental factors affecting 
flowering in herbaceous plants— photoperiod and temperature— also 
of course affect woody plants, and by similar mechanisms; however, 
the dominant factor here, that of maturity, appears to be internal. 
The lack of flowering in many trees until they have attained a 
given age is of great practical importance because it affects both 
food production and breeding programs, and also makes experi- 
ments slow and costly. Hence the effectiveness of some of the pro- 



Flowering in Woody Plants • 121 

cedures traditionally used in the hope of hastening flowering has 
only recently been confirmed in controlled experiments, and the 
value of some others is still uncertain. 

Further problems are presented by the fact that most trees and 
shrubs, at least in the temperate zone, are probably indirect- 
flowering plants unlike most herbs studied, so that conditions 
required for flower initiation may differ greatly from those favoring 
flower development, and the internal changes involved may differ 
as well. As an extreme example, the difficulties faced by the forest 
geneticist are evident in the fact that not only must most species 
of pine (Piniis) grow for some five or more years before flower 
initiation is possible, but then two and a half years are required 
to obtain seed. Flower primordia are formed in the spring of one 
year but do not develop further until the spring of the next, when 
pollination takes place. Then in the succeeding spring and summer 
cone elongation and actual fertilization finally occur, following 
which the seeds mature in the fall (see Stanley, 1958). Clearly, any 
way of reducing the age required for flowering and speeding up the 
reproductive cycle itself would be extremely helpful. 

A particular group of woody plants, the bamboos (Tribe 
Bambuseae of the grass family), provides the most striking exam- 
ples of long-lived monocarpic plants (Chapter One), which flower 
once and then die. As summarized by Arber (1934), there is abun- 
dant evidence that a bamboo will spend 5 to 50 years, the number 
being characteristic of the species, in vigorous vegetative growth. 
It then flowers, sets seed, and dies within a short time. Usually all 
plants of the species within a large area will flower at the same 
time, regardless of injury or even of destruction of all portions 
above ground by cutting or fire. Thus size alone does not appear 
to be a factor. Individuals transplanted to, say, the Kew Botanical 
Gardens still flower the same year as their fellows in the tropics, 
making it seem unlikely that periodic environmental changes such 
as droughts are the cause of such behavior— although this has been 
suggested. Possibly bamboos may provide instances of very long- 
term endogenous rhythms, but it will take a long-lived plant 
physiologist or a well-endowed research institute to find out. 
Certainly in no group of plants is the relation between age and 
flowering more evident and less understood. 

Most environmental factors affecting flowering in trees have 



122 • Age and Flowering 

been studied relatively little because of the obvious technical 
difficulties. Increased soil fertility may be of value (for experiments 
that deal with this possibility using pine, see Hoekstra and Mergen, 
1957). Fraser (1958) has correlated meteorological data with anatom- 
ical studies of spruce (Picea), and concluded that earlier reports 
that flower initiation is favored by high summer temperatures are 
probably correct. Reference to the discussions in the papers cited 
will indicate that, unfortunately, tree physiologists are generally 
uncertain about the importance of any particular soil or climatic 
factor. 

Photoperiodism affects largely the vegetative development of 
woody plants rather than flowering, at least according to present 
evidence. The rate of growth, its cessation and renewal, branching 
habit, leaf shape, and resistance to cold are among the characteris- 
tics affected (see Wareing, 1956; Nitsch, 1957). Such characteristics 
are often of great ecological significance, and their sensitivity to 
photoperiod frequently differs considerably within offspring of the 
same species gathered over a wide geographical area (see Vaartaja, 
1959). In certain crop trees, such as the SDP Cofjea arabico (coffee), 
flowering also is photoperiodically controlled (Piringer and Borth- 
wick, 1955), whereas the ornamental shrub Cestrum nocturnum has 
been previously discussed as an LSDP. 

Most work with economically important trees, however, sug- 
gests a minor role or none at all for photoperiodism in flower 
initiation. This is almost certainly true for pines (Mirov, 1956; see 
Mirov and Stanley, 1959), for peaches (Prunus), and probably for 
apples (Mains) (Piringer and Downs, 1959). One should note an 
indication of control by light in the last-named tree, however. In 
the paper cited, the variety used failed to flower at all on 16-hour 
photoperiods of which 8 hours were under fluorescent light, but 
flowered well if incandescent light was used. For such reasons, as 
well as because of the relatively few experiments done so far, it is 
impossible to guess whether or not photoperiodically controlled 
flowering is truly less common among woody plants than it appears 
to be among herbs. Certainly, however, even when photoperiodism 
is a direct factor, that of size or maturity is still of overriding 
interest both practically and theoretically. 

Because of effects on vegetative growth, photoperiodic treat- 
ment can indirectly hasten flowering. A species of birch, lietula 



Flowering in Woody Plants • 123 

verrucosa, normally requiring at least 5 years from seed in order 
to flower, was used by Longman and Wareing (1959) in a study on 
whether size was the major factor involved or whether a certain 
number of developmental seasonal "cycles" were necessary before 
flowering could take place. Some seedlings were kept constantly 
under long days or continuous light, in which vegetative growth 
continues rapidly. Others were allowed to make about 30 centi- 
meters of growth under such conditions, given short days to induce 
dormancy, and then kept in the cold for six weeks, following which 
they were returned to long days and the cycle repeated. There was 
also a control series under natural conditions. Fifty percent of the 
trees in the constant long-day conditions flowered within the first 
year, when 2 to 3 meters high, whereas none of the (smaller) control 
or "cycle" series flowered within two years. Hence in this tree at 
least, attainment of a certain size is crucial to flowering and can be 
speeded by constant long photoperiods, although the authors noted 
that the plants so treated were abnormally spindly. 

Although flowering may thus be hastened by speeding devel- 
opment to the requisite size, most of the traditional methods used 
by horticulturists involve operations or mutilations of some kind 
and bring about an inhibition of vegetative growth. Of these 
methods, one of the most widely favored is girdling— the removal 
of a ring of bark, including phloem, on an entire tree or on a 
branch. The immediate result is to prevent the translocation of 
photosynthate out of the girdled top or branch, so that materials, 
accumulate above the girdle. Naturally, this can thus result in the 
death by starvation of the root system if it is not permitted to heal 
over within a relatively short time. Girdling is often effective in 
causing flowering of plants too young to flower otherwise in species, 
as unrelated as Citrus (Furr et al., 1947), Pin us (Hoekstra and 
Mergen, 1957), and apples (Sax, 1957, 1958b). Related to girdling 
as a means of blocking phloem translocation is the technique of 
bark inversion, in which a ring ol, bark is cut out and regrafted 
in place upside down. Such procedures must be used before the 
period in which flower initiation would normally be expected to 
take place. In apples, bark inversion in June will affect flowering 
the following spring, even bringing it about in 2- or 3-year-old 
seedlings, whereas the same operation in late summer is ineffective 
(Sax, 1957, 1958b). 



124 • Age and Flowering 

With many fruit trees, grafting young scions onto dwarfing 
stocks is another method whereby both a promotion of flowering 
and an inhibition of growth are obtained. The stocks are usually 
varieties of the same or a closely related species, and may be used 
either as rootstocks or interstocks. The latter method involves first 
grafting the dwarfing stock onto a standard seedling rootstock and 
later grafting the variety to be dwarfed onto the developed dwarfing 
tissue, so that the latter is interposed between root and crown. 
The mechanism by which such procedures cause early flowering 
is not known, but may in some cases be related to the reduction 
of phloem transport out of the scions and thus analogous to 
girdling. However, the interactions between stock and scion in such 
grafts are often highly specific, and not all grafts that reduce growth 
or transport promote flowering. In addition, not all grafts that 
cause early flowering and dwarfing appear to involve inhibited 
phloem transport (Sax, 1958b). 

Another traditional method of handling fruit trees, the espalier 
technique, in which branches are bent horizontally or downward 
out of their normal direction, suggests that orientation with respect 
to gravity may affect flower initiation. This supposition was directly 
tested with young plants of several kinds of fruit trees by Wareing 
and Nasr (1958), who found marked effects on apples. Nineteen 
young shoots held in a horizontal position initiated a total of 116 
flower buds in contrast to a control series initiating 5. Smaller but 
similar effects were observed in cherries (Primus). Similar results 
have also been obtained by Longman and Wareing (1958) on young 
Japanese larch (Larix) trees. These are all, of course, reminiscent 
of results with pineapple and soybeans that may involve a changed 
auxin distribution, and it has also been suggested that the flower- 
promoting effects of bark inversion may be due to effects on auxin 
distribution, which then affect phloem transport (Sax, 1958b). 

As repeatedly noted, most of the methods described above 
have in common either a demonstrated or possible effect of causing 
the accumulation of photosynthate near the growing points affected. 
The promotion of flower initiation in some trees by the early 
removal of fruits might also be attributed to an increase in avail- 
able carbohydrates (for experiments of this kind dealing with Citrus, 
see Furr and Armstrong, 1956). The general hypothesis that ma- 
turity, and hence flowering, in many trees depends on a high level 



Flowering in Woody Plants • 125 

of carbohydrates is by no means unequivocally supported by the 
evidence at present, but it is attractive in view of Wetmore's (1953) 
observations, discussed in Chapter Five, that juvenility and maturity 
in fern leaf forms, and hence in the apex producing them, are 
clearly correlated to sucrose supply. On the other hand, more 
specific mechanisms of a hormonal nature may be involved in the 
flowering of trees. 

In view of the work with herbaceous plants leading to the 
florigen hypothesis, it is surprising how few experiments have been 
published on the flowering responses of young scions after grafting 
to mature, flowering plants. Sax (1958a) indicates that this tech- 
nique is common among tree breeders, but that there is no conclu- 
sive evidence for its effectiveness. Furr et al. (1947) found it com- 
pletely ineffective in Citrus. In this connection, results of the 
opposite kind of graft are also of interest. Freely flowering branches 
from mature trees have been grafted on young stocks in order to 
facilitate seed collection. Although Huber (1952) reports this tech- 
nique as successful in poplar (Populas), there are cases in which 
mature scions on young stocks revert to a nonflowering condition 
after several years (see Fraser, 1958). Whether this reflects an insuffi- 
cient supply of flower-promoting factors (florigen, carbohydrates) 
from stock to scion, or the movement of inhibitors, or some other 
relationship, is not known. 

The entire problem of juvenility is obviously closely related 
to the subject matter of this chapter. It is particularly relevant 
with regard to woody plants, but also probably important in herbs. 
This problem has attracted relatively little attention in recent 
years, but the interested reader should consult Sinnott (1960) for 
a consideration of the literature. One striking if somewhat atypical 
example, related to flowering, is provided by ivy (Hedera). The 
young plant is a vine, with lobed leaves and aerial roots. After 
10 or 12 years it produces branches that grow upward, bearing 
entire leaves and no aerial roots. Only these branches are capable 
of flowering. If they are cut off and rooted they grow into erect 
shrubs that may become very large and rarely if ever revert to the 
juvenile vine condition, although shoots produced from the base of 
old shrub (or arborescent) forms may be juvenile— a phenomenon 
observed also in apple and other trees with distinct juvenile forms 
(see Sax, 1958a). Recent work by Robbins (1957, 1960) has shown 



126 • Age and Flowering 

that reversion will occur after either heavy pruning or treatment 
with gibberellic acid, and also that it is possible to obtain forms 
intermediate between typically adult and typically juvenile. 
Gibberellic acid also causes the production of vegetative inflores- 
cences. However, the factors governing the attainment of the adult 
state in the first place are entirely unknown, and further work 
with this sort of organism should be valuable for an understanding 
of both flowering and differentiation in general. 



► 
► 



chapter eight t A Miscellany 



Several topics that have escaped the more systematic treatment 
in preceding chapters will be considered briefly in this one. The 
brevity does not imply that these topics are unimportant, but is 
a product of space limitations and the fact that this book, like most 
of the recent literature, is concerned with the circumstances bring- 
ing about flowering rather than with associated matters. In addition 
to the topics below, others connected with the physiology of flower- 
ing suggest themselves, notably the physiology of meiosis and of 
fertilization. These will be omitted entirely since an adequate 
consideration would require a general discussion of the physiology 
of reproduction, taking in material far beyond the scope of this 
survey. A few remarks on the future of the physiology of flowering 
conclude both chapter and book. 

ANTHESIS 

The culminating stage in flower development is the opening 
of the bud, anthesis, with which is often associated the attainment 
of the flower's characteristic color and scent. Most of the work on 
anthesis has been concerned with the precise diurnal timing often 
shown by this event. In the literature on endogenous rhythms, 
anthesis is considered as one of the many phenomena under such 
control. The effects of light and darkness on a number of plants 
support this view. 

Among the earlier studies, perhaps the most interesting are 
two papers by N. G. Ball on several plants whose flowers normally 

127 



128 • A Miscellany 

open early in the morning. For example, those of the tropical 
perennial herb, Turnera ulmifolia, open about two hours after 
dawn, then wither three or four hours later. This occurs in suc- 
cessive groups of buds even if the shoots are kept in darkness for 
several days so that they are isolated from the normal day-night 
changes. However, it is possible to prevent opening by illumination 
during the night, particularly during the second half of the night, 
and the anthesis-inhibiting effect of one such illumination lasts 
for the next three days. Air temperature and relative humidity 
changes appear to have little effect (Ball, 1933). 

Ball (1936) found similar inhibiting effects of night illumina- 
tion on morning anthesis in species of Campanula, Geranium, 
Cist us, and Ipomoea. He determined a crude action spectrum for 
this phenomenon, using filters, and found that red (6500-7000 A) 
was the most effective and blue the least effective color. With the 
advantage of twenty-five years, it is easy to interpret these results 
as representing the disturbance of a circadian rhythm originally 
"set" by the light-dark schedule through what is presumably the 
red, far-red system. However, this work was in a sense before its 
time, so the (for then) unusual effectiveness of red light attracted 
little attention. 

A paper by Arnold (1959) on Oenotliera (evening primrose) 
indicates that endogenous rhythms are also involved here, though 
relatively susceptible to modification. If the plants receive light 
from 6 a.m. to 6 p.m. the flowers open at about 6 p.m., as in nature; 
with an inverse cycle, they open in the morning. Anthesis of a bud 
that is ready occurs roughly 12 hours after a dark-to-light transition, 
which thus appears to "set" the timing mechanism, but the timer 
is easily perturbed by the length of the light period itself. On a 
schedule of 18 hours light-6 hours darkness anthesis is regularly 
later, and on 6 hours light-18 hours darkness regularly earlier, 
than would be predicted by the 12-hour rule. However, it is clear 
that there is an endogenous component to the timing since anthesis 
will not follow any arbitrary cycle of light and darkness. The cir- 
cadian periodicity of anthesis cannot be made into a 12-hour 
periodicity by schedules of 6 hours light-6 hours darkness, nor into 
a 48-hour periodicity by alternating 24-hour light and dark periods. 

According to Arnold's investigations the light-sensitive timer of 
Oenothera anthesis must be localized in the buds. In continuous 



Anthesis • 129 

darkness, anthesis occurs with circadian periodicity for several 
days, but only in those buds that had developed largely under 
normal day-night changes. Buds that develop from a young stage 
in total darkness are considerably delayed in anthesis, and finally 
open more or less at random. In addition, light must be given 
directly to the buds to reset or disturb the periodicity of anthesis— 
lighting schedules given to the leaves are ineffective. 

Other evening-blooming plants have been studied recently. 
Anthesis of the giant tropical water lily Victoria regia normally 
occurs soon after sunset (6 p.m.). It can be moved up as early as 
4 p.m. by darkening the buds with black paper for 30 minutes, but 
darkening earlier than this hour has no effect; therefore some endog- 
enous component, perhaps set by preceding illumination schedules, 
is involved in the sensitivity to darkness. Illumination during the 
night delays the opening of buds during the next days, but 
eventually they open even in continuous light (Gessner, 1960). 
The opening and odor production of the night-blooming jasmine, 
Cestrum nocturnum (an LSDP discussed earlier), show a circadian 
rhythm in constant light or darkness. In constant light, the period 
length is roughly 27 hours at 17° C; higher temperatures reduce 
it by several hours, and lower temperatures increase it (Overland, 
1960). 

Daily timing of anthesis is probably regulated in the ways 
indicated above, but much less is known about the control of 
anthesis in those indirect-flowering plants whose fully developed 
buds may remain dormant for a considerable period and then open 
in the course of a few days. Among temperate-zone plants this is 
usually the result of the breaking of dormancy by long cold exposure 
followed by periods of favorable temperatures for growth; as such, 
it resembles the breaking of other forms of dormancy by low 
temperature (see Chouard, 1960). Though this does not explain it, 
there is no need for further consideration as a separate topic here. 
Certain tropical plants, however, show the same extended bud 
dormancy, and the same explanation cannot hold for these. 

One of the few examples studied with any thoroughness is 
coffee, Coffea arabica. This is an SDP as far as flower initiation is 
concerned (Chapter Seven), but bud dormancy and anthesis appear 
to be controlled by moisture conditions. Under relatively dry con- 
ditions, rapid and uniform anthesis can be brought on by heavy 



130 • A Miscellany 

rains or irrigation— even by wetting the buds themselves. This 
suggests that the seasonal dormancy is simply due to a water deficit 
and disappears when water is supplied. But the situation is probably 
not this simple. Alvim (1960), working in a dry area where the 
water conditions on a plantation were completely controllable by 
irrigation, found that coffee plants irrigated at weekly intervals 
failed to reach anthesis over a long period of time. Others, allowed 
to remain dry for a shorter length of time and then given one good 
irrigation, responded with heavy anthesis within two weeks. It 
thus seems likely that a period of water deficit is required to break 
bud dormancy in this plant, so that anthesis is brought about by 
a thorough wetting after a dry period. Alvim suggests that this may 
be a major form of seasonal control of anthesis in tropical plants, 
a control in some respects ecologically analogous to that exerted in 
temperate-zone plants by low temperatures followed by warming. 

THE SEX EXPRESSION OF FLOWERS 

Flower primordia in a given species do not always give rise to 
identical structures, even if development is perfectly normal. 
Although probably the great majority of plants produce one kind 
of flower, with both functional stamens and functional pistils— a 
hermaphrodite or monoclinous flower— some do not. Unisexual (or 
diclinous) flowers, either staminate or pistillate, occur in many 
species. There are also intermediate conditions of various kinds. 
If staminate and pistillate flowers are borne on the same individual, 
the plant is said to be monoecious; if on separate individuals, 
dioecious. Until relatively recently, these phenomena of "sex ex- 
pression" have been studied largely from the morphological and 
genetic points of view, but they are frequently modifiable by 
environmental and chemical means as well. For a recent review of 
the genetic factors, see Westergaard (1958). A comprehensive 
review by Heslop-Harrison (1957) on the experimental modifica- 
tion of sex expression is the basis lor the general statements not 
otherwise documented in the discussion below. There is some 
controversy over the evolutionary origins of sex expression in plants 
and even over the proper terms in which to discuss it (see the 
references cited and also Heslop-Harrison, 1958). 

Consideration of the effects of light and temperature on sex 



The Sex Expression of Flowers • 131 

expression might best begin with a study by Nitsch et al. (1952) 
on a monoecious plant, the acorn squash (a variety of Cucurbita 
pepo). This plant produces one flower primordium at each node, 
and the primordia develop differently depending on their position 
in the sequence of nodes. The earliest give rise to underdeveloped 
staminate ("male") flowers; these are followed by normal staminate 
flowers that are followed in turn by normal pistillate ("female") 
flowers; interspersed among the nodes bearing the latter are nodes 
with inhibited staminate flowers. Still later, giant pistillate flowers 
occur, again interspersed with inhibited staminates; finally even 
larger pistillate flowers are produced that are parthenocarpic, pro- 
ducing fruits (but not seeds) without pollination. This trend of 
"feminization" occurs under all conditions, but the duration of 
each phase in terms of node number is easily modified. High 
temperatures and long days delay it, favoring the continued pro- 
duction of staminate flowers, whereas low temperatures and short 
days speed feminization greatly. Either daylength or temperature 
can be made the dominating factor depending on the values used. 
The control exerted is striking: for example, female flowers can 
be made to appear as early as the ninth, or as late as the hundredth 
node. 

It is not clear whether the effects referred to daylength are 
photoperiodic in the strict sense. Supplementary light of 1000 foot 
candles was used, and no low-intensity interruptions attempted. 
One observation in the paper suggests that lower intensities might 
not be as effective. In addition, some conclusions on the greater 
effectiveness of "night" than of "day" temperatures are weakened 
by the fact that the former were always given for 16 hours daily, 
the latter for only the 8 hours of daily sunlight employed, in each 
treatment, irrespective of supplementary light schedules. These 
points do not detract from the dramatic climatic effects reported, 
but the paper is chief among those usually cited as indicating 
control of sex expression by "photoperiod" and "thermoperiodicity," 
interpretations that may be overstated. 

Most other investigations with temperature, on both monoe- 
cious and dioecious plants, agree with the results described in 
showing low temperatures favoring pistillate development and 
high favoring staminate. The effects ol daylength, whether strictly 
photoperiodic or not, are more complex. Apparently the general- 



132 • A Miscellany 

ization holds that pistillate flowers represent a fuller intensity of 
flowering than staminate flowers; thus, with photoperiodic plants, 
prolonged short-day treatment favors pistillate expression relative 
to staminate in SDP, whereas long-day treatment does so in LDP. 
For example, in the LDP spinach, normally dioecious, short days 
following long-day induction cause the formation of some staminate 
flowers on plants that would normally produce only the pistillate, 
thus making the treated plants monoecious (see Heslop-Harrison, 

1957). 

The factors that affect sex expression in plants with diclinous 
flowers may affect even plants with hermaphrodite flowers in a 
similar fashion. One particularly interesting example, dealing with 
the effect of photoperiod, has recently been studied by J. and Y. 
Heslop-Harrison (1958a,b). The plant is Silene pendnla, an LDP 
in that flowering does not occur with 8 hours of daylight but is 
brought about by supplementing this to 21 hours with light of 
about 300 foot candles. Plants raised from germination on long 
days showed high male sterility, some 50 percent of the anthers 
being sterile; in addition, pistil development was excessive. Plants 
that had received some short-day exposure before being returned 
to long days, however, showed normal pistil development and 
fully fertile anthers. Hence this plant, while grossly an LDP in 
terms of mere flower initiation, is clearly an SLDP for normal 
flower development. 

Chemical control of sex expression has been studied in a 
variety of plants. The earliest clear-cut results with auxins (chiefly 
naphthaleneacetic acid) were obtained on monoecious cucurbits 
such as the cucumber, Cucttmis satimis, in which feminization is 
promoted (see, for example, Laibach and Kribben, 1950). Subse- 
quent work on other plants as well seems to bear out the 
generalization that high auxin levels favor pistillate and reduce 
staminate expression. As with other factors, such effects are not 
confined to plants with unisexual flowers. The Silene work discussed 
above also included studies of the effects of auxin application; 
these, like continual exposure to long days, caused male sterility 
and overdevelopment of the pistil. 

Other growth-regulating substances whose mechanism of action 
may be related to that of auxins also affect sex expression. Maleic 
hydrazide and 2,3,5-triiodobenzoic acid both may cause male 



The Sex Expression of Flowers • 133 

sterility and otherwise suppress anther development, but often only 
in conjunction with other strong morphogenetic effects. A feminiz- 
ing effect of carbon monoxide has been observed by J. and Y. 
Heslop-Harrison (1957) in a monoecious race of Mercurialis. This 
was accompanied by formative effects resembling those caused by 
auxins. 

Three other chemical effects should be mentioned. High 
nitrogen levels generally promote pistillate as opposed to staminate 
expression; this has been observed on monoecious species and on at 
least one hermaphrodite, the tomato. The question of whether mam- 
malian sex hormones may affect sex expression in higher plants has 
attracted surprisingly little attention. A single major investigation 
(Love and Love, 1945) with Melandrium showed highly significant 
effects in spite of high toxicity. Although similar work on a few 
other plants has found nothing of interest, the problem may still 
be worth pursuing. 

The gibberellins have so far been little studied with regard to 
these phenomena, but may prove to be of great importance. Galun 
(1959) has found that gibberellic acid, unlike auxin, causes a trend 
toward "maleness"— prolonged staminate and delayed pistillate 
expression— in the cucumber; this effect is partially counteracted 
by naphthaleneacetic acid. Moreover, certain cucumber strains that 
normally produce only pistillate flowers will produce staminate 
flowers as well following gibberellic acid treatment. Besides its 
theoretical interest, this result also holds promise for practical 
breeding work (Peterson and Anhder, 1960). 

So far, the only important hypothesis on the control of sex 
expression is that derived primarily from work with applied auxin; 
it envisages auxin level in the plant as the major controlling factor. 
Daylength, temperature, and other factors are considered to act 
through their effects on auxin level. Probably the most detailed 
statement is given by Heslop-Harrison (1957). In essence, optimum 
auxin levels for flowering are considered to be lower than those for 
vegetative growth; within the flowering range, the optimum for 
staminate expression is lower than that for pistillate expression. 
In a sense this hypothesis contradicts the suggestion, noted earlier, 
that the pistillate expression represents a more intense flowering 
condition than the staminate. As a working hypothesis, however, it 
has proved fruitful. Experiments on the relationships between 



134 • A Miscellany 

flowering, sex expression, and leaf form, for example in hemp, 
Cannabis sativa (J. and Y. Heslop-Harrison, 1958c), have provided 
further evidence in its favor. Work of this sort also has implications 
for the questions of juvenility and maturity mentioned in the 
preceding chapter, but cannot be discussed in detail here. In addi- 
tion, further information on the roles of other growth substances, 
notably the gibberellins, will certainly be required before a truly 
comprehensive hypothesis can be framed. 

GENETICS OF FLOWERING RESPONSES 

Flowering responses to photoperiod and temperature are of 
course genetically controlled, and from the relative ease with which 
"early" and "late" varieties of cultivated plants are bred, one might 
guess that this control is often quite simple. Although practical 
breeding work is not done with reference to narrowly defined 
physiological responses, a number of specific investigations confirm 

this guess. 

The SDP characteristic of Maryland Mammoth tobacco has 
been studied in crosses with Nicotiana tabacum var. Java. The ¥ 1 
generation is not homogeneous, suggesting that the dominance of 
Java's day-neutral (or, more accurately, weakly quantitative LDP) 
characteristic is incomplete. In the F 2 , however, the SDP character 
occurs in approximately one-fourth of the progeny, indicating 
dependence on a single recessive gene. The "mammoth" (essentially 
SDP) character apparently occurs frequently in various tobacco 
varieties as a single-gene mutation, but its expression is affected by 
other genetic properties of the variety. In the interspecific cross of 
Maryland Mammoth with the LDP Nicotiana sylvestris, the LDP 
character is completely dominant (Lang, 1948). In similar crosses 
between the SDP Coleus frederici and the quantitative LDP Coleus 
blumei the Fj plants are entirely SDP, indicating dominance of 
this characteristic (Kribben, 1955). 

The difference between winter and spring varieties of Petkus 
rye appears to be due to a single gene. In the ¥ 1 generation of a 
(toss, the spring (noncold-requiring) habit is dominant; the F., 
generation segregates in a spring:winter ratio of 8:1. However, 
the dispersion in flowering time within the spring and winter classes 
of the F 2 indicates that the situation may not be quite as simple 



Genetics of Flowering Responses • 135 

as the gross segregation suggests (Purvis, 1939). Sarkar (1958) has 
confirmed and extended earlier work on the cold requirement in 
Hyoscyamus niger. Here again, crosses between the annual and 
biennial strains indicate a single-gene difference in this regard, but 
there is no dominance. The F 3 is intermediate between homozygous 
annuals and homozygous biennials. The heterozygote will eventually 
respond to long days without a previous cold treatment, but does 
so more rapidly with it; a given cold treatment has a greater effect 
on the heterozygote than on the pure biennial; and the former 
reaches a vernalizable stage earlier in development than the 
latter. 

Not all vernalization requirements appear to depend on single 
genes. Napp-Zinn (1960) reports in one paper of a continuing study 
on Arabidopsis thaliana that the difference between summer and 
winter annual strains depends on at least two genes. In addition, 
the relation between developmental stage and susceptibility to 
vernalization is under further genetic control, which has not been 
completely analyzed. 

This brief survey will be sufficient to suggest the nature of 
such investigations. Two general observations are worth making in 
this connection. In the first place, it seems evident even from the 
little that is known that specific requirements for flowering are 
not necessarily genetically deep-seated, but may be easily acquired 
or lost. Hence conclusions about the distribution— geographical 
or geological— of species and families on the basis of the present-day 
response characters of certain members (for example, Allard, 1948), 
although stimulating, should be entertained with the greatest 
caution. Second, and perhaps more important, there is clearly room 
for much more work on the genetic control of flowering require- 
ments. Cold requirements, at least, are currently receiving con- 
siderable attention (see Napp-Zinn, 1960) but genetic studies are 
notably inconspicuous or absent in most of the recent literature on 
photoperiodism. The difficulties should not be underestimated— 
particularly those involved in finding SDP and LDP sufficiently 
closely related to allow crossing, a difficulty that in itself may be of 
great importance. However, with the increasingly precise knowledge 
that research in flowering may be expected to gain from investiga- 
tions as diverse as those on the red, far-red system and with chemical 
controlling agents, a biochemical genetics of flowering as envisaged 



136 • A Miscellany 

by Lang (1948) should be a perfectly attainable goal, and well worth 
the effort. 



FLOWERING AND DEATH 

In addition to providing a melodramatic heading, the relation- 
ship between these two processes is sufficiently intimate in some 
plants— the monocarpic— to warrant some further mention. 

One reason for death following heavy flowering might be 
simply morphological. If all the shoot meristems are converted to 
determinate structures, vegetative growth cannot continue— at least 
without the formation of adventitious buds. Whether this complete 
conversion of all meristematic areas into flowers ever actually occurs 
is of course another question, but the possibility can be envisaged. 

The usual explanation of death following flowering and fruit- 
ing is nutritional— death is seen as the result of metabolic patterns 
in which the flowers, fruits, and seeds in some way compete so 
successfully with the rest of the plant for energy sources and other 
materials that death is the eventual result. The evidence is largely 
from observations, so often made, that the life of annuals can be 
prolonged by removing flowers and young fruits. However, it has 
recently been pointed out that there may be other explanations for 
such results, such as the production of inhibitors at various stages 
of reproductive development. For example, senescence in staminate 
spinach plants can be put off for a long time by removing the 
flowers. Since no fruit or seed could be set by these plants under 
any circumstances, and the staminate flowers themselves do not 
appear to contain large amounts of reserves, the simple nutritional 
hypothesis seems very weak here (Leopold et al., 1959). The article 
cited contains additional experiments and references on this topic, 
which is largely unexplored. 

It has already been mentioned many times that there are close 
relationships between flowering and vegetative growth habit, de- 
pending upon the plant; it is usually unclear whether a given 
growth change is directly related causally to flowering or whether 
both express another underlying condition. The relationship in 
monocarpic plants thus represents another, and surely the ultimate, 
aspect of this more general problem. 



Prospects • 137 



PROSPECTS 

From time to time throughout this survey suggestions for 
future work have been briefly made. In an overall view, however, 
the directions of research in the physiology of flowering are hard 
to predict with any accuracy, and harder still to recommend with 
any assurance. The best thing may be simply to ruminate a little 
on the subject before going back to work. 

One can see that most of the large problems remain. Indeed, 
one of the major achievements of the research of the past few 
decades was to delineate these questions in the first place. Among 
them are the nature or natures of the persistent states induced by 
photoperiodic or cold treatments; the nature of the flower- 
controlling substances that move between plant parts or between 
grafted plants; whether or not endogenous circadian rhythms con- 
stitute the basic mechanism of photoperiodism; and the relation- 
ships between juvenility, maturity, and flowering. 

Some questions have been reduced to simpler forms. For 
example, a question on the role of light and darkness in photo- 
periodism can be reshaped, at least in part, much more sharply: 
What is the biochemical role of the red, far-red pigment? Some 
developmental questions— bolting in rosette plants, for instance- 
can now be asked, again at least in part, in terms of specific growth 
substances, the gibberellins. This increased concreteness obviously 
represents progress; and as long as the answers to such simpler 
questions are not mistaken for exhaustive explanations of all asso- 
ciated phenomena, they should increase that progress. 

A major goal— perhaps the only goal— of physiology can be 
stated as the understanding of growth and development in terms 
of simpler biochemical systems and their integration. This does not 
mean that physiology is or ought to be biochemistry; in a sense, 
the biochemist's job begins where the physiologist's ends, although 
in practice they necessarily overlap immensely. One can envision 
the physiologist as taking an organism apart into relatively large 
portions— speaking in terms of processes— that are then susceptible 
to biochemical investigation. Unfortunately, the general recogni- 
tion of the close relationship between physiology and biochemistry 
has occasionally led to almost meaningless work. For example, an 



138 • A Miscellany 

enzyme or other substance is assayed in tissues at two quite different 
stages of development; a difference is found, and this biochemical 
difference is now suggested as the cause of the developmental 
difference, in spite of the fact that it may be, and probably is, 
merely a correlation. Such work may be quite interesting, bio- 
chemically speaking, but the physiologist must always keep in mind 
the need of a causal analysis. This at the very least requires atten- 
tion to the kinetics— relations in time— of any two conditions, one 
of which is believed to cause the other. The physiology of flowering 
has had and will have its share of both sorts of biochemically 
oriented investigations, but probably only the kind of care with 
which Lang (1960) has started to analyze the relations between 
endogenous gibberellin level and bolting in Hyoscyarnus will pro- 
\ ide real understanding. 

Assuming, then, the goal of taking organisms apart bio- 
(hemically-as long as the "parts" so obtained fit together again, 
physiologically speaking— what other experimental approaches are 
available? A useful one in the past will continue to be so: the 
use of substances or conditions suspected of having relevant effects. 
Though easily mocked, in some forms, as "spray and weigh," this 
approach at least reduces the kinetics problem; the added substance 
or changed condition surely precedes the effect in a well-controlled 
experiment. However, the problem still remains of how directly 
the two are related. It is this kind of approach, in the broadest 
sense, that has led to the basic discoveries of photoperiodism and 
vernalization, as well as many others. Even genetic studies come 
into this general class. 

Advantages can be gained here from the use of more convenient 
experimental materials. Arabidopsis, Chenopodium seedlings, and 
Lemna are all small enough to be grown rapidly in aseptic culture 
under highly controlled conditions, and may thus partially replace 
the unwieldy Perilla and Xanthium of classical investigations. 
However, the full exploitation of tissue culture techniques should 
make the latter materials even more useful than ever for studies of 
florigen and the induced state. For some preliminary thoughts and 
results in this particular direction, see Chailakhyan (1961) and Fox 
and Miller (1959). 

An approach related to the two preceding has not been 
employed to any great extent. It involves following changes in 



Prospects • 139 

both meristems and other tissues with the most sensitive cyto- 
chemical and other microscopic techniques. Ideally, this sort of 
work could provide suggestions as to what biochemical changes to 
investigate with grosser methods. Even relatively traditional ana- 
tomical studies can give important information on the action of 
various growth regulators (for example, Sachs et al., 1959, 1960) 
and it would seem highly desirable to have such information as 
closely correlated as possible with that gained from other ap- 
proaches. Even some very simple-minded questions might have 
valuable answers: What are the differences, if any, in intracellular 
organization or content between induced and noninduced Perilla 
leaves, and how soon do they arise? During the time that florigen 
is believed to be moving from an induced leaf to a meristem, can 
changes be observed along its route? And so forth. 

In short, the field will undoubtedly continue to progress as it 
has in the past— through critically tested guesses, appropriate choice 
of experimental material, perseverance, and technical advances. It 
is obvious by now that the writer has no revolutionary improve- 
ments in approach to propose, which is hardly surprising since 
differentiation and development have yielded their secrets slowly 
to better minds than his. But the progressive understanding of 
these problems, representing as they do much of what is contained 
in that simple word, "life," is surely an enterprise worthy of 
the best. 



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index off plant names 



Adiantum 96 

Agax'e (century plant) 6 

Amaranthus 35, 73, 94 

Anagallis (pimpernel) 49 

Ananas (pineapple) 90, 107-108, 124 

Anemone 7 

Anethum (dill) 18, 37, 46-47, 112 

Apium (celery) 63 

Apple, see Malus 

Arahidopsis 49, 118. 135, 138 

Bambuseae (bamboos) 6, 121 
Barley, see Hordeum 
Bean, see Phaseolus 
Beta (beet) 74, 79 
Be tula (birch) 122-123 
Brassica 62, 112, 118 
Brussels sprouts, see Brassica 
Bryokalanchoe 82 
Bryophyllum 14, 82. 104 

Campanula (bluebell) 7, 62, 128 

Cannabis (hemp) 134 

Capsicum (pepper) 1 1 2 

Carrot, see Daucus 

Celery, see Apium 

Century plant, see Agave 

Cestrum (night-blooming jasmine) 14, 

93, 122, 129 
Chenopodium, facing 1, 18. 25. 27, 52, 

119, 138 
Cherry, see Prunus 
Chrysanthemum 37, 59-62, 64, 69-70, 

73, 105 
Circaea (enchanter's nightshade) 103 
Cistus 128 
Citrus 123-125 
Clover, see Trifolium 
Cocklebur, see Xanthium 



Cofjea (coffee) 122, 129-130 

Cole us 47, 49, 134 

Corn, see 7.ea 

Cosmos 4, 73-74 

Cruciferae (mustard family) 41 

Cucumis (cucumber) 132-133 

Cucurbita (squash) 131 

Dactylis (orchard grass) 61 
Datura (Jimson weed) 25 
Daucus (carrot) 101-102 
Dill, see Anethum 
Duckweed, see Lemna, Spirodela 

Echinocystis (wild cucumber) 101 
Eggplant, see Solanum 
Erigeron 71 

Flax, see Linum 

Fragaria (strawberry) 26, 64, 81 
110 



104, 



Geranium 128 

Glycine (soybean) 8, 12-13, 19. 23, 25, 

ni or. At\' k l TO HO O" HI 1 n. 1 in" 



31-32, 49, 51, 72-73, 87, 91, 
124 



101, 107, 



Hedera (ivy) 125-126 

Helianlhus (sunflower) 2, 16, 120 

Hemp, see Cannabis 

Hippeastrum 66 

Hordeum (barley) 13, 19, 32-33, 90, 107 

Humulus (hops) 11 

Hyacinthus 65 

Hyoscyamus niger (black henbane) an- 
nual 13, 15, 19, 22-23, 26-27, 32, 35. 
37, 41-42. 48-49, 51, 58, 70, 80, 91, 101, 
106. 108, 135; biennial 58-63, 82, 101, 
135 

159 



160 ■ Index of Plant Names 



Impatiens 51 

fpomoea (morning glorv, sweet potato) 

14,71-72, 128 
Ivy, see Hedera 

Kalanchoe 4-5, 13, 22, 25-27, 47-49, 51- 
52, 71, 74-75, 82-83, 92, 94, 105, 112 

Lactuca (lettuce) 34-36, 103 
Larix (larch) 124 

Lemna (duckweed) 18, 105, 113-115, 138 
Lepidium 90 
Lettuce, see Lactuca 
Linum (flax) 90 
Lolium 18, 119-120 

Lycopersicon (tomato) 15, 64, 109, 112- 
113 

Madia 47 

Mains (apple) 122-124 

Maryland Mammoth tobacco, see Nico- 

tiana 
Matthiola (stocks) 63 
Melandrium 133 
Mentha (mint) 7 
Mercurialis 133 
Millet, see Setaria 
Morning glory, see Pharbitis, fpomoea 

Narcissus 7 

Nasturtium, see Troparoluiu 
Nettle, see Urtica 

Nicotiana (tobacco) 12-13, 25-26, 70, 
80-82, 96, 134 

Oenothera 128-129 
Oryza (rice) 113 

Pea, see Pisum 

Peach, see Prunus 

Pepper, see Capsicum 

Perilla 22, 25, 42, 70, 76, 84-87, 112, 

119-120, 138-139 
Pharbitis (morning glorv) 18, 27, 38- 

39, 77-78, 92, 107. 119; see also Ipo- 

moea 
Phaseolus (bean) 44-45, 52 
Picea (spruce) 122 
Pineapple, see Ananas 
Pinus (pine) 121-123 
Piqueria (stevia) 77 
Pisum (pea) 6, 15, 57-58, 81-82, 101, 

110-111, 118 
Plantago (plantain) 24, 47 
Plum, see Prunus 
Populus (poplar) 125 



Prunus (cherry, peach, plum) 7, 122, 

124 
Pyrus (pear) 7 

Raphanus (radish) 111 

Rice, see Oryza 

Rudbeckia (coneflower, brown-eyed 

Susan) 26.28.71,90, 109 
Rye, see Sec tile 

Salvia 42 

Saxifraga 7 

Secale (rye) 14, 56-57, 59-62, 113, 117, 

134-135 
Sedum 71 

Sempervivum (houseleek) 11, 119 
Setaria (millet) 37-38 
Silene2S, 90, 101, 106, 132 
Solayium 1 12 
Soybean, see Glycine 
Spinacia (spinach) 18,47, 61, 90, 112, 

132, 136 
Spirodela (duckweed) 114 
Spruce, see Picea 
Squash, see Cucurbita 
Statice 90 

Stevia, see Piqueria 
Stocks, see Matthiola 
Strawberry, see Fragaria 
Streptocarpus 59, 92 
Sunflower, see Helianthus 
Sweet potato, see Ipomoea 

Taraxacum (dandelion) 7 
Tobacco, see Nicotiana 
Todea 96 

Tomato, see Lycopersicon 
Trifolium (clover) 61 
Triticum (wheat) 14 
Tropaeolum (nasturtium) 90 
Tulipa 7, 65-66 
Turnera 128 

Urtica (nettle) 103 

Victoria 129 

Washingtonia 109-110 
Wheat, see Triticum 

Xant hium (cocklebur) 13, 15, 17-21, 
25-28, 32, 35-39, 43, 50-51, 69-71, 76, 
79-80, 84-85, 87, 93, 97, 101, 105, 
107-110, 113, 117, 119-120, 138 

Zea (corn) 40 



subject index 



► 
► 
► 
► 
► 

► 



Acetylene, 108 

Action spectra, see Light-breaks; Light 
quality 

Age, and flowering in woody peren- 
nials, 120-126; of leaves, and photo- 
periodism, 117, 119-120; of plants, 
and response to cold, 118 — and 
photoperiodism, 5, 15-16, 118-120; 
see also Juvenility 

Altitude, 90 

Annuals, 6, 54, 136 

Anthesis, 7, 127-130 

Antiauxin, 106-109, 132-133 

Auxin, definition, 68; and induction, 
89-91; inhibition of flowering, 79, 
90-91, 106-108; promotion of flower- 
ing, 90, 106-108; and red, far-red 
system, 91; and sex expression, 132- 
133; and vernalization, 118; see also 
Antiauxin 

Bark inversion, 123-124 

Bending, 90-91, 124 

Biennials, cold requirements, 54, 58- 

59, 61-62, 118; definition, 6; genetics 

of, 135 
Bolting, caused by furfuryl alcohol, 

109; definition, 101; and gibberellin, 

101-104, 137 
Bulb plants, 64-66 

Carbohydrate, and devernalization, 60- 
61; -nitrogen ratio, 112, 119; promo- 
tion of flowering, 79; substitution 
for high-intensity light, 21; trans- 



location, and florigen translocation, 
73-77, 79 — and flowering in woody 
perennials, 123-125; and vernaliza- 
tion, 57 

Carbon dioxide, 21-22, 92 

Carbon monoxide, 133 

Cereals, winter and spring, devernali- 
zation of, 59-60; genetics of, 134-135; 
and gibberellin, 101; minimum leaf 
number in, 117; vernalization of, 54- 
57 

Chelating agents, 114-115 

Chlorophyll, 31-32, 41 

Circadian rhythms, see Endogenous 
circadian rhythms 

Cold requirements for flowering, of 
biennials, 54, 58-59, 61-62, 118; in 
bulb plants, 64-66; genetics of, 134— 
135; of perennials, 59; and plant 
age, 118, 135; relation to photo- 
periodism, 61-62; satisfaction of, by 
diffnsate, 111 — by gibberellin, 100- 
101— by short days, 61-62; of winter 
annuals, 54-58; see also Vernalization 

Cold treatments, of bulbs, 64-66; of 
developed plants, 54, 58-59, 61-64; 
- effects of, on dormancy, 62, 129 — on 
seed germination, 57 — on vegetative 
growth. 59-60, 64; of germinating 
seeds, 54-58, 62; seasonal control by, 
54, 129; see also Vernalization 
Copper, 115 
Cotyledons, 38-39, 119 

Critical daylength, definition and 
qualifications, 13, 15, 20, 22-24; and 

161 



162 • Subject Index 

light quality. 37-39; and red, far-red 
system, 37-40; and temperature. 25, 
43; see also Light and dark periods; 
Photoperiodism 

Critical nightlength, see Critical day- 
length 

Crown -gall, 97 

Cumulative-flowering plants, 7 

Dark periods, see Light and dark 

periods 
Darkness, see Light and dark periods 
Daylength, see Critical daylength 
Daylength-indifferent plants, definition, 

14-15; florigen production by, 72; 

genetics of, 134; nutrition and flower- 
ing of. 112-113 
Dayneutral, see Daylength-indifferent 

plants 
Devernalization, by gibberellin, M)l; 

by high temperature, 60-61; of 

perennials, 60-61 
2,4-Dichlorophenox\ acetic acid (2,4-D), 

107-108 
Diffusate, 110-111 
Direct-flowering plants, 7 

Endogenous circadian rhythms, and 
action of light-breaks, 47-50; as basis 
of photoperiodism, 42-44. 46-47, 52- 
53; and leaf movements. 44-45, 50; 
and light and dark period inter- 
actions, 51-53; and red. far-red sys- 
tem, 52; temperature effects on, 42- 
46. 52; in timing of anthesis, 127- 
129 

Ethylene. 108 

Eloral hormone or stimulus, see Flori- 
gen 

Florigen, activity, criteria of, 99 — of 
natural extracts. 109-110; concept 
examined, 78-82, 95-98; evidence for 
existence, 69-72; production by day- 
length-indifferent plants, 72; relation 
to induction, 85-87; transfer across 
grafts, 69-72, 82, 84-85, 125; trans- 
location, and carbohydrate trans- 
location, 72-77— rate of. 77-78 

Flower development and initiation, re- 
lation to vegetative growth, 8-9, 28; 



relations between, 6-7, 87, 94; sea- 
sonal occurrence of, 6-7, 10, 12, 54, 
129-130 
Flower opening, see Anthesis 
Flowering hormone or stimulus, see 

Florigen 
Furfuryl alcohol. 109 

Genetics of flowering responses, 134- 
136 

Gibberellin, devernalization by, 101; 
effects on short-day plants, 104-105; 
inhibition of flowering, 104-105, 126; 
long-day plants, 91, 101-104; promo- 
tion of bolting and flowering, 100- 
104, 137; promotion of staminate 
development, 133; and red, far-red 
system, 100; satisfaction of cold re- 
quirements. 100-102, 104, 118; and 
vegetative growth, 100-104 

Girdling, 123-124 

Grafts, ambiguous effects on flowering, 
79-82, 125; on dwarfing stocks, 124; 
transfer of florigen across, 69-72, 82, 
84-85, 125; transfer of induced state 
by, 84-85; transfer of vernalin 
across, 82-83 
Gravity, 90-91, 124 

Hormone, definition. 67; floral or 

flowering, see Florigen 
^-Hydroxyethvl hydrazine, 108 

Indirect-flowering plants, 7 
Induced state (Induction), and auxin, 
89-91; by cold, vernalization, 56-57, 
87-88; compared to crown-gall 
tumor. 97; defined, 17; fractional, 
24-25, 88; inhibition by dark periods 
in SDP, 20-22; and nucleic acids. 
92-93. 96; permanence of, 83-8S; 
quantitative nature of, 87-88, 94-95; 
transfer across grafts, 84-85; and 
vegetative growth, 28 

Induction, see Induced state 

Inflorescence. 2 

Iron, 113-114 

Juvenility, and carbohydrate, 96, 125; 
in woody plants, 116. 124-126; see 
also Age 



Subject Index • 163 



Lamarckism, 55 

LDP, see Long-day plants 

Leaf, age, and photoperiodic sensitiv- 
ity, 119-120; blades, photoperiodic 
perception by, 17; movements, and 
endogenous circadian rhythms, 44- 
45, 50; number, minimum, 117-118; 
true, compared to cotyledons, 38-39, 
119 

Light and dark periods, and endog- 
enous circadian rhythms, 47-52; 
lengths of, 10-11, 18-20, 22-24, 51- 
52; and red, far-red system, 35-39; 
roles in photoperiodism, 18-20; 
temperature interactions with, 25- 
27, 63-64 

Light -breaks, in action spectrum stud- 
ies, 30-33; definition, 19; and endog- 
enous circadian rhythms, 47-50; and 
red, far-red system, 35-39 

Light intensity, and criteria of photo- 
periodism, 11. 29, 131; high, re- 
quirement for, 20-22; low, photo- 
periodic effect of, 13, 19-20 — and 
red, far-red system, 39 

Light quality, action spectra, 30-33, 
52; and anthesis, 128; in main light 
periods, 40-42; and vegetative 
growth, 34-35, 39-40; see also Red, 
far -red system 

Long-day plants (LDP), definition, 13; 
and gibberellin, 91, 100-104; see also 
Critical daylength; Light; Photo- 
periodism 

Long-short-day plants, 13-14, 93 

Lysenkoism, 55 

Mercury, 115 

Meristem, age and flowering, 96, 120, 

124-126; organization and flowering, 

3-5 
Molybdenum, 114 
Monocarpic plants, 6-7 

Naphthaleneacetic acid (NAA), 97, 

107, 108, 118, 132 
Nightlength, see Critical daylength 
Nitrogen, see Carbohydrate, -nitrogen 

ratio; Nutrition, major element 
N-metatolylphthalamic acid, 109 
Nucleic acids, 92-93, 96 



Nutrition, and (lowering of daylength 
indifferent plants, 112-113; iron and 
trace metal, 113-115; major clement, 
111-113. 119, 133 

Perennials, cold requirements, dever- 
nalization, 54, 60-61; definition, 6; 
woody, 120-126 

Phasic development, 116-117 

Phloem, see Carbohydrate, transloca- 
tion; Plorigen, translocation 

Photomorphogenesis, 39 

Photoperiodism, classes of response. 
13-15; criteria, definitions of, 10-11, 
29, 131; discovery of, 11-13; effects 
on sex expression, 131-132; effects on 
vegetative growth, 27-28, 122-123; 
and endogenous circadian rhythms, 
42-53; induction by, 17-18, 83, 
87; and leaf or plant age, 15-16, 
117-120; and light intensity, 11, 20- 
22, 29; and light quality, 30-33, 35- 
39; and red, far-red system, 35-39; 
relation to cold requirements, 61-62; 
role of leaf in, 17, 84-87, 117-120; 
role of light and dark periods in, 
18-24; seasonal control by, 10-12, 
15; temperature effects on, 25-27; in 
woody perennials, 122-123 

Photosvnthate, see Carbohydrate 

Photosynthesis, 21-22, 29, 31-32, 41 

Photochrome, definition, 40; see also 
Red, far-red system 

Red. far-red system, and auxin, 91; 
and critical daylength, 37-40; effects 
on, of light and dark periods, 35-39 
— of light-breaks, 35-36 — of low- 
intensity light, 39; and endogenous 
circadian rhythms, 52; and gibberel- 
lin, 100; nature of, 39-40; and photo- 
morphogenesis, vegetative growth, 
39; and seed germination, 34-35; 
temperature effects on, 34, 36-37 

Respiration, 57, 91-92 

Rhythms, see Endogenous circadian 
rhythms 

Ripeness to flower, see Age; Juvenility 

Rosette plants, see Bolting 

Scotophile phase, 46 



164 • Subject Index 



SDP, see Short-day plant(s) 

Seasonal control, by cold treatments, 
54-55, 129; bv photoperiodism. 10- 
13, 15; by water, 129-130 

Seasonal flower initiation and develop- 
ment, 7 

Seed germination, 34-35, 57 

Sex expression, and auxin. 132-133; 
and gibberellin. 133; and photo- 
periodism, 131-132; and tempera- 
ture, 131 

Sex hormones, animal. 133 

Short-day plants (SDP) definition, 13; 
inhibition by long days in. 25; see 
also Critical davlength 

Short-long-day plants, 13-14. r>l-62 



Temperature, effects of, on bulb 
plants, 64-66 — on critical daylength, 
26. 43 — on endogenous circadian 
rhythms, 42-46, 52 — on photoperiod- 
ism, 25-27 — on red, far-red system, 
34, 36-37 — on sex expression, 131; 
high, devernalization by, 60-61; 
interactions with light and dark 
periods, 25-27, 63-64; see also Cold 
requirements; Cold treatments; Ver- 
nalization 

Thermoperiodism, 25-27, 63-64. 131 

Trees, see Perennials, woody 

2,3,5-Triiodobenzoic acid (TIBA), 107- 
109, 132-133 



Tropical plants, lack of cold require- 
ments in bulbs, 65-66; lack of 
knowledge about, G; water effects on 
seasonal anthesis, 129-130 

Ultraviolet radiation, 90 
United States Department of Agricul- 
ture, 11-13,32-39 

Vegetative growth, effects on, of cold, 
vernalization, 57, 59-60, 64 — of light 
quality, 39 — of photoperiodism, 27- 
28, 122-123; and gibberellin (stem 
elongation), 100-105; relation to 
flower development and initiation. 
8-9, 28; restraint of, 111; see also 
Age; Bolting; Meristem 

Vernalin, 82-83 

Vernalization, and auxin, 118; and 
carbohydrates, 57, 60-61; definition 
and qualifications, 55, 58, 62-64; 
induction by, 56-57, 87-88; and 
political ideology. 55; see also Cereals, 
winter and spring; Cold require- 
ments; Cold treatments; Devernali/a- 
tion; Vernalin 

Viruses, 78, 92 

Vitamin E, 110 

Water, 129-130 

Woody plants, see Perennials 

X-rays, 90