McGRAW-HILL PUBLICATIONS IN THE AGRICULTURAL AND BOTANICAL SCIENCES EDMUND W. SINNOTT, Consulting Editor GROWTH HORMONES IN PLANTS McGRAW-HILL PUBLICATIONS IN THE AGRICULTURAL AND BOTANICAL SCIENCES Edmund W. Sinnott, Consulting Editor Adams — Farm Management Bahcock and Clausen — Genetics in Relation to Agriculture Babcock and Collins — Genetics Laboratory Manual Belling — TheUse of the Microscope Boyle — Marketing of Agricultural Products Boysen Jensen and Avery and Burkholder — Growth Hormones in Plants Braun-Blanquet and Fuller and Canard — Plant Sociology Brown — Cotton Carrier — Beginnings of Agricul- ture in America Cruess — Commercial Fruit and Vegetable Products Cruess and Christie — Laboratory Manual of Fruit and Vegetable Products Curtis — The Translocation of Solutes in Plants Eames — Morphology of Vascular Plants Eames and MacDaniels — Intro- duction to Plant Anatomy Eckles, Combs and Macy — Milk and Milk Products Emerson — Soil Characteristics Fawcetl — Citrus Diseases and Their Control Fitzpatrick — The Lower Fungi — Phycomycetes Gardner, Bradford and Hooker — Fundamentals of Fruit Pro- duction Gardner, Bradford and Hooker — Orcharding GHumann and Dodge — Compara- tive Morphology of Fungi Hayes and Garber — Breeding Crop Plants There is also a series of McGraw Sciences, of which A. Franklin Shullis Heald — Manual of Plant Diseases Hill, Overholts and Popp — Botany Horlacher — Sheep Production Hutcheson and Wolfe — Production of Field Crops Jones and Rosa — Truck Crop Plants Loeb — Regeneration LOhnis and Fred — Textbook of Agricultural Bacteriology Lutman — Microbiology Maximov — A Textbook of Plant Physiology Miller — Plant Physiology Piper and Morse — The Soybean Pool — Flowers and Flowering Plants Rice — Breeding and Improvement of Farm Animals Seifriz — Protoplasm Sharp — Introduction to Cytology Sinnott — Botany: Principles and Problems Sinnott — Laboratory Manual for Elementary Botany Sinnott and Dunn — Principles of Genetics Smith — Fresh-water Algae of the United States Swingle — A Textbook of System- atic Botany Thatcher — The Chemistry of Plant Life Thompson — Vegetable Crops Waite — Poultry Science and Practice Weaver — Root Development of Field Crops Weaver and Bruner — Root Devel- opment of Vegetable Crops Weaver and Clements — Plant Ecol- ogy Wodehouse — Pollen Grains -Hill Publications in the Zoological Consulting Editor. J^ 7/ GROWTH HORMONES IN PLANTS AUTHORIZED ENGLISH TRANSLATION OF DIE WUCHSSTOFFTHEORIE UND IHRE BEDEUTUNG FUR DIE ANALYSE DBS WACH8TUMS TJND DER WACHSTUMSBEWEQUNGEN DER PFLANZEN BY P. BOYSEN-JENSEN Professor of Plant Physiology, University of Copenhagen Translated and Revised by GEORGE S. AVERY, Jr., and PAUL R. BURKHOLDER with the Collaboration of HARRIET B. CREIGHTON and BEATRICE A. SCHEER Department of Botany. Connecticut College Expanded to include 188 new contributions to the literature and 40 additional illustrations First Edition McGRAW-HILL BOOK COMPANY, Inc. NEW YORK AND LONDON 1936 Copyright, 1936, by the McGraw-Hill Book Company, Inc. PHINTED IN THE UNITED STATES OF AMERICA All rights reserved. This book, or parts thereof, ynay not be reproduced in any form without permission of the publishers. THE MAPLE PRESS COMPANY, YORK, PA. FOREWORD It is with the greatest satisfaction that I follow the increasing interest in America and England in the investigations concerning growth substances in plants; indeed, a series of important con- tributions to our knowledge about these substances originate from laboratories in these countries. It is, therefore, a great delight for me to have American friends translate my book "Die Wuchsstofftheorie " into English. I want to express my most sincere thanks to Professor George S. Avery, Jr., Dr. Paul R. Burkholder, Dr. Harriet B. Creighton, and Miss Beatrice A. Scheer, who have undertaken the troublesome and ungrateful work, and who have likewise revised the text and brought it up to date. I hope that the translation and revision may make the book more easily available to American and English colleagues and that it may also stimulate new studies on these promising problems, as there are still so many points needing further investigation. P. BoYSEN Jensen. Copenhagen, June, 1936. PREFACE It is with enthusiasm that we bring a translation and revision of Professor Boysen Jensen's "Die Wuchsstofftheorie " to botanists in English-speaking countries. It is the first comprehensive review of the literature dealing with the role of growth hormones in normal growth and tropisms of plants. In translating and revising the book to include the 1935 literature, it is our hope to stimulate sound progress in this important new field of plant physiology. Plant growth hormones have been recognized by many investi- gators as offering fruitful opportunities for study, and there is ample evidence in the contributions of the past year or two that research in this field is progressing at a rapid pace. One dis- covers in the literature that growth hormones promote cell enlargement in shoots of higher plants, initiate the development of roots (but at the same time inhibit their growth in length), inhibit the development of lateral buds, stimulate cell division in the cambium, and bring about callus formation. Such diverse effects are of great interest, but in many instances more evidence must be obtained to prove the theoretical views which we now possess. Professor Boysen Jensen's review of the subject makes clear numerous weaknesses and gaps in our knowledge and thus helps point the way for future research. The arrangement of certain chapters has been modified in the translation so that students not already acquainted with the literature may become familiar with techniques and general methods of procedure. Certain parts of the book have been condensed slightly, while others have been expanded to include data from the approximately 200 new citations added to the bibliography. The essential features of the controversial final chapter of the German edition have been included so far as possible in earlier chapters. In an attempt to make the American edition as useful as possible to students, we have added an index, a summary at the end of each chapter, and numerous illustrations. The following new figures have been inserted: 7, 8, 13-15, 17-20, vii viii PREFACE 23-26, 28-31, 33-44, 50, 55, 58-62; and Figs. 21 and 22 have been substituted for Figs. 10 and 11 of the German edition. The historical development of the subject has been presented in a series of diagrams (Figs. 1 and 2). Plant growth substances, that is, Wuchsstoffe, have been referred to by various workers as growth hormones, growth regulators, growth enzymes, phyto- hormones, and auxins. We have used all of these terms. A really satisfactory terminology, based on the chemical nature of the compounds in question, will have to wait until more is known. A selected list of titles dealing with the influence on plants of substances such as bios, folliculin, and other sex hormones, those affecting the growth of fungi, etc., has been added for the con- venience of students interested in these topics. They are not discussed in the text. It is a pleasure to express our appreciation of Professor Boysen Jensen's interest and cooperation in the effort to make this book available to English-speaking botanists. George S. Avery, Jr. Paul R. Burkholder. Connecticut College, June, 1936. W^i PREFACE TO THE GERMAN EDITION Twenty-five years have passed since the discovery of growth substance in the Avena coleoptile. The development of growth- substance research in this period of time is characterized by a continually growing mass of publications. The growth- substance literature, in a narrow sense, includes at present about 200 individual papers. Investigations in this field are appear- ing continually, so that important new discoveries must be taken into consideration each year. One can foresee great forward strides during the next few years in this line of study. What has been accomplished so far by this extensive activity? The most important contributions are as follows: The signifi- cance of growth substance for photo- and geotropic curvatures was demonstrated first in the Avena coleoptile. Then its effect on normal growth was investigated, and it was shown that growth substance plays an important part in the positive geotropic curvature of the main root. Next the general occur- rence of growth substances was demonstrated in higher and lower plants and in animals. Finally it became possible to prepare growth substance in a pure state. The main result has been a glimpse into the mechanism of the orientation of higher plants in space. Do the results that have been obtained justify the effort — has growth-substance research had its reward? Perhaps such a question should not be asked. I feel that it would be unfair to suppress the fact that, although great advances have been made, we are still far from an explanation of photo- and geotropic processes. It is satisfying to know that something has been attained that may be regarded as progress in our knowledge of life processes. One must become reconciled to the fact that in advanced physiological research, problems become so involved that every stride forward requires a great output of labor. The author has attempted to present the chief lines of investi- gation and the main results of research done in the last twenty- ix X PREFACE TO THE GERMAN EDITION five years. He has endeavored to present objectively the views of various investigators, to distinguish as clearly as possible between experimentally founded fact and more or less hypo- thetical views. Particular attention is given to the fundamentals of the growth-substance theory — and the author hopes, therefore, that his work will be useful to those carrying on growth-substance research. P. BoYSEN Jensen. Laboratory of Plant Physiology, University of Copenhagen. CONTENTS Page Foreword v Preface vii Preface to the German Edition ix CHAPTER I Introduction and Historical Sketch 1 Growth and growth substances 1 Historical sketch 5 Darwin — Wiesner — Rothert — Fitting — Boysen Jensen — Paal — Stark and Drechsel — Formulation of the growth-substance explanation. Summary 15 CHAPTER II Detection and Quantitative Determination of Growth Sub- stances 16 The test for the presence of growth substances with the Avena coleoptile 16 The culture of the Avena seedling for use as a phytohormone test object — Culture conditions — Culture methods — Preparation of the Avena coleoptile for use — Decapitation and unilateral application — Growth curs'ature of the coleoptile — Preparation of the material to be tested — Direct application of plant parts to the decapitated Avena coleoptile^Application of material to be tested in agar and in lanolin — Detection of growth substance in fluids — Detection of growth substance in pollen — Diffusion of growth substance into agar — Diffusion of growth substance into dextrose-agar — Diffusion of growth substance into water — Extraction of growth substance with alcohol — Extraction of growth substance with chloroform — Extraction of growth substance with water. Quantitative determination of growth substances 27 Determination of growth curvature produced by the unilateral action of a growth substance — Boysen Jensen's quantitative method — Went's quantitative method — Comparison of the units of different workers — Other quantitative methods — Growth in length of decapitated coleoptiles — Pea test method — Callus- forming effect of growth-substance pastes — Comparison of Avena with other test objects. Summary 38 xi 47664 xii CONTENTS I Page ] CHAPTER III ! Preparation and Properties of Growth Substances 40 j Preparation from plant and animal sources 40 ' Rhizopus, a source of growth substance — The identity of "rhizo- i pin" and 3-indole acetic acid — Aspergillus, a source of growth sub- stance— Substratum content and growth substance production — Urine, a source of auxentriolic acid (auxin a) — Maize oil, malt and other sources of auxenolonic acid (auxin 5)— Urine, a source of 3-indole acetic acid (heteroauxin) — Yeast, a source of heteroauxin. Properties of growth substances 50 The structural constitution of the auxins— Physical and chemical ^ characteristics of the auxins crystallized from plant materials — Physiological effectiveness of the auxins, their derivatives, and ; other compounds. Summary 55 CHAPTER IV The Occurrence and Formation of Growth Substances 56 Occurrence 56 j Higher plants — Growth substance in coleoptiles — Foliage leaves — Growth substance in hypocotyls, shoots and flower stalks — Roots —Pollen — Fruits and seeds— Lower plants — Plant products — i Human and animal organisms. The formation of growth substances 64 Lower plants — Higher plants — Production of auxin in human | urine. i Summary 69 CHAPTER V The Transport of Growth Substances 71 I The Avena coleoptile — The conducting tissue — Polarity of trans- port— Rate and amount of transport — Rate of transport — Amount of transport — X-irradiation and transport — Anaesthesia and transport — Growth-substance transport in shoots and roots — The mechanism of growth-substance transport — Diffusion — Proto- , plasmic streaming — Surface tension — Electrophoresis — Movement I of growth substance from agar blocks into decapitated Avena j coleoptiles. Summary 79 CHAPTER VI The Significance of Growth Substances for the Normal Growth OF Plants 81 General survey of the effect of growth substances upon growth. . . 81 The Avena coleoptile — Structure — Distribution of growth — Light- growth reaction — Geo-growth reaction — Growth substances and normal growth — Growth substances and the light-growth reaction CONTENTS xiii Page —Foliage leaves— Axial parts: hypocotyls, internodes, and flower stalks — Distribution of growth — Growth substances in relation to the distribution of growth — Roots— Lower plants— Animal cells — Plant tissue culture — Growth substance in relation to cell division. The mechanism of action of growth substances 103 Growth substance and the cell wall— Cell wall extensibility- Methods for measuring extensibihty — Hypotheses on the method of action: elasticity, plasticity, active growth — Discussion of hypotheses: growth substances and elastic extensibility, growth substances and plastic extensibility, growth substances and intus- susception— Acid-growth reaction and the growth hormone — Inhibition of growth in roots. Summary 115 CHAPTER VII The Significance of Growth Substances for Other Phenomena 117 Bud development — Tumor formation — Stomatal movement — Respiration — Cambial activity — Nastic movements — Root forma- tion— Miscellaneous. Summary 129 CHAPTER VIII The Significance of Growth Substances for Phototropism . . . 131 General discussion of phototropism with special reference to the Avena coleoptile 131 Stimulation and response — The light gradient — Distribution of sensitivity to light — Conduction of the stimulus — The quantity- of-stimulus principle — Positive and negative curv^atures — The primary positively phototropic curvature — The Blaauw theory — Evidence supporting the Blaauw theory — Evidence opposing the Blaauw theorj^ — Conclusions in regard to the Blaauw theory — The growth-substance explanation— The relation of growth substance to phototropism — The question of wound substances — Origin of the unequal distribution of growth substance — Contrasting theories of Boysen Jensen and Paal — Purdy's theory — The Went theory — Theories of Beyer, Cholodny, DuBuy and Nuernbergk — The displacement of growth substance — Growth-substance transfer and electrical potential — Conclusions in regard to the growth-substance explanation — Other theories on phototropism. Dicotyledonous stems 163 Distribution of phototropic sensitivity — Transmission of photo- tropic stimulus — Growth substance and phototropism in seedhng axes. The Phycomyces sporangiophore 166 Summary 167 xiv CONTENTS Page CHAPTER IX The Significance of Growth Substances for Geotropism . . . .169 1 o y The early investigations The Avena coleoptile ■ • Stimulation and response— Distribution of geotropic sensitivity- Transmission of the stimulus— The quantity-of-stimulus principle —The course of geotropic curvature— The growth-substance explanation— Growth substance and geotropic sensitivity— The unequal distribution of growth substance— The statolith theory- Electrical theories and experiments— Comparison of phototropic and geotropic curvatures— Recovery from geotropic curvature brings about equilibrium. Dicotyledonous hvpocotyls, shoots, etc Stimulation and response— Distribution of geotropic sensitivity— Negatively geotropic curvature in stems— The growth-substance explanation— Experiments with split stems— Growth-substance displacement— Electrical transport of growth substance— Inherit- ance of geotropic response— Geotropic response in nodes. Roots .■ ■ ... ., Stimulation and response— Distribution of geotropic sensitivity- Stimulus transmission— The quantity-of-stimulus principle- Growth in geotropic curvature— The growth-substance explanation of root curvature— Similarity of growth substance in the root and coleoptile— Extraction of growth substance from roots— Transverse distribution of growth substance— Mechanism of growth-substance displacement. Summary CHAPTER X The Significance of Growth Substances for Traumatic and Thigmatic Curvatures ,• ' u ' General survev of the phenomena— Amputations— Wounding by incisions— Chemical treatment— Stimulus transmission— Trans- mission in the Avena coleoptile— Transmission in roots— Transmis- sion in Mimosa— The growth-substance explanation— Amputa- tions—Wounding by incisions. ' ^^^ Summary 221 Bibliography 247 Supplementary Bibliography s 255 Index GROWTH HORMONES IN PLANTS CHAPTER I INTRODUCTION AND HISTORICAL SKETCH GROWTH AND GROWTH SUBSTANCES The growth of Hving organisms is of fundamental importance to all students of biology. It implies a permanent increase in the size of the whole organism or its parts as a result of the incorpora- tion of materials from the environment. Growth is due chiefly to the absorption of water, the synthesis of new protoplasm, extension of cellular boundaries, and increase in weight. As an organism grows, it becomes differentiated into parts that perform specific functions. Growth in plants results from the integration of many internal processes. In an attempt to analyze the discrete chemical reactions and physical conditions that contribute to growth, it may be useful to classify the substances concerned into two groups: nutritional substances and regulating substances. In the first group, considered in the broadest sense, belong water, minerals, gases, and the organic foodstuffs which supply energy for building the plant's structures. Following Huxley's sug- gestion (1935), the second group of regulating substances may be conveniently subdivided as follows: (1) localized chemical acti- vators whose range of influence may be limited strictly to intra- cellular activities {e.g., those concerned with various genie effects) or to a comparatively small sphere; (2) hormones which exercise specific effects upon cells or tissues other than those by which they are produced. To the latter group of substances belong those growth-regulating materials which are the subject of this book. 1 2 GROWTH HORMONES IN PLANTS The significance of chemical correlation in plant physiology and morphogenesis has captured the imagination of botanists ever since the discovery in plants of chemical substances which might be called hormones. The word hormone, derived from the Greek opMcico and meaning "I arouse to activity," was suggested by Hardy and first applied in animal physiology by Starling (1906) in discussing the substance secretin ; it was later defined by Starling (1914) as "any substance normally produced in the cells of some part of the body and carried to distant parts which it affects for the good of the body as a whole." It has been shown (Huxley, 1935) that all gradations exist between hormones and local activating substances and between the latter and ordinary by-products of metabolism wliich are less specific in regard to the nature of the structures acted upon. The word hormone was used for the first time in connection with plants by Fitting (1910), who found that a substance present in orchid pollen caused swelling of the gynostemium in the orchid flower. In dealing with the phenomena of growth, careful distinction has not always been maintained in the past between the sub- stances which may be correctly termed hormones and certain other types of materials. In comparatively recent years, a number of terms, more or less useful but not particularly well- defined, have been proposed to designate newly discovered functional materials, such as hormone, enzyme, vitamin, bios, Wuchsstoff, etc. Although these terms may overlap in meaning, they are temporarily useful until more information is available. The definition of a hormone is given above in biological terms because hormones play a role only in hving organisms; they are chemical substances which have a specific influence on correlation and differentiation of the organism. They are effective when present in minute amounts and control growth in plants in some way other than by direct nutritive means. The substances influencing growth through direct nutritive effects include vitamins (accessory or protective food factors in animals), hios (substances that apparently function much like vitamins in the growth of certain plants — Miller, 1930; Kogl, 1935, Mitt. XIV), etc. Enzymes are produced also by living organisms and promote chemical reactions either within or outside the organism and are not used up. In some instances a given sub- stance may fall into more than one category. Plant-growth INTRODUCTION AND HISTORICAL SKETCH 3 substances, i.e., Wuchsstoffe^, have been referred to by various workers as growth hormones, growth regulators, growth enzymes, phytohormones, and auxins. They inehide compounds that promote the growth of the Avena coleoptile and the hypocotyls, stems, and leaves of various dicotyledonous plants, but they apparently retard the growth of roots. They are known to be produced by Avena, Zea, Rhizopus, Aspergillus, various bacteria, and numerous other organisms. They are ether-soluble, sensi- tive to peroxide, and have an acid character. HISTORICAL SKETCH Botanists first became acquainted with growth substances through studies on tropisms, i.e., those growth curvatures that take place in response to unilateral stimulation of an organ by light or its displacement from the usual position of equilibrium with respect to the force of gravity, etc, A short historical survey of the earlier contributions to our knowledge of the growth phenomena concerned in photo- and geotropism is presented here as a background for the information that will follow. Darwin. — In a book entitled "The Power of Movement in Plants," Darwin (1881) recorded extremely valuable experi- ments and reflections upon the movements of plants in response to light. Among other things, he demonstrated a localization of the phototropic stimulus in certain plants. The main object on which he experimented was the coleoptile of Phalaris canariensis. When this organ was unilaterally illuminated, a strong positive phototropic curvature resulted. If the tip of the coleoptile was darkened by a tinfoil cap or a darkened glass cap, and only the lower part was unilaterally illuminated, curvature usually did not result. However, if the procedure was reversed, i.e., if the upper part of the coleoptile was unilaterally illuminated while the lower part was darkened (by means of moist sand), a photo- tropic curvature took place in the lower portion (Fig. 1). It was shown also that a coleoptile does not react phototropically when 2.5 to 4 mm. of the tip is removed (Fig. 1). Darwin concluded (1881, p. 474) "that when seedlings are freely exposed to a lateral light, some influence is transmitted from the upper to the lower part, causing the latter to bend." Localized sensitivity to light and conduction of a stimulus were observed also in the coleoptile 1 This refers to Wuchsstoffc A. The term Wuchsstoffe B refers to a different class of substances, such as Bios (see Nielsen, etc.. Supplementary Bibliography) . GROWTH HORMONES IN PLANTS A A Darwin, ISSO Light falling from one side upon the tip of a grass coleoptilc (Phalaris) causes some influence to be transmitted downward; the coleoptile curves toward the light. When the coleoptile tip is re- moved, phototropic response does not occur. Localized sensi- tivity to light and conduction of a stimulus was observed early in many kinds of plants. Fitting, 1907 In a room .saturated with water vapor, lateral incisions either on one or on both sides of the Avena coleoptile do not prevent its bending toward light from one side. A BoYSEN Jensen, 1910-1911 When an excised coleop- tile tip was replaced with gelatin inserted between it and the stump, phototropic curvature resulted as in nor- mal coleoptilcs; the tropic stimulus passed over the incision. £\ n a BoYSEN .Jensen, 1910-1(111 Insertion of mica plates on the shaded side pre- vented curvature following unilateral illumination of the tip. When the mica insert was made on the illumi- nated side, curvature resulted in the usual way. It was concluded that a substance migrates down the back side promoting growth curvature toward light. A Paal, 1918 When an excised tip is re- placed on one side of the Avena coleoptile stump, accelerated g:o" th beneath the tip results in curvature. \ r SUDING, 1925 Decapitation results in diminished growth of the Avena coleoptile, but when the excised tip is replaced, growth in length is renewed. a Negative curvature Stahk, 1917-1921 Seubekt, 1925 Expressed sap from Avena coleoptiles was put into agar blocks which were ■■ipplied unilaterally to coleoptile stumps; curvatures resulted. .Seubert (1925) found by using this method that some substances promote while others inhibit growth, as indicated by negative and positive curvatures. Positive curvature LOEB, 1910 The presence of vig- orous leaves on a hori- zontally placed Bryo- pliylluni stem increases geotropie bending (also the production of roots is stimulated). The action of hormones was suggested as the explanation. Went, 1928 When an Avena coleoptile is decapitated its growth in length cea.ses, a. The addition of a plain agar block, b, has no effect, but growth is renewed by the addi- tion of a block, c, containing juice extracted from the excised tip. a c fc 1 Fig. 1. — Historical outline of the early discoveries concerning plant growth hormones. INTRODUCTION AND HISTORICAL SKETCH Went, 1928 Growth hormone is given off from plant tissue (coleoptile tips) into agar. When a small block of this agar is placed unilaterally on a • decapitated Avena coleoptile, the resulting curvature, a, is proportional, within limits, to the concentration of growth hormone pres- ent, b. Went, 1928 When unilateral light falls upon an excised Avena coleoptile tip, a, placed in contact with two agar blocks, 6 and c, separated by a razor blade, d, growth hormone is displaced toward the shaded side; block 6 receives 65 per cent and block c 35 per cent of all the recoverable growth hormone given off from the tip. Went, 1928, ANb X.KN Der Weij, 1932 Transport of growth sub- stance from an agar block, f, through a segment from an Avena coleoptile into another agar block,/, takes place only toward the mor- phological base. Qualitative proof of the same phenome- non was given bv Beyer (1928). A V Van Overbeek, 1933 An agar block, a, containing ■ growth hormone is placed upon the . upper cut surface of a Raphanus hypocotyl segment standing upon " two plain agar blocks, 6 and c. . Exposure to unilateral light causes displacement of growth hormone 'toward the shaded side; the re- - coverable portion is present in the two blocks as indicated. Hormone Explanation of Phototropism The growth hormone is displaced by unilateral light into the shaded portion of a hypocotyl, petiole, or similar organ. Its presence in greater concentration promotes growth more rapidly there, and the organ bends toward the light. IIIU Gravitv DoLK, 1929 When an excised coleoptile tip, a, is placed in a horizontal position in contact with two agar blocks, b and c, growth hormone is displaced toward the lower side. It accumulates in greater concentration in block c. When a cylindrical segment of an Avena coleoptile is placed hori- zontally and supplied with growth hormone at the morphological apex, d, transport takes place toward the lower side of the morphological base, the hormone accumulating in block e. U 1 1 i 1 BoYSEN Jensen, 1933 When an excised root tip of Viaa Faba. a, is placed horizontally in contact with agar blocks, b and f, containing 10 per cent glucose, growth hormone is displaced to the lower side and accumu- lates in the lower block, c. illU IIUU DlJKMAN, 1934 Growth hormone supplied in agar to the cut apex of a [-— J segment of Lupinus by po- t's colyl, placed in a horizontal "tjy position, is transported to- ward the lower side of the morphological base. Hormone Explanation OF Geotropism Tropic bending results from dis- placement of hormone to the lower side of the plant axis. The shoot curves upward because its growth is promoted, and the root turns downward be- cause its growth is in- hibited by the hor- mone (Cholodny). 1 Hi 1 11 Fig. 2.— Outline of recent contributions to our knowledge of plant growth hormones. 6 GROWTH HORMONES IN PLANTS of Avena saliva, in the hypocotyls of Brassica oleracea and Beta vulgaris, and in the negatively phototropic movements of roots. Wiesner. — Although the significance of Darwin's investigations was recognized fully in the first edition of Pfeffer's "Plant Physiology," the conclusions were criticized adversely by Wiesner (1881). This criticism is of only historical interest, but it should be mentioned briefly. The curvature that occurs in the lower part of the hypocotyl of Brassica oleracea when the tip is illuminated was explained by a factor that Wiesner called a "traction growth " {Zugwachstum) . When the upper part curved under the influ- ence of unilateral light, its weight supposedly had a unilateral effect upon the lower part. The extended shaded side was interpreted as growing more rapidly than the compressed front side. This point of view does not seem at all convincing; moreover, only a few experiments were performed with grass coleoptiles. Yet, doubt was thrown upon the correctness of Darwin's con- clusions. At the suggestion of Pfeffer, Rothert (1894) investi- gated the problem of phototropic stimulus conduction in a very thorough manner at the Leipzig laboratory. Rothert. — The outcome of Rothert's work was a complete confirmation of that of Darwin. Conduction of the phototropic stimulus was demonstrated in a series of different plant organs, including coleoptiles of grasses, seedling axes of numerous dicotyledons, orthotropic leaves, petioles, etc. It developed, however, that the localization of phototropic sensitivity was not actually so marked as had been supposed from Darwin's experi- ments. Darwun had maintained that only the tip of the Avena coleoptile is phototropically sensitive; Rothert showed that a weak phototropic curvature resulted from unilateral illumination of the basal part. It was found also that sensitivity and mobility are very sharply separated in the seedlings of some grasses. In certain cases only the coleoptile was sensitive to light, and the curvature took place in the first internode below the coleoptile where sensitivity was completely lacking. With regard to the paths of stimulus conduction in the Avena coleoptile, Rothert noted the following: In the coleoptile cylinder two vascular bundles are situated opposite one another, not joined by cross-connecting bundles (Fig. 21). In an attempt to INTRODUCTION AND HISTORICAL SKETCH 7 determine whether or not these play an essential role in stimulus conduction, Rothert proceeded as follows: Both bundles were severed; the tip was exposed to light; and the lower wounded portion was darkened. A phototropic curvature occurred in this basal portion, and from this Rothert concluded that "it is proved that the hehotropic (phototropic) stimulus is conducted in the parenchyma of the fundamental tissue." Fitting. — Carrying the work of Rothert further, Fitting (1905- 1906, 1907) studied the fundamental processes of stimulus conduction. The question that he set out to investigate was stated as follows: "In tropism, how is the organ of perception so linked with the zone of reaction that the externally applied stimulus can indirectly determine the direction of the curvature?" To solve this problem, Fitting tried to ascertain whether the conduction of a stimulus on a certain side of the coleoptile is in any w^ay oriented with respect to the direction of light. The experiments, on Avena coleoptiles for the most part, were carried out in a room saturated with moisture at a temperature of 30°C. The influence of incisions upon growth and curvature of the Avena coleoptile was studied first. It was found that the growth rate remained practically unchanged by unilateral wounding. Weak curvatures of the coleoptiles were observed, first away from the wound and then toward it. The influence of unilateral transverse incisions upon longitudinal conduction of a stimulus in the Avena coleoptile was then investigated. Incisions were made midway between the base and the tip in coleoptiles measuring 1 to 1.5 cm. in length. The coleoptiles were darkened past the point of incision with tinfoil tubes or with collars made of black paper. Then, if the tip of the coleoptile was illuminated uni- laterally, a decided positive phototropic curvature resulted in the darkened basal portion under the incision, no matter what the orientation of the incision was with respect to the direction of light, i.e., whether the incision was on the illuminated or on the shaded side (Fig. 1). These experiments were modified in various ways, but the result was always the same. Even when tw^o incisions were made, one above the other and on opposite sides, the conduction of the phototropic stimulus met with no interference. Fitting concluded that the stimulus was conducted around an incision and transmitted exclusively through the Hving material. 8 GROWTH HORMONES IN PLANTS When two incisions were made one above the other, he thought that the stimulus conduction was not disturbed by the insertion of tinfoil, no matter how oriented with respect to the direction of light. However, if there were two transverse incisions with tin- foil or mica inserts one above the other, the phototropic curvature under the incisions was very slight. Fitting surmised that the absence of curvature in the latter case was due to drying out of the tip, since the vascular bundles had been severed. The phototropic curvature in the Avena coleoptile is caused by a difference in the rate of growth of the two sides, the darkened side growing more rapidly than the illuminated. Fitting observed that a positive phototropic curvature resulted whether an incision was made upon the lighted (front) or upon the shaded (back) side. This meant that if stimulus conduction occurred only in living tissue, it must take place in the former case on the back side and in the latter on the front side. The final result of stimulus conduction is the same in both cases; i.e., the shaded side of the Avena coleoptile grows more rapidly than the illumi- nated. Fitting explained this with the assumption . . . that the polar opposition (polarity), which is induced by an exter- nal stimulus in all parts (cells) of the organ of perception, is spread out through the living tissues in a physiologically radially symmetrical zone of response. There is no lateral polarity; all the parts (including all the cells) which made up the path of stimulus conduction become "polarized" (longitudinally) in the same way. Because of this the responding zone gives rise to a curvature, either positive or negative, which is determined solely by the direction of this polarity. The polar- ity is dependent indirectly upon the external stimulus. Curvature increases until this "polarity" is removed again, according to the circumstances. Stated in other words: The individual cells of the unilaterally illuminated tip become polarized so that a difference arises between the front and the back side, and this polarity is trans- mitted to the cells in the darkened basal portion. Boysen Jensen. — In 1907, before Fitting's work was published, Boysen Jensen began experiments on the processes of stimulus conduction in the Avena coleoptile. It was found that the con- duction of a stimulus from the unilaterally illuminated tip to the darkened basal portion could be arrested by an incision upon the back side, while this was not the case when an incision was made INTRODUCTION AND HISTORICAL SKETCH 9 -f^ II -Coleoptile tip I- Rubber band on the front. These investigations were continued in Pfeffer's laboratory in Leipzig in 1909 and were repeated later with the same results in the plant physiology laboratory in Copenhagen. The method of investigation (Boysen Jensen, 1910, 1911) was briefly as follows: Avena seedlings were grown singly in small glass vials (10 by 2.5 cm.) filled with soil at a temperature of 20°C. in a darkroom. When a length of 2 to 3 cm. was attained, the coleoptiles were used; the experi- ments were performed in a darkroom with a humidity of 50 to 60 per cent. The light source was a Nernst lamp placed approximately 100 cm. from the experimental plants. Rectangu- lar pieces of black paper about 9 cm. high were wrapped around the culture dishes to darken the basal portion of the plants (Fig. 3). Two screens held in place with a rubber band were placed around each glass; one screen extended to the bottom of the glass, while the other could be moved up and down and adjusted to the desired height. Even when these were not closed at the top, control experiments showed that light pene- trating from above did not produce a phototropic curvature. Transverse incisions were made with a sharp scalpel 2 to 3 mm. below the tip and extending about to the middle of the coleoptile. The basal portion of the coleoptile was darkened, and only the apical 1 to 2 mm. portion was illuminated vmilaterally. These light exposures produced phototropic curvature in the darkened basal portion only when the incision was on the front side of the coleoptile and not when it was on the back (shaded) side. Next it was shown that the absence of phototropic curvatures in coleoptiles with an incision on the side away from the light was not due to the fact that the experimental plants had lost either their sensitivity to light or their ability to respond in the basal region. Coleoptiles wounded on the side toward the light showed marked -Upper shade Lower shade Fig. 3. — Method of darken- ing Avena seedlings. The cul- ture dish is covered with cylindrical paper shades. The tip of the coleoptile protrudes through an opening in the top. 10 GROWTH HORMONES IN PLANTS stimulus conduction and phototropic curvature in tiie basal portion, pro\'ing that they had not been affected by the incision. Coleoptiles with cuts on the back showed curvature in the basal region when the tips were darkened and the bases illuminated from the front. Searching for an explanation of the disagreement between Fit- ting's investigations and those of Boysen Jensen, the latter repeated the experiments under the same conditions as those used by Fitting. The plants were kept in a saturated room, and their basal portions were darkened with the type of screen used by Fitting. Boysen Jensen then obtained the same result as Fitting, for the coleoptiles curved toward the light even when the incision was on the back side. Curvature did not occur, however, if a small piece of mica was inserted into the incision in the back side of the coleoptile (Fig. 1). If a thin transverse section from a Calamus stem was inserted in the cut instead of a piece of mica, then the stimulus was conducted past the incision. These experiments may be summarized as follows: When a transverse incision is made on the same side of the coleoptile that is unilaterally illuminated, there is invariably a conduction of the stimulus from the unilaterally illuminated tip to the darkened basal portion. If, however, the incision is made upon the back side, stimulus conduction takes place only when the coleoptile is in a saturated atmosphere or after the wound surfaces become closely pressed together. Even in a saturated atmosphere, con- duction is checked by the insertion of a thin piece of mica ; it is not checked by insertion of a thin section of the living Calamus stem, which has large bundles and permits the passage of water and dissolved substances. These results are readily explainable on the assumption that the stimulus is conducted upon the shaded side of the coleoptile and that it can be transmitted across an incision. Pfeffer was sceptical of the correctness of this theory, and so Boysen Jensen carried the investigations further. A deep incision was made upon the back side of the coleoptile so that only a very small connection remained between the tip and the basal portion on the front side. In this case, also, a transmission of the stimulus could be demonstrated, provided a close contact existed between the cut surfaces. Pfeffer maintained that so long as the tip is connected by any living substance with the basal region, INTRODUCTION AND HISTORICAL SKETCH 11 conduction of a stimulus over the incision is not proved beyond objection. There remained the problem of demonstrating that conduction of the stimulus can take place even when the living connection between the tip and base is completely destroyed. Accordingly, two wedgelike incisions were made in the coleop- tile about 1 cm. (or less) from the tip. Then the tip was removed, and the upper part of the foliage leaf was taken off to about 2 mm. above the womid. A small drop of gelatin solution was placed upon the cut surface of the coleoptile stump, and the tip was replaced in its original position, being held there by a ring of cocoa butter. When this replaced tip was unilaterally illumi- nated above the level of the cut, a decided positive phototropic curvature appeared in the darkened basal region (Fig. 1). This demonstrated beyond question that the stimulus could pass over an incision. Conduction of the phototropic stimulus was found to pass downward also when the cut surfaces were separated from each other by a thin section of Calamus. Similar experi- ments on the conduction of the stimulus in negatively geotropic curvature in the Avena coleoptile were carried out with the same result. From these investigations it became clear that conduction of the stimulus in phototropic curvature takes place by the down- ward movement of a growth-promoting substance upon the back (shaded) side of the coleoptile. Paal. — The correctness of Boysen Jensen's work was questioned by van der Wolk (1911). Later the experiments were repeated and confirmed by Boysen Jensen and many other investigators using somewhat different methods. Paal (1914, 1918) worked with coleoptiles separated from the seed and from the primary foliage leaf. After these empty coleoptiles had been placed in damp sand, the tips were cut off by smooth incisions and then replaced tightly. In about 88 per cent of the experiments a stimulus conduction from the unilaterally illuminated tip to the darkened basal region was demonstrated. Paal made the addi- tional discovery that if the excised tip was placed on only one side of the coleoptile stump (Coix), greater growth occurred on the side beneath the replaced tip, and the coleoptile exhibited a marked curvature (Fig. 1). He replaced a decapitated tip of Avena in its normal position, inserting a piece of mica across one half of the stump so that tip and base were in contact only on one 12 GROWTH HORMONES IN PLANTS side. A curvature developed toward the side with the mica plate. By inserting platinum foil between the tip and the base, transmission of the phototropic stimulus was inhibited. In this way, Paal proved that the stimulus was not electrical. Stark and Drechsel. — An advance in technique was reported by Stark (1921a) who appUed unilaterally to a coleoptile stump a block of agar which contained expressed sap from Avena coleop- tiles (Fig. 1). Curvature followed. Stark and Drechsel (1922) perfected another method in which the coleoptiles were unilat- erally cut a few millimeters from the tip with a sharp scalpel, the tip was then removed, and the exposed primary leaf was carefully pulled out, leaving the empty coleoptile attached to the seed. By this method they investigated transmission of the phototropic stimulus from a stimulated tip into the base of the same individ- ual or different individuals of the same species or different species and genera. The desirability of a chemical analysis of the growth-promoting substance and a comparative study of photo- and geotropic reactions as a means of obtaining a unified explana- tion of the two responses was recognized. Stark's investigations will be discussed at some length later, as will those of Seubert (1925), who found that saliva, diastase, and malt extract were growth promoting. Formulation of the Growth-substance Explanation. — After Boysen Jensen's results had been confirmed, there was some criticism of his experimental methods, and the suggestion was made that he had only hypothecated the existence of a growth- promoting substance in the Avena coleoptile, while Paal had demonstrated it. Boysen Jensen discusses this as follows. It is certainly not superfluous to see whether or not my experiments in the years 1909-1910 were without objection and what conclusions were drawn from these investigations. With regard to the first point, Padl maintained that the general set-up in my experiments with darkened coleoptiles was not reliable and that the experimental plants were insufficient in number. In considering the first point, it must be said that over one-half of my experiments were carried out with paper tubes as used by Rothert and Fitting. These are more reliable than the method used by Paal, and the usefulness of this method has been proved by controlled experiments both by Fitting and by myself. In addition, I could demonstrate a conduction of geotropic stimulus from the separated tip to the basal INTRODUCTION AND HISTORICAL SKETCH 13 This region even in plants that had not been subjected to light, objection can then surely be overruled. The next question is whether a sufficient number of experiments were carried out. I disregard here all incision experiments and confine myself to the experiments mth removed and replaced tips. Thirty-five plants were used for the experiments on phototropic stimulus con- duction; of these 2.5 reacted positively, 9 remained straight, and 1 showed Fig. 4. — Phototropic curvature in decapitated Avena coleoptiles. The tips were replaced upon the three plants at the left, while the two plants at the right served as controls. The plants with tips curved toward light. a weak negative curvature. Furthermore, a geotropic stimulus con- duction was demonstrated in 12 plants (number of experimental plants, 12). Information as to the strength of the curvatures is given by photographs in my papers of 1911 and 1913 (Figs. 4 and 5). The curvatures are so strong and the percentage of curved plants so high that the published material is sufficient to prove a stimulus conduction from the removed tip to the basal portion. Fig. 5. — Geotropic curvature iu decapitated Avena coleoptiles. The tips were replaced upon the two plants at the left, while the two plants at the right served as controls. The plants with tips curved away from the force of gravity. Relative to the conclusions that I drew from the experiments pub- lished in 1911, I should like to cite the following from that paper: "We are able, therefore, to represent the facts more or less as follows. Under the influence of the action of unilateral light there is produced a differentiation between the front and the back faces at the tip of the coleoptile (and not, as Fitting thought, in the individual cells of the 14 GROWTH HORMONES IN PLANTS coleoptile). We shall put aside provisionally the question of whether this differentiation is of a ' physical ' or ' chemical ' nature. The stimulus is transmitted from the back side of the tip down the length of the back side of the coleoptile. Since, as I have shown, there is no modification in the rate of growth on the front side of the coleoptile, the positive phototropic curvature must result from the accelerated growth rate which is induced by the light stimulus." In his discussion of the nature of the transmission, Boysen Jensen (1911) wrote further: "It seems to me that my studies of the transmission of the stimulus in the Avena coleoptile render it probable that in this case the trans- mission of the stimulus is of a material nature and produced by con- traction changes in the tip of the coleoptile. In every case it would seem necessary to resign oneself to a hypothesis according to which the transmission of the stimulus in Avena would be due to physical causes (changes of pressure, etc.), which is perhaps the case for Mimosa; in fact we have seen that the stimulus can be transmitted across an incision made in the coleoptile. For other reasons it is thought that the transmission of the stimulus is of a chemical nature. As may be recalled, the condition for transmission across an incision was that the edges of the wound were kept humid and held one against the other in a way to favor as much as possible transmission of a substance or of ions across the incision. Another reason: It has never been proved that the transmission of the stimulus could take place under water. The water under such a condition should prevent this transmission, which can be explained only in a hypothesis where the transmission of the stimulus would have been due to the migration of a substance or of ions, which would diffuse into the water and no longer act." It may be said, therefore, that in 1911, Boysen Jensen's conception of phototropic curvature in the Avena coleoptile was the following: Under the influence of unilateral light, a polarity is formed in the coleoptile tip which is associated with an unequal distribution of a substance upon the front and back side of the coleoptile. The substance in question migrates down the back side (he used the expression ''migration," since it seemed clear, even at that time, that it could not be a process of diffusion) and causes an acceleration of growth upon the back side in the basal region, which produces a phototropic curvature. This conclusion seemed the only possible one. The existence of a growth substance in the Avena coleoptile during photo- INTRODUCTION AND HISTORICAL SKETCH 15 tropic curvature was demonstrated, therefore, through these investigations. Contemporaneously with these studies, in the years 1909 and 1910, Fitting pubUshed two works in which it was shown that orchid polUnia contain a substance that produces a swelhng of the gynostemium. According to Fitting (1910), this substance is a hormone; and according to the more recent investigations of Laibach (1932) and of Laibach and Maschmann (1933), it is probably identical with the growth substance of the Avena coleoptile. In the past few years, marked advances have been made in our knowledge of the occurrence, movement, and quantitative determination of the plant-growth substances (Went, 1928a). Recent outstanding contributions to the chemistry of the subject (Kogl, Haagen Smit, and Erxleben, 1932-1935) have opened up new phases of the general investigation which may become valuable in horticultural practice (Bouillenne and Went, 1933; Hitchcock and Zimmerman, 1935; Cooper, 1935). Detailed dis- cussion of the more significant aspects of the growth-substance problem will be presented in the chapters that follow. SUMMARY The starting point for growth-substance investigations was the demonstration of a growth-promoting material in the tip of the Avena coleoptile, as shown by phototropic curvature. The brief historical sketch wliich has been presented here indicates that the growth-substance explanation of photo- and geotropism had its origin many years ago. About one-quarter of a century has elapsed since a hypothesis was suggested according to which a stimulus substance in the coleoptile was displaced by the effect of unilateral light, or gravity (Boysen Jensen). Other contribu- tions to the solution of the problem followed (Paal, Stark, and Seubert). A new impetus was given to the subject when Went (1927, 1928a) published his method of procedure for extracting growth substance and demonstrating the quantitative relation- ship between it and growth in the Avena coleoptile. An ever increasing fund of knowledge about hormone activity is con- tinually extending our understanding of tropisms and the whole problem of normal growth. CHAPTER II DETECTION AND QUANTITATIVE DETERMINATION OF GROWTH SUBSTANCES In demonstrating the presence of growth substances, the coleoptile of the Avena seedhng has been used almost exclusively as a test object. Its structure and sensitiveness to stimuli make it suitable for quantitative tests as well as qualitative demonstra- tions. A minute amount of a growth hormone applied unilat- erally near the tip of a coleoptile brings about increased growth on the side receiving the growth substance, and this produces a growth curvature. The amount of curvature can be used, within certain limits, to indicate the concentration of the appUed growth substance. In a similar way Cephalaria seedlings have been used as quantitative test objects (Soding, 1935a, h). Other methods and numerous other plant organs are equally useful for quaUtative demonstrations (see Figs. 17, 37, 39). THE TEST FOR THE PRESENCE OF GROWTH SUBSTANCE WITH THE AVENA COLEOPTILE The Culture of the Avena Seedling for Use as a Phytohormone Test Object. — A genetically uniform variety of Avena sativa has been used almost universally in the plant-hormone work of the past few years. It is obtainable from Dr. E. A. Akerman of Svalof, Sweden, and is known as siegeshafer, or victory oats. While other uniform strains may be used just as successfully, there is an important advantage in all workers having genetically comparable test material. The variety gul naesgaard is used in the Copenhagen laboratory. Culture Conditions. — The generally accepted culture method necessitates a darkroom for growing the Avena seedhngs and for carrying out the quantitative determinations, although Soding (1935a) has recently described a dayhght method. The arrange- ment of such a darkroom has been described repeatedly in the Uterature {e.g., Linsbauer, 1922; Went, 1928a; Nuernbergk, 16 DETECTION AND QUANTITATIVE DETERMINATION 17 19326). It is best to have controlled temperature and humidity; fluctuations of 0.5°C. and + 1 per cent relative humidity make little difference in most experiments. The laboratories that have such controlled conditions usually maintain the temperature at 25°C. and the relative humidity at 90 per cent. If such a laboratory is not available, a thermoregulator will provide temperature control for a darkroom, and a suitable humidity may be obtained by placing the experimental plants under bell jars. Light for the darkroom must be phototropically inactive, which o means that wave lengths longer than 5,500A. may be used. Ruby glass or filters such as Corning 246 or Schott OG-2 are satisfactory. The culture of the seedlings involves certain difficulties, perhaps the greatest of them being that at times the first internode ("mesocotyl" of the older literature — see Avery, 1930) elongates under the coleoptile and by its nutations makes a whole series of plants useless. Numerous factors have been suggested as the cause of this elongation, among them low temperatures (Blaauw, 1909), low moisture content of the soil (Noack, 1914), strong carbon dioxide content of the atmosphere (Maria de Vries, 1917), etc. Through the investigations of Lange (1927, 1929), Beyer (19276), duBuy and Nuernbergk (19296), and Hamada (1929, 1931) it has been shown that elongation of this internode in Avena can be suppressed by illuminating the seeds during the soaking period (see below) with bright daylight. Rothert (1894, p. 27) had already pointed out that temporary illumination was effective in checking the development of this internode. DuBuy and Nuernbergk (1929a) showed that its elongation can be checked also by heat radiation. It has been shown that nutations may occur in the coleoptile (Bremekamp, 1925; Lange, 1925; Pisek, 1926; Beyer, 19276; Lange, 1933), but these have no role in the usual culture diffi- culties. They become apparent only when the plants are put on the clinostat. These curvatures take place in the plane of symmetry of the plant, either away from the seed (e.g., \'ictory oats) or toward the seed {e.g., orien oats). In addition, occasional torsions may appear in the coleoptile (Beyer, 19276; Tammes, 1931) ; these have no special significance in the culture of experi- mental plants for hormone-test objects, nor have the photonastic curvatures described by Lange (1933). 18 GROWTH HORMONES IN PLANTS Culture Methods. — Whether the Avena seedlings are grown in soil, sawdust, or water culture, the preliminary treatment is about the same. The glumes may or may not be removed for soil or sawdust culture; the seeds are soaked in water for 2 to 4 hours, after which they are placed in petri dishes on moist filter paper and allowed to remain for about 36 hours. (If illuminated for a few hours at the beginning the first internode remains short.) For Boysen Jensen's soil-culture method, the seeds are removed from the petri dish and placed in glass vials, 20 by 100 mm., filled with screened, sandy garden soil (Fig. 6). The soil should be well-watered so that no further watering will be necessary but not Fig. 6. — Curvatures produced by placing Avena coleoptile tips unilaterally upon decapitated coleoptiles. too wet, for the plants then become less sensitive. Each vial contains but one seed, and 25 to 30 \'ials are held together by an elastic band. These are moved into the darkroom and placed in a petri dish, then covered with a small bell jar so that the air will remain saturated. Under the usual conditions in the dark- room at a temperature of 21 to 21.5°C., the coleoptile appears in two days. Then the bell jar is removed, and the coleoptiles continue their growth under low-humidity conditions for 24 hours. In this time they attain a length of 15 to 25 mm. and are ready for use. Navez and Robinson (19326) planted the seeds with glumes removed in sterile, purified, maple sawdust contained in glass vials 15 by 25 mm. The sawdust retains about 4.3 times its weight of water. The seed is planted dry, embryo side upward, and inclined about 10 deg. from the vertical. The end of the seed away from the embryo may or may not be allowed to protrude slightly above the level of the sawdust. In this method, germina- tion is allowed to proceed in a light-tight chamber at a tempera- DETECTION AND QUANTITATIVE DETERMINATION 19 ture of 22 to 22.5°C., and after 72 hours the seedUngs are ready for use; they are then 25 mm. long on the average. Went (1928a) used plants grown in water culture. After the preliminary treatment mentioned above, they are transferred to the darkroom and allowed to remain in the germinating dishes until the seedling roots are a few millimeters long. They are placed then in glass holders over zinc or glass trays of water, as indicated in Fig. 7. At a temperature of 25°C. and relative ^ Fig. 7. — Diagram illustrating the water culture method of growing Avena seedlings as test objects for making growth-hormone determinations. The oat seedlings are supported in glass holders held in brass clamps; these fit firmly into slots in a wooden block. The roots of the seedlings dip into a tray of water. Orientation of the coleoptiles may be accomplished by adjusting the positions of the brass clamps and glass holders. {Modified from Went, 1928a.) humidity of 90 per cent, the coleoptiles are ready for use after about 30 hours. Each of the methods described has its advantages and dis- advantages, which must be evaluated at the time when a partic- ular experiment is contemplated. Culture of the seedlings in \dals may facilitate working with indi\'idual plants but increases the number of manipulations when many tests are being made. Difficulty is encountered in properly orienting the coleoptiles for uniform application of the plant parts or agar blocks to be tested. The fixed position of the seedlings in the soil or sawdust, which raises this difficulty, turns out to be an advantage if much 20 GROWTH HORMONES IN PLANTS handling of the containers is necessary. Fine soil or sand may pack tightly enough to hinder proper aeration of the roots and thus inhibit growth of the seedlings. Sawdust, however, which provides excellent aeration and adequate room for root growth, must be boiled sufficiently to free it of toxic substances, such as resins and tannins. In using hquid culture methods, several factors must be considered. Roots immersed in solution may not be sufficiently aerated for vigorous growth. Attention should be given to the solution bathing the roots — whether it shall be distilled water or some nutrient mixture. In water culture it is necessary to handle the seedlings twice, once when they are placed in dishes to germinate and again when they are mounted in holders. This, however, provides an opportunity for selecting the uniform plants for use in testing, and those which are not satisfactory may be discarded. The advantages, which may outweigh the difficulties of the method are that the seedling holders allow for easy handling of many test plants, for perfect orientation of each seedling so that its coleoptile is vertical and for convenient photographing of the resulting curvatures. For any quantitative work these qualifications are of distinct advantage. For most qualitative studies, the less complicated methods of soil or sand culture are entirely adequate. Preparation of the Avena Coleoptile for Use. — The small quantities of growth substances in plant organs make difficult direct proof of their presence by chemical means. For the pur- poses of many biological experiments it is satisfactory to obtain indirect evidence of their existence by their activity in certain measurable growth reactions. Growth substances, in common with other hormones and activators, produce in the living organ- ism responses out of all proportion to the size of the stimulus. Although they have been extracted and purified from many plant sources, the usual method of detecting them is by means of biological indicators. For this purpose the Avena coleoptile has been used more extensively than any other organism. Its culture up to the time of the test has been described, and now some of the methods of procedure will be outlined. Decapitation and Unilateral Application. — Coleoptiles to be used for test purposes should have attained a length of 25 to 40 mm. before they are decapitated in the following way (Fig. SB) : DETECTION AND QUANTITATIVE DETERMINATION 21 <\ Fig. 8. — Technique of testing for growth hormone with agar blocks applied to the decapitated coleoptiles of Avena seedlings. A, diffusion of growth hormone into agar; a, agar plate: h, plant material (e.g., coleoptile tips) to be tested for the presence of growth hormone is placed in contact with agar for 2 hours; c, the agar plate containing growth hormone is cut into small blocks. {After Went, 1928a.) B, decapitation of Avena coleoptile and unilateral application of agar block. The tip of the coleoptile is removed and the foliage leaf is pulled out part way and cut off; a small portion is left extending from the apex of the coleoptile stump. An agar block containing the hormone to be tested is placed over a vascular bundle on one side of the cut apex (see Fig. 13). The ensuing curvature is proportional, within limits, to the concentration of growth hormone in the agar block. C, end view of decapitated coleoptiles in contact with agar blocks. The contact area is the same in both a and b though the volume of one block is 8 times that of the other. {After Thimann and Bonner, 1932.) 22 GROWTH HORMONES IN PLANTS a unilateral incision is made (Stark and Drechsel, 1922) 2 to 3 mm. from the tip with a sharp scalpel or razor blade; the tip then is removed by a slight jerk with forceps or the thumb and forefinger. Went (1928a) removed 5 to 8 mm. of the tip when decapitating. Various sorts of instruments have been made (Went, 1928a; van der Weij, 1931; duBuy, 1933) to aid in decapitation. The primary leaf protrudes from the cut surface of the coleoptile stump after decapitation. It may be pulled loose and carefully drawn out with a pair of forceps until only the basal 5 mm. of it remain inside the coleoptile; the protruding portion then is severed about 5 mm. above the tip of the coleop- tile stump. When the coleoptile has been prepared as above, the object to be tested, that is, a small plant organ, portion of an organ, or agar block, may be applied unilaterally as in Fig. 1 (Paal and Stark) and Fig. SB. If the object contains substances that influence growth, they migrate down one side of the coleoptile and cause a curvature. The actual procedure, from the time of decapitation on, varies with different workers: Immediately after decapitation (of 15 to 25 mm. coleoptiles), Boysen Jensen applies unilaterally the object that is being tested for the presence of growth sub- stance. He cautions that while curvature is taking place the humidity must not be so high that the plants guttate and disturb the object being tested or so low that the object dries up; plants grown in soil are best placed under bell jars which are partly lined on the inside with moist paper. The rapidity with which curvature takes place depends upon the temperature; in Boysen Jensen's laboratory (Copenhagen) the work is carried out at 21.5°C. At this temperature the maximum curvature occurs after 2^2 to 3 hours. The use of a longer experimental period is not recommended, since "physiological regeneration" of the tip can influence the reaction. In the Utrecht laboratory the coleoptiles are decapitated when 40 to 60 mm. long (Went, 1928a) and allowed to stand 40 min- utes; at the end of this time, all coleoptiles that are not perfectly straight are eliminated. Any guttation fluid that may appear at the tip of the decapitated coleoptile is removed by "blotting" with a small piece of filter paper. The object to be tested is unilaterally applied and allowed to remain for 120 minutes, at DETECTION AND QUANTITATIVE DETERMINATION 23 the end of which time the degree of curvature may be measured, as described later. The temperature of the laboratory at Utrecht is maintained at 22°C. and at a relative humidity of 90 to 95 per cent. If it is desirable to work with greater numbers of test plants, the coleoptiles may be decapitated a second time 60 or 90 minutes after the first decapitation (van der Weij, 1931). The time schedule mentioned is important only for the quantita- tive work discussed later. Growth Curvature of the Coleoptile. — The appearance of a curva- ture after the application of the unknown material is evidence for the presence of growth substance. A negative curvature (bend- ing away from the side with the applied object) indicates a growth-promoting substance, whereas a positive curvature shows that growth-retarding substances are present (Fig. 1) (Stark). Quantitative methods for determining the amount of growth- promoting substances present are described in the last part of this chapter. If no growth curvature occurs, it means either that the object being tested contains no growth substance or that there is some factor which disturbs either the transfer of the growth substance or its effectiveness after entrance into the Avena coleop- tile. Mention of cases in which the latter is true is made at the end of the chapter. Preparation of the Material to Be Tested. Direct Application of Plant Parts to the Decapitated Avena Coleoptile. — Many plant organs or parts of organs such as coleoptile tips, coleoptile cylinder segments, root tips, etc., may be tested for the presence of a growth substance by placing them (Figs. 6 and SB) unilat- erally upon decapitated Avena coleoptiles (see Paal, 1918; Stark, 19216; Nielsen, 1924). This is the simplest method and the first one employed in attempting to detect growth substances in some new object. Application of Material to Be Tested in Agar and in Lanolin. — Frequently it is not feasible or desirable to apply the plant parts to be tested directly to the Avena coleoptile. Other methods depend upon the fact that growth substances are soluble in water, alcohol, and ether, and that they are stable in agar and lanolin (wool-fat) paste. With some of these methods it is possible to concentrate the extract obtained from a quantity of plant material before making the biological test and thus demonstrate that growth substance is present even though only in small amounts. 24 GROWTH HORMONES IN PLANTS 1. DETECTION OF GROWTH SUBSTANCE IN FLUIDS. — If a fluid is to be tested for growth substance, the reaction must be weakly acid; one neutrahzes where necessary with sodium bicarbonate and adds a little citric or acetic acid (0.2 cc. per liter). If the solution is very pure, it may be necessary to add some potassium chloride (169 mg. per liter) (Kogl and Haagen Smit, 1931, Mitt. I). The substratum is prepared in the following way : A computed amount of agar is carefully washed with tap water for 24 hours; after- ward the agar plus the absorbed fluid is weighed again, and enough water is added to produce a 3 per cent agar. The solution Fig. 9. — Curvatures resulting from application of agar blocks containing saliva to one side of decapitated coleoptiles. (After Seubert, 1925.) which is being investigated for growth substance is then mixed with an equal amount of the substratum. After solidification, small blocks of equal size are cut out and placed unilaterally upon decapitated Avena coleoptiles (for size of blocks, see description under quantitative determination). Instead of mixing the solu- tion to be tested with melted agar, agar blocks can be placed in the solution for 1^^ hours. Through control experiments it can be shown that the agar substratum has no effect upon the Avena coleoptile. This method was originally used by Stark (19216) and later by Nielsen (1924), Seubert (1925), and others (Fig. 9). It is possible also to detect the presence of growth substance in a fluid by mixing it in various proportions with lanolin (Laibach, 19336). The lanolin growth-substance paste may be applied unilaterally to intact coleoptiles; if bending occurs, it may be concluded that growth substance is present (Fig. 37 A). DETECTION AND QUANTITATIVE DETERMINATION 25 2. DETECTION OF GROWTH SUBSTANCE IN POLLEN. — A mixture of pollen and 1 cc. water, weakly acidified with acetic acid, may be applied in a small chamber around the stump of a decapitated coleoptile. Growth may be measured interferometrically (Fig. 28) (Laibach and Kornmann, 1933a) or in any other suitable manner. Pollen mixed with agar (weakly acidified) may be cut into blocks and applied unilaterally to intact coleoptiles (Laibach and Kornmann, 1933a). Pollen may be suspended in lanolin by mixing thoroughly in the proportion of 50 mg. air-dried pollen, 1 cc. water (weakly acidified), and 1 g. anhydrous lanolin. The mixture may be applied to various kinds of test objects, where it will induce bending (Fig. 37) : intact or decapitated coleoptiles, epicotyls of Phaseolus multiflorus, aerial roots of various species, petioles of Coleus, etc. This method of preparation and applica- tion is very useful because the growth substance is given off into the plant very slowly, and the lanolin does not dry out (Laibach, 19336). 3. DIFFUSION OF GROWTH SUBSTANCE INTO AGAR. — Went (1928a) demonstrated that growth substance would diffuse out of decapitated coleoptile tips if the latter were allowed to remain standing on 3 per cent agar blocks for approximately 2 hours (Fig. 8A). Since this method was first described, growth sub- stances have been "diffused" out of numerous other plant parts. The agar blocks are then applied unilaterally to decapitated coleoptiles as described above. Details of Went's procedure are to be found under the discussion of quantitative determination. 4. DIFFUSION OF GROWTH SUBSTANCE INTO DEXTROSE AGAR. Boysen Jensen (19336) found that it was impossible to obtain growth substance from roots by standing the decapitated root tips on 3 per cent agar, but satisfactory demonstrations were made possible by the use of a dextrose salt agar of the following composition: 3 g. agar, 10 g. dextrose, 0.1 g. calcium nitrate, 0.025 g. potassium monohydrogen phosphate, 0.025 g. magnesium sulphate, a trace of ferric chloride, and 100 cc. water. The agar blocks must be made up fresh the day that they are to be used, and this is done most easily if 10 cc. of the agar mixture is spread out upon a warm glass plate (size 10 by 10 cm.). Blocks can be cut from this by using parallel knives; the size of the blocks used is 2 by 2 by 1 mm. Upon such blocks root tips of Zea mays or Vicia Faha are placed for 2 to 4 hours ; the blocks are 26 GROWTH HORMONES IN PLANTS then moistened with a sohition of 1 g. citric acid, 50 cc. alcohol, and 50 cc. water. If necessary, they may be kept for a time in the refrigerator and later applied unilaterally to decapitated Avena coleoptiles (Fig. 10). 5. DIFFUSION OF GROWTH SUBSTANCE INTO WATER. Another method of extraction has been described by Gorter (1932). The pieces of plant, for example, coleoptile tips, are placed upon a layer of sand which is soaked with water. After a time they are removed, the water filtered off, and the sand washed repeatedly. The filtrate and rinsing water are either evaporated in a vacuum or extracted with ether. The residue from the evaporated ether w) Iw ^ Fig. 10. — Curvatures of decapitated Avena coleoptiles resulting from applica- tion of agar blocks upon which root tips of Vicia Faba had been standing for 4 hours. (From Boysen Jensen, 1933.) extract is dissolved in water which contains 160 mg. potassium chloride and 0.2 cc. glacial acetic acid per liter; agar blocks are placed in the solution, and later these are tested for growth sub- stance in the usual way. 6. EXTRACTION OF GROWTH SUBSTANCE WITH ALCOHOL. Growth substance can be extracted also from plant parts with alcohol. The alcohol which is poured off is concentrated in a vacuum, and the residue dissolved in an optional amount of water; this solution is investigated either directly after mixing with agar or after it has been purified with ether (the latter is described later). 7. EXTRACTION OF GROWTH SUBSTANCE WITH CHLOROFORM. Thimann (1934) has described a method of chloroform extraction of growth substance from tissues. It consists, in brief, of killing the fresh material by immersing it in a small amount of chloro- form, adding 0.1 A'^ hydrochloric acid to the extent of about one- fifth the volume of the chloroform, and grinding the mixture thoroughly. The extract containing the growth substance is DETECTION AND QUANTITATIVE DETERMINATION 27 poured from the residue into, a small separatory funnel where the aqueous layer is drawn off and placed again with the ground tissue. A small amount of chloroform is added, and the mixture is ground again; the extract is placed in the separatory funnel as before, and the aqueous layer is again returned to the ground tissue. The same procedure is repeated a third time. The total chloroform-water mixture then is shaken thoroughly, and the chloroform layer separated off. The latter contains the growth hormone and is transferred to a small test tube and evaporated off. The minute amount of hpoidal material which remains is taken up in a very small volume of water, to which an equal volume of 3 per cent agar is added. If 0.15 cc. each of water and agar are used, the resulting 0.3 cc. may be cast into a small block 8 by 10.7 by 1.5 mm. (there is always some loss in volume, and the amount left will approximately fill a mold of this size), which in turn may be cut into 12 smaller blocks of equal size if quantitative determinations are desired (see Went's quantitative method) . 8. EXTRACTION OF GROWTH SUBSTANCE WITH WATER. Thi- mann also tried water extractions with fair success but found that the growth substance was rapidly inactivated by oxidizing enzymes. QUANTITATIVE DETERMINATION OF GROWTH SUBSTANCES Determination of Growth Curvature Produced by the Uni- lateral Action of a Growth Substance. — After demonstrating that growth substance would "diffuse out" of hving parts of plants into agar, Went (1928a) showed that if agar blocks were placed unilaterally on decapitated coleoptiles (after the method of Stark, 19216), there woidd result a curvature proportional within certain limits to the amount (later shown to be concentration) of growth substance in the block (Fig. 2) (Went). The investigations of Nielsen (1930a, h) and Dolk and Thimann (1932) have shown a similar direct relationship between concentration of growth substance and coleoptile curvature (Fig. 11), and it is upon this simple fact that quantitative determinations depend. The tests should be carried out in a darkroom under photo- tropically inactive light. Boysen Jensen's Quantitative Method. — This involves a deter- mination of the difference in length of the convex and concave 28 GROWTH HORMONES IN PLANTS sides of a curved coleoptile. The concentration of growth sub- stance necessary to bring about a certain difference in the length of the two sides is designated as one WAE (Wuchsstoff Avena 0.10 0,08 - 0,06 - 0,04 - 0.02 - 0 20 40 60 80 100 120 140 160 180 200 Fig. 11. — Curve showing relationship between amount of bending in the Avena coleoptile and the concentration of "rhizopin" applied unilaterally in agar blocks. Ordinate: d value. Abscissa: relative concentration of the growth substance. {After Nielsen, 1930.) Einheit — see 5, page 29). The method may be outlined briefly as follows: 1. The test plants are grown in soil cultures and decapitated as outlined under "culture methods" (p. 18). 2. The agar blocks 2 by 2 by 1 mm. containing the growth substance (see 4, p. 25) are applied unilaterally to decapi- tated coleoptiles and are allowed to remain for 3 hours. 3. The curvature produced by the unilateral effect of growth substance is the result of a difference in length between the convex and concave sides; this difference, d, is the result of unequal rates of growth. When measured in millimeters, this difference becomes a clear expression for the effect of growth substance l+d r t Fig. 12. — Diagram showing derivation of d value. /, length of coleoptile, inner curved side; d, difference in "PO" the coleoptile (if the approximate growth rate of decapitated coleoptiles is known). The d value may be determined by the method employed by Purdy (1921) (see Fig. 12). If Hs the length of the curved part of the Avena coleoptile, d the difference in length, t the diameter of the coleoptile, and r the radius of the curvature, the following equations are obtained: length between inner and outer curved side; I -\- d, length of outer curved side; t, diameter of the coleoptile; r, radius of curvature. I r +t l+d' l_ d' , tl a = — r DETECTION AND QUANTITATIVE DETERMINATION 29 I is determined with millimetric paper, and t with a micrometer screw; the diameter of the organ is usually about 1.5 to 1.7 mm. To measure r, arcs with various radii (0.6 to 10 cm.) are drawn upon paper. If the curved Avena coleoptile is compared with the arcs, the radius of curvature of the coleoptile can be measured, and the d value computed in millimeters from the equation given (p. 28). (For applications of the radius of curvature for curvature measurements, see Rothert, 1894.) 4. The number of experimental plants to be used for a determination depends, of course, upon the degree of accuracy that one wishes to achieve. Various experimental series of Nielsen (19306) give detailed information concerning the fluctuations in magnitude of curvature in the experimental plants. From these data it has been computed that when d is equal to 0.56 mm., the standard deviation of a single measurement is about ±0.09 mm. A mean error of about ±0.03 mm. has been found when using 9 plants; the error is reduced to approximately ±0.013 mm. with 50 plants. Measurements for general purposes of orientation can be made with about 6 to 8 plants; 10 to 12 and preferably 30 to 40 plants should be used for more exact measurements. 5. The unit of growth substance used in the Copenhagen laboratory (Boysen Jensen, 19316) is that amount, dissolved in 50 cc. water plus 50 cc. 3 per cent agar, which will produce a d value of 1 mm. when the curvature of the Avena coleoptile takes place at a temperature of 21 to 22°C., and the magnitude of this curvature is measured after 3 hours. This amount is designated as a growth-substance Avena unit (Wuchsstoff A-Einheit = WAE). The size of the blocks should be uniform (2 by 2 by 1 mm.), although small deviations have no influence upon the size of the curvature (as Nielsen (19306) and van der Weij (1932) have shown) since this is dependent upon the growth-substance concentration and not upon the amount of growth substance (see also van der Weij (1932) and Thimann and Bonner (1932) on concentration vs. amount); however, the amount of contact surface between the agar and the coleoptile should always be the same (Fig. 8C). The degree of curvature is much greater when the block is placed over a bundle than when it is placed on parenchymatous tissue (Laibach and Kornmann, 19336); hence the block should be placed in contact with a vascular bundle if consistent results are to be obtained (Fig. 13). 6. If the growth-substance content of a solution is to be measured in WAE, a number of dilutions are made from the solution, e.g., 3'2 (is., 1 cc. solution + 1 cc. agar), J4 (1 cc. solution, 1 cc. water, 2 cc. agar), etc., in order to find the dilution that produces a d value of about 0.5 mm. If, for example, the d value is 0.55 mm. with a 3^ dilution, then the original solution contains 4.4 WAE in 100 cc. 7. If it is desirable to determine how much growth substance moves from a plant organ into an agar block in a definite time, the block must have a very definite size, such as mentioned above. The following will serve as an example for computation of the amount of extracted growth substance: If a root tip is placed upon a block of dextrose agar 4 mm.^ in size and allowed to remain for 2 hours, and the block produces in the Avena coleoptile a curvature with a d value of about 1 mm., then the root tip has given off 30 GROWTH HORMONES IN PLANTS 1 /50,000 WAE per hour. However, in practice if the d value is much greater than 0.5 mm., the direct relationship between d and growth-substance con- centration (characterized by d values of 0.1 mm. or less up to 0.5 mm.) no longer holds. As a unit of the amount of growth substance given off into an agar block, the " tip hour " has also been proposed. The amount that comes from an Avena tip in one hour produces a curvature of about 15 deg. and A B Fig. 13. — Unilateral application of agar blocks containing growth hormone to the cut surface of Avena coleoptiles, either in contact with a vascular bundle, A, or parenchyma, B. (After Laibach and Kornmann, 19336.) corresponds therefore to about 1.5 AE (see duBuy and Nuernbergk, 1932). (For a definition of AE see p. 33.) Wenfs Quantitative Method. — This method involves a deter- mination of the angle of curvature of the decapitated coleoptile on which has been placed the object to be tested. The measure- ment is made by means of a protractor, equipped as shown in Fig. 14. The concentration of growth substance necessary to bring about a curvature of one or more degrees is designated in various ways, as discussed on page 32. The method has been modified slightly by several workers since 1928, and the current procedure, assuming that the test plants have reached the proper size, is as follows: DETECTION AND QUANTITATIVE DETERMINATION 31 1 . Three per cent agar plates are prepared from agar which has been tested previously and found to be free from growth substance. DuBuy (1931) has shown that curvatures are reduced when higher concentrations of agar are used. Two sizes of agar plates are in common use: 8 by 10.7 by 1.5 (Dolk, 1930) and 8 by 6 by 1.0 mm. (Went, 1935a). 2. The coleoptile tips, portions of leaves, buds, or other plant parts to be tested for growth substance are freshly severed from the plant and allowed to stand proximal end downward on the rectangular agar plates for a period of 2 hours (Fig. 8A). They should be covered with a bell jar lined with moist paper throughout the period of diffusion. 3. After diffusion the rectangular agar plates are cut up into 12 equal blocks by means of a special cutting device: Dolk proposes that the plates Fig. 14. — Method for determining the degree of curvature in an Avena coleop- tile. The transparent celluloid protractor is placed over a shadow picture (Fig. 15) of the curved coleoptile and the angle of curvature is measured directly by matching the thin line on the celluloid arm with the axis of the curved organ. {Modified after Went, 1928a.) 8 by 10.7 by 1.5 mm. be cut into 12 blocks, each 2.67 by 2.67 by 1.5 mm. (10.7 mm.'); Went suggests that the plates 8 by 6.0 by 1.0 mm. be cut into 12 blocks, each 2 by 2 by 1 mm. (4.0 mm.'). Kogl and his associates cut the agar into blocks 2 by 2 by 0.5 mm. or 2 mm.^ Since it has been shown that size of block is not of great importance as long as the amount of contact surface is the same, any of the foregoing sizes is satisfactory. The larger blocks used by Dolk do not dry out so readily. 4. These 12 blocks are applied unilaterally to 12 of the previously decapi- tated coleoptiles (40 minute interval between decapitation and application of blocks). The time allowed for curvature to take place is 110 (Dolk and Thimann, 1932) to 120 minutes (Went, 1928a). The same procedure is followed with agar blocks from Thimann's chloroform method (p. 26) or with agar blocks made up with different dilutions of growth substance (p. 24). In determining the growth-substance concentration of a solution it is possible also to immerse the blocks in the unknown solution for 1 to 1 J^ hours and then proceed in the usual manner. 32 GROWTH HORMONES IN PLANTS 5. The rack of 12 test plants with their curved coleoptiles is placed directly in front of a sheet of silver-bromide paper, and a shadow picture is taken of the 12 coleoptiles (Fig. 15). 6. After developing the print, the curvatures are determined by meas- uringthe deflection of the coleoptile tips in degrees, a method first intro- duced by Simon (1912); Went (1928a) suggests a simple measuring protractor (Fig. 14) for this purpose, and the photograph provides a permanent record which can be referred to later if desired. Soding (1934) has described a measuring method in which the photographic step is omitted. Each curved coleoptile is removed from the plant and placed upon a glass plate over a protractor. The angle is determined indirectly. Navez and Robinson (1932a) have described an automatic photographic method. Uiiinii Fig. 15. — Shadow pictures of Avena coleoptiles which have curved in response to unilateral application of agar blocks containing growth hormone. These curvatures may be measured with a protractor such as is shown in Fig. 14. Went (1928a) states that curvatures over a range of about 1 to 20 deg. are strictly proportional to the concentration of growth substance in the agar; hence if the mean curvature of 12 plants is 20 deg. or less, an accurate determination of the concentration is possible (Fig. 2, Went, and Fig. 11). If the curvature is much greater than 20 deg. ("maximum angle"), the direct relationship between curvature and concentration no longer exists. 7. METHODS OF EXPRESSING THE RESULTS. — Various uuits have been proposed by workers using this technique, each based upon the degree of curvature of the Avena coleoptile: One unit is that quantity of growth substance that has to be present in 1 cc. of solution to give, after mixing with 1 cc. agar, an angle of 1 deg. at a temperature of 25°C. and a relative humidity of 85 to 90 per cent. The blocks are prepared from the larger rectangular agar plates men- tioned on page 31, and each has a volume of a little over 10 mm.^ One block is applied to each of the 12 test coleoptiles. The average curva- ture of the coleoptiles, in degrees, is multiplied by 12. The product, then, may be expressed as plant units (Dolk and Thimann, 1932). One plant unit is the amount of growth substance applied in one agar block, as above, to give an angle of 1 deg. The growth substance has diffused from a plant part into the agar. These blocks are applied to 12 test plants also, so the average result is multiplied by 12, as in the above. In this case the actual amount of material in each block applied to the plant is but Moo of that present in 1 cc; hence a plant unit is J^oo unit (Dolk and Thimann, 1932). DETECTION AND QUANTITATIVE DETERMINATION 33 One Avena Einheit, or AE, is the amount of growth substance present in one block of agar 2 by 2 by 0.5 mm. that will cause a 10 deg. curvature at 22 to 23°C. and at a relative humidity of 92 per cent (Kogl and Haagen Smit, 1931, Mitt. I). Comparison of the Units of Different Workers. — The relation- ship of the d value used by Boysen Jensen and curvature as measured in degrees (Went and others) is as follows : If d is the difference in length of the two sides of the curved coleoptile, and (p the angle of curvature (Fig. 16), the relationship between them may be stated in the following way: d = . — Pore } } } H til H Fig. 24. — Detailed drawings of cells in longitudinal sections from different levels of a mature Avena coleoptile. The heavy line at the left indicates the relative length of the marked segments designated a, b, c, and d in proportion to the over-all length of the coleop- tile. Compare with Fig. 23. The drawings represent part of the apical end of segment a and small portions from the middle of segments b, c, and d. Compare the small size of the cells near the apex with the elongated cells in the lower portions. Note the extreme length of the epidermal cells. {Adapted after Avery and Burkholder, 1936.) 86 GROWTH HORMONES IN PLANTS 200 180 160 140 V) -! 120 u L. 100 o S 80 Z 60 40 20 SUBEPIDERMIS THIRD LAYER FOURTH LAYER INNER EPIDERMIS -* OUTER EPIDERMIS 10 20 30 40 50 COLEOPTILE LENGTH IN MM, Fig. 25. — Graph showing the number of cells from tip to base in the different layers of the Avena eoleoptile, at six stages in its growth. The outer epidermal cells do not increase in number, while the cells of the other layers multiply rapidly in the early period of development and then remain about constant in number. A definite gradient of cell-division intensity, decreasing inward from the subepidermis to the inner epidermis, is apparent for the first quarter of the growth period. During the last three quarters of its growth period, the increase in length of the eoleoptile is proportional to the elongation of its constituent cells. {From Avery and Burkholder, 1936.) ''{\' -A- - J ) 4 mm. Stage 12 mm. Stage 20 mm. Stage 29 mm. 37 mm. Stage Stage (at maturity) Fig. 26. — Diagrams of the Avena eoleoptile to show shift in position of the zone of maximum growth intensity at different stages of development. Coleop- tiles 4 ram. in length were marked into four 1 mm. segments (a, b, c, d) and the length of each was measured in several later stages of growth. The density of the dots indicates relative growth intensity. Note that the region of maxi- mum growth shifts from the base in the young eoleoptile (12 mm. stage) to the apical region in a maturing eoleoptile. (Adapted after Avery and Burkholder, 1936.) GROWTH SUBSTANCES FOR NORMAL GROWTH 87 rapidly throughout most of the coleoptile while it is young, the region of greatest elongation is basal (Fig. 26). As the coleoptile nears maturity, growth slows down throughout its length, ceases at its base, and becomes relatively greater near its apex. At the time the foliage leaf bursts through, all basal growth has ceased, but a localized region of slow elongation at the tip below the pore may persist for as much as two or three days after the leaf bursts through the coleoptile. These same facts apply, in gen- eral, to the coleoptile of Triticum, on which similar observations were made. Light-groivfh Reaction. — The inhibiting effect of light upon the rate of growth in plants was recognized in the older plant phys- 0» 234 5 6 7 89 Fig. 27. — The course of growth in the Avena coleoptile in different intensities of light. Ordinate: growth; abscissa: 12-hour periods, a, growth of coleoptiles in weak light for 4^-^ days; b-e, growth of other coleoptiles kept for 1 day in the same weak light as in a, and then transferred to different intensities of light, increasing successively from b to e. Greater light intensity shortens the period of growth and causes the size of coleoptiles to be smaller at maturity. (After Sierp, 1918.) iology literature (deCandolle, 1832; Sachs, 1874). Since the time when Blaauw (1914) attempted to use Ught-growth reactions as the foundation for a theory of phototropic curvatures, the role of light in growth has been the subject of many investigations. Although Blaauw did not perform any experiments on the light-growth reactions in the Avena coleoptile, such investigations were carried out by Vogt (1915), Sierp (1921), Lundegardh (1921, 1922), Koningsberger (1922), Renner (1922), Brauner (1922), Erman (1923, 1930), Went (1926, 1928a), Dillewijn (1925, 1927a, 6), Pisek (1926), Beyer (1926, 1927a, c, 19286), Priestley (1926c), Gradmann (1930), Bergann (1930), Nuernbergk and duBuy (1930), Cholodny, (1931rf, 19326, 1933a), and duBuy (1933). Sierp (1918) studied the development of the Avena 88 GROWTH HORMONES IN PLANTS coleoptile in darkness and when subjected to varying amounts of light. As may be seen in Fig. 27, he found that the rate of growth of the coleoptile is temporarily increased with increasing amounts of light. The point of maximum growth is reached sooner, and the final size of the coleoptile is not so great under conditions of increasing light. Van Dillewijn (1927a) illuminated the Avena coleoptile by placing a lamp vertically over the plant; the hght was reflected horizontally on to the experimental object by three obHque mirrors. He noted the influence upon the rate of growth of continued illumination as well as short periods of illumination with differing amounts of light. In some experiments only the tip was illuminated, in others, the subapical zones; or the entire coleoptile was supplied with hght. Light-growth reactions appeared in all cases. The reactions were sharply defined when definite zones, near the tip, were illuminated for a short time with a definitely determined amount of light. After a latent period there occurred a depression of growth during the course of which two types of response could be distinguished, one of short, the other of long duration (Sierp, 1921). In the short reaction, the maximum depression of the growth rate was reached after }/2 hour; in the long reaction, on the other hand, after 1}4 hours. The long reaction could be observed only when the tip was illuminated (Went, 1928a: tip response); the short reaction, when the basal zones were illuminated (Went: base response). Growth was accelerated again after tliis retardation. When the entire coleoptile was illuminated, these effects were summated. When illuminated plants were darkened, Sierp (1918) found that a dark- growth reaction took place also. Geo-growth Reaction. — The question whether gravity can produce fluctuations in the growth of the Avena coleoptile in a manner similar to that brought about by light has been investi- gated with contradictory results. Zolhkofer (1921) reported continually changing rates of growth in response to stimulation by gravity; while Koningsberger (1922) observed no geo-growth reaction during continued rotation on the clinostat, but a growth- promoting effect was produced by gravity in both erect and inverted coleoptiles. The dorsiventrality curvatures, however, mentioned elsewhere, can easily disguise growth changes when the Avena coleoptile is cUnostated. With this source of error GROWTH SUBSTANCES FOR NORMAL GROWTH 89 removed, Bremekamp (1925) and Dolk (1929a) showed that no geo-growth reactions appear in the Avena coleoptile. Navez and Robinson (19326) came to the same conclusion. Growth Substances and Normal Growth. — As has been mentioned previously, Paal (1918) showed that growth substance is being formed continuously in the nonilluminated coleoptile tip, whence it migrates into the more proximal portions of the coleoptile and promotes growth. Rothert (1894) and Stark (1917) showed that the removal of the coleoptile tip produces a retardation of growth in the coleop- tile stump, a fact that Soding confirmed when he investigated this same question (1924, 1925, 1929). The rate of growth (Table 3) Table 3. — Growth ix Length of Normal and Decapitated Avena coleoptiles The figures in the table are average values from Tables I to III of Soding, 1925 (p. 589) Treatment Increase in the first 5 hr. Increase in the fol- lowing 13 hr. A. Decapitated B. Decapitated, the tip replaced, and again' re- moved after 5 hr C. Intact control plants 2.57 1.65 3.40 in the first 5 hours after decapitation was only 42 per cent of that in normal seedlings. Furthermore, the rate of growth of the coleoptile stump was increased about 49 per cent in the first 5 hours when the removed tip was again replaced. Soding's experiments showed that the rate of growth of normal plants is not reached in decapitated plants in the first few hours, even with their tips replaced, probably because the transport of growth substance is retarded by the wound. After 10 to 14 hours, even ^^ithout replacing the tip, the rate of growth of decapitated coleoptiles became about the same as that of normal seedlings. This increase in growth was brought about by ''physiological regeneration" of the tip, which produced about the same amount of growth substance as the normal. It is clear from this that a substance is dispersed from the tip which promotes growth in the basal region. If, instead of replac- 90 GROWTH HORMONES IN PLANTS ing the tip after decapitation, one covers the wound with agar containing growth substance, the rate of growth can be increased far beyond the normal (Fig. 1) (Went). It has been found that when the growth-substance content of the agar amounts to 100 WAE (Boysen Jensen, 1933a), the coleoptile stump surpasses the enclosed leaf in growth, which normally never occurs. Soding (1929) investigated different portions of the coleoptile for growth substance and found that the amount decreased greatly from tip to base. This observation has been confirmed by the work of Thimann (1934). From these and other experiments, it has been concluded in the past that growth substance is formed exclusively in the tip under normal conditions and that it migrates from there into the more proximal portions of the coleoptile where it stimulates growth. In view of the upward movement of growth substance which has been demonstrated in certain plants by Zimmerman and Wil- coxon (1935), it appears equally probable that the hormone or its precursor is being formed in the endosperm (Cholodny, 19356) and moved upward in the vascular system to the tip, from which point it is dispersed downward. In fact, Pohl (1935) concludes from a series of important experiments that the coleoptile tip does not produce growth substance but can only activate the reserve stored in the endosperm. The phenomenon of "physi- ological regeneration" apparently could be explained by this interpretation. Further confirmatory evidence is found in the observation that physiological regeneration (Soding, 1929) takes place just as vigorously whether the coleoptile is decapitated at the tip or several milhmeters below and Heyn (1935) has found that physiological regeneration does not take place when the coleoptile is separated from the food stored in the seed. The hypothesis that the decrease in rate of growth after decapitation may be caused by lack of growth substance has been disputed by Priestley (1926rf) and by Tetley and Priestley (1927). When the coleoptile is decapitated, water exudes from the cut surface; this loss of water is, according to Priestley, the essential reason for the retardation of growth, and he contended that retardation must persist until the supply of water is rendered normal again by healing of the wound. The promotion of growth by replacement of the tip was explained by partial closing of the wound. It may be said here that Priestley's explanations GROWTH SUBSTANCES FOR NORMAL GROWTH 91 are no longer tenable in the light of the more recent discoveries concerning the role of growth substance. A question of importance is whether growth takes place in the Avena coleoptile when growth substance is completely absent. As shown by Soding's experiments, some growth takes place in the first 5 hours after decapitation, but Dolk (1930) showed that this occurs only because of the growth substance still Interferometer Coleoptile Solution Vaseline Fig. 28. — Stimulating effect of growth hormone (acidulated pollen extract) upon growth of a decapitated Avena coleoptile. A, diagram showing culture chamber in which coleoptile is grown immersed in solution. B, graph showing the increased rate of growth of a coleoptile treated with growth-hormone solution at the time indicated by arrow, as compared with a control plant (lower curve) which continues its growth in water. Growth was measured interferometrically. (After Laibach and Kornmann, 1933a.) present in the coleoptile stump. If the coleoptile is decapitated again 2 hours after the first decapitation, its growth ceases almost completely but can be renewed by supplying growth substance. From these experiments it may be concluded that normally no gi'owth substance is formed below the tip of the coleoptile and that the growth substance present in the stump at the time of decapitation is gradually used up. In any case, without growth substance there is no growth. 92 GROWTH HORMONES IN PLANTS An interesting technique was developed by Laibach and Kornmann (1933a) to demonstrate the accelerating effect of growth substance (extracted from pollen) upon growth in length of the decapitated Avena coleoptile (Fig. 28). Went (1928a) suggested reasons for the distribution of growth in the Avena coleoptile, stating that the rate of growth in the 10 0 _L _L 0 14 16 18 20 22 2 4 6 8 10 12 Time in hours Fig. 29. — Growth rate of the upper 10 to 15 mm. zones of Avena eoleoptiles with different amounts of growth hormone, a, the normal course of growth; b, growth of a group of eoleoptiles to which auxin paste was added at an early stage; c and d, other groups of eoleoptiles to which auxin was applied at later stages in growth. {After Went, 1935c.) basal portion is limited by the failing supply of growth substance ; on the other hand, growth in the tip is limited by the lack of organic material (supplied by the seed) which is necessary for cell elongation. The rate of growth reaches a maximum at that point where both food and growth substance are present in sufficient amount, and the water supply is adequate. DuBuy showed (1933) that growth in the coleoptile is gradually retarded when the endosperm is removed ; aging is also mentioned as one GROWTH SUBSTANCES FOR NORMAL GROWTH 93 of the factor complexes significant in its growth. Went has discussed the subject in a later paper (1935c) and concluded that growth substance is a limiting factor in the elongation of the coleoptile during its later stages of development. Artificially increasing the auxin supply in a coleoptile accelerates the growth rate either directly by promoting growth or indirectly by preventing senescence (Fig. 29). With a supply of food available, the addition of auxin brought about a revival of growth in the basal portion which had ceased to elongate; on the other hand, when the food supply was removed, further additions of auxin showed no growth-promoting effect. From the foregoing observations it seems clear that the rate and distribution of growth in the normally developing coleoptile are regulated by the supply of growth substance. Growth Substances and the Light-growth Reaction. — Went (1926) and van Dillewijn (1927a) conjectured that the growth reactions produced by complete illumination of the tip zone are the result of changes in the amount of growth substance given off, and Went (1928a) actually found a decrease of about 18 per cent in the amount of auxin given off when the tip was illuminated with 1,000 meter-candle seconds. According to duBuy (1933), weak blue light produces no change in growth-substance supply, and even strong white light (with heat and some of the red removed) may have no effect; on the other hand, white light plus the heat radiation decreases the supply of growth substance. General illumination of the lower zones of the coleoptile also produces light-growth reactions, as mentioned earlier, but it is not possible at this time to offer a satisfactory explanation of the phenomenon. Further data are discussed luider light-growth reaction in the stems of seedlings. That the influence of light on growth depends upon the kind of growth substance present has been shown by van Overbeek (1936a). When auxin a is applied unilaterally in agar blocks to Avena coleoptiles, curvature is less under illumination with white hght, than in darkness; when 3-indole acetic acid is similarly applied, no difference in growth is observed in darkness or in light. Foliage Leaves. — The presence of growth substance has been demonstrated in buds and foliage leaves of several species of dicotyledons (see chapter on the occurrence of growth sub- stances), and in Nicotiana (Avery, 1935) it has been shown that 94 GROWTH HORMONES IN PLANTS 20.4 -36.9 72 20.4 33.6 Fig. 30. — Diagrams of Nicotiana leaves showing growth-hormone content (expressed in plant units) at different ages and in different portions. A, young leaf. C and D, older leaves from plants grown in a greenhouse. B, leaf of same age as C, but kept in dark for 10 days, followed by 1 day in the light. The auxin concentration gradient shown in A and C is due to accumulation in the midrib and movement toward the base of the leaf. In contrast, leaf B shows less accumulation at the base (data in parentheses) . Note disappearance of growth hormone at the distal end of the older leaf, D. {After Avery, 1935.) = RELATIVE GROWTH INTENSITY = POLARIZED GROWTH A B Fig. 31. — Growth of the Nicotiana leaf. A, showing greatest growth intensity {localized growth) in marginal and basal regions, as indicated by the density of stippling. B, the segments indicated in the distal and proximal portions of the leaf show a relatively greater increase in length than in width. While this polarized growth is not pronounced at the apex toward the end of the growth period, it is very striking at the basal end of the leaf, where it is correlated with higher concentrations of growth hormone. {After Avery, 1935.) GROWTH SUBSTANCES FOR NORMAL GROWTH 95 the concentration is greater in young leaves, tending to decrease as the leaves mature. It has been shown, also, that there is a definite concentration gradient from the tip to the base of a leaf, the concentration being low at the distal end and increasing toward the base (Fig. 30). The increase toward the base is due to the accumulation of growth substance in the proximal end of the midrib and is correlated with greater longitudinal growth ("polarized growth") of the midrib in this region. Inasmuch as the application of growth-substance paste (lanolin method, Laibach, 19336) to large veins brings about a bending (differential growth) response, it may be assumed that it is the agent responsi- ble for promoting the normal growth in length of the midrib and larger lateral veins in the leaf; hence, growth substance is responsible, at least in part, for the normal growth pattern exhibited by the leaf {cf. Figs. 30 and 31). Axial Parts : Hypocotyls, Internodes, and Flower Stalks. Distribution of Growth. — Rothert (1894) investigated the distribu- tion of growth in the hypocotyls and epicotyls of dicotyledonous seedlings. In seedlings with epigeal cotyledons the hypocotyl usually elongates first. Enlargement of the growing point above the cotyledons begins only after the growth in length of the hypocotyledonary axis is completed. As long as the hypocotyl is very short, it grows throughout its entire length; later the basal portion ceases growing, and a growth zone of a rather constant length (1 to 4 cm.) is established in its upper portion. Following cessation of growth in this region the epicotyl begins to develop. The distribution of growth in the epicotyl of Phaseolus is shown in Fig. 32. The distribution of growth in stems with several elongating internodes is often quite comphcated. It would not be of value to discuss this question at length here, since the significance of growth substance for these growth processes has not been investigated. Very obvious light-growth reactions are exhibited by many seedling axes. According to Blaauw (1915), a decided retarda- tion of growth appears in Helianthus after brief illumination; this is followed later by an increase in growth enduring for a short period. According to van Overbeek (1933), the rate of growth in the hypocotyl of Raphanus is decreased to about one- half l)y illumination. 96 GROWTH HORMONES IN PLANTS The observations of Thimann and Skoog (1934), working with epicotyls of Vicia Faba, show that a growth-promoting substance is formed by the action of hght upon the green portions of the >> a •a u a O o Xw Zones 123456789 Fig. 32. — Distribution of growth in the seedling stem of Phaseolus multiflorus (hypogeal cotyledons) at 24°C. The stem was marked into nine 5 mm. zones, beginning at the level of the first foliage leaves. Ordinate: growth in millimeters in 24 hours; abscissa: 5 mm. zones. {After Boysen Jensen.) 0 I 2 3 4 5, TIME IN DAYS Fig. 33. — Growth of defoliated and decapitated plants of Vicia Faba in the Hght and in the dark, with and without growth hormone. A, growth in the dark, following the addition of 1600 units of growth hormone per cubic centi- meter. B, growth in the light with the same amount of growth hormone as in A. C, growth in the light following the application of plain agar. D, growth in the dark with plain agar. {Adapted after Thimann and Skoog, 1934.) plant. The rate of growth, however, is more rapid in darkness than in Hght in experiments where comparable amounts of growth substance are added (Fig. 33). GROWTH SUBSTANCES FOR NORMAL GROWTH 97 Growth Substances in Relation to the Distribution of Growth. — From the present evidence it is possible to distinguish two types of seedlings on the basis of the place of growth-substance forma- tion in their hypocotyls. Raphanus sativus may be cited as an example of the first type (van Overbeek, 1933), where the growth substance is formed in the cotyledons and moves from these into the hypocotyl. If the cotyledons are removed, the rate of growth falls off rapidly. After a time, the hypocotyl begins to grow again in its upper zones because the growing point has begun to form growth substance. That it is a growth substance which influ- ences the growth of the hypocotyl is clear, for when a block of agar containing growth substance is placed upon one of the petiolar stumps, a negative curvature in the hypocotyl is produced. The hypocotyl of Lu-pinus albus behaves chfferently. That its growth rate is influenced by growth substance was shown by Cholodny (1926) by boring out the middle portion of a 3 cm. segment so that only a hollow cylinder remained. Its rate of growth was greatly lessened by this operation, but if Zea coleop- tile tips were placed in the hollow region, approximately the same rate of growth was obtained as in untreated normal stems. The work of Dijkmann (1933) shows- that growth substance is found throughout the whole growing zone and is probably formed throughout. There seems to be no center for production of growth substance, and decapitation does not produce an immediate retardation of growth. Accordiaig to some unpub- lished experiments of Boysen Jensen, Phaseolus multiflorus belongs to the same type. The later work of Dijkmann (1934) indicates that the growth rate in the Lupinus hypocotyl is proportional, within certain limits, to the growth-substance concentration. According to van Overbeek (1933), the hght-growth reaction of the Raphanus hypocotyl is not caused by decreased production of growth hormone. It is explained by assuming a change in the abiUty of the organs (perhaps the cell walls) to react to growth substance. The rate of growth of various inflorescence stalks is influenced also by growth substance. Uyldert (1928) showed that the elongation of flower stalks of Bellis is retarded greatly by the removal of the inflorescence, but it could be increased again by 98 GROWTH HORMONES IN PLANTS the addition of growth substance. The rate of elongation of decapitated flower stalks is increased by rhizopin, also (Nielsen, 1930a). Soding (19326) showed that unilaterally applied Avena tips produce curvatures in flower stalks of Heliopsis laevis, Cephalaria tatarica, and Muscari ramosum; therefore, in these organs, also, the rate of growth is increased by growth substance. That the growth hormone present in the young internodes and nodes of grasses influences their elongation may be concluded from the fact that growth substance from coleoptile tips increases 4) S18 + 3 XT. 10 c lo «18- 0 E Fig. 34. — Comparison of the growth-hormone content and growth response of a normal race of Zea mays with the dwarf type "nana." A, coleoptile tips of the normal race yield almost double the amount of growth hormone given off by the dwarf "nana"; data from 400 plants of each type. B, growth curva- ture of coleoptiles of the normal race is about twice that of "nana" when a given amount of growth hormone is applied unilaterally in agar blocks; data from 200 plants of each type. (After van Overbeek, 1935.) the growth of young internodes. It has been shown to produce cell elongation even in mature nodes (Schmitz, 1933). The first internode (mesocotyl) in Zea mays, dwarf variety "nana," is appreciably shorter than the first internode in a normal race, although the coleoptiles are the same length. Van Over- beek (1935) has shown that the inhibited development of this first internode is due to the destruction of auxin, the amount produced being about the same in seedlings of nana and normal. A given amount of growth hormone applied unilaterally to the coleoptile stumps of normal and nana resulted in smaller curva- ture of the latter (Fig. 34), thus supporting the conclusion regard- ing destruction. This destruction is correlated with a greater catalase and peroxidase activity; hence van Overbeek concluded that the dwarf type of growth in this variety of maize must be due to a more active oxidation system. By raising the tempera- ture of normal seedlings to 60°C. for one hour, thus increasing the catalase activity, it was possible to make the normal into dwarf. GROWTH SUBSTANCES FOR NORMAL GROWTH 99 i.e., to induce the destruction of auxin and obtain a short first internode. Roots. — Roots are a continuation of the axial system of the plant but differ sharply from the shoot in structure and behavior. The region of primary growth in the root is so well-known that it is unnecessary to describe it here. With regard to the presence of growth substance in roots, there is considerable evidence indicating that it is formed by the root tip; lesser amounts are found proximally. Wiesner (1881) made the statement that the growth of roots in contact with water is accelerated by decapitation. This observa- tion was confirmed by Cholodny (1926); an increase in growth of 12 per cent took place after decapitation of the roots of Lupinus angustifolius. Then Biinning (1928) investigated the effect of decapitation upon the growth of the root; if the removed tip portion w^as not too long, there was a temporary retardation of growth in most roots, followed by an increase. Nielsen (19305) also showed an increase in root growth as the result of decapitation. To answer the question of how decapitation can produce an increase in growth, Cholodny assumed that the growth substance that is formed in root tips must be identical with that formed in coleoptile tips. Even though the growth substance increases the growth of the Avena coleoptile, it apparently retards the rate of growth of the root. If this is so, the removal of the root tip should bring about an increase in growth. Biinning (1927) con- cluded, on the other hand, that the growth changes described are to be construed as wound-growth reactions. It has been demonstrated repeatedly that growth substance can influence the rate of growth of roots. Cholodny (1924) showed that decapitated roots of Zea mays, upon which had been placed the coleoptile tips of the same plant, grew 36 per cent less rapidly than decapitated roots without tips. This indicates that the growth substance of the coleoptile retards growth of the root. This conclusion was confirmed by Nielsen (19306), who determined the growth increase of the root when the 2 mm. por- tion of the tip was first submerged in water and afterward in a rhizopin solution. He showed that the rhizopin completely inhibited the growth of roots of Lupinus alhus and Vicia Faha, whether they were intact or decapitated. Moreover, the roots 100 GROWTH HORMONES IN PLANTS were not permanently injured by the rhizopin, for if the rhizopin solution was replaced later by water, the roots immediately resumed their growth. In contrast with these results, Gorter (1932) concluded that growth substance had no influence upon the growth of roots of Pisum and maize. The roots were decapitated, and agar blocks containing growth substance from the coleoptile tips of maize were placed upon the wound surface. The experiment included six Pisum roots (three with agar and growth substance, three with agar without growth substance) and three maize roots. When growth substance was added, the rate of growth in Pisum was greater, in two cases, than when it was not present. Growth was greatest in maize without added growth substance. The author concluded from her experiments that growth substance has no influence upon the growth of the root. Her data were too few to permit a final conclusion on this matter. To clear up the existing disagreement in reports of the different workers, Boysen Jensen (1933c) carried out further experiments on the influence of growth substance upon the rate of growth in roots of Vicia Faba. In the first of these studies the methods of Nielsen were used. The rate of growth was determined for root tips, some of which were immersed in pure water and others in a growth- substance solution containing 2 WAE per 100 cc; in the latter case, growth decreases by about half. The use of Gorter's method also showed that growth substance influences the rate of growth of the root, but with her procedure it was necessary to use far higher concentrations of growth substance. In this method, the roots were decapitated 1.25 mm. back of the tip, and agar blocks were placed upon the wound surfaces, either without or with growth substance in concentrations of 25 to 50 WAE per 100 cc. The rate of growth was reduced about one-half by this con- centration of growth substance (see also Cholodny, 19336; Navez, 19336). Kogl, Haagen-Smit, and Erxleben (1934, Mitt. XII) have shown that 3-indole acetic acid when added to the culture solution in concentrations of 0.01 to 1 mg. per Uter inhibits root growth; auxin a and 6 have similar effects. The work of Meesters (1936) has shown further the inhibiting influence of 3-indole acetic acid on the growth of root hairs and roots of Agrostemma. Growth in length of the root hairs was retarded GROWTH SUBSTANCES FOR NORMAL GROWTH 101 by about 20 per cent in the presence of 0.5 mg. of 3-indole acetic acid per liter; almost complete inhibition of root elongation occurred with the same concentration of the hormone. Other solutions of the same pH value, obtained by the addition of acetic acid, did not show any inhibiting effects. It has been determined with certainty, therefore, that the rate of growth of roots is retarded by the addition of growth substance, and from this it might be concluded that growth substance is not necessary for the growth of roots. In support of this interpreta- tion is the fact that ageotropic roots, which can be obtained by treating the seed with eosin or erythrosin (Boas and Merken- schlager, 1925), often possess no demonstrable amount of growth substance (Boysen Jensen, 1934), although the rate of growth is not decreased. The evidence from normal distribution of growth substance in roots makes another interpretation equally plausible. Boysen Jensen (1933b) and Thimann (1934) both have shown that a concentration gradient exists at the growing end of the root, the tip possessing the most growth substance, and the concentration falling off in a proximal direction. From this it might be con- cluded that growth substance does take part in root growth and that elongation of the root is taking place in the region of opti- mum concentration. If this is the case, the optimum concen- tration for root growth must be very low. Why the root and shoot behave differently in the presence of a given concentration of growth substance remains to be explained. [Czaja (19356) discusses a possible explanation based upon the direction of streaming of growth substance in roots.] Lower Plants. — Heteroauxin is produced by many lower organisms, e.g., Aspergillus niger. This substance has a remark- able effect upon the rate of growth of the Avena coleoptile, and it is important to determine whether it has any demonstrable physiological significance for the growth of Aspergillus itself. Boysen Jensen (1932) showed that if Aspergillus is cultivated upon a glucose-nitrate solution, growth substance cannot be demonstrated either in the fungus mycelium or in the culture substratum. This would indicate that growth substance is not a necessity for the growth of Aspergillus. Whether it has any influence upon the growth of this organism was determined in the following way: Aspergillus niger was grown upon a glucose- 102 GROWTH HORMONES IN PLANTS nitrate-citric acid solution, in some experiments without growth substance, in others with it present in concentrations of 0.9, 9.0, and 21.0 WAE per 100 cc, respectively. After being cultured for 11 to 14 days at a temperature of 16°C., the mycelium was removed, dried, and weighed. It was found that the addition of growth substance always resulted in retardation of growth — in one case by as much as 50 per cent ; in other cases, by significantly less. This is in agreement with the results of Nielsen and Hartehus (1932), who found that rhizopin was without influence upon respiration or the production of dry matter in Aspergillus niger (see Nielsen, 1931a). Biinning (1934a, h) also found no furthering influence of the ether-soluble hormone of Aspergillus upon production of dry substance in this fungus. Biinning, Schopfer (1935), and others have shown the growth-stimulating effect of other substances on lower organisms. Schopfer reported that extracts from wheat embryos, orchid and other pollens, etc., stimulate the growth of numerous Mucorineae. Pure crystal- lized vitamin Bi promotes the growth of Phycomyces in such small amounts as 0.00057 per cubic centimeter of the culture medium. However, the extracts with which Schopfer has been working contain active substances which are not to be confused with the growth substances treated at length here. Animal Cells. — Since substances capable of promoting growth in plants can be extracted from animal sources, it is of interest to find out whether these substances have any influence on animal growth. According to the investigations of Fischer, the growth of heart fibroblasts in tissue cultures is not increased by auxin a or 6 (see Kogl, Haagen Smit, and Tonnis, 1933, Mitt. VIII), nor is the metamorphosis of tadpoles influenced by the addition of auxin (Kogl, Haagen Smit, and Erxleben, 1933, Mitt. VII; Sylven, 1933). Navez and Kropp (1934) obtained similar negative results when they appUed the plant-growth hormone to crustacean eyestalks; i.e., there was no activation of the chromatophores. Plant-tissue Culture. — LaRue (1935) removed pieces of the embryos of half-grown seeds of Taraxacum, Lycopersicon, Lactuca, etc., and cultured them on nutrient agar. White's (1934) culture solution was used but without the yeast extract. In Lactuca, complete plants developed from 0.5 mm. pieces of embryonic hypocotyl. Successful growth took place only in GROWTH SUBSTANCES FOR NORMAL GROWTH 103 cultures with 3-indole acetic acid (heteroauxin) (1 part: 20,000,000). Growth Substance in Relation to Cell Division. — The role of hormones in promoting growth by cell enlargement has been established by numerous investigations upon diverse plant materials. A limited number of observations have led to the suggestion that growth substances may also influence cell division in plants. Laibach, Mai, and Mliller (1934) obtained an ether-soluble, thermostable extract from orchid pollen and urine which when applied to the stems of Coleus and Tradescantia brought about increased frequency of cell division leading to callus formation. Further work (Laibach and Fischnich, 1935a) has shown that 3-indole acetic acid stimulates cell di^^sion in the epicotyls of Vicia Faba. More recently still, it has been found that applica- tion of purified auxin a and 3-indole acetic acid to the upper ends of decapitated Helianthus seedlings caused growth in thickness by cambial division (Snow, 19356). In a report of new experiments and a review of the literature, Jost (19356) has pointed out the stimulating effect of relatively high concentrations of various substances upon cell division in the pith of Vicia Faba, the main roots of Lupinus, etc. Popoff (1933) studied the influence of growth-substance extracts which were obtained from the coleop- tile and other parts of Zea seedlings and added to cultures of Euglena gracilis. It was reported that oxidation processes, cell division, and germination of the cysts were promoted by dilute concentrations of the extracts. Other workers have postulated the existence of other special hormones for cell division. The role of certain substances {e.g., bios) which apparently do not belong in the same category with the auxins has been described by many investigators (Wildiers, 1901; Miller, 1930; Schopfer, 1935; Dagys, 1934; Kogl, 1935, Mitt. XIV; etc.).^ The precise way in which the auxins and these other substances may regulate growth by means of their influence upon cell division has not yet been satisfac- torily explained. THE MECHANISM OF ACTION OF GROWTH SUBSTANCES In the foregoing survey of the discoveries concerning the effect 1 See supplementary bibliography on p. 247. 104 GROWTH HORMONES IN PLANTS of growth substances upon growth, we have found that they influence cell enlargement ("stretching growth") in diverse kinds of higher plants. The subject is not so simple because in some organs growth is stimulated, while in others it is inhibited by the presence of growth substance. In coleoptiles and portions of stems, growth is increased, while in roots, it is retarded; it is possible that growth substance may not be necessary for root growth, though its role in the neoformation of roots has been observed. Moreover, it appears to have little significance for the vegetative growth of Aspergillus and perhaps many other lower plants. The next step is to determine in what way growth substances exercise their growth-promoting effect. From investigations carried out by Went (1928a), van der Weij (1932), and duBuy (1933) on the transport of growth sub- stance, it can be seen that the hormone is actually used up in the growth of the Avena coleoptile. Van der Weij has shown that if two agar blocks, each with the same growth-substance concentra- tion, are placed on either end of a coleoptile cylinder 2 mm. long, a decrease in growth substance takes place in the upper block, but no increase can be demonstrated in the lower block. The most likely explanation of this and numerous similar observations is that it is consumed in growth. Growth Substance and the Cell Wall. — Up to the present, close quantitative relationships between consumption of growth substance and growth have not been demonstrated, and the present evidence is insufficient to prove that it participates stoichiometrically in the growth of the cell wall. Nielsen (1930a, h) showed that its effect is very great in proportion to its weight; and according to Kogl (1933, Mitt. Ill; see also Kogl, 1933a), a curvature of 10 deg. results in the Avena coleoptile from the action of less than one 50-millionth milligram of auxin a or b. Thimann and Bonner (1933) have computed that 2.31 X lO^i growth-substance molecules can produce a deposition of 6.8 X 10^^ CeHioOs molecules of glucose residues for the cellulose micelles, i.e., that one growth-substance molecule is active in the formation of 3.0 X 10^ CeHioOs molecules. Although these numbers are approximations, they are entirely adequate to show that growth substance does not participate as a "building stone" of the cell wall ; it must influence the growth of the cell in some other way. At this point in the discussion, it may be well to GROWTH SUBSTANCES FOR NORMAL GROWTH 105 review briefly certain studies on the composition and the micro- scopic structure of the cell wall. According to the investigations by Thimann and Bonner (1933), the dry matter of the cell walls of the coleoptile contains about 42 per cent cellulose, the remainder being made up of hemicelluloses, pentosans, and pectins. Recent observations have indicated that the cell wall consists of two different ele- ments, namely, micellae, i.e., little rods, which are probably crystalline, and an intermicellar substance, which fills the spaces between the micellae (Anderson, 1935). According to Heyn (19336; see also Kolkmeijer and Heyn, 1934), dehydration shrinks the cell wall to about one-third of its diameter when wet. Soding (1934) has assumed that the intermicellar substance is of greater volume than the micellae and that it is made up of a \'iscous, colloidal substance. Heyn (1933a, h, 19345) has studied the cell walls of the coleoptile under the microscope and by means of the Rontgen spectrograph. He states that the cell walls are smooth in intact cells, although the inner layers become wrinkled when dehydrated or released from tension ; the outermost layers of the external wall of the epidermis would be shorter than the inner layers when dried out. By means of the Rontgen spectrograph a difference can be found between younger and older cell walls. Heyn concluded the following from these experiments : If one assumes that the cellulose macromolecules of the young cell wall have not yet taken on the crystalline form described above, then the important role of the water held in these walls becomes under- standable. In older cells (fibers), aging of the walls is accompanied by progressive dehydration, while more macromolecules are continually taking on this crystalline form, until finally a pure cellulose pattern is obtained. When young walls, consisting of macromolecules, are dried, much more bound water remains between the single molecules not in crystalline form. Cell-wall Extensibility. — When considering the physical char- acteristics of the cell wall, its extensibility is of interest. Exten- sion can be either reversible {elastic extensibility) or not reversible (plastic extensibility). According to Pringsheim (1932), one can- not make a sharp distinction between these two types; the amount of wall substance is not changed in either case. Measure- ment of the degree of extensibility in plant organs is a difficult problem; plant cells can change their length easily without 106 GROWTH HORMONES IN PLANTS addition of wall materials. The walls can change their elastic and plastic properties by hardening, by increasing wall substance, and in many other ways. Methods for Measuring Extensibility. — A technique by which cell-wall extensibility can be measured may be described briefly as follows: A decapitated coleoptile or flower stalk can be sus- pended perpendicularly, and the changes in length in a definite region may be determined when a weight, for example, 2 to 10 g., is attached to the lower end of the organ. The plant part being tested must not be turgid, because the change in length resulting from a definite pull on a turgescent coleoptile is only a small fraction of the change in length of a coleoptile which is in a state of plasmolysis. For plasmolyzing, one may use a 50 per cent glycerin solution. The measurements are made with the horizon- tal microscope. If the original length of the region marked previously on the coleoptile is termed a, the length after attach- ment of the weight h, and after removal of the weight c, then the total increase in length (extension) is & — a (elastic and plastic extension), the remaining increase in length, c — a (plastic exten- sion) . If weights of 2 g. are used on the oat coleoptile, the exten- sion that remains is negligible; therefore, only elastic extensibility is measured (Soding, 1931). When a weight of 10 g. is used (Heyn, 193 1&), the increase in length remaining after the weight is removed (c — a) is about 30 per cent of the total length increase (6 - a). Heyn (19316) used the following method to measure plastic extensibility: Excised coleoptiles, with the primary leaf removed, were fastened at one end and placed in a horizontal position (Fig. 35). A weight of 250 mg. was placed upon the free end for a given length of time. After the weight was removed, the size of the bend formed in the coleoptiles was determined. This curvature was used as an index of plastic extensibility. It is possible, also, to measure the extension of the cell wall which is produced by osmotic pressure (Schmid, 1923). The plant part is first placed in water and afterward plasmolyzed. The difference in length before and after plasmolysis is a measure of the extension of the cell wall due to osmotic force. Hypotheses on the Method of Action. — Since growth substances may have a controlling effect upon the rate of growth, the relation that they have to the growth of the cell waU is closely bound GROWTH SUBSTANCES FOR NORMAL GROWTH 107 up with the fundamental process of growth. There are at least three different hypotheses concerning the nature of the first step in growth: 1. ELASTICITY. — Accordiiig to the first hypothesis, the growing cell wall must be extended elastically by turgor pressure first; it would at the same time, of course, become thinner. The original thickness is regained either by the incorporation of new particles h£ 2> b /f ]/ Fig. 35. — Method of determining elastic and plastic extensibility in Avena coleoptiles. The coleoptiles were cut away from the seed, the young foliage leaves pulled out, and the resulting hollow cylinders fastened on pins above metal plates. The tips were removed from series a one hour before the experi- ment started, thus depriving them of a growth-hormone supply. The tips were left intact in series b until the moment the experiment was begun, hence growth hormone was present (indicated by stippling). Then the tips were removed from series b and 250 mg. weights were placed on the ends of coleoptiles in both series. After one hour the curvature was about the same in both series (ai and bi). When the weights were removed, the curvatures decreased. Series a without growth hormone retained only 9.3° curvature (a;). Series b with growth hormone retained an angle of 17.3° (b-j). Hence, plastic extensibihty was greater in coleoptiles containing growth hormone. (Adapted after Heyn, 19316.) iintussusce'ption) or by the laying down of new layers {apposition) . The force that stretches the cell is, according to this hypothesis, produced by turgor, and the first step in growth is reversible and is not concerned with an increase in substance of the cell wall. A change in the rate of growth, whether general or unilateral, can come about by modification of the extending forces (turgor pressure) or of the elastic extensibility. 2. PLASTICITY. — In the second case, turgor pressure also may be considered the force behind the growth of the cell wall. While the extension considered in the first hypothesis is elastic, i.e., reversible, in this case it is plastic, i.e., not reversible. The first step in growth is not concerned with an increase of cell-wall 108 GROWTH HORMONES IN PLANTS substance which can take place later either by intussusception or by apposition. According to this hypothesis, then, a change in the rate of growth can result from changes in either turgor pres- sure or plastic extensibility. 3. ACTIVE GROWTH. — In the third place, the active growth of the cell wall has been considered as the primary step, a theory propounded by Pfeffer (1904). According to this hypothesis, new particles are laid down between those already present in the wall. Although the turgor pressure is significant in so far as it is necessary to keep the protoplasm in connection with the cell wall, it is not regarded as the source of the energy; rather, this is the result of forces that are active in the secretion of new ingredients for the cell wall. The first step in growth is concerned, there- fore, with an increase in the substance of the cell wall; it is not a reversible process and is not influenced by changes in turgor pressure. Discussio7i of Hypotheses. — A consideration of the influence of growth substances upon these processes may make it possible to determine the primary step in growth. 1. GROWTH SUBSTANCES AND ELASTIC EXTENSIBILITY. HcyU (19316) and Soding (1931), at practically the same time, found that a far-reaching parallel exists between growth and elastic extensibility in the oat coleoptile, and Soding made the same observation on flow^er stalks. Elastic extensibility remains practically constant during the normal growth of the coleoptile, but it decreases when growth is retarded either by decapitation or by complete removal of the coleoptile from the seedling. In decapitated seedlings, the decrease in extensibility may be partly annulled by the addition of growth substance. From this it might be concluded that elastic extension is the primary step in growth and that growth substances make growth possible by increasing the elastic extensibility. Heyn, however, did not interpret his results in this way. The elastic extensibility of excised coleoptiles (removed from the seed and not growing), with growth substance applied, in some of the experiments was greater than the extensibility of excised coleoptiles without applied growth substance. The difference was not great, how- ever, and in both cases the elastic extensibility was far less than in growing coleoptiles. The fact that extensibility in nongrowing coleoptiles cannot be increased to any great extent by the addi- GROWTH SUBSTANCES FOR NORMAL GROWTH 109 tion of growth substance led Heyn to conclude that changes in elastic extensibility are not the primary cause of growth. Soding (1931, 19326, 1934) came to the same conclusion. He investi- gated the extent to which curvature, produced by the unilateral application of a tip to a decapitated Avena coleoptile, can be removed by plasmolysis. It was found that while the early part of the growth curvature persists almost entirely, the later part disappears to some extent. Soding concluded from this that the first step in growth is not reversible; hence it cannot be brought about by a difference in elastic extensibility of the cell walls or by changes of turgor pressure or of osmotic concentration. This conclusion is sound as far as can be judged at present. 2. GROW'TH SUBSTANCES AND PLASTIC EXTENSIBILITY. Accord- ing to Pfeffer's original idea, the limit of elasticity is not reached in turgor extension, thus excluding plastic growth. More recent investigations by Overbeck (1926), Pringsheim (1931), and Heyn (19316) have shown that saturation with water produces an overextension of the cell wall. The question arises whether this plays a part in normal growth. Went (1928a) suspected that the effect of growth substances involves an increase in the plastic extensibihty of the cell walls; Heyn (1930, 19316, 1934c) came to a similar conclusion. The latter determined plastic extensibility according to the method outlined above. It was found that the amount of bending was far greater when the coleoptiles were treated with growth substance before the experiment (see Heyn and van Overbeek, 1931). In coleoptiles which were not growing, plasticity was increased following the addition of growth sub- stance. Similar results were obtained from experiments with hypocotyls of Lupinus (Heyn, 19316, 1934a) (Fig. 36). He found, furthermore, that the turgor pressure itself is sufficient to produce an irreversible increase in the surface of the cell wall when the growth substance has increased the plasticity suffi- ciently. Heyn concluded, therefore, that the primary cause of growth is the plastic expansion of the cell wall. This brings about a decrease in the thickness of the cell wall, which is com- pensated for by an increase in substance. Gessner (1934) has shown, also, that a close relationship exists between growth and wall extensibility in Helianthus. He concluded that change in wall extensibility is the cause for a change in the rate of growth, not the result of it or an accompanying phenomenon of it. no GROWTH HORMONES IN PLANTS Although Soding did not state that plastic extensibility cannot assist in growth, he concluded that this is neither the only cause of growth nor the main one. He found that the plasticity of flower stalks is decreased only sHghtly by topping, although the growth of the stalks suffers a great decrease. Moreover, the dif- ferences in the plasticity of individual stalks are considerable. He concluded it improbable "that such a variable and continually changing property as plasticity is the single cause of a regularly occurring process." This implies the assumption that the Avena J <*■ b O] 61 02 62 Fig. 36. — Method of determining elastic and plastic extensibility in portions of Lupinus hypocotyls. An agar block without growth hormone was applied to a segment from the hypocotyl as shown in a. A similar agar block containing growth hormone (indicated by stippling) was applied to another hypocotyl segment, h. After 23^2 hours the hypocotyl segments were bent by mechanical force to an angle of 45° where they were held for 5 minutes (ai and ?)i). Upon removal of the force, the hypocotyls returned part way to their original vertical position; the segment treated with growth hormone retained an angle of curvature of 22.9° (62), while the control retained an angle of only 12.5° (02). Hence, plas- tic extensibility is nearly twice as great in the hypocotyl treated with growth hormone. (Adapted after Heyn, 1934a.) coleoptile behaves in a fashion similar to the inflorescence stalks. In a growth-substance curvature the plasticity of the convex side is greater than that of the concave side, and this difference still remains after the curvature has reached its maximum point, i.e., when no further growth is taking place. The fact that extensibility in nongrowing coleoptiles is less than in growing coleoptiles is yet to be explained. It is influenced neither by growth substance nor by low temperature (0°C.). Heyn originally concluded that it was brought about by an increase of the cell-wall matter, perhaps by intussusception. Later he showed that the decrease in extensibility is due to a reduction of the extension capacity of the elastically extended outer layers of the wall. Soding considered that the decrease in extensibihty may be conditioned by a hardening of the wall and, conversely, that the increase in elastic extensibility of the wall GROWTH SUBSTANCES FOR NORMAL GROWTH 111 during growth may be brought about by a softening of the plastic intermicellar substance. 3. GROWTH SUBSTANCES AND INTUSSUSCEPTION. If plastic extensibiUty is not the primary cause of growth, as Soding con- tends, the only possibility remaining is that of intussusception. Pfeffer tried to show that this is of decided importance in the growth of the cell wall. He demonstrated that growth occurs in the root only when the turgor pressure is compensated by an opposing pressure; the growth force must be supplied, therefore, by intussusception. Soding's hypothesis regarding the growth of the cell wall is this: A viscous intermicellar substance embedded between the micellae can be plastically extended by turgor. This extension, although a part of the growth process, is of only secondary significance. The essential step consists of the addition of new glucose particles from the intermicellar substance to the micellae. At the same time, new intermicellar substance is being formed from the protoplasm, and this increase of matter in the cell wall necessarily is associated with cell elongation. The forces effective in this process are so great that turgor pressure is of secondary significance. It is clear that we have not yet been successful in determining the primary cause of growth, and it seems best to leave this question open for the present. With regard to the effect of growth substances upon wall growth, there are apparently two possibilities : They influence either plastic extensibility or growth by intussusception. The first possibility is upheld by Heyn. His view is sum- marized in the following statement : In view of the evidence, it might be expected that the protoplast acts as a mediator in the process of growth-substance activity on the wall. Therefore, the protoplast may be important for the supply of growth substance to a particular portion of the wall, or growth substance may have an effect not directly upon the cell wall but upon the protoplasts which subsequently produce changes in the condition of the wall. The other possibility — that the growth substance is concerned in growth by intussusception — is upheld by Soding. He assumed . . . that the hormone has an effect upon the intermicellar substance directly or indirectly (perhaps through the mediation of the protoplasm) 112 GROWTH HORMONES IN PLANTS and stimulates the formation of the structure necessary for growth as well as the whole process of intussusception. Since a greater plasticity of the cell wall (dependent upon the intermicellar substance) occurs in actively growing oat coleoptiles, it may be concluded that the increased plasticity is conditioned physically by this "growth mechanism," i.e., intussusception. It could be considered then that growth is prepared for by the hormone and that increased plasticity of the walls follows. This may be seen from numerous experiments by Heyn (1931a, b; 1932a, b). According to this hypothesis, the essential function of the hormone is not to make the wall plastic (Heyn) — if this were the case, only a subordinate process in growth would be influenced (at least in flower stalks) — but its role lies in the regulation of intussusception growth. Soding presented his views on the mechanism of stretching growth in the following manner: stimulative substance: Stage of preparation: Stage of elongation: Main process Hormone Preparation for growth Elongation by intussus- ception Secondary processes Increase in wall extensibility Plasticity Plastic turgor extension Elastic extensibility Elastic turgor extension Although a clear picture of the effect of growth substance must await further evidence (see Bonner and Thimann, 1935), several other points may be mentioned in this connection. It is unlikely that growth substance spreads itself out in a monomolecular layer over the growing cell wall and in this way influences growth ; the computations of Thimann and Bonner (1933; see also Kogl, 1933, Mitt. Ill) show that insufficient growth hormone is present to form even a monomolecular film on the growing cell walls; hence, any kind of hormone action through increase of permeabil- ity seems improbable. Further work on this problem by Bonner (19346) has shown that cell elongation may not be attended necessarily by a corresponding amount of wall formation. A given amount of elongation may be accompanied by more than the usual amount of wall deposition, as when the tissue is grown in fructose solution; or practically no wall may be laid down, as occurs at 2°C. He concluded that the increase of wall area GROWTH SUBSTANCES FOR NORMAL GROWTH 113 probably is not due primarily to active intussusception of new material, at least in the case of the Avena coleoptile. Growth of the wall, according to Bonner (1935), appears to come about by turgor extension of the plastic w'all and incorporation of definitely oriented cellulose micellae into the wall. Bonner and Heyn (1935) have tried to determine the influence of growth substance upon the electrical properties of materials composing the cell wall. Suspensions of Avena-coleoptile cell walls, ground and washed in water, were placed in an electro- phoresis apparatus, and their movement in an electrical circuit was measured. Apparently the electrical properties of the particles depended chiefly upon constituent proteins. Electro- phoresis of material obtained from normal coleoptiles and from coleoptiles that had been decapitated for 2 hours gave no indica- tion of differences in charge. Furthermore, addition of 100 units of 3-indole acetic acid per cubic centimeter of suspension produced no effect upon the charge of the particles. Since neither a direct nor an indirect effect of growth substance upon the charge of the coleoptile cell-wall particles could be demonstrated, it was con- cluded that the role of growth hormones in promoting cell-wall elongation probably is not exercised through any great modifica- tion of the electrical. properties of the cell wall. The theory that growth is promoted through increased plastic- ity due to the direct action of growth substance upon the cell wall seems untenable because of the small amounts of the hormone involved. It seems probable that the growth stimulus is con- cerned in some w'ay with the processes of the living protoplasm (Bonner, 1933a). The many diverse \dews and the scarcity of sound information concerning the mechanism of growth- hormone activity permit no definite conclusions at the present time. Acid -growth Reaction and the Growth Hormone. — Another growth theory has been propounded by Strugger (1932, 1933, 1934). He considered as the primary causes of growth all factors that can change the colloidal condition of the protoplasts. It was shown that Helianthus hypocotyls freed of growth sub- stance by decapitation can be influenced to renewed grow^th by physicochemical treatment of the protoplasts; this was accom- plished by immersion of the tissues in suitably buffered solutions, usually by the apphcation of acids. The same effect could be 114 GROWTH HORMONES IN PLANTS obtained also in neutral tap water covered with paraffin oil. According to Strugger, the renewal of growth is brought about by acidification ; in the case of tap water and paraffin, by acidification resulting from the lack of oxygen. He summarized this view of growth processes in the following scheme: The primary impetus in growth by elongation is: An acidity-gradient present within the tissue A plasmatic gradient as a result of the above (change in ionization of the protoplasmic colloids) Change of swelling ' Nonosmotic increase of inter- nal pressure by the pressure of swelling 1. Elastic extension Becomes fixed for the greater part, by active growth of the membrane. The possibility of overextension exists to some degree Change in characteristics of the membrane by the protoplasm Change in volume, the turgor pressure remaining the same? 2. Plastic extension Intervention of suction-force regulation and of osmotic regulation Growth of the cell wall? Strugger (1933) interprets the significance of growth substances for growth by elongation as follows: "It is clear, therefore, that growth substance obviously does not influence the protoplasm and membrane directly but rather that it regulates the acidity gradients in the course of metabolism and therefore the course and intensity of stretching growth." Following up the work of Strugger, Bonner (1934a) showed that in the Avena coleoptile the effect of acid on growth is not a direct effect upon the physical properties of protoplasm. His study indicates that the "acid-growth reaction" consists primarily in setting free a certain amount of active auxin acid from the inac- tive salt form of the growth hormone already present in the coleoptile. The growth stimulation by acidification is propor- tional to the concentration of the free auxin acid. It had been GROWTH SUBSTANCES FOR NORMAL GROWTH 115 shown previously that auxin is ineffective at pH 7, more active at pH 6, and about equally active at pH 4 and 5 (Dolk and Thimann, . 1932). From these facts it seems quite certain that Strugger's acid-growth reaction is little more than a consequence of the setting free of the auxin acid from its inactive salt. Inhibition of Growth in Roots. — Up to this point we have observed only the promoting effect of growth substance. It must not be forgotten that the rate of growth in roots is retarded by growth substance. How the same compound can produce the opposite effects in stems and roots has not been explained satisfactorily. Cholodny (1931a, e) has proposed the hypothesis that growth substance promotes the rate of development of growing cells but shortens the length of their individual life cycles. Elongation in the root lasts only a short time, and the period of growth is shortened still more by growth substance; i.e., if the latter is added, the cells mature quickly without elongating. Growth in length of the root, therefore, is retarded by growth substance. In the stem, on the other hand, the zone of cell stretching is greater, and growth continues for a relatively longer time; the period of growth in stems is shortened (i.e., the rate is accelerated) by the addition of growth substance. There is insufficient evidence to support this hypothesis at the present time. SUMMARY Numerous experiments have shown that without growth hormones, growth of the shoots of higher plants cannot take place. Although hormones are not the only important factors concerned in growth, they are essential for the normal enlargement of cells. In the Avena coleoptile, Lupinus hypocotyl, and the foliage leaf of Nicotiana, growth intensity has been shown to be correlated with the differential distribution of hormones. Regions of stems, leaves, etc., where such hormones are present, always appear to undergo greatest growth in one dimension; i.e., growth is polarized. The same substances that are essential for the growth of coleoptiles, foliage leaves, hypocotyls, and stems inhibit the elongation of roots over a wide range of concentration. The mechanism by which hormones promote growth is not well- understood. There is some evidence that they are used up not as 116 GROWTH HORMONES IN PLANTS "building stones" but as activators, influencing in some way the deposition of materials in cell walls. The primary effect of the hor- mone has been regarded by some investigators as making the cell walls plastic. The stretching that takes place, due to turgor, is accompanied by the incorporation of new wall materials. The result is a permanent increase in size. Other investigators hold that the primary influence of growth hormone is upon the deposi- tion of new wall materials by intussusception and that the forces concerned are so great that turgor pressure is of secondary importance. Whether these or other explanations are valid cannot be decided without more evidence. Growth must be regarded as a function of living protoplasm. The increase in cell- wall boundaries is only one manifestation of the fundamental ability of an organism to build itself out of the materials of its environment. CHAPTER VII THE SIGNIFICANCE OF GROWTH SUBSTANCES FOR OTHER PHENOMENA The significance of hormones for the growth and development of plants appears to be exercised mainly through some effect upon the enlargement of cells. By regulating the increase in cell size, growth substances control the growth of tissues and organs. Recent investigations have extended the role of these substances to include the initiation of roots, the production of tumors, the stimulation of cell divisions in the cambium, and many other important physiological and morphogenetic processes. Bud Development. — The phenomenon of apical dominance and the inhibition of buds lower down on the shoot axis has been interpreted variously as being due to differential distribution of the food supply, the electrical pattern, or chemical regulators. In recent years, the evidence in favor of some sort of chemical regulation has come to the front. Since growth substance is a necessary factor for stem elonga- tion, one might consider that the dormancy of resting plant organs, for example, of buds, is caused by a lack of the growth substance. If this hypothesis is correct, then the substance might function to promote growth in dormant tissues. The presence of growth substance in the periderm of dormant potatoes would suggest that other factors must be involved also. In order to throw more light on this question, the results of some unpublished experiments (by Boysen Jensen) dealing with the influence of growth substance upon resting buds will be discussed. A difficulty in the experimental set-up was encoun- tered in bringing the growth-substance solution into the vicinity of the buds. It was found that Forsythia is suitable for the pur- pose, since there are diaphragms across the stem at the nodes. If one bores into the stem, the interior can be filled with growth- substance solution. Another method involved removal of the tips from twigs of Salix, Syringa, and Aesculus. The basal end of 117 118 GROWTH HORMONES IN PLANTS each twig was connected with the lower end of a perpendicularly suspended funnel 20 cm. long. When this was filled with growth- substance solution, the fluid flowed slowly through the twig and exuded from the apical-cut surface. These experiments were carried out with different concentrations of growth substance during the winter when the buds were still in a resting condition. In no case was it possible to observe any ''forcing" effect as a result of the treatment. Various investigators have studied the retarding effect which the axis of the shoot has upon the development of the axillary buds. According to Snow (1925a, 1929a, h, 1931a, h, 1932a), this is caused by a specific retarding substance. Thimann and Skoog (1933) and Skoog and Thimann (1934) have reported that this substance may be identical with growth substance. When the terminal buds were removed from seedlings of Vicia Faba, the lateral buds developed rapidly. This activity could be retarded by placing agar blocks with relatively large amounts of auxin upon the cut surfaces. These investigators concluded, therefore, that the growth substance formed by the terminal bud in normal, nondecapitated plants is the retarding factor in the development of lateral buds. When these start to develop, growth substance is formed, and this has its effect upon their further development. If the hypothesis of Thimann and Skoog is correct, it follows that the growth substance formed by buds can both inhibit and pro- mote their development. Uhrova (1934) found that a substance diffusible into agar or gelatin was present in the leaves of Bryophyllum crenatum and inhibited lateral bud development. Hormone from Avena cole- optiles, diastase, and saliva, as well as acids had the same effect. Czaja (1934) and Soding (1935a) have demonstrated growth substance in the developing buds of many woody plants, and Avery (1935) has found it in the young growing leaves of Nicoti- ana. The relationship between the processes involved in bud development and the role of growth substance has not been discovered. Further studies by Hitchcock (19356) have shown that lateral bud inhibition can be brought about by the applica- tion of indole-acetic and indole-propionic acids or ethylene and propylene gases to decapitated tobacco plants. Bauguess (1935) has found inhibition effects with /3-3-indole pyruvic, /3-3-indole-oximinopropionic, and iS-3-indole acryHc acids. GROWTH SUBSTANCES FOR OTHER PHENOMENA 119 Tumor Formation. — Growth substances not only function by regulating normal growth but also, under certain conditions, bring about abnormal swellings and intumescences by hyper- trophy of the tissues of stems and leaves. Cholodny (1931a, e) placed coleoptile tips laterally upon the root tips of maize. He found that tumor formation occurred and that the zone of elongation of the swollen roots was significantly shortened. Anatomical investigation revealed considerable enlargement of the cortex. The number of cell layers was not changed, but radial and tangential dimensions of the single cells were remark- ably increased, and they were less susceptible to dyes than normal root cells. According to Loeb (1924), callus formation occurs only at the basal, never at the apical, end of a stem of Bryophyllum. A small piece of stem without leaves forms little or no callus, while a piece of stem of the same mass with a leaf attached to it forms considerable callus. Callus formation has been found to result from the application of lanolin paste containing an extract obtained from orchid pollinia (or human urine) to the internodes of Tradescantia and Coleus (Laibach, Mai, and Muller, 1934). The active agent, supposedly promoting cell division, was termed meristine. Since it was found to be soluble in ether and water and is thermostable, it was considered identical wdth auxin. Laibach and Fischnich (1935a) devised a quantitative method of testing the callus- forming action of 3-indole acetic acid paste (and other substances) which brings about an increased rate of cell division in Vida Faba epicotyls (Fig. 18A). Laibach (1935) reported that urine, pollinia, and corn-flower pastes, when applied to decapitated Vicia Faba epicotyls and to Coleus stems, all brought about callus formation in 3 days. Czaja (1935c) has criticized Laibach's conclusion that a cell- division effect has been demonstrated for growth substance in callus formation. He found that if the growth substance is apphed on the side of the axis so that it moves inward in opposi- tion to the normal stream of the substance, swelling results below the place of its application. These swellings arise by the trans- verse stretching of the cells. Complete disorganization of the normal polarity leading to cambial activity and the subsequent formation of masses of tracheids on the apical end of the organ 120 GROWTH HORMONES IN PLANTS <) -i^ i i i i Gravity ^^S^ZZ^ (a^ abed a b Fig. 37. — Various phenomena brought about by the application of growth hormone to plants. The paste containing the hormone is indicated by stippling. A, unilateral application of 3-indole acetic acid paste, as shown in a, induces curvature in the Avena coleoptile {b and c). (After Laibach, 19336, 1935.) B, growth curvature in roots may be regulated by the unilateral application of growth hormone: a, when applied to a vertical root, lateral bending occurs, as shown in b; c, primary roots in the horizontal position normally curve down- ward, but growth hormone applied on the upper side causes negative geo tropic curvature, d; applied on the lower side of a horizontal root it promotes positive geotropic curvature, e. (After Koch, 1934.) C, young leaves of Nicotiana and other plants exhibit hyponasty, a, and epinasty, &, as a result of the addition of 3-indole acetic acid paste. (After Avery, 1935.) D, Mimosa petioles bend upward, a, or downward, b, upon application of 0.01 per cent 3-indole acetic acid paste to the lower or upper sides of the primary pulvini. (Burkholder and Pratt, 1936.) E, application of growth-hormone paste around the hypocotyl of a Helianthus seedling nullifies polarized growth in the long axis: a, normal seedling; b, with ring of hormone paste; c, untreated seedling; d, treated seedling, after 7 daiys. (After Czaja, 1935a.) F, if growth hormone is applied unilaterally to a young stem, as shown in a, bending occurs away from the side of application, b. (After Zimmerman and Wilcoxon, 1935.) GROWTH SUBSTANCES FOR OTHER PHENOMENA 121 were thought to arise as secondarily induced phenomena follow- ing the artificial addition of substances causing cell enlarge- ment. In another paper, Czaja (1935a) reported the results of further investigations dealing with the effects of growth substance over a considerable range of concentration when applied to Helianthus, Avena, and other plants (Fig. 37 E). Additional evidence was obtained for the way in which cells increase their 0 20;u. 0 20/J. A _ B Fig. 38. — Longitudinal sections of Helianthus hypocotyls, showing effects of the application of 3-indole acetic acid on polarized growth. A, the normal cortex is comprised of cells markedly elongated in the direction of the long axis of the organ. B, cells in a hypocotyl treated with growth-hormone paste become nearly isodiametric, and have a much greater volume than those in the normal hypocotyl. (After Czaja, 1935a.) dimensions under the influence of growth-substance supply. The lateral application of relatively high concentrations brought about retardations of growth in length and increase in thickness of roots, stems, etc. (Fig. 38). It was concluded that the direc- tion of growth by cell enlargement is controlled by the direction of transport of growth substance. Recently LaRue (1935) has investigated the role of the auxins in the development of intumescences on poplar leaves. The injection of heteroauxin into twigs or its application in lanolin directly upon the leaves brought about proliferation of tissue. Cell outgrowths from the mesophyll of Mitchella repcns were produced by the feces of insect larvae or applied droplets of 0.0005 per cent 3-indole acetic acid. 122 GROWTH HORMONES IN PLANTS Stomatal Movement. — In an attempt to find out whether growth substance influences the production of starch in the guard cells and therefore possibly affects the opening of the stomata, Boysen Jensen removed the petioles of leaves of Sinapis and Sambucus and placed them in water and in growth-substance solutions of different concentrations. Although the growth- substance solution was taken up by the leaves in abundant amounts, no effect upon the degree of opening of the stomata could be distinguished, nor could any increase or decrease of starch in the guard cells be observed. Respiration. — Boysen Jensen and Nielsen (1925) published some experiments dealing with the effect of decapitation on the intensity of respiration in Avena coleoptiles. It was found to be practically the same in decapitated and nondecapitated coleop- tiles, and no effect of growth substance upon respiration could be shown. Nielsen and Hartelius (1932) also found that rhizopin was without effect in this respect. According to the more recent experiments of Bonner (1933a, h), however, growth substance may influence the intensity of respiration of the Avena coleoptile. Coleoptile cylinders 3 mm. long were placed in growth-substance solution, and the intensity of respiration was determined. In the lower concentrations the rate was promoted about 27 per cent; and at higher concentrations there was a retardation. When the growth substance was inactivated by treatment with peroxide, no promoting effect could be observed. Biinning (19346) concluded from his observations on Aspergillus that the assimilation of nitrates was increased by the promoting effect of growth substance on respiration. The effect was brought about by moving the pH in an alkaline direction, which increased conidia formation. Cambial Activity. — The stimulating influence of foliage leaves upon cambial activity has been suggested by a number of workers (Coster, 1927; Thoday, 1933). Kastens (1924) had suggested that the stimulus for cambial activity may be a hormone. The observations of Snow (1933) made it seem probable that the stimulus contributed by developing buds might be some chemical substance in the nature of a hormone. Snow and LeFanu (1935o) obtained indications of increased cell division in the cambium of young Helianthus plants which had been decapitated and treated with urine extracts. In more detailed experiments these same GROWTH SUBSTANCES FOR OTHER PHENOMENA 123 workers (Snow and LeFanu, 19356; Snow, 1935a, 6) used purified auxin a and 3-indole acetic acid in concentrations of 1 or 2 p. p.m. When aqueous solutions of these substances were applied to the upper ends of decapitated Helianthus seedlings, cambial division was stimulated. The demonstrated presence of growth substances in sprouting buds and young leaves, and the effectiveness of these substances in promoting cambial activity, lead to the conclusion that the earlier suggestions of a hormone stimulus passing from the leaves to the cambium in the stem is probably true. The precise way in which the auxins bring about increases in both cell size and number in different plants under different circumstances is not yet clear. Nastic Movements. — The great volume of evidence concerning the role of growth substance in tropic curvatures suggests strongly that some similar mechanism may be involved also in nastic responses. The differential growth of bilaterally symmetrical organs, in response to stimulation by light, temperature, electric- ity, touch, gases, etc., has been studied in detail (Hennings, 1930; Zimmermann, 1931, 1932; Zeltner, 1932; Schmitz, 1934; etc.), but only recently has the growth-substance explanation actually been tried out experimentally in this connection. Avery (1935) showed that epinasty or hyponasty could be produced reachly in Nicotiana by applying a small amount of growth substance in lanolin to the adaxial or abaxial surface of the petiole (Fig. 37C). The well-known movements of Mimosa have been found to be remarkably influenced also by the application of 3-indole acetic acid to the puKdni (Fig. 37 D) (Burkholder and Pratt, 1936). Nastic movements of Coleus leaves following treatment with 3-indole acetic acid (0.5 per cent in paste or solution) have been studied in detail by Fischnich (1935). The amount and duration of response varied directly with the concentration of the applied substance. Hitchcock (1935a, h) has demonstrated recently that a number of different substances, when applied to leaves of tobacco and tomato, are capable of bringing about epinastic movements. The decreasing order of effectiveness for a series of compounds producing epinasty was as follows: naphthalene acetic and indole acetic acids, indole butjo-ic and indole propionic acids, phenylat-etic, phenylpropionic, and phenylacrylic acids. The production of leaf epinasty by ethylene, acetylene, propylene, 124 GROWTH HORMONES IN PLANTS and carbon monoxide gases (Crocker, Hitchcock, and Zimmer- man, 1935) and by a-naphthalene acetic acid, ;S-naphthalene acetic acid, acenaphthyl-5-acetic acid, fluorene acetic acid, anthracene acetic acid, and a-naphthyl acetonitrile (Zimmerman and Wilcoxon, 1935) is of great interest. Bauguess (1935) has reported similar epinastic effects with other organic acids. Further investigations are needed to discover the mechanisms that lead to these manifestations. Root Formation. — The influence of many kinds of substances upon the initiation and growth of roots has been studied by numerous workers, but only in comparatively recent years has it become apparent that the old hypothesis of root-forming sub- stances, proposed by Sachs (1882a), may possibly have some basis in fact (see also Morgan, 1903). Loeb (1916, 1917, 1924) per- formed some very instructive experiments which led to the obser- vation that a stem segment of Bryophyllum forms roots and bends geotropically more readily when a vigorous leaf remains attached to the stem (Fig. 1, Loeb). As a result of his observa- tions, he wrote: ''All these facts suggest a close association if not identity between the root-forming substances and the substances (or hormones) causing geotropic curvatures." The significance of this statement was not realized until the more recent develop- ments in the field of plant hormones. The work of van der Lek (1925, 1934), showing that the presence of leaves or buds promotes the formation of roots at the morphological base of a cutting (Fig. 39), led to further investiga- tions of the possible role of hormones in the initiation of roots. Following this line of attack, F. W. Went (1929) found that a non- specific, heat-resisting substance could be extracted from leaves and germinating barley which, when applied to cuttings, promoted the development of new roots (Fig. 39). F. A. F. C. Went (1930) investigated the root-forming substance in Bryophyllum calycinum. The function of root-forming substances was studied further in Impatiens and Acalypha by Bouillenne and Went (1933) (see Bouillenne, 1933). Laibach, Miiller, and Schafer (1934) demon- strated the formation of roots by urine extracts applied to inter- nodes of Tradescantia, Helianthus, and Ligustrum. Laibach (1935) and Fischnich (1935) obtained similar results with 3-indole acetic acid on Coleus (Fig. 39). Went (1934a) named the substance that stimulates root formation rhizocaline and GROWTH SUBSTANCES FOR OTHER PHENOMENA 125 \'nn Her Lek, 1925. The presence of a developing bud or young leaf promotes root formation in a woody cutting. HV«(. 1929. A substance stimulating root production is contributed to a stem by a grafted leaf, c, or agar con- taining boiled diastase solution, h; the control stem, a, remains without roots. Went, 1934b. Thimrnin and Koepfli, 1935. A quantitative test for substances stimulating root production may be made by immersing the apex of a split stem in solution for 15 hours, ."ifter two weeks' growth in water, the number of roots gives a measure of the concentration of the substance. (See Fig. 40 for further details.) With this method, it was established that rhizopin, auxins, and "rhizocaline" are identical. Lfnbacli,MuUcr,nnd Sthdfir, 1934. Lnilmrh. 1935. Applica- tion of 3-indole acetic acid paste to a Coleus stem cau.ses roots to be produced in great numbers. Zimmerman and Wilcoxon, 1935. Injection of indole acetic acid, naph- thalene acetic acid, indole butyric acid, etc., into plant stems stimulates root production. Hitchrnck and Zimmerman, 1935. Addition of 3-indole acetic acid or any one of several other synthetic growth substances to the soil stimulates root production on aerial parts of Lycopcrsicon and Nicotiana. Zimmerman, Croeker, and Httchcock. 1933. Treatment of Tageles plants with 1.0 per cent carbon monoxide mixed with air for 2 to 10 days stimulated the production of adventitious roots on the stem. Zimmerman and Hitelieoek, 1933. Tageles: a, roots were pro- duced six days after a 72-hour treatment with 0.25 per cent acetylene; b, a second treat- ment with gas caused a change in the direction of growth and the production of root hairs. Fig. 39. — Rooting phenomena. 126 GROWTH HORMONES IN PLANTS worked out the proportionality existing between its concentration and the number of roots formed in pea seedlings (Fig. 39). The details of the method may be obtained from Fig. 40. Thi- mann and Went (1934) found that the active substance was present in large quantities in the crude auxin extracts obtained from Rhizopus and also from urine. Then it was found that the u + Jl20 KMn04 4 hrs. 4 hrs. V Many per bottle C D Y s c n u + 5 cc. 1 cc. Solution „ to be tested 2 per cent sugar ,j5 hrs. -Ji>ft 6 days Five per bottle G 5 cc. H2O 7 days j H I f) A+ Fig. 40. — A method of testing for the presence of substances stimulating the production of roots. The number of roots formed indicates the approximate effectiveness of the solution. The test is made in a darkroom at 25°C. and at a relative humidity of 60 to 70 per cent. A, etiolated pea seedling 7 days old, cut above first scale leaf and again just below the tip, gives test plant B. Basal end of shoot marked +. After standing in water 4 hours, C, and in potassium permanganate 4 hours, D, the shoot, E, is split longitudinally at the apical end with a sharp razor blade, and the base rinsed with water. It is immersed 15 hours in the solution to be tested, then rinsed with water and placed in sugar solution where it is allowed to stand for 6 days, G. Next it is removed from the sugar solution, rinsed with water and placed in water for 7 days, H. Two weeks after stage A, the test plant I is examined for the number of roots present. (After Went, 19346.) pure auxins prepared by Kogl and coworkers were effective in root formation (Thimann and Went, 1934) as well as in growth promotion. Thimann and Koepfli (1935) showed that 3-indole acetic acid stimulates root production; hence substances causing cell elongation are also effective in root formation. The role of pollen and urine extracts in root formation has been studied further by Muller (1935) and by Laibach and Fischnich (19356). GROWTH SUBSTANCES FOR OTHER PHENOMENA 127 The latter workers have shown that in horizontally placed stems there is a displacement of root-forming substances from the upper to the lower side just as is the case for the cell-elongation sub- stances. Initiation of roots is brought about in a region where longitudinal transport of the substance is prevented, e.g., by a wound. The synthesis of root-forming substances in leaves through the action of orange-red light and their polar transport Fig. 41. — Rooting effects obtained by applying synthetic substances to plants. A, Nicotiana. An overhanging slit portion of the stem is immersed in 0.05 per cent indole butyric acid. The acid travels upward in the transpiration stream and causes adventitious roots to be produced along the stem. B, Lycopersicon, adventitious roots on stem 14 days after treatment with 2.0 per cent 3-indole acetic acid in lanolin. {From Zimmerman and Wilcoxon, 1935.) have been pointed out recently by Went (19356). Attention should be called to the fact that the same substances that cause formation of roots also inhibit their growth in length. That the auxins are not unique in their ability to produce new roots has been shown by a series of contributions from the Boyce Thompson Institute. The initiation and stimulation of adven- titious roots by treatment with appropriate doses of ethylene, acetylene, propylene, and carbon monoxide gases have been demonstrated in some 15 species and varieties of plants (Fig. 39) (Zimmerman and Hitchcock, 1933; Zimmerman, Crocker, and 128 GROWTH HORMONES IN PLANTS Hitchcock, 1933a, h). The effects of 16 different "growth sub- stances" appUed to stems and leaves either in the form of paste or Fig. 42. — Rooting and nastic responses obtained by applying synthetic sub- stances to Lycopersicon. A, left: control plant, decapitated; right: plant with surface of cut stem treated with 1.0 per cent a-naphthalene acetic acid; photo- graphed after 8 days. B, left — control plant; right — plant after injections of 0.01 per cent indole butyric acid, photographed after 14 days. Note induced epinasty of leaves and adventitious roots. {From Zimmerman and Wilcoxon, 1935.) in sohitions have been considered in relation to the local initiation of adventitious roots on these organs (Figs. 41 and 42) (Zimmer- man and Wilcoxon, 1935). a-Naphthalene acetic acid and GROWTH SUBSTANCES FOR OTHER PHENOMENA 129 indole butyric acid were the most effective substances for the stimulation of new roots. The mechanism of action of these different compounds is not known. Miscellaneous. — Since the discovery of growth substances in plants, the effect of these substances when externally applied to plant organs has revealed a wide variety of interesting phenomena. The influence of growth substance upon regeneration and the forming of wound tissue has been studied by Mrkos (1933). The fluid obtained from cultures of Rhizopus suinus, excised Zea coleoptile tips, and portions of Bryophyllum leaves was found to promote cell division in the wounded mesophyll of Bryophyllum leaves. These studies seem to support the hypothesis of "wound hormones" proposed by Haberlandt (1921, 1922). Mai (1934) investigated the significance of growth substance for the elonga- tion of petioles and reported a prolonged period of petiole growth due to the application of pollen paste to Coleus, Acer, Viburnum, and other plants. Laibach (1934) demonstrated the curling of leaves by applying growth-substance paste to young leaves. LaRue (1935) found that when the blades were removed from the petioles of Coleus, abscission soon followed. Applications of agar blocks or lanolin containing exudate of leaves or pollen of Populus grandidentata, 2 per cent unne, or 0.0005 per cent hetero- auxin caused the petioles to remain on the stems from 35 to 141 per cent longer than in the controls. SUMMARY It has been shown in Chap. \T that hormones regulate the normal growth of plants by promoting cell enlargement in expand- ing organs. Many other effects have been attributed to these growth substances. One of the most interesting of these is concerned with the phenomenon of chemical correlation within the plant body. Growth substance apparently is formed in expanding buds and rapidly growing leaves whence it moves into other regions of the plant, exercising some degree of control over the behavior of potential centers of growth. It has been shown further that auxin a and heteroauxin may stimulate cell division in the cambium; hence it is probable that cambial activity in dicotyledons is stimulated by the growth hormones supphed from growing leaves and buds. 130 GROWTH HORMONES IN PLANTS Under the proper circumstances, growth substance may bring about the formation of tumors, the building of callus tissue, and the initiation of new roots. The substances present in the coleoptiles of maize, orchid pollen, urine, and also pure hetero- auxin have been shown to bring about an increase in the bulk of the tissues near the site of their application in both roots and stems. Such hypertrophy may be brought about either through an increase in the number of cells or by a shift in the direction of growth. Proliferation of cells has been demonstrated, too, by the application of heteroauxin to mesophyll tissue. The role of the auxins in the neoformation of roots has been demonstrated in numerous instances, as has the fact that these same substances also inhibit the growth in length of roots. That the auxins are not unique in their ability to produce new roots has been shown by the effectiveness of many other substances in this respect, such as, a-naphthalene acetic, indole butyric acid, etc. The mechanism of action of these different compounds, whether direct or through some influence upon the auxins or in still other ways, is not known. The differential growth on the two sides of bilaterally sym- metrical organs leading to nastic movements has been shown to be influenced by the local application of auxins and divers other substances. For example, heteroauxin caused marked nastic responses in the leaves of such plants as Nicotiana, Mimosa, etc., and a number of other substances have been shown to bring about epinasty in Nicotiana and Lycopersicon. Pro- longation of the growth period and prevention of petiole abscis- sion by auxins, also, have been demonstrated in several species. Future investigations may provide an explanation of the funda- mental mechanism of growth-substance activity in connection with these diverse manifestations of hormone-controlled growth. CHAPTER VIII THE SIGNIFICANCE OF GROWTH SUBSTANCES FOR PHOTOTROPISM When illuminated from one side, plant shoots usually turn toward the light. This response is brought about by differential growth, in such a way that curvature results. Though the direction of movement is usually positive, there are some instances in which organs turn away from the light; in such cases, they are said to be negatively phototropic. Inquiries into the nature of phototropism led to the discovery of substances that regulate growth in plants; continued studies on this subject have helped elucidate the mechanism of tropic responses. GENERAL DISCUSSION OF PHOTOTROPISM WITH SPECIAL REFERENCE TO THE AVENA COLEOPTILE The benchng responses to light have been studied in many kinds of plants, but the favorite object for investigations dealing with the mechanism of phototropic curvature is the coleoptile of Avena sativa. Certain distinct advantages in its use arise from the fact that seeds from pure lines of the species can be obtained practically every^^here, the plants can be grown easily in any laboratory, and reproducible results can be obtained under controlled environmental conditions. Stimulation and Response. The Light Gradient.— When an Avena coleoptile is illuminated unilaterally, light is absorbed by the tissues, and a descending gradient of light intensity across the organ results. Under these circumstances, that part of the coleoptile nearest the source of light naturally receives a greater amount of light than the portion on the shaded side of the organ. This gradient of the light stimulus, which is of considerable importance for the theory of phototropism, has been measured with great care by several investigators. Lundegardh (1922) tried to determine the magnitude of the light decrease in unilaterally illuminated oat seedlings by employ- ing photometric methods. It was found that the light in the 131 132 GROWTH HORMONES IN PLANTS coleoptile tip is diminished by only one-tenth when the back side is not shaded by the primary leaf. In other words, the shaded side receives nine-tenths of the light which falls upon the side nearest the source of light. In the basal region, however, the far side obtains only one-twentieth to one-fiftieth the amount of the light received by the front side. Van Dillewijn (1927a) found, in rather good agreement with these measurements, that the back side receives about one-thirtieth as much light as does the front. Nuernbergk (1927) since has found lower absorption values in unilaterally illuminated coleoptiles than were indicated by earlier workers. When the broad side was illuminated, the light was decreased to about one-seventh in the basal region; when the narrow side was illuminated, the light value on the far side was reduced to one-tenth of the incident amount. The decrease in intensity in the apical zones depended somewhat upon whether or not the primary leaf was within the coleoptile. With the leaf present, the front side received 4 to 8 times as much light as the back. With the leaf not acting as a screen, the front side received 1.3 to 1.5 times as much light as the back when illuminated on the broad side and 2.6 to 3 times as much when illuminated on the narrow side. This difference in the absorption values for the nar- row and wide dimensions of the organ is probably due to the difference in thickness of the tissues through which the light passes. As shown in Fig. 21, the walls of the coleoptile are much thicker in the narrow portions containing the bundles than in the other parts of the organ. Bergann (1930) also determined the decrease of light in the Avena coleoptile by microphotometric measurements. It was found that the blue rays passing through the coleoptile tip from the broad side were 2.2 times and from the narrow side 3.0 times more intense on the front than on the back side. In the basal portion of the coleoptile the intensity was 33 to 37 times greater on the exposed side. However, the stimulating effect of light arises mainly by its action upon the apical portion of the tip. It should be mentioned that the height of the primary leaf in the hollow cylinder of the coleoptile has in itself no significance for the course of phototropic curvature, although the presence of the leaf, acting as a light screen, has a decided influence upon the hght gradient across the coleoptile. Du Buy (1934) studied the GROWTH SUBSTANCES FOR PHOTOTROPISM 133 light gradient and phototropic curvature in Avena coleoptiles which were filled with water or with air. Curvatures were less in the water-filled coleoptiles owing to a smaller light gradient. Distribution of Sensitivity to Light. — Early studies upon the question of relative phototropic sensitivity of different zones of the oat coleoptile established the fact that not all regions are equally sensitive to light (Darwin, 1899; Rothert, 1892, 1894). Rothert concluded from the results of his studies that "the apical region of high phototropic sensitivity is not longer than 3 mm., and only in the extreme 1 to 1.5 mm. portion of the tip is the sensitivity to light particularly great." Since the studies by Rothert, this same question has been investigated with improved technique. Sierp and Seybold (1926) used adjustable screens in order to partially darken an exactly determinable portion of the tip. The presentation time was then determined when 0.25, 0.50, 0.75, to 2 mm. portions of the tip were darkened. From the figures obtained in this manner, a curve was constructed which showed an increase in presentation time with an increase in the length of the darkened apical region. Sierp and Seybold stated their results as follows : In conclusion it can be said that the sensitivity to light in the Avena saliva coleoptile is greatest in the J^ mm', at the tip, from which point downward it decreases rapidly. In the zone of about K to K tarn. lying directly below the tip region, the sensitivity is only J-^o of that at the extreme tip. At a distance of 2 mm. from the tip, sensitivity decreases to 1/36,000 of that of the uppermost region; and from this point on to the bottom, it remains about constant. At about this time, Lange (1927) studied the distribution of sensitivity to light in the coleoptile tip. He thought that the method of darkening used by Sierp and Seybold could cause half- shadow formations which would impair the accuracy of the measurements. To avoid this, he illuminated each transverse zone to be investigated by means of a slit, the width of which could be diminished to 50 microns. The light value was determined as the product of the intensity of light X time of illumination X area of the illuminated surface. The threshold values necessary to elicit phototropic response were determined for different zones of the coleoptile. From the formula: Sensitivity to light = threshold hght value' V^^^oi^^ iulLIIRARY ^ 134 GROWTH HORMONES IN PLANTS where k can be any desired constant, the sensitivity to light may be computed. A comparison of the experiments of Lange and of Sierp and Seybold is given in Table 4, where the values obtained Table 4. — Relative Sensitivity to Light in Different Zones of the AVENA CoLEOPTILE Zone, millimeters from Light sensitivity, as calculated from the experi- ments of apex Sierp and Seybold Lange 0 - M 1 -iH lM-2 33,948 564 34.7 8.42-24.2 38,870 2,870 253 24.7 by the latter authors have been recomputed on the basis of Lange's formula. Even though the two series of numbers deviate con- considerably from each other in the second and third half- millimeter zones, the results are essentially in agreement. Since Lange worked with zones as narrow as 50 microns, he has given us very exact information concerning the distribution of sensitivity to light in the extreme tip region. It is clear that the uppermost zone, which is only about the width of a single cell, is the point most sensitive to light (see Table 5). Phototropic Tabi,e 5. — Relative Sensitivity to Light in the Upper Half Millimeter OF the Tip of the Avena Coleoptile Zones, Microns Sensitivity 0- 50 7,600 50-100 6,422 100-200 5,665 200-300 4,106 300-400 3,046 perception diminishes rapidly when the tip is removed (Koch, 1934). The reduction in sensitivity becomes more marked with the removal of tip pieces up to 1 mm. in length, just as has been observed by darkening cUfferent zones. With still further decapitation, the increase is much more than that found by the darkening method. After some time has elapsed following decapitation, a "physiological tip" is regenerated; at the same time, phototropic sensitivity is increased, and the ability to react GROWTH SUBSTANCES FOR PHOTOTROPISM 135 to light stimuli is restored (Dolk, 1926; Reinders, 1934). It should be mentioned that general illumination of colcoptiles during the growth period lowers their sensitivity to subsequent unilateral lighting (Filzer, 1930). Interesting data on the differential response of the tip and base of the Avena coleoptile to varied intensity and controlled wave lengths of radiation have been obtained recently by Haig (1935). The extreme tip portions (1.5 mm.) of some plants and the sub- apical regions of other plants were exposed to white light for short periods, and the reaction time was measured for positive phototropic curvature. The speed of the initial reaction-time process was found to be proportional to the logarithm of the light intensity up to about 1,000 meter-candle seconds, above which there was a marked decrease in the rate of response. The reaction-time curves for the tip and for the rest of the coleoptile yielded separate loci suggestive of two distinct photoreceptive systems. With white light, blue-green, and minus-blue regions of the spectrum, the tip was phototropically more sensitive than was the base. These results support the earlier work of Went (1926), who reported a distinct difference in the light sensitivity of the tip and base of the Avena coleoptile. The phototropic sensitivity of the Avena coleoptile to different regions of the spectrum has been determined in recent years by several investigators (Bachmann and Bergann, 1930; Johnston, 1934). It seems to be well-established that the shorter wave lengths of visible light are most active in causing phototropic curvature, while the longer wave lengths in the red region are practically inactive (Fig. 43). The relative effectiveness in the region of greatest sensitivity {i.e., in blue light) is represented by a bimodal curve with maxima at about 4,400 and 4,800A. (Johnston, 1934). As a matter of interest it may be mentioned here that a similar bimodal curve with maxima in the same regions (Fig. 44) has been found for the light inhibition of germinating lettuce seed (Flint and McAlister, 1935). The phenomenon of differential spectral sensitivity would appear to be of considerable significance in connection with an analysis of phototropism and growth. Conduction of the Stimulus. — The early studies (Rothert, Fitting, Boysen Jensen) on the conduction of the phototropic stimulus in the Avena coleoptile were described in the first chap- 136 GROWTH HORMONES IN PLANTS 6200 5780 5460 4360 4050 3660 Wave length in Angstroms Fig. 43. — Spectrum sensitivity curve of phototropic response (solid line) and protoplasmic streaming (points) in the Avena coleoptile. The agreement between the phototropic response and the effect of light on retarding proto- plasmic streaming suggest an interrelationship. (After Bottelier, 1934.) {Curve of phototropic response from Blaauw, 1909.) 100 '' ^ "^ / / \\ \ \ \ \ \ \ \ \ 80 / N / / \ \ \ \ 1 ^ / / ' ""' / \ \ M / / / / \ \ c / / \ \ % 60 — / / \ \ """ "€ / / \ \ ^ ' /^N / \ \ > \ \ \ \ '■^ y/ \ / \ \ ■? 40 / ' \ / — & / / / / ^^^ \ \ 20 > / / / / \ 0 X --4-' 1 1 1 1 1 5400 5200 5000 4400 4200 4000 4800 ^ 4600 Wave length in Angstroms Fig. 44. — Curve of inhibition of Lactuca seed germination (solid line) in the violet-green region compared with the curve of phototropic response of Avena coleoptiles (broken line). {After Flint and McAlister, 1935.) (Curve of photo- tropic response from Johnston, 1934.) GROWTH SUBSTANCES FOR PHOTOTROPISM 137 ter. In these experiments, transverse incisions were made upon the front (lighted) or back (shaded) side of the coleoptile a few milHmeters below the tip. In some cases, mica plates were inserted in the cuts. Then the tip was unilaterally illuminated above the incision, and it was determined whether the cut had interfered with the conduction of the stimulus to the growing portions below. Boysen Jensen's experiments showed that the stimulus is conducted on the back (shaded) side of the Avena coleoptile. Later, Purdy (1921) carried out quantitative studies deahng with the localization of the stimulus conduction. In order to eliminate the stimulus resulting from making the incision, the experimental plants were used 24 hours after the operation, when they were again entirely straight. Pieces of mica were then inserted into the incisions, and the tips of the plants were unilaterally illuminated, with the pieces of mica facing either toward the front or the back. The inconsequential curvature, which was caused in some oases by the insertion of the mica pieces, was measured and taken into account. When the incision was on the front side, a phototropic curvature resulted with a d value (see Chap. II) of 0.61 mm.; in other cases, of 0.10 mm. (Fig. 45). Purdy concluded from her experiments "that the strongest tendency is for transmission of the stimulus to take place in a longitudinal direction, mainly locaHzed in the side of the coleoptile farthest from light." The importance of the vascular bundles in conduction of the phototropic stimulus from the tip downward has been indicated in the experiments of Baffet et al. (1933) with glass plates inserted transversely in the Avena coleoptile. Beyer (1928a) tried to show that conduction of the phototropic stimulus can take place upon the front as well as upon the back side. In his experiments, the basal portion of the plants were darkened with sand. According to Cholodny (1929a), sand is translucent and is not sufficient to protect the basal portion of the coleoptile entirely from light (see also Reinhard and Bro, 1933). Conduction of a stimulus upon the front side of the coleoptile, although a weak one, has been repeatedly observed, first by Purdy and later by others. From the standpoint of the growth-sub- stance explanation, such a phenomenon might be expected, but the stimulus transmission that takes place upon the front side is of an entirely different nature from that occurring on the back 138 GROWTH HORMONES IN PLANTS side. By unilateral illumination, as will be shown later, the growth substance in the tip is moved to the back; therefore, less growth substance flows upon the front side than is normally Fig. 45. — Transmission of phototropic stimulus in the Avena coleoptile; A, incision on the darkened side. B, incision on the lighted side. C, control plants. {From Purdy, 1921.) the case, and the growth rate of the front side is retarded. The "stimulus transmission" on the front side, therefore, is condi- tioned by the decrease of the growth-substance supply from the tip to the basal region. GROWTH SUBSTANCES FOR PHOTOTROPISM 139 An important question is whether or not the phototropic stimulus can be transmitted in an acropetal direction as well as in a basipetal one. Rothert (1894) and van der Wolk (1911) found that it could not, but von Guttenberg (1913) demonstrated that, when coleoptile tips of plants whose basal portions had been previously illuminated unilaterally were lighted from the opposite side, the tip curvature which might have been expected was either absent or exhibited feebly. The author concluded that a stimulus is conducted from the unilaterally illuminated basal portion to the tip. This is not consistent with the in^-estigations on growth-substance transport which ha^'e shown that its trans- mission takes place only in a basipetal direction. Arisz (1915) criticized the work of von Guttenberg on other grounds. Lange (1927) suggested that during unilateral illumination of the base, some of the light might fall upon the tip and in this way influence the transmission of stimulus. Reinhard and Bro (1933) have pointed out still other possibilities. Quaniity-of-stimulus Principle. — It has been held for a long time that in certain photochemical reactions the amount of applied energy is of importance in bringing about a constant effect. Bunsen and Roscoe (1862) proposed a quantitative rule for the effect of light upon a sensitive photographic plate, where the product of the exposure time X intensity of light = a con- stant value. Froschel (1908, 1909) and Blaauw (1909) showed that a definite amount of light must be applied to a plant organ in order to produce a threshold phototropic response. The amount of light is the product of two factors: the intensity of light and the dura- tion of illumination. That the quantity-of-stimulus principle holds over a wide range for phototropic curvature in the Avena coleoptile may be seen in Table 6, wliich is taken from the work of Blaauw. The fact that such a short illumination as 1/1,000 second can call forth a phototropic curvature is of importance for the comprehension of the induction process. Certainly the matter of photic stimulation is an excellent example of the general rule regarding the excitation of irrital)le protoplasm, i.e., that a relatively small amount of applied energy is capable of setting into action a chain of processes which leads eventually to a comparatively large response. The significance of the Blaauw theory will be discussed in greater detail later on. 140 GROWTH HORMONES IN PLANTS Table 6. — The Quantity-of-stimulus Principle in the Phototropic Response of the Avena Coleoptile (Blaauiu, 1909) Illumination, meter- Amount of light, meter- candles Duration of illumination candle seconds 0.00017 43 hours 26.3 0.000439 13 hours 20.6 0.000609 10 hours 21.9 0.000855 6 hours 18.6 0.001769 3 hours 19.1 0.002706 100 minutes 16.2 0.004773 60 minutes 17.2 0.01018 30 minutes 18.3 0.01640 20 minutes 19.7 0.0249 15 minutes 22.4 0.0498 8 minutes 23.9 0.0898 4 minutes 21.6 0.6156 40 seconds 24.8 1 . 0998 25 seconds 27.5 3.02813 8 seconds 24.2 5.456 4 seconds 21.8 8.453 2 seconds 16.9 18.94 1 second 18.9 45.05 % second 18.0 308.7 %5 second 24.7 511.4 3^2 5 second 20.5 1,255 1-5 5 second 22.8 1,902 J-ioo second 19.0 7,905 yioo second 19.8 13,094 J^oo second 16.4 26,520 H.ooo second 26.5 Along with a knowledge of the amount of light necessary to produce a threshold curvature, it has been found equally profita- ble to study the course of phototropic curvatures with greater amounts of light. Different observers have found that as the amount of light is increased progressively above the threshold- stimulus value, various types of positive and negative bending may occur. Positive and Negative Curvatures. — Pringsheim (1909) observed that when an Avena coleoptile is unilaterally illuminated with increasing amounts of light, no curvature is produced so long as the amount of light is below the threshold. Above the minimum GROWTH SUBSTANCES FOR PHOTOTROPISM 141 quantity of light necessary for excitation, positive curvatures are evoked; i.e., the stimulated organ bends toward the light over a considerable range of stimulus values. These curvatures become more pronounced and disappear less rapidly with increasing amounts of light. If the amount of light is increased still more within a certain range, a stage of indifference appears, and after some time an opposing movement may take place so that weak negative reactions can be observed. With still larger amounts of light, positive curvatures are again brought about. Pringsheim presented graphically the influence of the amount of applied light upon the manner of reaction of the seedlings, and these conclu- sions were verified by Clark (1913). It is clear from the investi- gations of Pringsheim and Clark that three different modes of response can be distinguished in the Avena coleoptile: the first positive curvature, the first negative curvature, and the second positive curvature. Arisz (1915) investigated carefully the manner of reaction of the Avena coleoptile to different amounts of light in an attempt to determine at what quantities of light the various curvatures appeared. The results of his experiments (Table 7) indicate that Table 7. — Size of Curvature in the Avena Coleoptile in Response to Different Amounts of Unilateral Light Amount of Light, m.c.s. Size of Maximum Curvature* 7.6 0.7 12.4 1 18.1 1.6 26.4 2.3 45 3 65 3.3 75 4 100 5 140 4.7 237 5.4 560 4 1,500 3 2,800 1-2 * Curvature in millimeters deviation from the vertical position. Coleoptiles were about 25 mm. in length. the magnitude of curvature increases with the amount of light applied until a maximum curvature is reached with about 100 meter-candle seconds. After this point, curvature decreases with increasing amounts of light. With about 6,000 to 40,000 meter- 142 GROWTH HORMONES IN PLANTS candle seconds, the first positive curvature disappears entirely, and a pure negative curvature appears. With still greater amounts of light, the negative curvature also disappears, and a positive curvature appears immediately — the so-called second positive curvature. The negative curvatures appear only when the tip is illuminated and cannot be definitely demonstrated when the base is unilaterally illuminated. These results have been confirmed by other investigators. Lundegardh (1922) found that the first purely positive reaction appeared with light values up to 10 meter-candle seconds, the maximum being at 10 meter-candle seconds. The range of the negative curvature lay between 800 and 500,000 meter-candle seconds, with a minimum at 4,000 to 10,000, the second maximum being at about 2,000,000 meter-candle seconds. The reaction decreased again with still greater amounts of light. The experiments of Pringsheim, Clark, Arisz, an^ Lundegardh were all carried out with mixed white light. Du Buy and Nuern- bergk (19296) investigated the course of phototropic response using monochromatic blue light of wave length 4,360 A. Only the apical 2 to 3 mm. portion of the coleoptile was illuminated. The amount of light was measured in ergs per square centimeter per second. For the sake of comparison with the other investi- gations cited, 1 erg at wave length 4,360A. may be considered as corresponding to about 10 meter-candle seconds (Table 8). Du Buy and Nuernbergk believe that they are able to distinguish Table 8. — Positive and Negative Curvatures in the Avena Coleoptile IN Response to Different Amounts of Radiation Seconds Ergs Response Curvature Mo 12.2 Soon, very obvious + Ks 24.2 Soon, fairly obvious + H 122 Later, weak — 1 610 T-ater — 5 3,050 Later, weak + 12 7,320 Soon, weak + 25 15,250 Later — 100 61,000 Soon — 300 183,000 Soon, good + 600 366,000 Later, weak + 900 549,000 Later, good + GROWTH SUBSTANCES FOR PHOTOTROPISM 143 five types of curvatures, viz., three different positive curva- tures, which are separated from each other by two cHfferent negative curvatures, or indifferent stages. A comparison of the data given in Table 8 with the results of Arisz shows that the second positive curvature of du Buy and Nuernbergk is new, appearing first at about 30,000 to 70,000 meter-candle seconds, i.e., in the range that, according to Arisz and Lundegardh, gives negative curvatures. The first positive curvature of du Buy and Nuernbergk corresponds to the first positiv^e curvature of Arisz, and the third positive curvature of du Buy and Nuernbergk corresponds to the second positive curvature of Arisz. The Primary Positively Phototropic Curvature.— Some analyses of the growth processes which occur during the curvatures will now be reviewed. It can be shown experimentally that the first positively phototropic curvature is not connected with a change in the average rate of growth. For such experiments, Cholodny (1930) used a micropotometer. An excised Avena coleoptile from which the primary leaf had been removed was plugged with lanolin at the bottom and placed in the enlarged opening of a capillary tube bent at right angles so that the intake of water could be measured over a period of time. The coleoptile was placed in a saturated moist chamber to check transpiration. It was assumed that all the water taken up under these conditions was used for the volume increase of the coleoptile during growth, and the intake of water, therefore, was used as a measure of the growth rate. It was found that no appreciable change in the rate of growth could be shown in coleoptiles that were unilaterally illuminated with a light value of 500 to 2,000 meter-candle seconds and afterward darkened, although decided phototropic curvatures resulted in the course of 1}4 to 2 hours. Du Buy and Nuernbergk obtained similar results at a later date. In these investigations, the course of growth of both the front and back sides of an Avena coleoptile was recorded during the first positive curvature produced by unilateral illumination of the tip with 3.55 ergs at a wave length of 4,360A. for 2 seconds. From the curves it may be seen that the average rate of growth of the coleoptile is not changed during the curvature, so the first posi- tive curvature involves an increase in the rate of growth of the back side and a corresponding decrease in the growth of the lighted side. The increase and decrease in the rate of growth of 144 GROWTH HORMONES IN PLANTS the two halves just balance, so that no change in the growth rate of the whole organ takes place under conditions leading to phototropic curvature. The course of the first positive curvature has been investigated by Lundegardh (1922), Went (19286), du Buy and Nuernbergk (19296), Dolk (19296), and du Buy (1933). Dolk illuminated Avena coleoptiles unilaterally with 50 meter-candles for 10 seconds and recorded the course of the tropic curvature cinemato- graphically. The radii of curvature of the individual growth zones were measured on enlarged photographs. The results of his investigations are given in Fig. 46. The ordinates give the size of curvature, i.e., the reciprocal value of the radius of curva- ture, and the abscissae represent the time in minutes. The curvature begins almost simultaneously in the first three zones, that is, about 40 minutes after illumination, and proceeds down- ward. In the tenth zone, curvature becomes apparent only after 100 minutes. In the meantime, the curvature has increased continuously in the apical region, reaching its maximum in the first two zones after 170 to 180 minutes. That the average rate of growth is not changed during the second positive curvature has been shown by the investigations of Beyer (1927c), who employed three series of experimental plants: series A, illumi- nated bilaterally 1 hour and bilaterally 2 hours; series B, 1 hour bilaterally illuminated and 2 hours darkened; and series C, 1 hour illuminated bilaterally and 2 hours unilaterally (50 candles at a distance of 60 cm.). The growth of the lighted and darkened sides of series C during the phototropic curvature was then com- pared with that of both the other series. The linear growth in series A was 0.20 cm.; and in series B, 0.23 cm.; in C on the light side it was 0.11 cm., and on the dark side, 0.33 cm., which gives an average of 0.22 cm. The average rate of growth during photo- tropic curvature appears to be the same as it is in plants in the dark or in those bilaterally illuminated. Du Buy and Nuernbergk (19296) obtained similar results when they followed the course of the second positive curvature under strong illumination. Although the second positive curvature corresponded to the course of the first positive response, a sudden increase in growth could be observed during illumination. The authors attributed this increase to cell-wall extension brought about by the strong illumination. GROWTH SUBSTANCES FOR PHOTOTROPISM 145 20 40 60 80 lOO 120 140 160 180 200 Fig. 46. — Course of curvature in different zones of the Avena eoleoptile after phototropic stimulation with 50 m.c. for 10 seconds. Ordinate: size of curvature; abscissa: time in minutes. Zone 1 is at the tip, zone 10 at the base. (From Dolk, 19296.) 146 GROWTH HORMONES IN PLANTS In conclusion, it can be said concerning the course of photo- tropic curvatures that (1) there are three (or five) different types of reaction, two (or three) positive and one (or two) negative curvatures; (2) the curvature begins in the tip and proceeds in a basipetal direction; (3) the average rate of growth is not changed during the primary positively phototropic curvature. During the second phototropic curvature there is, however, according to du Buy and Nuernbergk, a generally lessened rate of growth. Apparently there is no agreement concerning the precise influence of light upon the second positive curvature. The Blaauw Theory. — Among the different theories which have been proposed to explain the phenomena of phototropic curvature, perhaps the best known is that of Blaauw. The Blaauw theory has been discussed so often and so thoroughly in phototropic literature that it seems unnecessary to treat it here in great detail. This theory has been regarded occasionally as an explanation for all curvature phenomena, but actually it was applied for a time only to the phenomena of phototropism, and even here decisive proof was not obtained. The main point of the theory is that phototropic curvature in the separate regions of a plant organ comes about by an unequal rate of growth which is caused by an unequal distribution of light in the organ. Each part of the plant grows separately, according to the amount of light with which it is provided. Blaauw (1919) has described his theory in the following words: "The light-growth reaction is the primary phenomenon; phototropism is the secondary one which necessarily follows from it, when locally different light-growth reactions arise from locally different conditions of illumination." The ideas of Blaauw coincide, in the main, with the theory pro- posed earlier by de Candolle (1832). The application of the Blaauw theory to phototropism in the Avena coleoptile is difficult, because of the transmission of the stimulus which causes a phototropic curvature to take place in a region of the organ which is not directly illuminated. Evidence Supporting the Blaauw Theory. — The test of the valid- ity of the Blaauw theory of phototropic curvature in the Avena coleoptile begins with the determination of whether or not a phototropic curvature can be derived from the summation of the light-growth reactions of the illuminated and shaded sides under conditions of unilateral lighting. Sierp (1921) investigated the GROWTH SUBSTANCES FOR PHOTOTROPISM 147 effect of light upon the rate of growth in the coleoptile and con- chided that the Blaauw theory is an adequate explanation of the observed light-growth response. Brauner (1922) also found a general agreement between the course of phototropic curvature and the light-growth reaction for small amounts of light, thus supporting the validity of the Blaauw theory (also see Brauner, 1927a). In these experiments, however, the distribution of light in the coleoptile was not taken into consideration; therefore, these attempts to derive the laws of phototropic curvature from the light-growth reaction of the front side alone appear to be illogical. Evidence that might favor the Blaauw theory is to be found in the work of Bergann (1930). He observed that the order of phototropic effectiveness dechnes from the blue to the orange regions of the spectrum in the same way as does the light- growth response, which consists of a depression of the growth rate. Van Dillewijn (1927a) made quantitative determinations of the course of the light -growth reaction on the front and back sides of the coleoptile and attempted to describe the course of the phototropic curvature. In van Dillewijn's computations, the light-growth reactions for different amounts of light were deter- mined on the assumption that the illumination of the dark side was about one-thirtieth of that of the light side. Computations of growth curvatures for various methods of illumination yielded good qualitative agreement between the experimental results and what might have been expected on the basis of Blaauw's theory. The weakness in van Dillewijn's reasoning is the fact that he did not actually observe the phototropic curvatures computed from the light-growth reactions but compared the latter with the curvatures obtained by Arisz. When an accurate quantitative comparison is made between the phototropic curvatures which are to be expected from light-growth reactions and those actually appearing, no agreement between light-growth reaction and curvature can be demonstrated. Evidence Opposing the Blaauw Theory. — Evidence in opposition to the Blaauw theory has been obtained by Lundegardh (1922), von Guttcnberg (1922), Pisek (1926, 1928), Beyer (1927c, 19286), Went (1928a), Boysen Jensen (1928), du Buy (1933), and others. Pisek determined the difference in the growth reactions of coleop- tiles subjected to intensities of 2.5 and 80 meter-candle seconds and at the same time measured the difference in length between 148 GROWTH HORMONES IN PLANTS the convex and concave sides resulting from unilateral illumina- tion with 80 meter-candle seconds, after different reaction times. In this experiment, the front and back sides of the unilaterally illuminated coleoptile are exposed to the same two light intensities as the two coleoptiles mentioned, owing to the light gradient through it. This should yield information of value as to whether the differential growth occasioned by this difference in the inci- dent light can account for phototropic curvature. It is clear from the data (Table 9) that the growth changes in the light- Table 9. — Comparison of the Light-growth Reaction in the Avena Coleoptile Subjected to General Illumination of Two Inten- sities, and Growth on the Two Sides of a Similar Coleoptile Unilaterally Illuminated So That the Lighted and Shaded Sides Are Subjected to the Same Two Intensities Difference After 1 hr. After 2hr. After 2M hr. In growth reactions of 2.5 and 80 m.c Between the convex and concave sides of a curvature at 80 m.c 7.8 22.1 15.9 67 19.6 83 growth reactions under the two different intensities are not sufficient to produce a curvature of the size that is actually obtained in the unilaterally illuminated coleoptile. With a somewhat different approach, Beyer came to the same conclu- sion. In Beyer's experiments, three series of plants were illumi- nated bilaterally for one hour. At the end of the hour, the illumination was continued unchanged in series A; in series B, both lamps were turned off; in series C, one lamp was turned off. Phototropic curvature resulted in the plants of the C series by decreasing and not by increasing the light intensity. According to the Blaauw theory, one would expect that the lighted side Ci of the curved plants should grow just as fast or certainly not any more slowly than that of a plant in series A and that the shaded side Cg should grow as fast as, or no faster than, a plant in series B. The results may be expressed in the following way: A - Ci = 0.07 and 0.08, and C, - 5 = 0.12 and 0.11. Since a decrease in the rate of growth takes place on the lighted side, while an increase occurs on the darkened side, the data are not in accordance with Blaauw's theory. Bergann's (1930) GROWTH SUBSTANCES FOR PHOTOTROPISM 149 criticism of the matter will be mentioned later. Cholodny (193 Ir/, 19326, 1933a) has pointed out additional discrepancies between the light-growth reaction and phototropism. He demonstrated that coleoptiles, immersed in water, can curve phototropically with very brief periods of illumination, without showing any kind of light-growth reaction. In an entirely different way, Boysen Jensen (1928) showed the inadequacy of Blaauw's theory for phototropism. The coleop- tile was split lengthwise, and a rectangular platinum plate was inserted in such a way as to divide the organ into two halves, so that each could be illuminated by itself. If Blaauw's theory were applicable, a phototropic curvature should have been produced by suitably decreasing both light sources. The front half was illuminated with 51, the back half with 0.9, 1.6, and 2.6 meter- candles, so that the light decrease from the front to the back was about 51:1, 32:1, and 20:1. Only minimal phototropic curva- tures were obtained. In other experiments, a glass plate was inserted instead of a platinum one, and the tip was illuminated unilaterally at right angles to the plate. Here, again, only a minimal phototropic curvature resulted, although the distribution of light was the same as in intact plants. These experiments show plainly that the phototropic curvature does not come about through separate reactions of the single parts of the tip. To test the validity of Blaauw's theory, Li (1934) conducted experiments with decapitated Avena coleoptiles which were exposed to different amounts of unilateral light. It was found that immediately following decapitation, short exposures of 10 minutes even at 28,800 meter-candle seconds were incapable of inducing bending, while in exposures of 30 minutes curvature was caused by 3,600 meter-candle seconds. When the exposure was extended to 3 hours, even an intensity of 100 meter-candle seconds could elicit a response. It is quite clear that Blaauw's theory does not apply to decapitated coleoptiles. In many cases, a rather far-reaching parallel can be found between the light-growth reaction and the course of phototropic curvature. By a method of compensation whereby the coleop- tile is bilaterally illuminated, the stimulus values of different regions of the spectrum were obtained (Bergann, 1930). It was found "that illumination from all regions of the spectrum (except red and infrared), when applied in corresponding intensi- 150 GROWTH HORMONES IN PLANTS ties, produce equal light-growth reactions. Different wave lengths, when applied in suitable intensities, also produce equal curvature reactions (first positive, negative, and second positive)." In spite of agreement of this sort, it must be said that phototropism and light-growth reaction in Avena are two fundamentally different processes. The reader is referred to Biinning (1929) for further discussion of the matter. Conclusions in Regard to the Blaauw Theory. — It is highly probable that the light-growth reaction which appears in unlocal- ized illumination also exists in unilateral illumination and there- fore may have some significance in the production of phototropic curvature. In view of the available data on the subject, it is certain that Blaauw's theory is not sufficient to explain the observed changes in the rates of growth during the phototropic response of the Avena coleoptile. It can be shown that photot- ropism is connected with the transverse transport of a growth substance, Mobile such a phenomenon is not concerned in Hght- growth reactions which appear with unlocalized illumination. The Growth-substance Explanation. — The historical develop- ment of the growth-substance explanation of tropisms has been sketched in some detail in the first chapter. Sachs (1882a) early postulated the existence of formative substances which were supposed to control growth and development in plants. Definite evidence in favor of special growth substances was not forthcom- ing until some thirty or forty years later when, in connection with certain studies on phototropism, it was demonstrated that a growth-promoting substance is dispersed from the tip of the Avena coleoptile. (See Figs. 1 and 2 for a graphical story of the discovery of growth substances.) The Relation of Growth Substance to Phototropism. — In a paper entitled "Das Problem des Phototropismus und sein Ende," Blaauw (1919) wrote: The problem of phototropism in itself has become empty. Further theoretical observations on this problem will only keep us still further from the investigation of the actual and therefore significant phenomena of growth. Surely there is no problem in phototropism itself, since it is a pure growth phenomenon. Growth, however, as a phenomenon of life, is a problem of great depth. The growth-substance explanation has been proposed for both phototropic and geotropic phenomena. The application of this GROWTH SUBSTANCES FOR PHOTOTROPISM 151 explanation to phototropic curvature in the Avena coleoptile is presented with emphasis upon the positive curvatures arising from unilateral illumination of the tip. Since the phototropic curvature in the basal region results from a difference in the rate of growth upon the back and front sides, and since also the growth of the basal region is known to be regulated by the growth- substance supply from the tip, it can be concluded that the curva- ture must arise as a result of more growth substance flowing down the back side than the front side. The Question of Wound Substances. — The growth-substance explanation of phototropic curvature of the Avena coleoptile rests upon the proof that such a curvature can be produced by a growth-promoting substance, migrating down the shaded (or back) side of the coleoptile. After Paal and Soding showed that a growth-promoting substance migrates from the tip into the basal region in dark-grown plants, the question arose as to whether this growth substance was identical with the growth sub- stance acting during the phototropic reaction or whether special tropism hormones — " tropohormones " (Cholodny) — exist. Since growth substances in the coleoptile could be demonstrated only by their effects upon the rate of growth, it was not easy to determine whether the observed phenomena were due to one growth substance or many. Stark and Drechsel (1922) held that special tropism hormones exist. They carried out experiments on the transmission of the phototropic stimulus when excised tips of one species or genus were placed on the bases of other species or genera. When these were stimulated with unilateral illumination, it was found that bases with foreign tips applied reacted much more slowly than with tips of their own kind. It was concluded that the photo- tropic compatibility decreases with increased distance of natural relationship. These experiments were interpreted to mean that the stimulating substances are, to a certain degree, specific. Later, this hypothesis was advocated by Beyer (1928a) also, who held that no quantitative relationship exists between the regeneration of growth substance in a decapitated coleoptile and the restoration of the phototropic sensitivity. Similar conclusions have been reached by Li (1934), who found that the decapitated coleoptile is sensitive to light immediately following decapitation and that "physiological regeneration," 152 GROWTH HORMONES IN PLANTS VI leading to increased sensitivity, is concerned only with the production of growth substance and bears no relation to phototropic sensitivity. The existence of special tropohormones has been disputed by other investigators, viz., Cholodny (1927) and Went (1928a). Cholodny emphasized the fact that the rate of growth during phototropic curvature remains un- changed, which indicates that during unilateral illumination the growth- substance production is unchanged also, and that probably no new sub- stances are formed. Under critical examination, the experiments of Stark, Drechsel, and Beyer in no way demon- IV strate the existence of special photo- tropohormones. Cholodny (1929a) has pointed out that the experiments of Beyer are consistent with the assump- tion that there is but one growth sub- stance in the Avena coleoptile. It seems quite certain that no special photo- (or geo-) tropohormones are present in the Avena coleoptile, for Fig 47.-Diagram of the experiments which are to be mentioned tip of an Avena coleoptile ^ showing different ways in later show that Unilateral light or which unequal distribution of gravity can produce a displacement of growth hormone might occur ^ ^ ^^ >- as a result of unilateral the growth substance and therefore an illumination. {From Boysen unequal distribution of it. These ex- Jensen.) periments show beyond question that photo- and geotropic curvatures are brought about by a growth substance which is also present in the tip of plants in the dark and not by special hormones. The question, however, as to whether or not other tropohormones, for example, chemo- tropohormones, are concerned in traumatic curvatures is not so easily settled. Origin of the Unequal Distribution of Growth Substance. — The amount of growth substance given off by the tip in unilaterally illuminated plants as well as in plants grown in the dark is probably conditioned by the growth-substance concentration in the tip. If more growth substance is supplied by the tip to the GROWTH SUBSTANCES FOR PHOTOTROPISM 153 back than to the front side during the phototropic curvature, the growth-substance concentration upon the back side of tlie tip will be greater than that upon the front side. There are several other possible ways of explaining the origin of this difference in con- centration. Some of these are shown in Fig. 47. The line D in the figure shows the growth-substance concentration in the tip of plants grown in the dark, while the different possibilities of its distribution which can lead to positive curvatures are shown by the lines I to VI. Co7itrasting Theories of Boysen Jensen and Padl. — As a result of studies on the _ transmission of the stimulus in the Avena • I I coleoptile, Boysen Jensen concluded that phototropic curvature is brought about by an increase in the rate of growth upon the back side of the coleoptile, caused by a downward migrating substance (line VI in Fig. 47). In opposition to this view, Paal (1918) presented the hypothesis that the phototropic curvature may be caused by a retardation of growth upon the front side. He held that the growth-promoting sub- stances, which show an unlocalized migra- tion from the tip of plants in the dark, are either partly destroyed by the light on the front side of the coleoptile tip or are impeded in their movement in such a man- ner as to produce a growth retardation upon the front side of the coleoptile. Paal's suggestion can be represented by the line I or II in Fig. 47. Paal's theory is not consistent with the investigations of Boysen Jensen, and Purdy (1921), who demonstrated that the transmission of the stimulus can be almost completely inhibited by a transverse incision upon the dark side. Furthermore, the theory is refuted by certain experiments of Boysen Jensen and Nielsen (1925). In these experiments with coleoptiles 2 to 3 cm. long, a 4 mm. tip and the vipper portion of the primary leaf were removed. The empty part of each coleoptile was then split, and a thin, rectangular piece of platinum was inserted in the incision. Two coleoptile tips were then placed symmetrically I I Fig. 48. — Diagram of a decapitated Avena cole- optUe with two coleoptile tips applied symmetri- cally. A platinum plate is inserted vertically in the stump between the two tips. This permits illumination of a single tip from one side. {From Boysen Jensen.) 154 GROWTH HORMONES IN PLANTS upon the coleoptile stump (Fig. 48), and one of these was unilat- erally illuminated in the usual fashion. Curvature toward the light was produced as shown in Fig. 49. This experiment indicates that the flow of growth substance upon the darkened side is increased above the normal, and from this it follows that the growth-substance concentration upon the back side of the tip is increased by unilateral illumination. Purdy's Theory. — What is known regarding the growth- substance concentration upon the front side of a unilaterally lighted tip? According to one hypothesis (indicated by line VI Fig. 49. — Avena coleoptiles as diagrammed in Fig. 48. Light came from the right-hand side, so only the tips on the right were illuminated; positive curva- tures resulted. {From Boysen Jensen and Nielsen, 1925.) in Fig. 47), the phototropic curvature may arise solely through the increase in the growth-substance concentration upon the back side, while it remains unchanged upon the front side. The experiments of Purdy, however, indicate that even when the transmission of the stimulus upon the back side is blocked, a slight phototropic curvature occurs. To explain this, one must assume that the rate of growth upon the lighted side is slightly retarded. According to this hypothesis (represented graphically by line V in Fig. 47), the growth-substance concentration upon the front side is slightly decreased. The Went Theory. — Went (1928a) determined the amount of growth substance given off on the illuminated and shaded sides of unilaterally illuminated coleoptile tips by placing the tip upon two agar blocks, so that the lighted side stood upon one block and the darkened side upon the other. It was found that less growth substance was given off by the lighted side (about 46 per cent as much as from the darkened side). According to Went, the GROWTH SUBSTANCES FOR PHOTOTROPISM 155 distribution of growth substance in the illuminated tip should be represented by line III in Fig. 47. Theories of Beyer, Cholodmj, chi Buy, and Nuernhergk. — The total concentration of growth substance present in the coleoptile during phototropic curvature, in relation to the normal concentra- tion, is slightly increased according to the hypothesis of Boysen Jensen (as represented in hne V). On the other hand, according to the hypothesis of Went (Hne III), the amount of growth sub- stance is shghtly decreased. The investigations of Beyer, Cholodny, and du Buy and Nuernbergk have shown that the average rate of general growth during phototropic curvature is not demonstrably changed, and one must conclude, therefore, that none of the foregoing hypotheses is entirely correct but rather that the growth-substance distribution in the tip during phototropic curvature is proportionately decreased on the illuminated side and increased on the shaded side, as represented by line IV in Fig. 47. It has been shown that different types of phototropic curvature may occur in response to different amounts of light. The ques- tion arises as to whether or not the distribution of growth sub- stance in the tip can be correlated with the two different positive curvatures. Since, according to du Buy and Nuernbergk, the rate of growth is unchanged in both curvatures, it may be con- cluded that the distribution of growth substance is substantially the same in both cases. What the growth-substance distribution may be during the negative curvatures, remains to be investigated. The Displacement of Growth Substance. — The origin of the unequal distribution of growth substance during phototropic induction remains an important point for further discussion. Various suggestions have been offered to explain the growth- substance distribution as presented diagrammatically in line IV in Fig. 47. One might conclude that the rate of formation of growth substance in the tip is governed by the application of light, so that at certain light intensities the amount present is increased, while at other intensities it is decreased. However, such a notion would seem to refer us back to Blaauw's theory which has been proved inadequate as an explanation for phototropic curva- ture in the Avena coleoptile. The assumption of an unequal distribution of growth substance brought about by an exchange of material between the light and 156 GROWTH HORMONES IN PLANTS dark side of the coleoptile tip is supported by certain of Boysen Jensen's investigations (1928). When the tip of an Avena coleoptile was split and a thin glass plate was inserted into this incision parallel to the direction of light, normal phototropic curvature resulted in response to unilateral illumination. On the other hand, the curvature was very weak when the plate was oriented perpendicularly to the direction of light. If both halves of the tip, without the insertion of a glass plate, were held together closely either with a platinum spiral or with a thin glass tube, a phototropic curvature took place even when the incision was oriented perpendicularly to the direction of the light beam. The result of this experiment can be explained only on the assumption that an exchange of material between the two halves of the tip takes place during the photic induction. The question arises, What kind of substance is transported during the induction? At least two possibilities present them- selves : It could be assumed that the growth substance itself was displaced in a transverse direction during the action of unilateral light. Or, on the theory that a constant formation of growth substance takes place in the coleoptile tip, it could be assumed that the growth substance itself was not transported, but rather another substance which in turn influences growth-substance production. The displacement of such a substance should then cause a lessened formation of growth substance upon the front side and an increased formation of growth substance upon the back side. Since both of the above were possibilities, the author did not venture to draw definite conclusions from his investigations, though the first of these two assumptions was without doubt the simpler. At practically the same time as the publication of Boysen Jensen's (1928) experiments, Cholodny (1927) and Went (1928a) expressed the idea that the unequal distribution of growth sub- stance in the coleoptile comes about by a growth-substance displacement. Cholodny proceeded with his investigations on the assumption that tropic stimuli do not influence the produc- tion of growth substances. Went supported his ideas with experiments which seemed to show that after phototropic induc- tion, the amount of growth substance given off by the lighted side of the tip was lessened, while that of the darkened side was increased. The results of his experiments are given in Table 10 GROWTH SUBSTANCES FOR PHOTOTROPISM 157 in a very reduced form . If the amount of growth substance given off by the tip, in darkness, is adjusted to equal 100 per cent, then, on this scale, the growth substance given off by the shaded side of a unilaterally illuminated tip is increased from the expected 50 to 67 per cent. By another method of calculating the results given Table 10. — Relative Amounts of Growth Substance Recovered from AVENA-COLEOPTILE TiPS PlACED UPON AgAR BlOCKS AND KePT Either in Darkness or Exposed to Unilateral Light See Fig. 2 (Went) In darkness Illuminated with 1,000 meter-candle seconds Experiment number Time on agar, min- utes Lighted side Shaded side Total 360-363 364-367 368-374 379-381 383-388 100 100 100 100 100 70 60 90 80 120 38 26 6 32 33 57 51 62 60 57 95 77 68 92 90 Average 100 84 27 57 1 84 in Table 10, Went (1935) adjusted to 100 per cent the total amount given off into agar by the illuminated tip; then 65 per cent of this is dispersed into the block beneath the shaded side, and 35 per cent into that under the illuminated side (Fig. 2) (Went). There is some doubt whether one can conclude from these experiments that the amount of growth substance given off on the shaded side is increased by unilateral illumination. This method does not give entirely satisfactory evidence for the displacement of growth substance in the tip. It does not seem possible to present proof for the displacement of growth substance in the coleoptile tip, since new substance is continually being dispersed from there. In the basal portion of a decapitated coleoptile, however, none is present for some time after decapitation, and it should be possible here to decide whether a displacement of growth substance can take place. Dolk (19296) showed that in horizontal coleoptile cyHnders of Avena which were supplied with growth substance at one end, 158 GROWTH HORMONES IN PLANTS more growth substance was conducted through the lower than through the upper half. Although it is possible that unilateral changes in permeability may be produced by the action of gravity, whereby differences in the conduction of the substance might arise, still these experiments show decidedly that the growth substance can actually be displaced transversely by the action of gravity. The effect of unilateral light upon the distribution of growth substance in the coleoptile stump of Avena has been investigated by Boysen Jensen (1933a). When an Avena coleoptile was decapitated, and the cut surface covered with a block of growth- substance agar, the growth substance proved to be unlocalized in its downward flow. When the upper part of such a plant was unilaterally illuminated, and the lower portion was darkened, a positive phototropic curvature arose not only in the upper, lighted portion but also in the lower, darkened portion. It would appear, therefore, that in the shaded part of the coleoptile, more growth substance flows downward along the back side than upon the front side. The difference in the conduction of growth substance upon the front and the back side of the lighted portion of the coleoptile could be explained by the destruction of the growth substance upon the front side, by the lowering of the permeability upon the front side, or by the displacement of growth substance from the front to the back under the influence of light. Displace- ment of growth substance was directly proved by a comparison of the phototropic curvature in coleoptile stumps with and with- out growth substance present. Some decapitated coleoptiles were covered with growth-substance agar and some with plain agar blocks for a sufficient period of time to permit the intake of substances present in the agar. Then the blocks were removed, and the coleoptile stumps were illuminated with continuous light. It was found that in coleoptile stumps receiving growth substance, the phototropic curvature appeared about 2 hours earlier than in those without growth substance. This result can be explained by the assumption that the growth substance present in the coleoptile is displaced in a transverse direction by the unilateral effect of light. In some instances, the growth-sub- stance content of the coleoptile stump can be so great that a phototropic curvature does not appear, the reason being that an ample supply of the growth substance remains upon the front GROWTH SUBSTANCES FOR PHOTOTROPISM 159 side (in spite of its displacement) to produce a maximum rate of growth under the prevaihng conditions. Although a displace- ment of growth substance in the coleoptile tip has not been proved directly, it may be supposed, on the basis of these experiments with subapical portions, that growth substance is displaced by the action of unilateral light in the tip. Growth-substance Transfer and Electrical Potential. — Proof of the accumulation of growth substance upon the back side of the unilaterally illuminated tip has supplied a link in the chain of phototropic response, which may be considered as the concluding link in the process of induction. There remain for consideration the preceding factors in induction, i.e., how the effect of unilateral light can produce a displacement of growth substance; and the subsequent factors, i.e., how the unequal distribution of growth substance can produce the positive phototropic curvature in the Avena coleoptile. A consideration of the first steps in the process of induction leads back to the problem of growth-substance transport in the plant. The difficulties of constructing a plausible theory for the longitudinal transport of growth substance have been discussed in Chap. V. It is equally difficult to explain the transverse transport of growth substance in a satisfactory way. In a discussion of the theory of transverse transport, it should be pointed out that illumination for a mere fraction of a second can produce a curvature. Effective displacement of growth sub- stance naturally cannot take place so rapidly. It must be concluded, therefore, that illumination creates a condition in the coleoptile tip which is the primary cause of the displacement of growth substance. Since growth substance is an acid, one might suppose that the unilateral accumulation of it could be brought about by differ- ences in electrical potential induced by illumination of the tip. On this supposition, the back side would have to be electro- positive with respect to the front side. According to the investi- gations of Waller (1929) and Bose (1928), a potential difference actually exists in unilaterally illuminated stems in such a way that the shaded side is positive. Whether the observed differ- ences in potential are sufficient to explain the transverse dis- placement of growth substance is discussed more fully under geotropic curvatures. 160 GROWTH HORMONES IN PLANTS Recently, Koch (1934) has demonstrated the transport of growth substance across the Avena coleoptile toward the positive side in an artificially produced electric field. Similarly, Kogl (1933, Mitt. VI) and Ramshorn (1934) have shown the electrical transport of growth substance in Avena saliva and other species of plants. There are various obstacles in the way of an electrical explanation. Not all organs that contain growth substances react phototropically. A theory adequate to explain the transverse transport of growth substance must take into con- sideration the negative as well as the positive responses. Conclusion in Regard lo the Growth- sub slance Explanation. — If an unequal distribution of growth substance has taken place in the tip itself, a curvature must of necessity follow in the lower portions of the organ, since growth-substance transport in the basal region takes place almost exclusively in a longitudinal direction. According to du Buy (1933), the following three factors must be taken into consideration for the evolution of a phototropic curvature: (1) the production of growth substance, (2) the transport of growth substance, and (3) the effect of growth substance. Actually, the methods that one has for the measurement of these individual components are still far from perfect. Even in the simplest case of unilateral illumination of the tip, the quantitative relationship between the curvature and the distribution of growth substance (not to mention the amount of light applied) does not hold. When the entire coleoptile is illuminated, the difficulties become still greater. The phototropic curvature is determined not only by the amount of growth substance given off from the tip but also by its dis- placement throughout the basal region. Other factors must be considered also, such as the effect of light upon the activity of the growth substance and modifications in the inherent capacity for response. The importance of fight absorption for phototropism may be emphasized by brief reference to the peculiar action of certain dyes introduced experimentally into the fiving tissues of plants. Blum and Scott (1933) have demonstrated the photosensitizing effect of dilute erythrosin upon wheat roots grown in a nutrient solution. In the presence of 1:500,000 erythrosin, the uni- laterally illuminated roots exhibited relatively greater growth rates on the shaded side, so that bending occurred toward the GROWTH SUBSTANCES FOR PHOTOTROPISM 161 light. In the absence of dye, these roots were normally not phototropic. This response was attributed to the light absorp- tion of the dye in a manner similar to that exhibited by the naturally occurring porphyrins in plants that are generally photodynamic. On the other hand, the presence of certain dyes in plants has in some instances been found to destroy the phototropic response without having any great effect upon the average rate of growth (Boas, 1933; Schweighart, 1935). Boas found that seedlings of Lolium perenne treated with dilute eosin exhibited no photo- tropic curvature in response to unilateral illumination. The eosin seemed to affect both sensitivity to the stimulus and the distribution of the growth substance, though, obviously enough, the mechanism is still imperfectly understood. The growth substance which is active in phototropic cur- vatures is identical with the growth substance of normal growth. By unilateral illumination of the coleoptile tip, the rate of grow^th-substance dispersal is scarcely changed, but a displace- ment of it in a transverse direction takes place with the result that its concentration is greater upon the shaded than upon the illuminated side. The flow of growth substance into the basal region on the front side of the coleoptile is decreased, and that upon the back side is increased; therefore, the rate of subsequent growth on these two sides is roughly proportional to the amounts of controlling growth substance present. The average rate of growth over-all is either not changed or only slightly altered. Blaauw's theory assumes that the individual parts of the coleoptile grow at rates inversely proportional to the differential amounts of light and that the phototropic curvature results from separate reactions of the individual regions. According to the growth-substance explanation, or theory, the coleoptile tip reacts as a whole, and a difference between the lighted and shaded sides of the organ is created by a displacement of growth substance in the unilaterally illuminated tip. Herein lies the fundamental difference between the two theories. The growth- substance theory properly interpreted is capable of explaining all the observed facts of phototropic response in the Avena coleoptile. Other Theories on Phototropism. — The theory of Fitting (1907), mentioned in the introduction, holds that a polarization 162 GROWTH HORMONES IN PLANTS of the single cells of the organ of perception arises from unilateral illumination and that this polarization is conducted along paths of living tissues to the region of reception. This theory- might be questioned because, during phototropic induction, not only are the single cells polarized but also the tip as a whole. The recent findings of Rosene (1935) lend support to the previ- ously expressed idea that the electrical potentials observed in plant organs result by the algebraic summation of the electro- motive forces of polarized cells. Brauner (1922, 1924) assumed that the phototropic curvature comes about by retardation of growth upon the illuminated side of the organ. He concluded that unilateral illumination increased the permeability of the lighted side and in this way promoted migration of the substances from their source at the tip down the front side, producing a retardation of growth on this side. The theory fails because the substances dispersed from the tip have a promoting and not an inhibiting effect upon the growth of the coleoptile. However, the fact remains that modifications in tissue permeability do occur as a result of illumination (Brauner 1924, 1935), and it is conceivable that the translocation of growth substance may be influenced thereby. According to Priestley and Tetley (Priestley 1926a, b, c, d; Tetley and Priestley, 1927), the growth changes brought about by decapitation are not caused by growth substances but by changes in water supply. It was argued that cell elongation is conditioned by water intake. When the coleoptile is decapitated, water is exuded, and growth is decreased until the original condi- tions are again obtained by healing of the wound. Phototropic curvature was held to arise "by the increased resistance to stretching induced by the action of hght upon the walls and by the increased entry of water and rapidity of the movement of sap generally, which is the result of the increased tissue permea- bility that follows exposure to light." Tetley and Priestley close their discussion with the following words: "There seems no necessity to assume that any hypo- thetical substances are diffusing from the apex, and until such hormones are experimentally demonstrated they may quite well be dispensed with in theories of tropic response." This require- ment is now fulfilled, and Priestley's explanation is no longer tenable. GROWTH SUBSTANCES FOR PHOTOTROPISM 163 Gradniann (1930) proposed a complicated theory of growth and of phototropic curvature which, in the main, is in accord with the growth-substance explanation. However, Gradmann's theory assumes the presence of two different growth substances. One of these substances {A) is formed only in the tip and flows in an unlocalized fashion into the basal region. It is identical with the growth substance described above. The other {B) arises along the entire length of the coleoptile and on up into the tip. Each of these substances alone is considered as being ineffective, but together they form a substance {AB) which increases growth. Photo- and geotropic stimuli cause more growth substance B to be formed on the shaded, or under, side; hence more of the compound AB is produced, thus increasing the rate of growth upon this side. With the increase of substance 5 in a particular region, a greater use of A follows. A flows to this region of deficit, and the diversion of the stream of growth substance A strengthens the primary effect considerably. With this hypothesis, Gradmann tried to combine Blaauw's theory with the growth-substance explanation, to account for the photo- tropic response. In accord with Blaauw, the unequal origin of B is the primary reaction, and it should be sufficient in itself to bring about a curvature. The main objection to Gradmann's theory is that growth and growth changes in the Avena coleoptile can be much more easily explained by the growth-substance explanation alone. The Gradmann postulate is directly refuted by Boysen Jensen's (1933a) experiment in which phototropic curvature was hindered by the application of very great amounts of growth substance although the coleoptile grew vigorously. In spite of the displace- ment of growth substance, a' sufficiently large amount of it remains on the front side to produce a maximum rate of growth permitted by the prevailing conditions. This experiment could not be explained, by Gradmann's theory, because if the curvature came about by unequal distribution of the substance B, then an excess of substance A should not hinder the phototropic curvature. DICOTYLEDONOUS STEMS The great majority of investigations on the question of photo- tropism have dealt with the Avena seedling, and, without doubt, many of the conclusions reached in these studies concerning 164 GROWTH HORMONES IN PLANTS phototropic stimulus and response apply equally well to photo- tropism in other plants. Many valuable contributions to our knowledge of phototropic curvature, particularly in recent years, have developed out of studies on the response of dicotyledonous shoots to light. Distribution of Phototropic Sensitivity. — Darwin found long ago that only the tip of the grass coleoptile was sensitive to light and that this apical region determined the phototropic curvature of the lower portion. Similar results were obtained also with the stems of Brassica oleracea. However, Darwin's conclusions were not entirely convincing because of the considerable variabil- ity in his experiments. Later on, Rothert (1894) came to differ- ent conclusions regarding the phototropism of stems of Brassica napus, Agrostemma, Vicia, and other dicotyledonous plants. The sensitivity to light was found to be irregularly distributed in the stem, being particularly strong in a relatively short region near the tip and weaker in the basal region. This differential distribution of light sensitivity was found to decrease with age in Vicia. In other seedling stems, such as those of Tropaeolum, Solanum, and Coriandru.m, the sensitivity appeared to be regularly distributed over the stem, while the condition in Daucus and Linum formed a transition between these two groups. Transmission of Phototropic Stimulus. — Rothert demonstrated the transmission of a phototropic stimulus in various seedling stems of dicotyledonous plants. When the upper region of the stem of Brassica napus was unilaterally lighted, and the lower region was darkened by dry earth, paper aprons, or paper tubes, phototropic curvatures appeared in the darkened region of the stem. Similar results were obtained with Agrostemma, Tropae- olum minus, and some other species. It was relatively difficult to demonstrate any transmission of stimulus in Vicia. A study of Rothert's photographs for the conduction of the phototropic stimulas shows that the curvatures which are obtained in the darkened basal region in dicotyledonous seedlings are smaller than those obtained in Avena similarly treated. It appears, therefore, that the region over which the stimulus is transmitted is comparatively small in these plants. A decided phototropic stimulus transmission, extending over several centimeters and at times of marked strength, has been demonstrated in the stems of Linum usitatissimum, Brodiaea congesta, and Galium purpureum. GROWTH SUBSTANCES FOR PHOTOTROPISM 165 The results of these experiments make it probable that growth substances, at least in certain cases, are also concerned in the phototropic response of stems. Growth Substance and Phototropism in Seedling Axes. — Van Overbeek (1933), as mentioned before, studied the significance of growth substance for normal growth and the photogrowth reaction in Raphanus sativus. He demonstrated the formation of growth substance in the cotyledons and its subsequent move- ment into the hypocotyl. When the upper end of a hypocotyl cylinder was completely covered with growth substance and unilaterally illuminated, more growth substance was extracted from the basal regions on the dark side than on the hghted side (Fig. 2). This shows that it was displaced by the action of unilateral light, just as it is in the Avena coleoptile. It seems reasonable to conclude that the growth substance dis- tributed from the cotyledons is displaced toward the back of the hypocotyl by unilateral light. In addition, van Overbeek showed that the light-growth reac- tion was very strong in dark-adapted plants. With general illumination he found a growth retardation which amounted to over 50 per cent. This phenomenon could be explained neither by changes in growth-substance transport nor by the destruction of growth substance through the action of light, and van Over- beek assumed that the tonus of the seedling stem with reference to growth substance must be changed by the application of light. The importance of this phenomenon for phototropism in seedlings is clear. Under conditions of unilateral illumination, the light intensity on the lighted side was 5.2 times greater than that on the shaded side of the Raphanus hypocotyl. Since the rate of growth is retarded so greatly by the application of light, then unilateral illumination must be sufficient to produce a phototropic curvature in accord with Blaauw's theory. Boysen Jensen (1936) extracted with chloroform the growth substance from the front and back sides of unilaterally illuminated seedling axes of Phaseolus. During phototropic curvature the growth substance was more concentrated on the side away from the light. Van Overbeek (1932) was able to show that the quantity of growth substance transported through a portion of Raphanus hypocotyl was about the same in the light as in the dark. The presence of the tip was not essential for bringing about a lateral 166 GROWTH HORMONES IN PLANTS displacement of growth substance with illumination on one side, for it was found that the substance could be drawn toward the shaded side of a decapitated hypocotyl cylinder. This investigator came to the conclusion that the phototropic curvature of the Raphanus hypocotyl is brought about by the combined action of two different effects: (1) by the differential retardation of growth, which is inhibited more upon the lighted side, and (2) by the displacement of growth substance to the back, which must produce an increase in growth upon the dark side and a retardation of growth upon the lighted side. He concluded, further, that the Blaauw theory and the growth-substance theory are not to be considered as antitheses but that the fundamental ideas of both are complementary. The question remains how these two effects share quantita- tively in producing the curvature. The miequal distribution of growth substance and of light must be considered together with the fact that the retarding effect of hght is influenced by the growth-substance concentration. Although the computations become somewhat involved, it may be concluded that the curva- ture comes about mainly through the fact that growth on the lighted side is checked markedly, while growth of the darkened side is slightly increased in comparison with the normal. THE PHYCOMYCES SPORANGIOPHORE The growth-substance explanation has not yet been applied to positive phototropism in the sporangiophores of Phycomyces. Another explanation, however, has been attempted on the basis of the unequal distribution of light wdthin the cylindrical stalk bearing the sporangium. Blaauw (1914, 1919) studied the light- growth reaction of Phycomyces and found that illumination increased the rate of growth. Wiechulla (1932) found that colored lights of equal phototropic effectiveness produce about the same size and type of accelerative growth response. Blaauw explained the positive (not negative) response which follow^s unilateral illumination, by pointing out that the parallel rays of light are concentrated on the back side owing to the lenticular effect of the sporangiophore. In agreement with this theory is the well-known experiment of Buder (1918), who immersed the fungus in paraffin oil to overcome the lens effect and cause the front side to be more strongly illuminated than the back side. A GROWTH SUBSTANCES FOR PHOTOTROPISM 167 negative instead of a positive curvature resulted under these conditions. According to Oehlkers (1926), the curvature is not brought about by any concentration of the Hght upon the back side but rather by the fact that the Hght rays penetrate farther in the back than in the front. Castle (1930, 1933a, h) has investigated the Avhole question thoroughly. His computation of the path of light rays in the sporangiophore led to the conclusion that the light in the back half, owing to refraction, is 1.26 times as great as it is in the front half of the organ. When the absorption coefficient is not greater than 6, more light is absorbed in the back half of the cell than in the front. From this, Castle concluded that the difference in the amount of light absorbed causes the back side to grow more strongly than the front side ; hence bending occurs toward the light. Up to the present time, nothing is known concerning the signif- icance of growth substance for the phototropic curvature of sporangiophores. It can be demonstrated easily that Phy- comyces forms growth substance in culture, and it is possible, therefore, that this substance may be concerned in the photo- tropic curvature exhibited by the fungus. Heyn (1935) has extracted a growth substance from the sporangiophores of Phycomyces nitens which, on the basis xjf the coefficient of diffu- sion, appears to be 3-indole acetic acid. SUMMARY The observations of de Candolle (1832) led to an appreciation of the fact that differential growth is involved in the phototropic and geotropic responses of plants. These tropisms may be regarded as specialized cases of normal growth, where unequal stimulation on the two sides of an organ leads to greater enlarge- ment on one side. The bending of plant organs toward light is the result of more rapid growth on the shaded side than on the side toward the light. Since plants elongate less rapidly in the light than in darkness, Blaauw and others concluded that light must have a retarding effect upon growth. Subsequent detailed investigations of phototropic curvature in the Avena coleoptile and organs of other plants showed that (1) bending can be induced in the darkened part of an organ only when some other portion is illuminated; (2) the depressing action of light upon growth is not sufficient to 168 GROWTH HORMONES IN PLANTS account for the curvatures induced by the same amount of unilateral light ; (3) when the apical portion of an erect coleoptile is separated into two halves by the median lengthwise insertion of a thin glass plate oriented at right angles to the beam of incident light, the characteristic phototropic curvature does not occur. Such evidence demonstrated clearly that the Blaauw theory is inadequate to account for phototropism. Experiments performed by many different investigators indi- cated that some chemical substance must be responsible for tropic growth. Such a conclusion was substantiated by experi- ments which showed that unequal growth on the two sides of a light-stimulated organ is due to unequal concentration of hormone on the two sides. Since the average rate of growth in length did not change during phototropic curvature, it followed that the active concentration probably was not affected by light. Further investigations proved that the concentration of the growth hormone is decreased on the illuminated side and increased on the shaded side of the bending organ. The growth- hormone explanation of phototropism, based on studies of the Avena coleoptile, may be summarized as follows: The hormone is distributed from the distal end of the organ and flows downward into the elongating regions below. Unilateral illumination scarcely affects its formation but brings about displacement toward the shaded side during the course of its downward move- ment. The subsequent rate of growth on each side is propor- tional, within hmits, to the concentration of the hormone present. The displacement of growth substance in a direction away from light has been demonstrated also in the seedUng stems of certain dicotyledonous plants. It has been shown, in addition, that its role in promoting cell elongation is hindered by light. The mechanism by which light exercises a controlling influence upon growth-hormone distribution and activity is not well understood, but one is led to the conclusion that phototropism results from (1) a direct retarding effect of light upon growth, possibly brought about by its influence upon the molecular structure of cell walls, and (2) the differential accumulation of growth hormone within the organ. The directional movement of growth hormone leading to this differential accumulation is brought about by light through its influence on protoplasmic streaming, permeabihty, and electrical potential. CHAPTER IX THE SIGNIFICANCE OF GROWTH SUBSTANCES FOR GEOTROPISM The bending of the organs of a plant toward or away from the earth is a well-known phenomenon. This response to gravity eventually brings about a position of equilibrium with respect to the earth. Perception of the gravitational stimulus and the course of the processes which lead to the geocurvature have long been the subject of scientific inquiries. Much of the older litera- ture pertinent to the question of geotropism has been discussed in reviews by Schober (1899), Christiansen (1917), Zimmermann (1927), and Rawitscher (1932). Since much that is known about growth substances was learned in the study of tropisms, a few of the more outstanding original contributions will be discussed for purposes of orientation in this field. THE EARLY INVESTIGATIONS Knight (1806, 1803-1812) demonstrated by experiments in centrifuging that the force of gravity caused an upward bending of stems placed in a horizontal position and a downward curva- ture in horizontally placed main roots. He attempted to explain how the same stimulus could produce these opposite responses in the shoot and the root on the basis of a purely mechanical effect of gravity. Growth in the root takes place by the continual laying down of new tissues at the vegetative tip. In seedlings, the food for growth comes in solution from the cotyledons. It was assumed that gravity affected this fluid and the tender, flexible fibers and bundles in such a way as to cause bending of the root tip in a downward direction. The curvature was supposed to be a passive, downward movement of the tip, due to its weight. Such a hypothesis cannot be used as an explanation for the upward curvature of the stem, for Knight pointed out that it grows by the stretching of the region back of the tip, the amount of extension being in proportion to the food supply. It was held that an 169 170 GROWTH HORMONES IN PLANTS accumulation of food takes place on the lower side of the hori- zontally placed stem owing to the effect of gravity and that this causes more growth upon the lower than upon the upper side. A negatively geotropic curvature results. The theory did not stand under experimental tests, for Johnson in 1828 showed that during geotropic curvature the root can over- come a resistance which is at least one hundred times the weight of the root tip. Furthermore, Pinot found in 1829 that a root can bend into mercury, in spite of the fact that the specific gravity of the root tip is much less than that of the mercury. It may be seen from these investigations that work is performed in the positive geotropic curvature of a root and that this curva- ture is active rather than passive. In spite of these investigations. Knight's theory was taken up anew from the anatomical standpoint by Hofmeister more than j&fty years later. No anatomical differences between the root and the stem could be found as a basis for explaining these different reactions. Hofmeister concluded that the cause must be some fundamental difference or differences in regions where the curva- tures actually occur. In the root, the zone in which the down- ward bending takes place is made up of similar cells which constitute "a region capable of curvature following the effect of gravity like a drop of viscous liquid." On the other hand, the portion of the stem which curves geotropically consists of differ- entiated elements which are stretched taut next to each other. Hofmeister (1867) concluded from a few experiments that the upward geotropic curvature of the stem probably results from the fact that "in the lower longitudinal half of the organ, the extensibility of the cell membranes is increased." The explana- tion of increased extensibility is that when the stem is in a horizontal position, more water can penetrate the membranes of the cells on the underside than those on the upper side. Frank (1868^.; see Rawitscher, 1932) and other investigators opposed this hypothesis of Knight and Hofmeister, because they were convinced that the downward curvature of the root is an active process. The experiments and conclusions of Johnson and Pinot formed the basis for a long controversy which was finally decided in favor of Frank. The principal point of Frank's theory is that positive as well as negative geotropic curvatures are vital processes which result from changes in growth. GROWTH SUBSTANCES FOR GEOTROPISM 171 This interpretation of geotropic curvatures, established by Frank, was then rounded out by Sachs (1873-1882), as the result of numerous investigations which form the foundation of our knowledge of geotropic processes. Sachs evolved the general concept that any valid explanation of geotropism must explain both positive and negative curvatures. His idea may be stated as follows: A theory of geotropism is satisfactory only when it explains both positive and negative curvatures at the same time ; when it shows why the same external cause produces the opposite effect in structurally similar cells and organs, i.e., the promotion of growth upon the underside in the stem and inhibition of growth upon the underside in the root. The effect of gravity upon root and stem can be explained only by the assumption "that the inner organization (even though submicroscopic) of the different regions determines the different manner of reaction to the same external stimuli." Sachs formulated a theory to account for opposite reactions of the root and stem in the plant. The geotropic processes he viewed as stimulation phenomena whose course is determined partly by external force and partly by the organization of the plant organ concerned. If the course of a stimulus phenomenon is determined by the internal organization of the plant, then the final response must be the result of the combined action of various single reactions in a chain. The problem is to analyze the chain of reactions which begin with the direct effect of gravity upon the plant cells and end with a positively or negatively geotropic response. The contribution of the theory of growth substance to the analysis of geotropism will be taken up for different plants separately, in the same manner as has been done for phototropic curvatures. THE AVENA COLEOPTILE The Avena coleoptile is negatively geotropic, because when displaced from the vertical position it grows away from the center of the earth. This geotropic curvature is the result of unequal rates of growth on the upper and lower sides of the displaced organ. Stimulation and Response. Distribution of Geotropic Sensitiv- ity.— In the case of phototropism, a local induction of the stimulus can be obtained easily by screening the remaining regions of the 172 GROWTH HORMONES IN PLANTS coleoptile from the light. Geotropic stimulation of the coleop- tile locally, however, is an extremely difficult matter owing to the constant action of gravity on all points. The distribution of geotropic sensitivity, therefore, is difficult to determine. Since the course of the geotropic curvature in the Avena coleop- tile begins at the tip and gradually proceeds toward the basal region, Rothert (1894) concluded that the tip is most sensitive to geotropic stimulation, just as it is in the case of phototropic induction. F. Darwin (1899) investigated the distribution of geotropic sensitivity in the coleoptiles of Setaria, Phalaris, and various other grasses by means of a new method : The coleoptile 1st intemode . . i i • i , i tip was placed m a glass tube and was permanently fixed in a Coleoptile''' '"Glass tube horizontal position. Negative Fig. 50.— Diagram of a Setaria geotropic curvature appeared in seedling, showing coUing response to the first internode (or in the geotropic stimulation brought about i i • c ±\ i ^-i \ by fixing the coleoptile tip horizon- t»asal region ot the colcoptile) tally in a glass tube; after 7 days, and progressed along the Organ, (Adapted from Darwin, 1899.) r- n i • i -i imally producing several coils below the coleoptile (Fig. 50). Darwin concluded from his experiments that a geotropic stimulus was given off constantly from the fixed part of the coleoptile to the lower regions, inducing this portion to curve. The experiments of F. Darwin were not entirely comdncing for several reasons, and the problem was investigated further by other workers. The distribution of geotropic sensitivity in the Avena coleop- tile was first described with certainty by employing the method of Piccard (1904). In this technique, the plant is fixed upon a centrifuging apparatus in a sloping position, so that the axis of the apparatus meets the plant organ at an exactly determinable distance from the tip. Then, by centrifuging, the tip and the basal region are influenced in an opposite direction by the centrif- ugal force, and from the reaction one can estimate the distribution of sensitivity in the plant organ. With this method, Darwin (1908) showed that the curvature appeared in response to stimula- tion of the coleoptile {Sorghum sp.). From this, it was concluded that geotropic sensitivity resided almost exclusively in this organ and that the geotropic stimulus is transmitted from there into the first internode. Thorough investigations on the distribution GROWTH SUBSTANCES FOR GEOTROPISM 173 of geotropic sensitivity in the coleoptile of Avena sativa, Hordeum vulgare, and Phalaris canariensis were carried out by von Gntten- berg (1912). It was found that a short tip region in these plants (3 mm. in Avena and 4 mm. in Hordeum and Phalaris) is far more sensitive than the lower regions of the coleoptile, though the basal region is not entirely insensitive. Dolk (19296) came to a similar conclusion by another method and found that the length of the sensitive zone in the Avena coleoptile is about 5 mm. From what has been said, it is clear that geotropic as well as phototropic sensitivity is localized in the tip of the Avena coleoptile. However, there seems to be a difference between the two loci of stimulation, since phototropic sensitivity is mainly confined to the upper 0.5 mm. and geotropic sensitivity to the upper 3 to 5 mm. Transmission of the Stimulus. — The geotropic stimulus is transmitted from the tip into the basal region just as is the photo- tropic stimulus. Investigations on the pathway of stimulus conduction were carried out by Boysen Jensen using methods described in the section on transmission of the phototropic stimulus. When a mica plate is inserted into a transverse inci- sion made upon the upper side of a horizontal Avena coleoptile, a strong geotropic curvature results; but when the incision faces downward, the curvature is very weak. From this kind of experi- ment it can be concluded that the stimulus is transmitted upon the underside of the coleoptile. The problem was investigated also by Purdy (1921) ; she found that after the stimulation due to woimding had subsided, the size of the negatively geotropic curvature yielded a d value of 1.73 mm. when the incision faced upward and a d ^•alue of 0.74 mm. when the incision faced down- ward. It is obvious that the stimulus transmission is far stronger on the lower side, though a significant conduction of stimulus can be demonstrated upon the upper side (Fig. 51). Since the basal region of the grass coleoptile, as well as the tip zone, is affected by gravity, any explanation of curvature that comes about in response to geostimulation of the tip must take into consideration also the direct effects of gra\aty upon the basal region. Geotropic experiments are probably not so satisfactory as those dealing with phototropism. Zollikofer (1926) showed that transport of growth substance from the coleoptile to the first internode in grass seedlings can be retarded by burning the first 174 GROWTH HORMONES IN PLANTS internode. The georesponse of the coleoptile is increased when this is done. The transmission of a geotropic stimukis is bound up with the transport of growth substance in much the same way as the transmission of a phototropic stimulus. In a horizontally placed coleoptile, the migration of growth substance from the tip Fig. 51. — Negatively geotropic curvatures in Avena coleop tiles. The plants of series A and B were placed in a horizontal position for geotropic stimulation. A, when a mica plate was inserted into a transverse incision made upon the lower side of each coleoptile, only a weak curvature resulted. B, when the incision was made upon the upper side, a strong geotropic curvature took place. C, control plants. {After Purdy, 1921.) along the underside is increased, while on the upper side it is lessened. The Quantity-of-stimulus Pnnczp/e.— Rutten-Pekelharing (1910) demonstrated experimentally that the amount-of-stimulus rule holds for the geotropic curvature of the Avena coleoptile. From Table 11 it may be seen that the product of the centrifugal force and the presentation time is constant within the range of stimulation that has been investigated (in producing a given curvature). GROWTH SUBSTANCES FOR GEOTROPISM 175 Table 11. — The Quantity-of-stimulus Principle in the Curvature of the avena coleoptile in response to centrifugal force Presentation time, Centrifugal force Product, seconds g g X seconds 7,800 0.04 312 160 2.24 358 150 2.244 337 120 2.88 346 110 2.907 320 110 3 330 100 3.15 315 90 3.82 334 17 21 . 66 368 8 40.9 327 7 46.08 322 The Course of Geotropic Curvature. — Before discussing the course of geotropic curvature in detail, it will be well to mention the question of the total growth taking place during the curvature. With the help of the micropotometric method, Cholodny (1930) compared the rate of growth during the period of geotropic curvature with that of a normal upright-growing Avena coleop- tile. He concluded that "all these experiments clearly point to the fact that geoinduction has absolutely no influence upon the growth rate of a coleoptile." It is true that Cholodny 's figures for half-hour growth increments fluctuate considerably, and it is probable that small growth changes cannot be demonstrated by this method. By means of cinematographic photographs, Weber (1931) investigated the course of growth in barley seedlings which lay continually in a horizontal position. Immediately after having been placed in the horizontal position, a difference between the rate of growth of the two sides of the coleoptile became apparent ; an increase on the lower side and a decrease on the upper side occurred. The total growth was not demonstrably changed (Fig. 52), When barley seedlings were stimulated by being placed in a horizontal position for 30 minutes, a negative curva- ture of the coleoptile appeared, followed by several back-and- forth curvatures in the upper zones, where growth promotion on one side alternated with growth retardation on the other. 176 GROWTH HORMONES IN PLANTS Fig. 52. — Course of geotropic curv^ature in coleoptiles of Hordeum placed permanently in a horizontal position. The dotted line shows the growth of the lower side; the dash line, the upper side. Note the increased growth of the lower side. {After Weber, 1931.) Fig. 53. — Course of geotropic curvature in coleoptiles of Hordeum in the first thirty minutes after placing them in a horizontal position. Dotted line shows the growth of the lower side; dash line the growth of the upper side. Note the alternation of the positive and negative growth responses. (After Weber, 1931.) GROWTH SUBSTANCES FOR GEOTROPISM 177 ZONE 10 _ ZONE 9 _ ZONES ZONE? 20NE6 20 40 60 80 100 120 140 160 Fig 54 —Course of curvature in different zones of the Avena coleoptile after geotropic stimulation (30 minutes in a horizontal position). Ordinate: the size of curvature (/..., the reciprocal values of the radii of curvature) ; abscissae time in minutes. Zone 1 is at the tip, zone 10 at the base. (From Dolk, 1929b.) 178 GROWTH HORMONES IN PLANTS During these pendulum-like movements, the average rate of growth appeared to be unchanged (Fig. 53). The course of the geotropic curvature of the Avena coleoptile was investigated by Dolk (19296), who determined the magnitude of curvature in different zones of the organ during response. The seedlings were geotropically stimulated in a horizontal position for 30 minutes, after which they were placed upon the intermittent clinostat in a horizontal position so that the curva- tures continued unaffected by the further opposing action of gravity (Fig. 54). Comparison of these curvatures wdth those resulting from phototropic stimulation (Fig. 46) brings out the following: Curvatures begin in both cases in the tip region, but the geotropic curvature sets in much earlier than the phototropic ; in both cases, the curvature proceeds from the tip to the base. The Growth-substance Explanation. Growth Substance and Geotropic Sensitivity. — The suggestion of growth-substance activ- ity in geotropic curvature of the Avena coleoptile goes back to the investigations of Boysen Jensen (1911), w^ho showed that the geotropic stimulus from an excised and replaced tip can be trans- mitted into the stump. These experiments were confirmed and expanded by Stark (1924), who found that a geotropically stimu- lated tip, which was transferred to a nonstimulated stump, could cause the latter to curve. These studies showed that a growth substance is concerned in the geotropic curvature of the Avena coleoptile (see Brauner, 1923). The same problem now arises that was mentioned in the discussion of growth substance in relation to phototropic curva- ture, i.e., whether only materials involved in normal growth are concerned in geotropic curvature or a special geotropic hormone is formed. The constancy of the rate of growth during geotropic curvature seems to warrant disregarding the latter possibility for explaining negatively geotropic curvature in the Avena coleoptile. By direct measurement, Dolk (1929a) demonstrated that the amount of growth substance given off during the curvature is not changed : some coleoptile tips were removed before and some after a geotropic stimulation; both kinds were placed upon agar blocks, and the amount of grow^th substance given off into the agar blocks was determined in the usual way. The results of the experiments are given in Table 12. Although the figures are not always consistent, it may be concluded that the amount of growth GROWTH SUBSTANCES FOR GEOTROPISM 179 Table 12. — Amount of Growth Substance Given Off by Geotropically Stimulated Avena-coleoptile Tips in Comparison with Controls Test curvat ures, degrees Number of tips Time on agar, minutes stimulation, minutes Tips from horizontal Tips from control coleoptiles coleoptiles 30 8 60 10.3 10.1 33 8 61 8.4 8.4 30 8 60 5.6 6.4 30 8 63 2.2 2.8 30 8 60 7.8 7.9 substance given off from a coleoptile tip is not changed by geotropic stimulation. The Unequal Distribution of Growth Substance. — Just as photo- tropic curvature is caused by an unequal distribution of the growth substance present in the coleoptile tip, so geotropic curvature must be also. Dolk (1929a) showed that the geotropic stimulus alters the distribution of growth substance in Avena coleoptiles if they are placed in a horizontal position. By placing them in such a position for 30 minutes, then removing the tip and extract- ing the growth substance from its upper and lower sides, he was able to show that the lower side gives off considerably more than the upper side (Table 13). With a similar technique, Navez and Table 13. — Amount of Growth Substance Given Off from the Upper AND Lower Sides of Geotropically Stimulated Avena-coleoptile Tips Time of stimulation, seconds Number of plants Time on agar, minutes Test curvatures, degrees Upper side Lower side 1,800 1,800 1,800 1,800 7 8 8 8 60 60 60 60 4.4 3.0 3.2 2.4 7.0 6.2 4.0 9.0 Robinson (19326) found that a shift in distribution of growth substance in the Avena tip is brought about by change in position of the coleoptile. 180 GROWTH HORMONES IN PLANTS Koch (1934) found that when a lateral half of the tip of an A vena coleoptile (1.5 mm. long) is cut away, making the growth- substance supply unilateral, then both geo- and phototropic effects can be compensated for, so that no curvature results (Fig. 55). For example, when the side with the tip present was oriented upw^ard in the horizontally placed coleoptiles, 18 out of 22 showed no negative geotropic bending. A y^ i 1 1 i i Gravdty IP (I J3 be- ^ ib^ D i 1 i 4 i Gravity B Fig. 55. — Diagrams showing that excision of half portions of the tips of Avena coleoptiles affects their tropic response to light and gravity. A, when the half tip is removed from the side nearest the light, strong phototropic curvature takes place, due to downward movement of the growth hormone from the half tip which remains on the back side. B, when the half tip is cut out from the side away from the light, either no response takes place (due to radially sym- metrical distribution of the hormone) or a small negative curvature results, presumably from the slightly greater amount of growth hormone on the side beneath the intact half tip. C, growth-hormone displacement due to gravity is diminished when the half-tip portion is removed from the lower side. D, negatively geotropic curvature occurs when the half tip is oriented below. {After Koch, 1934.) The problem of how this unequal distribution of growth sub- stance occurs was investigated by Dolk (19296). On the apical end of each of several decapitated Avena coleoptiles was placed a block of agar containing growth substance. The coleoptiles then were placed in a horizontal position, and the growth sub- stance exuding from the basal end was collected separately from the upper and lower sides (Fig. 2). The results (Table 14) indicate that the lower side gives off more growth substance than the upper side. This difference might arise from unilateral changes in permeability due to the action of gravity. According to Nuernbergk (1933), wounding {e.g., by decapitation) retards GROWTH SUBSTANCES FOR GEOTROPISM 181 Table 14. — Comparison of the Amount of Growth Substance Recov- ered Separately in Two Agar Blocks from the Upper and Lower Halves op an Avena-coleoptile Cylinder Which Had Been Supplied Artificially with the Hormone at the Distal End See Fig. 2 (Dolk) Time in a horizontal Number of Amount of growth sulistance Half the orig- inal amount position, minutes cylinders Upper side Lower side of growth substance 120 6 6.3 9.8 11.7 120 6 7.5 10.3 11.7 120 6 4.0 8.8 9.5 120 6 6.5 8.0 9.5 120 5 6.3 8.8 16.3 120 5 3.4 6.8 16.3 120 5 3.8 8.8 16.3 the transverse transport of growth substance ordinarily induced by the unilateral action of gravity. He concluded that trans- verse transport is brought about by the increased resistance to longitudinal transport on the upper side when the Avena coleop- tile is placed in a horizontal position (see also du Buy, 1933). On the other hand, Dolk's work indicates that the unilateral effect of gravity brings about displacement of growth substance in the geotropically stimulated tip. With recognition of the displacement of growth substance in the coleoptile tip as a link in the geotropic stimulus chain, it becomes necessary to examine the separate steps in the process, i.e., how gravity can produce this displacement and how the negatively geotropic curvature arises in the Avena coleoptile as a result of the unequal distribution of growth substance. The Statolith Theory. — Contemporaneously with certain theoretical observations by Noll (1900), two investigators, Nemec and Haberlandt, suggested that movable grains of starch function as statoliths. If the organ is in a position of geotropic equilib- rium, the pressure of the starch grains cannot produce a stimulus. If the organ is taken out of its position of equilibrium, the starch grains accumulate in layers on one side of the cell and exert a pressure upon the plasma membrane of the tangential (and perhaps also radial) walls. This pressure is supposed to produce 182 GROWTH HORMONES IN PLANTS a geotropic reaction which results in bringing the organ back into a position of equiUbrium. Nemec (1901) showed that movable starch grains are present in the parenchymatous tissue of the Avena-coleoptile tip. Whether or not they are significant for the perception of the geotropic stimulus was investigated by von Guttenberg (1912), who found that their presence is associated with geotropic sensitivity, although the relationship does not constitute definite proof for the statolith theory. The literature on this theory has been brought together in an extensive monograph on geotropism by Rawitscher (1932), who came to the following conclusion: If we look back over the results of the main experiments, we must admit that a very close relationship seems to exist between the presence of starch and georeception. We must not overlook the role that carbo- hydrate metabolism plays in the reception of the stimulus of gra\aty. Whether the starch grains actually function in the sense of the statolith theory as conveyors of pressure has become extremely doubtful in view of more recent observations. If starch grains actually function as statoliths, it still remains to be explained how their pressure can lead to the unequal distribu- tion of growth substance. Electrical Theories and Experiments. — Whatever the explana- tion of the response to gravity may be, the primary reaction is initiated by a traction or pressure effect. According to the electrical theories of geotropic stimulation, differences in potential are produced by the action of gravity upon electrically charged particles which presumably initiate the chain of reactions leading to geotropic response. Small's explanation (1920a, h) was based upon the colloidal nature of protoplasm in relation to isoelectric points. The assumption was made that the protoplasmic particles in the root are electropositive while those of the stem are electronegative. When the plant organs are placed in a hori- zontal position, these particles rise to the upper side and in this way bring about radial potential differences. Cholodny (1918, 1923a, b, c, d, 19316) assumed that negatively charged microsomes move downward under the influence of gravity. The difference in potential which arises therefrom was supposed to displace the metal ions and lead to a change in the relationship between GROWTH SUBSTANCES FOR GEOTROPISM 183 univalent and bivalent ions. The unequal distribution of the earth alkalies, which tend to accumulate on the negatively charged side, modifies the swelling properties and the permeability of the protoplasmic membrane and hence, also, the rates of growth. Various objections to these theories can be raised, the main one being that the differences in potential postulated by Small and Cholodny do not agree with those actually found. In recent years, differences in electrical potential have been described with considerable accuracy in horizontally placed organs (Brauner, 1926, 19276, 1928; Brauner and Amlong, 1933). Brauner discovered the fact that when plant organs are placed in a horizontal position, a difference in potential arises so that the lower side is positively charged with respect to the upper side. This has been observed in both roots and stems. These so-called "geoelectric phenomena" can be demonstrated in dead organs and also in model experiments with parchment-paper membranes. The magnitude of the potential in plant organs fluctuates between 4 and 9 millivolts, according to Brauner; but Amlong (1933) found potentials as high as 34.6 millivolts. Dolk (1930) and Cholodny (1931c) investigated the possible relationship between these phenomena and the transverse dis- placement of growth substance. Since the latter is an acid, it ought to migrate toward the positively charged pole, i.e., toward the lower side of the root and stem. Although Dolk was unable to discover any movement of growth substance in a potential gradient, several recent investigators have been more successful (Koch, 1934; Ramshorn, 1934; Kogl, 1933, Mitt. VI). The results of experimental investigations on its displacement by gravity are in accord with the findings with respect to electrical potential. The question that remains is whether the potential gradients shown to exist in illuminated or horizontally placed plant organs are adequate to displace growth substance and thus produce a tropic curvature. In a preliminary work, Brauner and Biinning (1930) reported on studies of the behavior of Avena coleoptiles in an electric field. The experimental objects were placed in a moist chamber and fastened between two aluminum plates so that the lines of force went through the organ. A field of continuous current with a maximum of 640 volts per centimeter was obtained by charging these plates. The coleoptiles curved somewhat weakly toward 184 GROWTH HORMONES IN PLANTS the positive pole (Fig. 56). Both the coleoptile of Avena and the root of Vicia were found to be positively charged on the lower side when placed in a horizontal position (Fig. 57). At about the same time, Hartmann (1932) obtained somewhat different results. In experiments with 3,000 volts per centimeter, Avena coleoptiles showed a weak bending toward the negative plate at first and later exhibited curvatures toward the positive pole. These curvatures were always very weak. Amlong (1933) since has studied the electrotropic curvature of Helianthus seedlings according to the method of Letellier, using a field of 1,000 volts per centimeter applied to seedlings growing in a moist chamber. The seedlings curved away from the negatively charged plate. Koch (1934) found that when an electric current is applied through upright coleoptiles placed in conductivity water, the coleoptiles do not conduct the current; hence, within the coleoptile a negative pole is induced on one side, and the coleoptile curves toward it (Fig. 60). When an electric current coleoptile toward the positive pole in -^vas applied tO rOOts of Pisum an electric field. (From Brauner and , i i • i ^^ • j Biinning, 1930.) placed horizontally m conduc- tivity water, it was found that they conducted the current and curved to the positive pole, away from the force of gravity (Fig. 60). A good qualitative agreement has been found between the course of geotropic and electrotropic curvatures, although the former curvature is significantly stronger than the latter. It may be conjectured that the geoelectric phenomenon is probably essential but not the only necessary factor for the displacement of growth substance. It should be remembered that although growth substance may be present, its displacement does not always occur under conditions of unilateral stimulation by light or gravity. Avena GROWTH SUBSTANCES FOR GEOTROPISM 185 From what has been said it is clear that the first step in the geotropic response in the Avena coleoptile is the displacement of growth substance. The manner of its transport and the changes in rate of growth in the basal region remain to be discussed. Comparison of Phototropic and Geotropic Curvatures. — As soon as an unequal distribution of growth substance has taken place in the tip of the Avena coleoptile as a result of the action of gravity, a geotropic curvature in the basal region must of neces- _ + + jt + + + + A B Fig. 57.— Diagram showing distribution of ions in geotropism and electro- tropism. A, Avena coleoptile. B, root of Vicia. When the plant parts are placed in an electric field, in a moist chamber, an internal polarity results by induction. The coleoptile bends toward the positive pole, while the root bends toward the negative. It is assumed that growth hormone accumulates in the tissue regions which are electropositive, and there promotes growth (in the coleoptDe) or inhibits it (in the root). Geotropic response of these organs has been explained on the basis of electrical potentials induced by gravity, as shown at the top of the figure. (From Brauner and Banning, 1930.) sity result (because of the longitudinal transport of growth sub- stance which follows). From the time displacement of growth substance occurs in phototropism and geotropism, the two types of curvature are essentially the same. There is one difference between the two which might be cited as an objection to the growth-substance explanation, and it may be worth while to examine it more closely (see du Buy and Nuernbergk, 1930). A comparison of the curves (Figs. 46 and 54) showing the course of phototropic and geotropic curvature in the Avena coleoptile indicates that in both cases the curvatures begin in the tip and proceed toward the base; the geotropic curvature sets in much earUer than does the phototropic. Two different conditions may 186 GROWTH HORMONES IN PLANTS be pointed out in explanation of this : (1) The phototropic stimukis lasted only 10 seconds in the experiments cited, while the geotropic stimulus lasted 30 minutes. It seems probable that the further steps in the reaction (which take place after the stimulation) probably are beginning during the lengthened period of geotropic stimulation. (2) Since the geotropically sensitive zone is signifi- cantly longer than the phototropic, the unequal distribution of growth substance resulting from stimulation may set in more rapidly in the former. In geotropic movement, the curvatures reach a maximum and then decline; in phototropic stimulation, the curvatures of the single zones increase gradually to a maxi- mum and then disappear slowly. In order to explain this differ- ence, it must be recalled that according to the growth-substance explanation, the course of curvature is conditioned by the duration and the magnitude of the unequal distribution of growth substance. Since the first phases of the geotropic and the photo- tropic stimulus processes are not identical, it is unreasonable to expect a complete agreement in the course of the curvatures. It is not known how long after the cessation of the stimulation the displacement of growth substance continues ; possibly the unequal distribution of growth substance is more rapidly equalized when it is produced by gravity than when brought about by light. From the curvatures in Figs. 46 and 54, it can be con- cluded that a geotropic stimulation for 30 minutes is far stronger than a phototropic stimulation obtained by illumination with 500 meter-candle seconds. This may cause a difference in the course of the curvatures. Recovery from Geotropic Curvature Brings About Equilib- rium.—Dolk (19296) investigated the opposing geotropic curva- ture (the straightening of the tip) which starts when the rate of growth of the concave side becomes greater than that of the convex. The conditions necessary for the appearance of such a response are that growth substance in the tip be again equally distributed and that an unlocalized supply of it be furnished by the tip to the basal region. With the extraction method, he showed (1930) that more was present in the lower than in the upper side of the tip after the coleoptile had been in a horizontal position for 30 minutes. When the coleoptile was placed again in an upright position, the amount of growth substance extracted from the upper and under side was equal after 60 minutes (see GROWTH SUBSTANCES FOR GEOTROPISM 187 Table 15). By removing the tip of the Avena coleoptile immedi- ately after geotropic stimulation, Dolk (19296) found that the opposing reaction was greatly retarded. Li this way, he demon- strated that a continuous supply of growth substance is necessary Table 15. — Comparison of the Amounts of Growth Substance Extracted from the Upper and Lower Halves of Avena Coleoptiles Which Had Been Stimulated Geotropically and Allowed to Recover Time in a horizontal position. Number of tips Rotation time on the clinostat, minutes Time on agar, minutes Amount of growth sub- stance, in degrees curvature minutes Upper side Lower side 30 6 64 105 5.0 4.7 30 7 60 90 5.8 5.6 30 7 60 90 6.7 7.0 30 7 60 90 6.8 6.6 30 7 60 90 5.3 5.7 30 7 60 90 11.0 10.6 30 7 60 90 12.0 12.5 for the reaction to take place. It appeared after 150 minutes, which is the same length of time required for renewed appearance of growth substance in this region. During the opposing growth curvature which leads to straight- ening of the organ, the concave side grows more rapidly than the convex. If one does not assume a shift in the distribution of growth substance, making concentration for a time greater upon the concave than upon the convex side, then one must explain how an equal distribution of growth substance can bring about an unequal rate of growth. Went (1928a) had hypothecated a cell- stretching material which, together with growth substance, is necessary for growth. Dolk assumed that more of it is used on the convex side and, therefore, that the concave side where more stretching material remains should grow more rapidly with an equal supply of growth substance. It should be mentioned, too, that the individual cells go through a long period of growth and that the stage of development of the cells also influences the rate of growth of the organ as a whole. On the convex side, the cells may be in a later stage of development than upon the concave 188 GROWTH HORMONES IN PLANTS side, and this may be the reason for different rates of growth on the two sides, although the supply of growth substance is equal. Some observations of a geotropic curvature in the barley coleoptile cannot be explained by the unequal distribution of growth substance (Weber, 1931). When the coleoptile is placed in a horizontal position, a difference in the growth rates of the upper and lower sides arises throughout its whole length. If it is placed in an upright position after it has been geotropically stimulated for 30 minutes, there follow, after the first negative curvature, several positive and negative curvatures which appear to be due to alternating retardation and promotion of the rate of growth in the upper zones. These may be identical with the pendulum-like movements which appear in all negative geotropic curvatures before the position of equilibrium is attained. No curvatures appear in the basal portion of the coleoptile where the vigorous growth is evenly distributed on both sides. Still other cases are known where no growth-substance displacement or geotropic curvature is observed, even though growth substance is present and growth takes place. The young internodes of grasses, for example, behave in this fashion. Further discussion of these cases may be found elsewhere. DICOTYLEDONOUS HYPOCOTYLS, SHOOTS, ETC. The main stem of the higher plants is almost always negatively geotropic. Experiments with hypocotyls and stems of dicoty- ledons have resulted in substantial contributions to our knowl- edge of the role of growth hormones in geotropism. Stimulation and Response. Distribution of Geotropic Sensi- tivity.— Satisfactory data on the distribution of geotropic sensi- tivity in seedling axes have been obtained by Herzog (1925) with the Piccard (1904) method. The following hypocotyls were investigated: Vicia sativa, Brassica napus, Linum usitatis- simum, and Lepidium sativum. All of these were shown to be geotropically sensitive only in the apical portion, and the length of the zone in each species was 11, 18, 16, and 12 mm., respec- tively. The sensitivity appears to be evenly distributed through- out this region. The ability to respond extends beyond the limits of the sensitive zone, which means that a part of the curva- ture is induced by a conducted stimulus ; the length of this region is about one-third of the entire portion which has the capacity GROWTH SUBSTANCES FOR GEOTROPISM 189 for curvature. More precise evidence for the transmission of the geotropic stimuhis in stems is lacking. In BeUis, geotropic sensitivity extends over the greater part of the flower stalk but is not uniformly distributed. An apical zone of 7 mm. is more sensitive than the remaining portion. Negatively Geotropic Curvature in Stems. — Sachs (1872, 1888) measured the upward curvature of various stems in order to discover the course of negatively geotropic curvatures. The zone of growth and curvature of the stem was found to be as long as 15 to 40 cm. At the beginning, the strongest bending was in the vicinity of the tip. As the bend moved downward, the upper originally curved zones straightened; finally, the basal, slowly growing zone often showed a sharp curvature. Sometimes the tip curved back and forth several times before a final position of equilibrium was reached. Sachs showed that negatively geo- tropic curvatures in stems arise by an increased rate of growth on the lower and an accompanying retardation of growth on the upper side. Sachs did not determine whether the average rate of growth changed during bending. Cholodny (19296), using a micropotometer, concluded that the hypocotyls of Lupinus and Helianthus grow at the same rate, whether in the horizontal or in the erect position. The Growth-substance Explanation. — In order to show that the geotropic curvature of an organ is produced by the function- ing of growth substance, it is necessary to prove (1) that a growth substance is present in the plant organ in question and (2) that the organ responds to the application of the growth substance, the rate of growth being either increased or decreased. If proof of these two points can be obtained, it must be shown, in addition, that gravit}^ brings about an unequal distribution of growth substance. This differential distribution can occur in two difTerent ways: either growth substance may be displaced to the lower side as in the Avena coleoptile under the unilateral effect of gravity, or increased formation of growth substance may take place upon the lower side. It is possible, also, that the unequal distribution of another substance influences the action of the uni- formly present growth substance. Experiments with Split Stems. -^Avaong the early investigations concerning the role of growth hormones in negatively geotropic curvature of stems, those of Loeb (1916, 1917) should be men- 190 GROWTH HORMONES IN PLANTS tioned. In a series of experiments with Bryophylhim calycinum, it was found that geotropic growth takes place on the lower side of horizontally suspended stems and that the response is much greater if a leaf is present on the stem (Fig. 1). From his numerous experiments, Loeb postulated the presence of hormones as a plausible explanation for the observed phenomenon. Gradmann (1925) tried to determine the presence of growth substance during geotropic curvatures in different plants belong- ing to the Labiatae and Scrophulariaceae. Whole intern odes of Mentha and other plants were placed in a horizontal position, and the epidermis was removed from the top and bottom sides. Other internodes were split lengthwise, and the cut surfaces placed in contact with the exposed surfaces of the whole inter- nodes. After a time, the central internode curved toward the upper contact surface. This and other similar experiments led Gradmann to the conclusion that growth-promoting substances are present in the lower half. However, the experiments were complicated by the fact that traumatic effects were introduced by the removal of the epidermis. The experiments were criticized by Cholodny (1927, 1929a, b, 1931c, /; see Gradmann, 1931), but the various hypotheses and auxiliary hypotheses which were propounded scarcely warrant discussion here, especially since no final decision concerning the interpretation of the Gradmann experiments has seemed possible. An improved method would be very desirable for clarifying the situation. If Gradmann's experiments could be considered as conclusive evidence, it would follow that the growth-substance content of the lower halves is greater than that of the upper halves, even when both are isolated. This difference cannot be attributed to growth-substance displacement. Both halves would react sepa- rately during the geotropic reaction, and a harmonic combined effect would not result. It is of fundamental significance to know whether this assumption is actually correct, and from this point of view it might be profitable to study geotropic response in spHt stems. When intact barley coleoptiles are placed in the horizontal position, they show an increased growth rate on the lower side and a retarded growth rate on the upper side (Weber, 19266). If the coleoptiles are split lengthwise, an exchange of growth substance GROWTH SUBSTANCES FOR GEOTROPISM 191 between the upper and lower sides cannot take place, and one would expect that the halves would grow at the same rate at which they do when in a vertical position. According to Weber, this is not the case, for although the lower half grows at approxi- mately the normal rate, growth is retarded in the upper half. In both cases, the growth substances apparently accumulate on the side that is downward. Since the cut surface of the upper half is on the under side, it might be assumed that wounding influences the effectiveness of the growth substance and decreases the rate of growth, whereas no effect of this kind is present in the under half. This agrees with some of Cholodny's (1931c) experiments, in which the retarding influence of unilateral wounding upon the rate of growth depended upon how the wound surface was oriented with respect to the direction of gravity. The rate of growth in Hehanthus hypocotyls, with the wound on the top, w^as 1.5 to 2 times as great as that in plants with the wound on the under side. The circumstances that exist in split dicotyledonous stems should be considered briefly. It is known that a median longi- tudinal cut retards the rate of growth appreciably (Sachs, 1873 ; Schtscherback, 1910) and that when a split stem is brought into a horizontal- position, the under half grows more rapidly than the upper half (and faster than a control half which is placed in a vertical position). In an investigation of geotropism in many plants, Ahrens (1934) found that when hypocotyls were halved lengthwise, only the lower halves reacted geotropically. The results of these experiments, however, are influenced so greatly by traumatic effects that it is impossible to draw final conclusions about the production of the geotropic curvature. Loeb (1917) marked portions of Bryophyllum stems into 1 cm. segments with India ink. Each stem portion possessed one or more vigorous leaves and was placed in a horizontal position. After several days, geotropic curvatures took place as a result of stretching growth of the cortex in the lower half. In other experiments, the stem was split lengthwise into two equal halves, each having one vigorous leaf left at the apex. The stem portions were suspended horizontally in moist air, one with the cortex below, the other with the cortex above (Fig. 58). Only that half stem with the cortex oriented downward showed geotropic bending. It may be pointed out that the tissue into 192 GROWTH HORMONES IN PLANTS which growth-promoting substances are transported must of necessity be capable of growth response in order to accomphsh geotropic bending. In the Bryophyllum half stems, no growth curvature can be produced when the woody tissue and pith are oriented downward. Many variables enter into all these experi- ments with spHt stems which possibly vitiate the results, e.g., the errors in halving the stem, the unequal degree of drying of the surfaces, etc. Growth- substance Displace- ment.— Dijkmann (1934) inves- tigated the geotropic curvature of the Lupinus hypocotyl and demonstrated the presence of growth substance there. It was present over the whole growing zone; the growth rate was pro- portional to its concentration, within certain limits. Growth- substance displacement toward the lower side of a stem placed -Bryophyllum stem split horizontally was also studied. longitudinally, each half possessing a . , j ■! j. xu leaf at its apex. The split halves were 1 he results mdlCated that the suspended horizontally in moist air. \q^q^ ^{^q contains morC growth A, with the cortex below. B, cortex . above. Geotropic curvature occurred SUbstance than the upper Side, only in the half stem with the cortex ^ut the results of these experi- below. {After Loeb, 1917.) , ^ i xi, ments are not so clear as those on hypocotyls. If a hypocotyl cyhnder placed in a horizontal position is completely covered v/ith growth substance on the apical end, it is possible after a time to extract more growth sub- stance from the lower than from the upper half of the morpho- logically basal end (Fig. 2) (Dijkmann). From these experiments, it can be concluded that the geotropic curvature of the Lupinus hypocotyl results from displacement of growth- substance to the under side and that the behavior of this organ fits in with the growth-substance explanation. Van der Laan (1934) investigated the same problem, using the epicotyl of Vicia Faba. When it was oriented horizontally, more growth substance could be extracted from the under than from the upper side, an indication that displacement had occurred (Fig. 59). When the seedlings were treated with ethylene, the Fig. 68. GROWTH SUBSTANCES FOR GEOTROPISM 193 greater part of the growth substance disappeared, as did geo- tropic sensitivity, and the shoots grew horizontally. The small amount that was left w^as unequally distributed, more being found in the upper half. Since the quantity remaining after treatment with ethylene was very slight, the measurements may not be significant. 14 V2 Time in hours PiQ. 59.— Negatively geotropic curvature and differential distribution of growth hormone in horizontally placed seedling stems of Vicia Faba. A, the course of curvature. B, the growth hormone is equally distributed in the upper and lower halves during the first half hour in the horizontal position; in the ensuing 15 minutes there is a shift in the concentration, so that the lower side contains 65 per cent, the upper side, 35 per cent. The higher concentration on the lower side is correlated with the negatively geotropic curvature. (.AJter Van der Loan, 1934.) Boysen Jensen (1936), using the chloroform extraction method, investigated the distribution of growth substance in seedling axes of Phaseolus and Vicia during geotropic curvature. In accord with previous work, he found a greater concentration in the under side than in the upper, although the difference in concentrations was not so great as might have been expected from the observed rates of growth on the two sides. According to Beyer (1932), geotropic curvatures observed in stalks of Taraxacum, hypocotyls of Impatiens and Cucurbita, and 194 GROWTH HORMONES IN PLANTS Electrical potential and growth hormone transport in coleoptiles Electrotropism in roots ^ C 6 T^ (PiR- Electrotropism Photo ■ and electrotropism Mil Gravity Geo- and electrotropism H i i i i Gravity Fig. 60. — Electrical potential in relation to growth curvatures. In accordance with its acid character, the active radical of the growth-hormone molecule bears a negative charge; hence it should be moved toward the positive electrode in an electrical circuit. Accumulated hormones in the relatively electropositive tissues should regulate growth in agreement with the theory of growth-hormone activity. Density of stippling indicates the presence and relative concentration of the growth hormone. The diagrams show the setup and the resulting growth response in each experiment. A, effect of an electrical field upon growth curvature. Upright coleoptiles immersed in conductivity water bend toward the positive electrode in a circuit. This may be explained on the assumption that the coleoptile with its cuticle GROWTH SUBSTANCES FOR GEOTROPISM 195 hypocotyls and epicotyls of Helianthus cannot be brought directly into accord with the growth-substance explanation. In Taraxa- cum, during the course of curvature, growth increased and often was renewed in stalks that had previously ceased elongating. It was shown that these organs may curve geotropically after growth in length has stopped. A shortening of the upper side was reported in HeHanthus. Beyer did not investigate growth- substance content or distribution, so it is entirely possible that formation of growth substance could have been renewed in these horizontally placed organs and thus account for the observed revivals of growth. The investigations of Gundel (1933) showed that in geotropi- cally curv'ed parts of a plant, the acidity upon the convex side is heightened while that of the concave side remains unchanged. Metzner (1934) fomid increased acidity and plasticity of the cell walls upon the convex side of Helianthus hypocotyls. In accord with the investigations of Stnigger (see page 113), this observation might be of significance for the comprehension of geotropic curvatures in seedling stems of dicotyledonous plants. It is not known what influence the accumulation of is not a conductor, hence by induction the internal polarity is positive on the side toward the negative electrode. B, electrical polarity and longitudinal transport. Decapitated Avena coleop- tiles with growth substance applied in agar blocks on the apical cut surface were put in series ^-ith a small battery by attaching wet silk threads to the agar blocks and closing the circuit at the other end where the roots dipped into the water of the culture vessel. Transport of the auxin anion into the stump (as determined by the amount of the ensuing curvature) was retarded by making the base of the coleoptile negative to the tip, as shown in a, and promoted by ha%'ing the base positive to the tip, as in h. The potential employed was 80 miUi- volts and the current 0.0008 milliampere. C, effect of an electrical circuit upon geotropic curvature in roots. The main root of Pisum. placed in a horizontal position normally curves downward, a. When immersed in conducti\'ity water in an electrical circuit with the positive pole above the root, as in h, the root conducts the current and bends toward the positive pole, even away from the force of gravity, c. D, effect of applied potential upon erect Helianthus hypocotyls; they curve away from the positive electrode. E and F, influence of applied potentials upon phototropism. E, when the negative electrode is on the shaded side of a unilaterally illuminated Helianthus hypocotyl, the electrotropic stimulus is stronger than the phototropic, and the h>T>ocotyl bends away from the light. F. when the shaded side is made positive, normal phototropic curvature is augmented. G and H, influence of applied potentials upon geotropism in hypocotyls of Helianthus. G, in a horizontally placed hypocotyl, negatively geotropic curva- ture is augmented by making the lower side positive. H, normal geotropic curvature does not occur when the upper side is made positive. {B after Kogl, 19336; a^l others after Koch, 1934.) 196 GROWTH HORMONES IN PLANTS acids may have on releasing growth substance from an inactive state. (See further discussion on pages 113-115.) Electrical Transport of Growth Substance. — -In this connection, the investigations of Koch (1934) throw some Hght upon the problem. It was found that the hypocotyls of Helianthus moved toward the positive pole when an artificial potential was applied to them (Fig. 60). The geotropic response was easily com- pensated for electrotropically by inducing a positive charge on the upper side of the horizontally placed hypocotyl. The negatively geotropic response could be increased considerably by applying the positive lead from a battery to the lower side. The author concluded that displacement of growth substance, in accordance with its acid character, took place in the direction of the positively charged region, with consequent effects upon growth and tropic curvature (Fig. 60). Along this same line, also, Ramshorn (1934) showed correla- tions between growth intensity and electrical potential which seem to be in accord with the growth-substance explanation. Brauner (1935) found that characteristic potential differences appeared in all the plant tissues investigated when they were in a horizontal position. The under side becomes electropositive to the upper side, showing maximum potentials of 35 millivolts. This geoelectric effect is independent of the geotropic sensitivity of the organ and its life conditions. Working with suitable membrane models, Brauner and Amlong (1933) suggested a possi- ble origin of these potentials in the influence of gravity upon diffu- sion potentials (see Brauner, 19276) . Brauner's (19276) statement that the geoelectric effect is independent of living processes, while photoelectric potentials arise only in living tissues, points to a marked difference in their origins. Further than this, Brauner (1935) concluded that although electric potential causes growth- substance transport leading to geotropic response, quite a differ- ent mechanism (such as the movement of growth substance by modification of cell permeability) is concerned in phototropism. Inheritance of Geotropic Response. — Comparative studies of geotropism in different species of plants have yielded some interesting information with respect to the possible inheritance of the mechanism of response to gravity. In a study of the geotropic response of Capsicum fruit stalks. Kaiser (1935) found that a single gene difference is responsible for the dominance of GROWTH SUBSTANCES FOR GEOTROPISM 197 pendant over erect fruit in the Fi generation. Van Overbeek (1936a) studied geotropic response in a variety of maize commonly referred to as "lazy." He reports that when grown in darkness the young plants up to 5 or 6 days old are negatively geotropic, as are normal seedlings; when grown in light, the plants are ageotropic. Brain (1933) observed a seasonal variation in the gravitational irritability of Lupinus arhoreus and L. polyphyllus, while L. alhus appeared to be equally responsive throughout the year. The first two species were termed "physiologically zygomorphic" because their sensitivity to gravity was greater for the cotyledonary than for the intercotyledonary plane, while the last was equally sensitive for both planes of the hypocotyl. These and other variations between closely related plants seem to indicate clearly that certain tropic properties may be inherited. Geotropic Response in Nodes. — It is well-known that in many plants (particularly in the grasses) portions of the axis in the region of the nodes remain potentially embryonic long after the rest of the internodal parts have differentiated into mature tissues. When such plants are displaced from their normal position with respect to gravity, negatively geotropic curvatures occur. The geotropic response of mature grass nodes was investigated by Sachs (1872) and DeVries (1880). When grass stems were placed in a horizontal position, growth in length of the nodes was renewed on the under side, while the upper side became com- pressed. Two effects of gravity should be distinguished in this case: (1) the resumption of growth and (2) the unilateral distribu- tion of growth, Elfving (1884) made the noteworthy discovery that growth was resumed under the unlocalized action of gravity, attained when the plant was rotated on a clinostat. As has been mentioned in the chapter on normal growth, Schmitz (1933) demonstrated the presence of growth substance in young growing internodes. In spite of possessing growth substance, the young internodes do not curve geotropieally, nor can accumulation of growth substance on one side be demon- strated. Young nodes also contain growth substance, but the mature ones do not. However, when a plant with mature nodes is rotated on a clinostat, formation of growth substance occurs apparently as a result of the unlocalized action of gravity. 198 GROWTH HORMONES IN PLANTS Growth substance is formed anew under the unilateral action of gravity, but it is found only on the under side. When geotropi- cally stimulated nodes were excised, split lengthwise, and placed unilaterally upon decapitated Avena coleoptiles, a curvature resulted from use of the under halves only. These experiments were carried out with nodes of Triticum, Secale, Lolium, Holcus, and Setaria. In an experiment using the under halves from 40 test plants, there were 27 negative, 10 straight, and 3 positive curvatures ; using the upper halves of nodes from 26 plants, there were 3 negative, 20 straight, and 3 positive responses, A clear difference exists between the growth-substance content of the upper and lower halves of geotropically stimulated nodes. There are two possibilities concerning the origin of growth substance as a result of the unilateral effect of gravity. Either the growth substance is formed exclusively in the cells of the under half of the node, or else it is formed in all cells and is imme- diately displaced to the under side as a result of gravity. Schmitz deals with the first possibility in the following words: "Growth substance is formed anew upon the under side of a node when it is unilaterally stimulated by gravity, but it is formed in the whole node when it is subjected to unlocalized stimulation on a clino- stat." The second possibility of general formation followed by unilateral displacement is the most probable. The growth and curvature in halved nodes lend strong supporting evidence. As deVries originally showed, the upper as well as the lower half of nodes can curve geotropically which must mean that growth sub- stance is formed in the upper half also, when it is in a horizontal position. The fact that it cannot be demonstrated there in intact nodes can be explained as due to displacement to the lower side. While the nodes of grasses act independently in geotropic curvature, in other plants, such as Tradescantia, there is some relationship between the curvature of one node and the node next above it. Kohl (1894) showed that the negatively geotropic curvature of a node of Tradescantia could be suppressed by removal of the node above. He concluded that the stimulus is received in the upper node and is transmitted from there to the lower node where the reaction occurs. Miehe (1902) showed that this view was not correct, for, if the internode between the two nodes was bent so that the upper node was vertical and the lower GROWTH SUBSTANCES FOR GEOTROPISM 199 one horizontal, a curvature still appeared at the lower node. Stimulus reception and response both appear exclusively in the lower node. Schumacher (1923) objects to Miehe's experiments because the vertical position of the upper node is essentially a position of stimulation, since the shoots are plagiotropic. Accord- ing to investigations by Uyldert (1931), the influence of the upper node upon the lower one is brought about by the giving off of growth substance. Actually, the presence of growth substance was not demonstrated in the organs in question, but when growth and geotropic curvature of the node was checked by removal of the internode, an application of growth substance brought about the usual response to gravity. ROOTS Roots are very sensitive to the force of gravity, but not all roots exhibit the same type of response. Primary roots grow toward the earth's center, i.e., they are ^positively geotropic; branch roots frequently grow out at an angle and assume a position more or less transverse to the direction of the earth's force, i.e., they are diageotropic. Recent investigations indicate that growth hor- mones, similar in nature to those occurring in leaves and stems, control in a pecuUar way the processes of growth and tropic curvature in roots. Stimulation and Response. Distribution of Geotropic Sensi- tivity.—In roots of Vicia Faba, the geotropic curvature at first is evenly distributed over the 3 mm. at the tip of the root (Sachs, 18826). After 23 hours, the curvature in the extreme tip has disappeared, the region just behind is only sUghtly bent, and most of the curvature is back about 3 mm. This means that the response is confined to a very short region in contrast to the dis- tribution of the negatively geotropic curvature of the stem. It was shown by Ciesielski (1872) and Darwin (1881) that the ability of the root to curve geotropically could be checked by removal of the root tip. From this, Darwin concluded that the stimulus is perceived in the tip and transmitted from there to the growing zone where the reaction takes place. Keeble and Nelson (1935) found that amputation of the tip 1 mm. nearly always removed the capacity for geotropic curvature even when the root remained in a horizontal position for 24 hours. That this decapitated root could still perceive the stimulus of gravity was 200 GROWTH HORMONES IN PLANTS shown by the fact that when reheaded and placed vertically, the root curved toward the side that had been lowermost when the root was horizontal (Keeble, Nelson, and Snow, 1929). Although it has been shown that the growth of the root is in no way retarded by decapitation, the possibility still exists that the ability of the growing zone to react is lessened by decapitation. It would be far more satisfactory to have the localization of sensitivity in the tip demonstrated by experiments with intact plants, but such a requirement is much harder to fulfill for geotropic than for photo- tropic stimulation. Czapek (1895) tried to obtain data along this line with the use of his well-known tube experiments. Haber- landt (1908), Jost (1912), and Dewers (1914), using the method of Piccard, showed that if the tip and basal regions of a root were stimulated in opposite directions on a centrifuge, the whole root reacted uniformly in response to the stimulation of the tip, pro- vided the length of this tip was 1.5 to 2 mm. It may be con- cluded that the tip of the root is more sensitive to the effect of gravity than the proximal regions and that the stimulus must be transmitted from the tip into the zone of reaction. Stimulus Transmission. — Snow" (1923, 1924a) gave the first direct proof for transmission of stimulus in the root. He showed that when the root tips of Vicia Faba were removed after geotropic stimulation and then replaced on the cut surfaces with gelatin, geotropic curvatures took place. Of 76 roots treated in this way, 45 showed positively geotropic curvatures after 15 to 24 hours, the mean size of the curvature amounting to about 30 deg. Of 32 decapitated roots, on which the tip was not replaced, 27 remained straight, 4 showed a weak positive curvature, and only 1 exhibited a strong curvature. Snow concluded that the geotropic stimulus can be transmitted over a wound surface, and his work was confirmed by Boysen Jensen (1933c). In decapitated roots of Vicia Faha (variety Windsor White), the d value of the geotropic curvature which took place in 24 hours amounted to 0.04 mm. (average of 36 plants) ; in decapitated roots with replaced tips, it amounted to 0.44 mm. (average of 39 plants). Snow determined upon which side of the root the stimulus was transmitted. A transverse incision was made either upon the upper or on the under side of roots of Vicia Faha, and a mica plate was inserted in the wound in order to check the transmission of the stimulus over the wound. The roots were placed in a hori- GROWTH SUBSTANCES FOR GEOTROPISM 201 zontal position, and the size of the geotropic curvature was measured in degrees. This value was corrected for the wound curvatures caused by the incision. The following numbers were obtained: Incision on the upper side. . . (— 39.7°) + ( — 7.0°) = —46.7° Incision on the lower side.... (-27.7°) - (-7.0°) = -20.7° Snow concluded that the stimulus can be transmitted on the upper side as well as on the lower side, although greater trans- mission of the stimulus takes place on the under side of the root. Keeble and Nelson (1935) showed that although the secretion of a growth substance inhibiting root elongation must be localized in the 1 mm. tip zone of Zea mays, the region capable of receiving t/ V. \J V B Fig. 61. — Transverse insertion of mica plates into roots of Zea mays, and their influence upon resulting curvatures. A, when the plate is inserted near the tip, bending occurs away from the wound, due presumably to hindrance of growth- hormone transport from the tip into the elongation region. B, if the plate is inserted farther back from the tip, curvature occurs toward the wound, due presumably to the accumulation of hormones in the elongating region. {After Keeble and Nelson, 1935.) the stimulus of gravity extends possibly into the whole elongating region. By inserting small mica plates transversely halfway through the root at different distances from the tip, it was possible to demonstrate that (1) negative curvature takes place when the semisection hinders growth substance from being transported to the elongation region on the cut side and (2) positive curvature (toward the wound) results when the section hinders growth substance from escaping from the region of elongation on the cut side (Fig. 61). The Quantity-of-stimulus Principle. — A definite amount of stimulus is necessary for the attainment of a minimum reaction in the geotropic curvature of the root. The geotropic stimulus intensity can be changed by altering the direction of perception of gravity. The effect may be computed in this case as g sine (p, where (p is the angle between the root and a vertical axis. Another 202 GROWTH HORMONES IN PLANTS method of controlling the stimulus is by the use of centrifuging, which can be accurately graded by changing the rate of revolution or the length of the radius; Pekelharing (1909) investigated the threshold reaction of Lepidium roots (see Table 16) by this Table 16. — The Threshold Value of Centrifugal Force for Curvature of Lepidium Roots, Illustrating the Quantity-of- sTiMULUS Principle Presentation time, seconds Centrifugal force g Product, g X seconds 1,260 0.284 358 780 0.44 343 560 0.67 375 315 1.14 359 120 3.15 378 60 6.20 372 30 12.8 384 method. It appears from her data that the product of the pre- sentation time by the force is a rather constant value, within certain limits. Growth in Geotropic Curvature. — For the production of a mini- mum curvature, a definite amount of stimulus is necessary. If the amount of stimulus is increased, the size of the reaction is also increased. Lundegardh (1918) made a quantitative study of curvature in the root tip after different amounts of geotropic stimulation (Table 17). The relationship between the increase Table 17. — Increase in Amount op Curvature of Avena Coleoptiles WITH AN Increase in the Amount of Centrifugal Force Amount of Stimulus, g Minutes Curvature, Degrees 5 12 10 22 20 47.8 of curvature with increasing quantity of stimulus is approxi- mately linear. With great amounts of stimulus, a negative instead of a positive curvature takes place in the root (Jost and Stoppel, 1912; Jost and Wissmann, 1924). It is important to know whether or not the average rate of root growth during geotropic curvature remains constant. Sachs (1887) concluded that the geotropic reactions in seedhng roots GROWTH SUBSTANCES FOR GEOTROPISM 203 "are essentially the same as those involved in the upward curva- ture of the shoot axis." "During the curvature, a decrease in the rate of elongation appears in the growing axis; while the convex side grows more rapidly, the concave side grows less than would be the case in undisturbed growth in a vertical direction." That seedling roots show no noticeable change in growth rate when they are placed upon the clinostat was shown at about the same time by Schwarz (1881) and Elfving (1883). Later, Lux- burg (1905) investigated the same question and found that the individual difference in the experimental plants was too great to make possible a definite decision. It seemed highly probable that the growth increase in the median zone cannot be large. A retardation of growth in relation to the normal took place in 9 out of 13 roots, while in the remaining ones an increase in growth occurred. More recently Keeble, Nelson, and Snow (1931a) have studied the growth rate of maize roots placed in both horizontal and vertical positions. In all experiments, they found that hori- zontally oriented roots grew considerably more rapidly than vertical ones. These results, however, could not be confirmed by Cholodny (1932a). Navez (1933a) repeated these experiments and interpreted the results as follows: When excess water was present so that drops were allowed to form at the tips of roots grown in a vertical position, the rate of growth of these roots was retarded. On the other hand, if surplus water was not present, a difference in the rate of growth could not be shown. The evidence does not permit a definite conclusion regarding the rate of root elongation during the geotropic response. The Growth-substance Explanation of Root Curvature. — In a series of experiments with Vicia Faba roots. Snow (1923) demon- strated that a geotropic curvature can be produced after decapita- tion and replacement of the tip. This shows that the stimulus can be transmitted over a wound, and it can be concluded that transmission of the stimulus in the root is bound up with the transport of a substance. The geotropic responses of roots have been studied by Cholodny (1924, 1926, 1927, 1928a, 1929a, 1931e, 19336), who made (1924) the interesting discovery that decapi- tated roots of Lupinus angustifolius produced a positively geotropic curvature when coleoptile tips of Zea mays were placed on them. The growth rate of the decapitated roots, so treated, was retarded 204 GROWTH HORMONES IN PLANTS about 36 per cent. Then Nielsen (1930a) showed that rhizopin (3-indole acetic acid) can completely inhibit root growth in Lup- inus albus and ViciaFaha without permanently injuring the roots. Other work by Cholodny (1926) supplied the information that although decapitation has a retarding effect upon the rate of growth of the coleoptile, it has a promoting effect upon the growth of the root. The increase amounted to only about 12 per cent. Accord- ing to Keeble, Nelson, and Snow (1930), the growth-retarding sub- stance accumulates in the wound when the root is decapitated. When the substance is removed by washing, growth of the decapi- tated root again takes place. If the excised tip is replaced, the rate of growth is decreased again. All these observations conform with the theory of positively geotropic curvature in the root as pro- pounded by Cholodny (1927). According to Cholodny's theory, a growth substance, which is identical with that formed by the coleoptile tip, is secreted by the root tip. This growth substance has a retarding effect upon the rate of growth of the root. The movement of it is influenced by gravity in such a way that it accumulates upon the lower side of horizontally placed roots. As a result, the rate of growth of the root is retarded on the under side, and a geotropic curvature results. In 1932, Hawker published some experimental data which pointed to the presence of growth substance in the root of Vicia Faha. Geotropically stimulated root tips were cut longitudinally into an upper and lower half. Four such pieces (all either upper or lower) were placed on each of a series of gelatin blocks. After one hour the half tips were removed, and the gelatin blocks were placed unilaterally upon decapitated, vertically suspended Vicia Faha roots. Curvatures appeared, due to bending toward the side with the gelatin block. The curvatures were greatest in those roots with gelatin blocks which had obtained something from the lower halves of the original root tips. In a study of the influence of temperature upon geotropism in seedlings of Vicia Faha, Hawker (1933) demonstrated conduction of the geotropic stimulus into the root from excised and replaced root tips. Excised tips from plants grown at 20°C. were found capable of causing greater geotropic bending than tips taken from plants which had grown at the same temperature but had been kept at 5°C. for 24 hours immediately preceding the experiment. A period of 24 hours in the cold apparently decreased the produc- GROWTH SUBSTANCES FOR GEOTROPISM 205 tion of growth substance in the root tips (Fig. 62). It may be considered, therefore, that displacement of growth substance is responsible for the geotropic curvature in roots. Similarity of Growth Substance in the Root and Coleoptile. — The experimental evidence shows that the growth substance of the coleoptile has a retarding effect upon the growth of the root ; B> r> i i i A i^ 1 i i B ^^^^:> ^^^^> '. C/iem., 46:469-473. , A. J. Haagen Smit, and H. Erxleben. 1933. VII. Mitteilung. Studien uber das Vorkommen von Auxinen im menschlichen und im tierischen Organismus, Hoppe-Seyl. Zeitschr. Phys. Chem., 220: 137-161. A. J. Haagen Smit, and B. Tonnis. 1933. VIII. Mitteilung. tJber das Vorkommen von Auxinen und von Wachstumsstoffen der "Bios"-Gruppe in Carcinomen, Hoppe-Seyl. Zeitschr. Phys. Chem., 220: 162-172. BIBLIOGRAPHY 233 — , H. Erxleben, and A. J. Haagen Smit. 1934. IX. MitteilunK. tJber die Isolierung der Auxine a und b aus pflanzlichen Materialien, Hoppe-Seyl. Zeitschr. Phys. Chem., 225: 215-229. and . 1934. X. Mitteilung. tJber die Konstitution der Auxine a und b, Hoppe-Seyl. Zeitschr. Phys. Chem., 227: 51-73. , A. J. Haagen Smit, and H. Erxleben. 1934. XI. Mittcilung. tJber ein neues Auxin (Heteroauxin) aus Ham, Hoppe-Seyl. Zeitschr. Phys. Chem., 228:90-103. , , and . 1934. XII. Mitteilung. tjlier den Einfluss der Auxine auf das Wurzelwachstum und liber die chemische Natur des Auxins der Graskoleoptilen, Hoppe-Seyl. Zeitschr. Phys. Chem., 228 : 104-112. and D. G. F. R. Kostermans. 1934. XIII. Mitteilung. Hetero- auxin als Stoffwechselprodukt niederer pflanzlicher Organismen. Isolierung aus Hefe, Hoppe-Seyl. Zeitschr. Phys. Chem., 228: 113-121. . 1934. liber Auxine, Pharm. Zeitschr., 79: 1272-1273. . 1935. XIV. Mitteilung. Uber Wuchsstoffe der Auxin- und der Bios-Gruppe, Ber. Deut. Chem. Ges., Abt. A., 68: 16-28. and H. Erxleben. 1935. XV. Mitteilung. Synthese der " Auxin- glutarsaure" und einiger Isomerer, Hoppe-Seyl. Zeitschr. Phys. Chem., 235:181-200. and D. G. F. R. Kostermans. 1935. XVI. Mitteilung. Uber die Konstitutions-Spezifitat des Hetero-auxins, Hoppe-Seyl. Zeitschr. Phys. Cheyn., 235: 201-21Q. 1935. Untersuchungen iiber pflanzliche Wuchsstoffe, Naturwis- sensch., 23: 839-843. Kohl, F. G. 1894. "Die Mechanik der Reizkriimmungen," Marburg, 94 pp. Kolkmeijer, N. H., and A. N. J. Heyn. 1934. The hydration film of cellulose in cell-walls, Proc. K. Akad. Wetensch. Amsterdam, 37: 92-93. KoNiNG, H. C. 1933. Het winden der slingerplanten, Diss. Utrecht, 74 pp. Koningsberger, V. J. 1922. Tropismus und Wachstum, Rec. Trav. Bot. Neerl, 19: 1-136. Kornmann, p. 1935. Die Aufhebung der Wuchsstoffwirkung durch lebende Pflanzenteile, Ber. Deut. Bot. Ges., 53 : 523-527. KtJSTNER, H. 1931. Hormonwirkung bei den Pflanzen und Hormon- steigerung durch rotes Licht, Klin. Wochenschr., 10: 1585. Laan, p. a. van DER. 1934. Der Einfluss von Aethylen auf die Wuchs- s'toffbildung bei Avena und Vicia, Rec. Trav. Bot. Neerl, 31: 690-742. Laibach, F. 1929. Untersuchungen iiber die Postfloration tropischer Orchideen, Planta, 9 : 341-387. . 1932. PoUenhormon und Wuchsstoff, Ber. Deut. Bot. Ges., 50: 383-390. . 19330. Wuchsstoffversuche mit lebenden Orchideenpolhnien, Ber. Deut. Bot. Ges., 51: 336-340. . 19336. Versuche mit Wuchsstoffpaste, Ber. Deut. Bot. Ges., 51: 386-392. . 1934. Zum Wuchsstoffproblem, Der Ziichter, 6 : 49-53. 234 GROWTH HORMONES IN PLANTS . 1935. itber die Auslosung von Kallus- unci Wurzelbildung durch /3-Indolylessigsaure, Bcr. Deut. Bot. Ges., 53: 359-364. and O. FiscHNiCH. 1935o. tjber eine Testmethode zur Prtifung der kallus-bildenden Wirkung von Wuchsstoffpasten, Ber. Deut. Bot. Ges., 53 : 469-477. - and . 19356. Kiinstliche Wurzelneubildung mittels Wuchs- stoffpaste, Ber. Deut. Bot. Ges., 63: 528-539. , and . 1936. tJber Blattbewegungen unter dem Einfluss von ki'mstlich zugefiihrtem Wuchsstoff, Biol. Zentralbl., 56: 62-68. and P. KoRNMANN. 1933a. Zur Methodik der Wuchsstoffversuche, Planta, 19 : 482-484. and . 19336. Zur Frage des Wuchsstofftransportes in der Haferkoleoptile, Planta, 21 : 396-418. , G. Mai, and A. Muller. 1934. tTber ein Zellteilungshormon, Naturwissensch., 22 : 288. and E. Maschmann. 1933. JJher den Wuchsstoff der Orchideen- pollinien, Jahrb. Wiss. Bot., 78 : 399-430. and F. Meyer. 1935. tJber die Schwankungen des Auxingehaltes bei Zea mays und Helianthus annuus im Verlauf der Ontogenese, Senckenbergiana, 17 : 73-86. -, A. Muller, and W. Schafer. 1934. Uber wurzelbildende Stoffe, Naturwissensch., 22: 588-589. Lange, S. 1925. tJber autonome Kriimmungen der Koleoptile von Avena auf dem Klinostaten, Ber. Deut. Bot. Ges., 43 : 438-443. . 1927. Die Verteilung der Lichtempfindlichkeit in der Spitze der Haferkoleoptile, Jahrb. Wiss. Bot., 67: 1-51. . 1929. tJber den Einfluss weissen und roten Lichtes auf die Entwicklung des Mesokotyls bei Haferkeimlingen, Jahrb. Wiss. Bot., 71: 1-25. . 1933. Die Dorsiventralitatskriimmungen der Haferkeimlinge, Jahrb. Wiss. Bot., 78: 157-202. LaRxje, C. D. 1935. The role of auxin in the development of intumes- cences on poplar leaves; in the production of cell outgrowths in the tunnels of leaf-miners; and in the leaf-fall in Coleus, Abstract in Amer. Jour. Bot., 22 : 908. *Leeman, a. C. 1933. Growth regulating substances, Pamphl. So. Afr. Biol. Soc, 6: 10-14. Lek, H. a. a. van der. 1925. Over de wortelvorming van houtige stekken, Diss. Utrecht, 230 pp. (English summary, pp. 211-230.) . 1934. (On the influence of the buds on root-development in cuttings), Meded. Landbouwhoogesch. Wageningen, 38(2): 95 pp. (Dutch with Enghsh summary, pp. 74-82.) Letellier, M. a. 1899. L'electricite k I'etat statique exerce une action directrice sur les racines de la feve vulgaire. Bull. Soc. Bot. France, 46 : 11-23. Li, Tsi-Tung. 1930. The appearance of the new physiological tip of the decapitated coleoptiles of Avena saliva, Proc. K. Akad. Wetensch. Amsterdam, 33: 1201-1205. BIBLIOGRAPHY 235 1934. Phototropism of decapitated coleoptile of Avena saliva, Sci. Rep. Nat. Tsing Hua Univ., Ser. B, Biol. & Psychol. Sci., 2:1-10. LiNSBArER, K. 1908. tjber Reizleitungsgeschwindigkeit und Latenzzeit bei Mimosa pudica, Wiesner-Festschrift, 396-411. 1922. Methoden der pflanzlichen Reizphysiologie: Tropismen und Nastieen, Handb. Biol. Arbeitsniethoden, herausg. von Abderhalden, Abt. 11, Teil 1: 191-308. 1929. Fortschritte der pflanzlichen Reizphysiologie, Sammel- bericht, Osterr. Boi. Zeitschr., 78: 81-93. LoEB, J. 1916. On the association and possible identity of root-forming and geotropic substances or hormones in Bryophyllum calycinum, ^Science, 44: 210-211. . 1917. Influence of the leaf upon root formation and geotropic curvature in the stem of Bryophyllum calycinum and the possibility of a hormone theory of these processes, Bot. Gaz., 63 : 25-50. 1924. "Regeneration — from a physico-chemical viewpoint," McGraw-Hill Book Company, Inc., New York, 143 pp. LoEWE, S. 1933. Analyse der Pflanzenhormone, Handb. Pflanzenanalyse {Klein), 4: 1005-1041. LuNDEGARDH, H. 1918. Ubcr Beziehungen zwischen Reizgrosse und Reaktion bei der geotropischen Bewegung und iiber den Autotropismus, Bot. Notiser, 65-118. . 1921. Die Beziehungen zwischen der Lichtwachstumsreaktion und dem Phototropismus, Ber. Deut. Bot. Ges., 39: 195-200. 1922. Ein Beitrag zur quantitativen Analyse des Phototropismus, Ark. Bot., 18 : No. 3, 62 pp. LuxBURG, H. 1905. Untersuchungen iiber den Wachstumsverlauf bei der geotropistischen Bewegung, Jahrb. Wiss. Bot., 41: 399-457. Mai, G. 1934. Korrelationsuntersuchungen an entspreiteten Blattstielen mittels lebender Orchideenpollinien als Wuchsstoffquelle, Jahrb. Wiss. Bot., 79: 681-713. Majima, R., and T. Hoshino. 1925. Synthetische Versuche in der Indol- Gruppe. VI. Eine neue Synthese von /3-Indolyl-alkylaminen, Ber. Deut. Chem. Ges., 58 : 2042-2046. ♦Malowan, S. L. 1934. Wuchsstoffe und Pflanzenwachstum, Proto- plasyna, 21 : 306-322. Maschmann, E. 1932. Der Wuchsstoff bosartiger Geschwlilste, Natur- vrissensch., 20: 721-722. and F. Laibach. 1932. f'ber Wuchsstoffe, Biochem. Zeitschr., 255 : 446-452. . and . 1933. Das Vorkommen von Wuchsstoff in tierischem und pflanzlichem Material, Naturivissensch., 21: 517. Meesters, a. 1936. The influence of hetero-auxin on the growth of root hairs and roots of Agrostemma githago L., Proc. K. Akad. Welensch. Amsterdam, 39 : 91-97. Metzner, p. 1929. flier die Wirkung der Langskraft beim Geotropismus, Jahrb. Wiss. Bot., 71: 325-385. 236 GROWTH HORMONES IN PLANTS . 1934. Zur Kenntnis der Stoffwechselanderungen bei geotropisch gereizten Keimpflanzen, Ber. Dcut. Bot. Ges., 52 : 506-522. Meyer, F. 1936. tJber die Verteilung des Wuchsstoffes in der Pflanze wahrend ihrer Entwicklung, Diss. Universitat zu Frankfurt am Main, 39 pp. MicHENER, H. D. 1935. Effects of ethylene on plant growth hormone, Science, 82: 551-552. MiEHE, H. 1902. Tiber correlative Beeinflussung des Geotropismus einiger Gelenkpflanzen, Jahrb. Wiss. Bot., 37 : 527-593. Miller, W. Lash. 1930. Bios, Jour. C/iem. ^duc, 7 : 256-267. MoissEJEWA, M. 1928. (Zur Frage nach den Wuchshormonen des Getreides.) Visnik. Kiivsk. Bot. Sadu {Bull. Jard. Bot. Kieff), 7/8: 36-51. (Ukrainian with German summary.) Morgan, T. H. 1903. The hypothesis of formative stuffs. Bull. Torr. Bot. Club, 30: 206-213. Mrkos, O. 1933. tJber den Einfluss des Wuchsstoffes auf die Regenera- tion und Wundgewebebildung, Planta, 21: 206-210. MtJLLER, A. M. 1935. tJber den Einfluss von Wuchsstoff auf das Aus- treiben der Seitenknospen und auf die Wurzelbildung, Jahrb. Wiss. Bot., 81: 497-540. MtJNCH, E. 1930. "Die Stoffbewegungen in der Pflanze," Gustav Fischer, Jena, 234 pp. Navez, a. E. 1933a. "Geo-growth" reaction of roots of Lupinus, Bot. Gaz., 94: 616-618. . 19336. Growth-promoting substance and elongation of roots, Jour. Gen. Physiol, 16 : 733-739. . 1933c. Growth-promoting substance and illumination, Proc. Nat. Acad. Sci., 19: 636-638. and B. Kropp. 1934. The growth-promoting action of crustacean eye-stalk extract, Biol. Bull., 67: 250-258. and T. W. Robinson. 1932a. Automatic recording of movements of plant organs. Jour. Gen. Physiol., 16: 125-131. and . 19326. Geotropic curvature of Avena coleoptiles, Jour. Gen. Physiol, 16 : 133-145. Nemec, B. 1901. tJber die Wahrnehmung des Schwerkraftreizes bei den Pflanzen, Jahrb. Wiss. Bot., 36 : 80-178. Neukirchen, J. 1930. Uber die Beeinflussung des tropistischen Reiz- verhaltens von Gramineenkoleoptilen durch chemische Vorbehandlung des Saatgutes, Planta, 12: 505-531. Nielsen, N. 1924. Studies on the transmission of stimuli in the coleoptile of Avena, Dansk Bot. Arkiv, 4(8). . 1928. Untersuchungen liber Stoffe, die das Wachstum der A ycna- Koleoptile beschleunigen, Planta, 6: 376-378. — ■ . 1930a. Unders0gelser over et nyt Plantevaekststof Rhizopin, Diss. Kopenhagen, 95 pp. . 19306. Untersuchungen liber einen neuen wachstumsregulier- enden Stoff: Rhizopin, Jahrb. Wiss. Bot., 73: 125-191. BIBLIOGRAPHY 237 — . 1931a. The effect of rhizopin on the production of matter of Aspergillus niger, Compt. Rend. Labor. Carlsberg, 19(5): 1-10. — . 1931b. tJber Wuchsstoffe der Hefe, Biochcm. Zeitschr., 237: 244-246. . . 1932. tJber das Vorkommen von Wuchsstoff bei Boletus edulis, Biochem. Zeitschr., 249: 196-198. and V. Hartelius. 1932. The separation of growth-promoting substances, Compt. Rend. Labor. Carlsberg, 19(8): 1-17. NoACK, K. 1914. Die Bedeutung der schiefen Lichtrichtung fiir die Hehoperzeption parallelotroper Organe, Zeitschr. Bot., 6: 1-79. Noll, F. 1900. tJber Geotropismus, Jahrb. Wiss. Bot., 34: 457-506. NuERNBERGK, E. 1927. " Untersuchungen uber die Lichtverteilung in Awno-Koleoptilen und anderen phototropisch reizbaren Pflanzen- organen bei einseitiger Beleuchtung; ein Beitrag zur Kritik der Beweis- fuhrung der Blaauwschen Theorie." Gustav Fischer, Jena. (Bot. Abh., hrsg. von K. Goebel, 12). . 1929. Phototropismus und Phototaxis bei Pflanzen, Handb. Norm. Path. Physiol, 12 : 36-59. . . 1932a. Phototropismus und Phototaxis bei Pflanzen, Handb. Norm. Path. Physiol, 18: 310-314. . . 19326. Physikalische Methoden der pflanzHchen Lichtphysi- ologie, Abderhalden, E. Handb. Biol. Arbeitsmethod., 11(4): 739-950. . 1933. tJber den Auxin-Quertransport und den Geotropismus der Awena-koleoptile: Einfluss der Dekapitation, Flora, 28(n.s. 28): 99-110. and H. G. du Buy. 1930. tJber Methoden zur Analyse von Wachstumserscheinungen, Rec. Trav. Bot. Neerl, 27: 417-520. and . 1932. Die Analyse von pflanzlichen Wachstums- vorgangen mit kinematographischen Registriermethoden, Handb. Biol Arbeitsmeth. herausg. von Abderhalden, 11(4): 951-1014. Oehlkers, F. 1926. Phototropische Untersuchungen an Phycomyces nitens, Zeitschr. Bot., 19: 1-44. OosTERHUis, J. 1931. Der Einfluss der Knospen auf das Stengelwachstum von Asparagus plumosus und A. Sprengeri, Rec. Trav. Bot. Neerl, 28: 20-74. OvERBECK, F. 1926. Studien uber die Mechanik der geotropischen Kriimmung und des Wachstums der Keimwurzel von Vicia faba, Zeitschr. Bot., 18: 401-451. —. . 1934. Beitrage zur Kenntnis der Zellstreckung (Untersuchungen am Sporogonstiel von Pellia epiphylla), Zeitschr. Bot., 27: 129-170. OvERBEEK, J. VAN. 1932. An analysis of phototropism in dicotyledons, Proc. K. Akad. Wetensch. Amsterdam, 35: 1325-1335. . 1933. Wuchsstoff, Lichtwachstumsreaktion und Phototropismus bei Raphanus, Rec. Trav. Bot. Neerl, 30: 537-626. . 1935. The growth hormone and the dwarf type of growth in corn, Proc. NaL Acad. Sci., 21: 292-299. . 1936a. "Lazy," an a-geotropic form of maize, Jour. Hered., 27: 93-96. 238 GROWTH HORMONES IN PLANTS . 19366. Different action of auxin-a and of heteroauxin (Pre- liminary note), Proc. Nat. Acad. Sci., 22: 187-190. Paal, a. 1914. tiller phototropische Reizleitungen, Ber. Deut. Bot. Ges., 32 : 499-502. . 1918. Uber phototropische Reizleitung, Jahrb. Wiss. Bot., 58: 406-458. Pekelharing, C. J. 1909. " Onderzoekingen over de perceptie van den zwaartekrachtprikkel door planten," Diss. Utrecht, J. V. Boekhoven, 106 pp. Perry, J. T. 1932. A possible hormone-secreting region in the grass coleoptile, Science, 76(1966): 215-216. Pfeffer, W. 1904. " Pflanzenphysiologie : ein Handbuch der Lehre vom Stoffwechsel und Kraftwechsel in der Pflanze. Bd. II: Kraftwechsel," W. Engelmann, Leipzig, 1881; 2d. ed., 1904. PiccARD, A. 1904. Neue Versuche iiber die geotropische Sensibilitat der Wurzelspitze, Jahrb. Wiss. Bot., 40: 94-102. PiSEK, A. 1926. Untersuchungen liber den Autotropismus der Hafer- koleoptile bei Lichtkriimmung, liber Reizleitung und den Zusammen- hang von Lichtwachstumsreaktion und Phototropismus, Jahrb. Wiss. Bot., 65: 460-501. . 1928. Beitrag zu einem quantitativen Vergleich von Lichtwachs- tumsreaktion und Phototropismus der Haferkoleoptile, Jahrb. Wiss. Bot., 67 : 960-980. . 1929. Wuchsstoff und Tropismen, Osterr. Bot. Zeitschr., 78: 168-186. PoHL, R. 1935. Uber den Endospermwuchsstoff und die Wuchsstoff- produktion der Koleoptilspitze, Planta, 24 : 523-526. PopoFF, M. 1933. Uber die pflanzlichen Auxine und ihre Wirkung auf Einzellige, Biol. Zentralbl, 53: 661-668. Priestley, J. H. 1926o. Light upon growth. Naturalist, pp. 37-44, 71-80. . 19266. The negative phototropic curvature of the root. Natu- ralist, pp. 103-105. . 1926c. Light and Growth. III. An interpretation of phototropic growth curvatures, New Phytol., 25: 213-226. 1926(i. Light and Growth. IV. An examination of the photo- tropic mechanism concerned in the curvature of coleoptiles of the Gramineae, New Phytol., 25: 227-247. Pringsheim, E. G. 1909. Studien zur heliotropischen Stimmung und Prasentationszeit, Beitr. Biol. Pflanzen (Cohn), 9: 415-478. . 1912. "Die Reizbewegungen der Pflanzen," Julius Springer, Berlin. . 1931. Untersuchungen iiber Turgordehnung und Membran- beschaffenheit, Jahrb. Wiss. Bot., 74: 749-796. 1932. Botanische Mitteilungen, Naturvoissensch., 20: 594-598. PuRDY, H. A. 1921. Studies on the path of transmission of phototropic and geotropic stimuli in the coleoptile of Avena, Danske Videnskab. Selskab. Biolog. Medd. 3(8). 29 pp. BIBLIOGRAPHY 239 Raalte, M. H. van. 1936. On the influence of glucose on auxin produc- tion by the root tip of Vicia Faba, Proc. K. Akad. Wetensch. Amsterdam, 39: 261-265. Ramaer, H. 1926. Phototropical curvatures of seedlings of Avena which appear when reaction of the distal side is excluded, Proc. K. Akad. Wetensch. Amsterdam, 29: 1118-1121. Ramshorn, K. 1934. Experimentelle Beitrage zur elektrophysiologischen Wachstumstheorie, Planta, 22 : 737-766. Rawitscher, F. 1932. "Der Geotropismus der Pflanzen," Gustav Fischer, Jena, 420 pp. Reinders, D. E. 1934. The sensibility for light of the base of normal and decapitated coleoptiles of Avena, Proc. K. Akad. Wetensch. Amsterdam, 37: 308-315. Reinhard, a. W., and L. Bro. 1933. Zur Frage der Leitung des photo- tropischen Reizes, Jahrb. Wiss. Bot., 79: 1-8. Renner, O. 1922. Die Wachstumsreaktionen bei Licht- und Schwer- kraftreizung, Zeitschr. Bot., 14: 449-462. RiccA, U. 1916. Solution d'un probleme de physiologic: la propagation de stimulus dans la Sensitive, Arch. Ital. Biol., 65: 219-232. . 1926. Transmission of stimuli in plants, Nature, 117 : 654-^55. Rosene, H. F. 1935. Proof of the principle of summation of cell E.M.F.'s, Plant Physiol, 10: 209-224. RoTHERT, W. 1892. tJber die Fortpflanzung des heliotropischen Reizes, Ber. Deut. Bot. Ges., 10: 374-390. . 1894. Uber Heliotropismus, Beitr. Biol. Pflanzen {Cohn), 7: 1-212. Rutten-Pekelharing, C. J. 1910. Untersuchungen uber die Perzeption des Schwerkraftreizes, Rec. Trav. Bot. Neerl, 7 : 241-348. Sachs, J. 1872. Langenwachstum der Ober- und Unterseite horizontal- gelegter sich auf warts krtimmender Sprosse, Arb. Bot. Inst. Wiirzburg, 1: 193-208. . 1873. liber Wachstum und Geotropismus aufrechter Stengel, Flora, 56: 321-331. . 1874. "Lehrbuch der Botanik," W. Engelmann, Leipzig, 4th ed., 928 pp. . 1882a. Stoff und Form der Pflanzenorgane, Arb. Bot. Inst. Wurzburg, 2: 452-488; II, 2: 689-718. . 18826. "Textbook of Botany," Clarendon Press, Oxford, 2d ed., 980 pp. . 1887. Vorlesungen liber Pflanzenphysiologie, W. Engelmann, Leipzig, 2d ed. 1888. Erkliirung der diesem Hefte beiUegenden Tafeln I bis VII ■~? (Beispiele geotropischer Kriimmungen aufrecht wachsender Spross- achsen), Arb. Bot. Inst. Wurzburg, 3 : 553-558. Sakamura, T., and T. Yanagihara. 1932. Zur Bildung des Wuchsstoffs bei Aspergillus niger, Proc. Imp. Acad. Tokyo, 8: 397-399. Salkowski, E. 1885. tJber das Verhalten der Skatolcarbonsaure im Organismus, Hoppe-Seyl. Zeitschr. Phys. Chem., 9 : 23-33. 240 GROWTH HORMONES IN PLANTS ScHMiD, G. 1923. Das Reizverhalten kiinstlicher Teilstiicke, die Kon- traktilitat und das osmotische Verhalten der Oscillatoria Jenensis, Jahrb. Wiss. Bot., 62: 328-419. ScHMiTZ, H. 1933. Uber Wuchsstoff und Geotropismus bei Grasern, Planta, 19: 614-635. . . 1934. Die periodischen Bewegungen der Blatter von Coleus Penzigii, Zeitschr. Bot., 27: 353-411. ScHOBER, A. 1899. Die Anschauungen liber den Geotropismus der Pflanzen seit Knight. Geschichtliche Studie eines physiologischen Problems, Wiss. Beilage Ber. Realsch. Eilbtck., 50 pp. ScHOPFER, W. H. 1935. Facteurs de croissance et vitamines chez les champignons, Mitt. Naturf. Ges. Bern., 1934: 35-37. ScHTSCHERBACK, J. 1910. Die geotropische Reaktion in gespaltenen Stengeln, Beih. Bot. Centralbl, Abt. I. 25: 358-386. Schumacher, M. 1923. Dekapitation und geotropische Kriimmungs- fahigkeit von Sprossen, Jahrb. Wiss. Bot., 62: 420-448. ScHWARZ, F. 1881. Der Einfluss der Schwerkraft auf das Langenwachs- tum der Pflanzen, Untersuch. Bot. Inst. Tubingen, 1 : 53-96. ScHWEiGHART, O. 1935. Eosiu und Keimpflanzen, Beih. Bot. Centralbl, Abt. A, 53: 217-292. Seubert, E. 1924. Uber Chemotropismus bei Avena, Biochem. Zeitschr., 150 : 93-100. . 1925. tJber Wachstumsregulatoren in der Koleoptile von Avena, Zeitschr. Bot., 17: 49-88. SiERP, H. 1917. Uljer den Einfluss des Lichtes auf das Wachstum der Pflanzen, Ber. Deut. Bot. Ges., 35: (8)-(20). . 1918. Ein Beitrag zur Kenntnis des Einflusses des Lichtes auf das Wachstum der Koleoptile von Avena sativa, Zeitschr. Bot., 10: 641-729. . 1919. Neuere Arbeiten iiber Photo- und Geotropismus, Zeitschr. Boi., 11: 510-537. . 1921. Untersuchungen liber die durch Licht und Dunkelheit hervorgerufenen Wachstumsreaktion bei der Koleoptile von Avena sativa und ihr Zusammenhang mit den phototropischen Krlimmimgen, Zeitschr. Bot., 13: 113-172. and A. Seybold. 1926. Untersuchungen liber die Lichtempfind- lichkeit der Spitze und des Stumpfes in der Koleoptile von Avena sativa, Jahrb. Wiss. Bot., 65: 592-610. Simon, S. V. 1912. Untersuchungen liber den autotropischen Ausgleich geotropischer und mechanischer Krummungen der Wurzeln, Jahrb. Wiss. Bot., 51: 81-178. Skoog, F. 1934. The effect of x-rays on growth substance and plant growth. Science, 79 : 256. . 1935. The effect of x-irradiation on auxin and plant growth, Jour. Cell. Comp. Physiol, 7: 227-270. and K. V. Thimann. 1934. Further experiments on the inhibition of the development of lateral buds by growth hormone, Proc. Nat. Acad. Sci., 20 : 480-485. BIBLIOGRAPHY 241 Small, J. 1920a. A theory of geotropism; with some experiments on the chemical reversal of geotropic response in stem and root, New Phytol, 19 : 49-63. 19206. Preliminarj' notes on additional evidence for the hydrion differentiation theory of geotropism. New Phytol, 19: 208-212. Snow, R. 1923. The conduction of geotropic excitation in roots, Ann. BoL, 37 : 43-53. , 1924a. Further experiments on the conduction of tropic excita- tion, A?in. Boi., 38: 163-174. 19246. Conduction of excitation in stem and leaf of Mimosa pudica, Proc. Roy. Soc. London, Ser. B. 96: 349-374. . 1925a. The correlative inhibition of the growth of axillary buds, Ann. Bot., 39: 841-859. . 19256. Transmission of stimuli in plants. Nature, 115 : 82-83. . 1925c. Conduction of excitation in the leaf of Mimosa Spegazzinii, Proc. Roy. Soc. London, Ser. B. 98: 188-201. . 1929o. The transmission of inhibition through dead stretches of stem, Ann. Bot., 43: 261-267. . 19296. The young leaf as the inhibiting organ. New Phytol, 28: 345-358. . 1931o. Experiments on growth and inhibition. I. The increase of inhibition with distance, Proc. Roy. Soc. London, Ser. B. 108 : 209-223. _. 19316. Experiments on growth and inhibition. II. New phe- nomena of inhibition, Proc. Roy. Soc. London, Ser. B. 108 : 305-316. 1932a. Experiments on growth and inhibition. III. Inhibition and growth promotion, Proc. Roy. Soc. London, Ser. B. Ill: 86-105. . 19326. Growth-regulators in plants. New Phytol, 31: 336-353. . 1933. The nature of cambial stimulus, New Phytol, 32 : 288-296. . 1935a. Substances causing cambial growth, Proc. Int. Bot. Congress Amsterdam, 2 : 269. . 19356. Activation of cambial growth by pure hormones. New Phytol, 34:347-360. and B. Le Fanu. 1935a. Activation of cambial gro\vth, Nature, 135: 149. and . 19356. Activation of cambial growth by pure hormones. Nature, 135 : 876. SoDiNG, H. 1924. Werden von der Spitze der Haferkoleoptile Wuchs- hormone gebildet?, Ber. Deut. Bot. Ges., 41 : 396-400. . 1925. Zur Kenntnis der Wuchshormone in der Haferkoleoptile, Jahrb. Wiss. BoL, 64 : 587-603. . 1926. tJber den Einfluss der jungen Infloreszenz auf das Wachs- tum ihres Schaftes, Jahrb. Wiss. Bot., 65: 611-635. . 1927. tJber Wuchshormone, Zellstimulations-forschungen, 2: 381-392. . 1929. Weitere Untersuchungen fiber die Wuchshormone der Haferkoleoptile, Jahrb. Wiss. Bot., 71 : 184-213. . 1931. Wachstum und Wanddehnbarkeit bei der Haferkoleoptile, Ja;ir6. Wiss. Bot., 74: 127-151. 242 GROWTH HORMONES IN PLANTS * . 1932a. Hormone und Pflanzenwachstum, Beih. Bot. Centralbl. Erganzungsbd., 49: 469-481. . 1932b. tjber das Wachstum der Infloreszenzschafte, Jahrb. Wiss. Bot., 77 : 627-656. . 1932c. tJber das Streckungswachstum der Zellwand, Ber. Deut. Bot.Ges., 50: 117-123. . 1934. liber die Wachstumsmechanik der Haferkoleoptile, Jahrb. Wiss. Bot., 79 : 231-255. , 1935a. Die Ausflihring des Wentschen Auxintestes am Tages- licht, Ber. Deut. Bot. Ges., 53: 331-334. . 19356. 1st der Wuchsstoff unspezifisch?, Proc. Int. Bot. Congress Amsterdam, 2: 272-273. . 1935c. tJber den Wuchsstoff in der Basis der Haferkoleoptile, Ber. Deut. Bot. Ges., 53 : 843-846. 1936. Wirkt der Wuchsstoff artspezifisch?, Jahrb. Wiss. Bot., 82 : 534-554. Stark, P. 1916. Experimentelle Untersuchungen liber das Wesen und die Verbreitung der Kontaktreizbarkeit, Jahrb. Wiss. Bot., 57: 189-320. . 1917. Beitrage zur Kenntnis des Traumatotropismus, Jahrb. Wiss. Bot., 57: 461-552. . 1919. tJber traumatotropische und haptotropische Reizleitungs- vorgange bei Gramineenkeimlingen, Ber. Deut. Bot. Ges., 37: 358-363. . 1921a. Neuere Erfahrungen fiber das Wesen pflanzlicher Reiz- leitungsvorgange, Naturwissensch. Monatsh. Biol. Chem. Geogr. Geol. Unterr., 20(n.s. 3): 101-110. . 19216. Studien uber traumatotrope und haptotrope Reizleitungs- vorgange mit besonderer Beriicksichtigung der Reiziibertragung auf fremde Arten und Gattungen, Jahrb. Wiss. Bot., 60: 67-134. . 1924. Geotropische Reizleitung bei Unterbrechung des organ- ischen Zusammenhangs, Ber. Deut. Bot. Ges., 42: 125-130. . 1926. Vergleichende Physiologie der Tangoreceptoren bei Pflanzen. (Haptotropismus, Seismonastie, Traumatotropismus usw. bei Pflanzen), Handb. Norm. Path. Physiol., 11: 84-93. * . 1927. Das Reizleitungsproblem bei den Pflanzen im Lichte neuerer Erfahrungen, Ergebnisse Biol., 2: 1-94. and O. Drechsel. 1922. Phototropische Reizleitungsvorgange bei Unterbrechung des organischen Zusammenhangs, Jahrb. Wiss. Bot., 61:339-371. Starling, E. H. 1906. Die chemische Koordination der Korpertatig- keiten, Verh. Ges. Deutsch. Naturf. Arzt., Stuttgart, 78(1): 246-260. . 1914. Discussion on the therapeutic value of hormones, Proc. Roy. Soc. Med. 7, Part III (Sec. Therap. Pharmac): 29-31. Strugger, S. 1932. Die Beeinflussung des Wachstums und des Geo- tropismus durch die Wasserstoffionen, Ber. Deut. Bot. Ges., 50 : (77)-(92). . 1933. tlT^er das Wachstum dekapitierter Keimpflanzen, Ber. Deut. Bot. Ges., 51: 193-209. . 1934. Beitrage zur Physiologie des Wachstums. I. Zur proto- plasmaphysiologischen Kausalanalyse des Streckungswachstums, Jahrb. Wiss. Bot., 79: 406-471. BIBLIOGRAPHY 243 Sylvan, B. 1933. Growth experiments on tadpoles with cystine and auxine, Skand. Arch. Phydol., 67: 129-136. Tammes, p. M. L. 1931. iTjer den Verlauf der geotropischen Kriimmung bei kiinstlich tordierten Koleoptilen von Avena, Rec. Trav. Bot. Neerl., 28: 75-81. Tendeloo, N. 1927. On the so-called traumatotropic curvatures of oat seedlings, Proc. K. Akad. Wetensch. Amsterdam, 30: 756-760. Tetley, U., and J. H. Priestley. 1927. The histology of the coleoptile in relation to its phototropic response, New Phytol., 26: 171-186. Thimann, K. V. 1934. Studies on the growth hormone of plants. VI. The distribution of the growth substance in plant tissues, Jour. Gen. Physiol, 18: 23-34. . 1935a. Growth substances in plants, Ann. Rev. Biochem., 4: 545-568. . 19356. On the plant growth hormone produced by Rhizopus suinus. Jour. Biol. Chem., 109: 279-291. . 1935c. On an analysis of the activity of two growth-promoting substances on plant tissues, Proc. K. Akad. Wetensch. Amsterdam, 38: 896-912. and J. Bonner. 1932. Studies on the growth hormone of plants. II. The entry of growth substance into the plant, Proc. Nat. Acad. Sci., 18: 692-701. and . 1933. The mechanism of the action of the growth substance of plants, Proc. Roy. Soc. London, Ser. B., 113: 126-149. and H. E. Dolk. 1933. Conditions governing the production of the plant, growth hormone by Rhizopus cultures, Biol. Zcntralbl., 53 : 4^-66. and J. B. Koepfli. 1935. Identity of the growth-promoting and root-forming' substances of plants. Nature, 135: 101-102. and F. Skoog. 1933. Studies on the growth hormone of plants. III. The inhibiting action of the growth substance on bud development, Proc. Nat. Acad. Sci., 19 : 714-716. — and . 1934. On the inhibition of bud development and other functions of growth substance in Vicia faba, Proc. Roy. Soc. London, Ser. B, 114: 317-339. — and F. W. Went. 1934. On the chemical nature of the root- forming hormone, Proc. K. Akad. Wetensch. Amsterdam, 37: 456-459. Thoday, D. 1933. Some physiological aspects of differentiation. New Phytol, 32 : 274-287. Uhrova, a. 1934. tiber die hormonale Natur der Hemmungswirkung der Blatter bei Bryophyllurn crenatum, Planta, 22: 411-427. Umrath, K. 1925a. Uber die Erregungsleitung im Blatte von Mimosa pudica, Sitzungsh. Akad. Wiss. Wien Math.-Naturw. Kl, Abt. I. 134: 21-44. . 19256. tTber die Erregungsleitung bei Mimosen, Sitzungsh. Akad. Wiss. Wien Math.-Naturw. Kl, Abt. I. 134: 189-208. . 1928. tJlier die Erregungsleitung bei sensitiven Pflanzen, mit Bemerkungen zur Theorie der Erregungsleitung und der elektrischen Erregbarkeit im Allgemeinen, Planta, 6 : 274-324. 244 GROWTH HORMONES IN PLANTS . 1929. liber die Erregungsleitung bei hoheren Pflanzen, Planta, 7: 174-207. Uyldert, I. E. 1928. The influence of growth-promoting substances on decapitated flower stalks of Bellis perennis, Proc. K. Akad. Wetensch. Amsterdam, 31: 59-61. . 1931. De invloed van groeistof op planten met intercalaire groei, Diss. Utrecht, 78 pp. VoGT, E. 1915. tJber den Einfluss des Lichts auf das Wachstum der Koleoptile von Avena saliva, Zeitschr. Bot., 7: 193-270. Vries, H. de. 1880. tJber die Aufrichtung des gelagerten Getreides, Landw. Jahrb., 9: 473-520. . 1885. tJber die Bedeutung der Circulation und der Rotation des Protoplasma flir den StofTtransport in der Pflanze, Bot. Zeitung 43: 17-26. Vries, M. S. de. 1917. tJber die Ursache des Auswachsens des Hypo- kotyls bei Keimlingen von Avena sativa, Rec. Trav. Bot. Neerl., 14: 109-118. Waller, J. C. 1929. Plant electricity. II. Towards an interpretation of the photo-electric currents of leaves. New Phytol., 28: 291-302. Warner, T. 1931. tJber den Einfluss von Salzen auf die geotropische Reaktion, Planta, 12 : 635-669. Weber, U. 1926o. Probleme des Geotropismus, Verh. Physik. Med. Ges. Wurzburg, 51 : 14-17. . 19266. Untersuchungen liber Wachstum und Krlimmung unver- letzter und halbierter Koleoptilen nach geotropischer Reizung, Jahrb. Wiss. Bot., 66: 35-108. 1931. Wachstum und Krlimmung einzelner Zonen geotropisch gereizter Gerstenkeimlinge, Jahrb. Wiss. Bot., 75: 312-376. Wehnelt, B. 1927. Untersuchungen liber das Wundhormon der Pflanzen, Jahrb. Wiss. Bot., 66: 773-813. Weu, H. G. van der. 1931. Die quantitative Arbeitsmethode mit Wuchsstoff, Proc. K. Akad. Wetensch. Amsterdam, 34: 875-892. . 1932. Der Mechanismus des Wuchsstoff transportes, Rec. Trav. Bot. Neerl, 29: 379-496. . 1933a. On the occurrence of growth substance in marine algae, Proc. K. Akad. Wetensch. Amsterdam, 36: 759-760. -. 19336. Uber das Vorkommen von Wuchsstoff bei Meeresalgen, Pubbl. Staz. Zoolog. Napoli, 13: 172-179. . 1933c. tJber Wuchsstoff bei Elaeagnus angustifolius, Proc. K. Akad. Wetensch. Amsterdam, 36: 760-761. ■. 1934. Der Mechanismus des Wuchsstofftransportes. II, Rec. Trav. Bot. Neerl., 31 : 810-857. Weimann, R. 1929. Untersuchungen liber den Traumatotropismus der A!;cwa-Koleoptile, Jahrb. Wiss. Bot., 71 : 269-323. Went, F. A. F. C. 1929. Plant movements, Proc. Int. Congress Plant Sci. {4th) Ithaca, 1926. 1: 1-12. . 1930. Uber wurzelbildende Substanzen bei Bryophyllum, calydnum Salisb., Zeitschr. Bot. (Festschr. Oltmanns), 23: 19-26. BIBLIOGRAPHY 245 — . 1932a. Over groeistoffen bij planten, Chem. Weekbl, 29 : 316-317. — . 19326. Pflanzenwachstum und Wuchsstoff (Auxin), Forschungen und Fortschritte, 8 : 371-372. — . 1933a. Die Bedeutung des Wuchsstoffes (Auxin) fur Wachstum, photo- und geotropische Krummungen, Naturwissensch., 21 : 1-7. . 19336. Recent progress in the study of growth substance (auxin) in plants, Rept. Brit. Assoc. Adv. Sci., 103: 555-556. Abstract in Nature, 133 : 452-453. 1933. . 1934. Hormone bei Pfianzen, Verh. Schweiz. Naturf. Ges. Zurich, 1934 : 220-240. 1935. The investigations on growth and tropisms carried on in the botanical laboratory of the university of Utrecht during the last decade, Biol. Rev. Cambridge Phil. Soc, 10: 187-207. and S. KosTYTSCHEW. 1931. "Lehrbuch der Pfianzenphysiologie," Bd. 2, "Wachstum und Bewegung," Julius Springer, Berhn. Went, F. W. 1926. Concerning the difference in sensibility of the tip and base of Avena to light, Proc. K. Akad. Weiensch. Amsterdam, 29: 185-191. . 1927. On growth-accelerating substances in the coleoptile of Avena sativa, Proc. K. Akad. Weiensch. Amsterdam, 30: 10-19. Dutch version in Versl. Gew. Vergad. Afd. Natuurk., 35: 723-732. . 1928a. Wuchsstoff und Wachstum, Rec. Trav. Bot. Neerl, 25: 1-116. . 19286. Die Erkljirung des phototropischen Kriimmungsver- laufs, Rec. Trav. Bot. Neerl., 25A: 483-489. . 1929. On a substance, causing root formation, Proc. K. Akad. Wetensch. Amsterdam, 32 : 35-39. . 1932. Eine botanische Polaritatstheorie, Jahrh. Wiss. Bot., 76: 528-557. . 1934a. A test method for rhizocaline, the root-forming substance, Proc. K. Akad. Wetensch. Amsterdam, 37 : 44^-455. . 19346. On the pea test method for auxin, the plant hormone, Proc. K. Akad. Wetensch. Amsterdam, 37 : 547-555. * . 1935a. Auxin, the plant growth-hormone, Bof. i?ey., 1: 162-182. 19356. Hormones, involved in root formation. The phenomenon of inhibition, Proc. Int. Bot. Congress Amsterdam, 2: 267-269. 1935c. Coleoptile growth as affected by auxin, aging and food, Proc. K. Akad. Wetensch. Amsterdam, 38 : 752-767. White, P. R. 1934. Potentially unlimited growth of excised tomato root tips in a liquid medium. Plant Physiol, 9: 585-600. WiECHULLA, O. 1932. Beitrage zur Kenntnis der Lichtwachstumsreaktion von Phycomyces, Beitr. Biol. PJkmzen (Cohn), 19: 371-419. WiESNER, J. 1881. "Das Bewegungsvermogen der Pflanzen." Holder. Vienna, 212 pp. WiLDiERS, E. 1901. Nouvelle substance indispensable au developpement de la levure. La Cellule, 18: 313-333. WiTSCH, H. VON. 1934. Untersuchungen liber die Beeinflussbarkeit des Plagiogeotropismus von Seitenwurzeln, Jahrh. Wiss. Bot., 79: 790-812. 246 GROWTH HORMONES IN PLANTS WoLK, P. C, VAN DER. 1911. Investigation on the transmission of light stimuli in the seedlings of Avena, Proc. K. Akad. Wetensch. Amsterdam, 14: 327-342. *Zeller, a. 1932. Wuchs-, Wund- und Reizstoffe bei Pflanzen, Schrift. Ver. Verbr. Naturw. Kenntn. Wien, 72 : 73-97. Zeltner, H. 1932. tJber Elektronastie und andere Reizbewegungen der Ranken, Zeitschr. Bot., 25: 97-172. Zimmerman, P. W., W. Crocker, and A. E. Hitchcock. 1933a. Initia- tion and stimulation of roots from exposure of plants to carbon monoxide gas, Contrb. Boyce Thompson Inst. Plant Res., 5: 1-17. , ^ and . 19336. The effect of carbon monoxide on plants, Contrb. Boyce Thompson Inst. Plant Res., 5: 195-211.. and A. E. Hitchcock. 1933. Initiation and stimulation of adven- titious roots caused by unsaturated hydrocarbon gases, Contrb. Boyce Thompson Inst. Plant Res., 5: 351-369. and . 1935. The response of roots to "root-forming" substances, Contrb. Boyce Thompson Inst. Plant Res., 7: 439-445. and F. Wilcoxon. 1935. Several chemical growth substances l^y' which cause initiation of roots and other responses in plants, Contr. Boyce Thompson Inst. Plant Res., 7: 209-229. *Zimmermann, W. 1927. Die Georeaktionen der Pflanze, Ergebnisse Biol., 2: 116-256. -. 1931. Die Orientierung von Pflanze und Tier im Raume. I. Botanischer Teil, Biol. Zentralbl., 51: 21-36. 1 932 . Orientierungsbewegungen der Pflanze und ihre Beziehungen zur Polaritatsanalyse, Mediz. Klinik, 28: 16-18. Zollikofer, C. 1921. tJber den Einfluss des Schwerereizes auf das Wachstum der Koleoptile von Avena sativa, Rec. Trav. Bot. Neerl., 18: 237-321. . 1926. Uber geotropische Krummungen von Paniceen-coleoptilen bei gehemmter Reizleitung, Planta, 2 : 10-18. . 1928. tiber Dorsiventralitatskriimmungen bei Keimlingen von Panicum und Sorghum und den Einfluss der Koleoptile auf das Meso- cotylwachstum, Rec. Trav. Bot. Neerl, 25A: 490-504. SUPPLEMENTARY BIBLIOGRAPHY Selected references pertaining to other hormones and similar substances affecting plant growth (bios, animal hormones, growth substance B, vita- mins, pantothenic acid, etc.); these are not discussed in the text. Almoslechner, E. 1933. tJber Hefewuchsstoffe in Boletus edulis und im Ham, Anz. Akad. Wiss. Wien Math.-Naturw. Kl., 70: 236-238. . 1934. Die Hefe als Indikator fiir Wuchsstoffe, Planta, 22: 515-542. Behrens, W. U. 1935. Die Wirkung von technischem Progynon auf den Pflanzenwuchs, Zeitschr. Pflanzenerndhr . Diing. u. Bodenk., 39: 140-145. Boas, F. 1934. Beitrage zur Wirkungsphysiologie einheimischer Pflanzen. I, Ber. Deut. Bot. Ges., 52: 126-131. ■ . 1935. Vergleichende Untersuchungen iiber Wachstumsanreger in einheimischen Pflanzen, Ber. Deut. Bot. Ges., 53: 495-511. BoTTOMLEY, W. B. 1914. Some accessory factors in plant growth and nutrition, Proc. Roy. Soc. London, Ser. B, 88 : 237-247. BtJNNiNG, E. 1934a. Die physiologische Bedeutung des Wachstums- regulators bei Aspergillus niger, Naturvxissensch, 22: 291. • . 19346. Wachstum und Stickstoffassimilation bei Aspergillus niger unter deni Einfluss von Wachstumsregulatoren und von Vitamin B, Ber. Deut. Bot. Ges., 52: 423-444. BuRGEFF, H. 1934. Pflanzliche Avitaminose und ihre Behebung durch Vitaminzufuhr, Ber. Deut. Bot. Ges., 52 : 384-390. BusTON, H. W., and S. Kasinathan. 1933. The accessor}- factor neces- sary for the growth of Nematospora gossypii. III. The preparation of concentrates of the second accessory factor, Biochem. Jour., 27: 1859-1868. and B. N. Pramanik. 1931rt. The accessory factor necessary for the growth of Nematospora gossypii. I. The chemical nature of the accessory factor, Biochem. Jour., 25: 1656-1670. and . 19316. The accessory factor necessary for the growth of Nematospora gossypii. II. The relation of the accessory factor to "Bios," Biochem. Jour., 25: 1671-1673. Chouard, p. 1934. Action de la folliculin et de thyroxine sur les Reine- Marguerite (Callistephanus sinensis, Nees = Aster sinen-sisL,.). Compt. Rend. Soc. Biol. Paris, 117: 1180-1183. Dagys, J. 1934. t'ber B-wuchsstoffe in embryonalen Geweben und in Blutungssafte, Anz. Akad. Wiss. Wien Math.-Naturw. Kl., 71: 251-255. . 1935. Wuchsstoffe der Mikroorganismen in embryonalen Ge- weben und im Blutungssafte, Diss. Univ. Gras., 91 pp.; also in Proto- plasma, 24: 14-91. Davis, E. E. 1934. Influence of thyroxin on the growth of plants, Plant Phy.nol, 9: 377-384. These references are not discussed in the text. 247 248 GROWTH HORMONES IN PLANTS Eastcott, E. V. 1928. Wildiers' Bios. The isolation and identification of "Bios I," Jour. Phys. Chem., 32: 1094-1111. Edwards, R. L. 1933. Some observations on the nature and properties of "Bios," Jour. Soc. Chem. Ind., 52: 271-272. EuLER, H. VON, D. BuRSTROM, and H. Larsson. 1934. Versuche iiber den Einfluss von Wuchsstoffen auf Gersten-Mutanten. II, Ark. Kemi, Min. Geol., 11B(4), No. 39, 5 pp. ^ , and M. Malmberg. 1934. Versuche liber den Einfluss von Wuchsstoffen auf Gersten-Mutanten. I, Ark. Kemi, Min. Geol., 11B(4), No. 38, 6 pp. , H. Hellstrom, and J. Hagen. 1934. tjber die aus Gersten- mutanten Albina 1 und 3 gewonnene Indolbase und ihre Umwandlung, Ark. Kemi, Min. Geol. 11B(4), No. 36, 6 pp. and T. Philipson. 1932. Wasserlosliche Wachstumsfaktoren, Biochem. Zeifschr., 245: 418-430. and B. Zondek. 1934. FoUiculin und Pflanzen. Versuche an Hyacinthen und an Hefe, Biochem. Zeitschr., 271 : 64-73. Flancowna, p. 1933. Einfluss von Koleoptilen-u. Radiculae-Wuchs- stoffen von Zea mays auf die Stickstoffbindung verschiedener Stamme von Azotobacter chroococcum Beij., Acta Soc. Bot. Polon., 10: 521-535. (German summary.) Florentin, p., and M. Ehrenfeld. 1935. Action de la thyroxine sur la croissance des racines d' Allium cepa, Compt. Rend. Soc. Biol. Paris, 118 : 1003-1005. Fric, a. V. 1934. Zur Hormon-Frage, Mailer's Deut. Gdrtn.-Zeit., 26 : 304. Gad-Andresen, K., and E. Jarl0v. 1934. Can sexual hormones influence the germination and growth of plants?. Act. Med. Scand., 84: 241-252. Hall, H. H., and L. H. James. 1933. Yeast growth-promoting substances in white sugars, Jour. Bact., 25: 67. , and , and L. S. Stuart. 1933. Yeast-growth stimulants in white sugars, Ind. Eng. Chem., 25: 1052-1054. Harder, R., and I. Stormer. 1934. tJber den Einfluss des Follikel- hormons auf das Bliihen von Pflanzen, Jahrb. Wiss. Bot., 80 : 1-19. Hartelius, V. 1933. tJber das Vorkommen von Wuchsstoff B, Biochem. Zeitschr., 261: 89-91. . 1933. The occurrence of growth substance B in urine, Compt. Rend. Trav. Lab. Carlsberg, 19(18): 1-20. Havaz, L., and J. Caldwell. 1935. Some experiments on the effects of animal hormones on plants, Ann. Bot., 49: 729-747. Hykes, O.-V. 1933. De I'influence de quelques hormones sur la feuillaison et le developpement des vegetaux, Compt. Rend. Soc. Biol. Paris, 113 : 629-632. Imshenetskogo, a. a. 1932. Influence of hormones on yeasts, molds and bacteria, Izv. Akad. Nauk SSSR (Bull. Acad. Sci. URSS), Ser. 7, No. 10: 1559-1578. (Russian with English summary.) Ito, S., and S. Shimada. 1931. On the nature of the growth-promoting substance excreted by the "Bakanae" fungus, Ann. Phytopath. Soc. Japan, 2 : 322-338. SUPPLEMENTARY BIBLIOGRAPHY 249 Janot, M.-M. 1934. Action des hormones cristallis^es fem^lles sur le developpement de quelques vegetaux, Compt. Rend. Acad. Sci. Paris, 198: 1175-1178. . 1935. Action de la folliculine et de I'equilenine sur le developpe- ment de la jacinthe, Compt. Rend. Acad. Sci. Paris, 200: 1238-1240. Knight, B. C. J. G., and P. Fildes. 1933. A vitamin necessary for the growth of B. sporogenes: its relation to auxin and other growth factors, British Jour. Exp. Path., 14: 112-124. KoGL, F. 1935. Uber Wuchsstoffe der Auxin- und der Bios-Gruppe. XIV. Mitteilung iiber pflanzliche Wachstumsstoffe, Ber. Deut. Chem. Ges., Abt. A, 68: 16-28. Lacroix, H. 1929. tJber den Einfluss wachstumsfordernder Substanzen vom Charakter des Vitamins D auf die Hefezelle, Zentr. Bakt., Para- sitenk. Abt. II, 76: 417-428. Lazar, O. 1935. L'influence du carotene sur la neoformation des racines et le developpement de la gemmule chez Impatient balsamina, L., Compt. Rend. Soc. Biol. Paris., 120: 1374-1376. See also pp. 799-804. Lepeschkin, W. W. 1924. The influence of \dtamins upon the develop- ment of yeasts and molds. (Contribution to the bios problem), Am£r. Journ. Bot., 11: 164-167. Li, T.-T., and T. Shen. 1934. The effect of "pantothenic acid" on the growth of the yeast and on the growth of the radical of Ginkgo embryo in artificial media, Sci. Rep. Nat. Tsinghua Univ., Ser. B, Biol. & Psychol. Sci., 2: 53-60. LoEWE, S., F. Lange, and E. Spohr. 1927. ITber weibliche Sexualhormone (thelytropine). XII. Mitteilung: Brunsterzeugende Stoffe (Thely- kinine) als Erzeugnisse des Pflanzenreiches, Biochem. Zeiischr., 180: 1-26. Mattei, G. E. 1931. (Morphogenesis of Ricinus communis subjected to the action of suprarenal cortex preparation) , Scritti Biol., 6 : 33-35. Ital. McBuRNEY, C. H., W. B. BoLLEN, and R. J. Williams. 1935. Panto- thenic acid and the nodule bacteria-legume symbiosis, Proc. Nat. Acad.