MARINE BIOLOGICAL LABORATORY. » » —— Received P.e.c.. X.7., 19.37 48912Accession No. /* Given by Mac mil. Ian Go Place, ^.^'''^...XPJ^ Q.X.ty *n* No book or pamphlet is to be removed from the Laboratory without the permission of the Trustees. ¥ ft 4.0^ PHYTOHORMONES EXPERIMENTAL BIOLOGY MONOGRAPHS Editors: Philip Bard, Johns Hopkins University; L. R. Blinks, Stanford University; W. B. Cannon, Harvard University; W. J. Crozier, Harvard University; J. B. Collip, McGill University; Hallowell Davis, Harvard University; S. R. Detwiler, Columbia University; Seliq Hecht, Columbia University; Hudson Hoagland, Clark University; J. H. Northrop, Rockefeller Institute for Medical Research; G. H. Parker, Harvard University; Gregory Pincds, Harvard University; L. J. Stadler, The University of Missouri; SewALL Wright, University of Chicago. PACEMAKERS IN RELATION TO ASPECTS OF BEHAVIOR. By Hudson Hoagland NEUROEMBRYOLOGY. By Samuel R. Det- wiler THE EGGS OF MAMMALS. By Gregory Pincus AUTONOMIC NEURO-EFFECTOR SYSTEMS. By Walter B. Cannon and Arturo Rosen- BLUETH PHYTOHORMONES. By F. W. Went and Kenneth V. Thimann Other volumes to follow PHYTOHORMONES BY F. W. WENT, Ph.D. Professor of Plant Physiology California Institute of Technology AND KENNETH V. THIMANN, Ph.D„ Assistant Professor of Plant Physiology Harvard University NEW YORK THE MACMILLAN COMPANY 1937 CoPTRiaHT, 1937, Bt the macmillan company ALL RIGHTS RESERVED NO PART OF THIS BOOK MAT BE REPRODUCED IN ANY FORM WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER, EXCEPT BY A REVIEWER WHO WISHES TO QUOTE BRIEF PASSAGES IN CONNECTION WITH A REVIEW WRITTEN FOR INCLUSION IN MAGAZINE OR NEWSPAPER Published July, 1937. SET UP AND BLBCTROTTPBD BT T. MORBT A 80N PRINTED IN THE UNITED STATES OF AMERICA Dedicated to the memory of F. A.F. C. Weiit and Herman E. Dolk Oats, peas, beans and barley grow, Oats, peas, beans and barley grow, Can you, or I, or anyone know How oats, peas, beans and barley grow? —Old English Nursery Rhyme ACKNOWLEDGMENT It is a pleasure to express our thanks to the many colleagues who have aided in one way or another the preparation of this monograph. Dr. J. van Overbeek and Mr. C. L. Schneider have carried out a number of special experiments; Dr. J. Bonner, Dr. J. van Overbeek, and Dr. A. J. Haagen Smit have made numerous valuable suggestions and criticisms of the manuscript; and Miss E. M. Wallace has spent much time in the preparation of the drawings. Finally a number of co-workers and students have been kind enough to allow the citation of their unpublished results. In many cases this has made possible the discussion of papers pubhshed later than the autumn of 1936, although we have made no attempt at completeness beyond this date. Thanks are due to Prof. F. Kogl for the samples of auxin and biotin used in some of the unpublished experiments cited here. Thanks are also due to the authors of a number of papers and the editors of the journals in which they appeared for permission to reproduce figures. The source of these is acknowledged below the figures in each case. F. W. Went Kenneth V. Thimann June, 1937 vu TABLE OF CONTENTS CHAPTER ^^°^ I. INTRODUCTION 1 A. Outline of the Book 1 B. Definitions 3 C. Previous Reviews of the Field 4 II. DEVELOPMENT OF THE HORMONE CONCEPT . . 6 A. Correlation and Formative Substances 6 B. Tropisms 9 C. Birth of the Growth Hormone Concept 11 D. Isolation of the Growth Hormone 13 E. Other Hormones in Higher Plants 17 III. THE TECHNIQUE OF AUXIN DETERMINATIONS . 21 A. Morphology of the Avena Seedling 21 B. Evolution of the Avena Test Method 24 C. The Avena ISIethod in Its Present Form 27 1. Dark Room and Equipment 27 2. Preparation of Test Plants 28 3. Preparation of the Agar 34 4. Technical Modifications 36 5. Evaluation of Results 38 6. Positive Curvatures 42 7. The Maximum Angle 45 8. Variability of the Test 49 D. Other Methods of Auxin Determination 51 1. Straight Growth 51 2. The Pea Test 54 3. Epinastic Responses 56 4. Other Methods 56 IV. FORMATION AND OCCURRENCE OF AUXINS . . 57 A. The Formation of Auxin in the Plant 57 B. The Auxin Precursor 64 C. Distribution of Auxin in the Plant 67 D. Auxin in Animal Material 70 E. Production of Auxin by Microorganisms 71 V. THE RELATION BETWEEN AUXIN AND GROWTH . 73 A. Auxin and the Growth of Coleoptiles 73 B. The Role of the Food Factor 79 C. The Limitation of Size in the Plant 80 D. Applications of the Two-Factor Scheme 81 E. Auxin Inactivation and Dwarf Growth 86 F. Radiation and Its Effects on Auxin 88 ix 4891^ X TABLE OF CONTENTS CHAPTER PAGE VI. AUXIN TRANSPORT AND POLARITY 90 A. Auxin Transport in General 90 B. Polarity in General 91 C. Polarity of Auxin Transport 93 D. Other Views on the Transport of Auxin 98 E. Possible Mechanisms of Auxin Transport 101 VII. THE CHEMISTRY OF THE AUXINS 105 A. Early Work 105 B. The Isolation of Auxins a and 6 106 C. Discovery of Indole-3-Acetic Acid as an Auxin . . . 110 D. Identity of the Native Plant Growth Hormone . . . 113 E. Activity of Compounds Related to Indole-Acetic Acid 114 VIIL THE MECHANISM OF THE ACTION 118 I. The Physiological Approach 119 A. Definitions 119 B. Nature of Growth 119 C. Effect of Auxin on the Cell Wall ...... 121 D. Structure of the Growing Cell Wall 124 E. The Intermediate Processes 127 F. The Effect of Acid on Growth 130 11. The Chemical Approach 132 G. Primary and Secondary Activity 132 H. The Relation between Structure and Activity . . . 136 IX. THE GROWTH OF ROOTS 141 A. The Effect of the Tip in Root Growth 141 B. The Effect of Auxin on Root Growth 142 C. Auxin Production in the Root 144 D. The Role of Auxin in the Growth of Roots .... 148 X. TROPISMS 151 I. Tropisms in General 151 A. Historical Introduction 152 B. The Cholodny-Went Theory 154 II. Geotropism 157 C. Geotropism of Shoots 157 D. Geotropism of Roots 161 E. The Mechanism of Geotropic Perception .... 164 HI. Phototropism 166 F. Auxin Redistribution 166 G. Light-Growth Reactions 171 H. Comparison of Phototropic and Geotropic Curvatures . 176 rV. Other Tropistic Responses 178 J. Traumatotropism 178 K. Electrotropism 179 L. Chemotropism 181 M. Nastic Movements 181 TABLE OF CONTENTS xi CHAPTER PAGE XI. ROOT FORMATION 183 A. Root Formation as a Correlation Phenomenon . . . 183 B. Hormones and Root Formation 185 C. Nature of the Root-Forming Substance 190 D. Effect of Light on Root Formation 192 E. Effects of Factors Other than Auxin 194 F. Root Formation on Other Organs 202 G. Practical Applications 204 XII. BUD INHIBITION 207 A. Bud Inhibition as a Correlation Phenomenon .... 207 B. Bud Inhibition Caused by Auxin 209 C. Possible Mechanisms of Bud Inhibition 213 XIII. OTHER ACTIVITIES OF AUXINS 217 A. Auxin and Cell Division 217 B. Cambial Growth 218 C. Callus and Stem Swellings 222 D. Miscellaneous Effects 227 XW. GENERAL CONCLUSIONS 230 A. Quantitative Relations between Auxin and Growth . 231 B. Comparison with Animal Hormones 232 C. Comparison with Growth Substances of Lower Plants . 235 D. Regeneration 238 E. Organization 242 F. The Stimulus Concept and the Nature of Auxin Action . 244 G. Abnormal Growth 247 H. Outlook 248 BIBLIOGRAPHY 251 AUTHOR INDEX 285 SUBJECT INDEX 289 ujlLliRARY ^ PHYTOHORMONES ^^^^i/ m VV CHAPTER I INTRODUCTION The field of plant hormones is perhaps now at the stage of its most rapid development. The number of facts is becoming so large, and their distribution through the literature so scattered, that there is a danger of losing sight of the general trend. We shall attempt not so much to give a detailed historical account as, rather, to present the field from the point of view of workers in it. Where matters of hypothesis are concerned, our personal views will necessarily be emphasized, but opposing views will be given an opportunity for the reader's consideration. In matters of fact, as also in regard to credit and priority, every attempt will be made to give as fair and accurate an account as possible, both of the experiments and of the concepts of the different workers. For the sake of completeness many new experiments have been included. These are designated as u (unpublished). Some idea of the amount of work which has been done in this field may be gained from the statement that the contents of this book are based on actual measurements of the responses of about one million plants. The growing interest in the field is exemplified by the fact that the bibliography includes references to 77 pubhcations dated 1936. Since equally detailed and critical treatment cannot be accorded to all of this material, we have naturally laid emphasis on w^hat appears to be the most important work. A. Outline of the Book Our re\dew will deal only with the hormones of higher plants. We shall first trace the development of the leading 2 PHYTOHORMONES idea that correlations in plants are due to the influence of special substances (Chapter II), ^ We shall try to show how experiments along four different and apparently unrelated lines, —correlation proper, organ formation, tropisms, and normal growth,—have gradually come together and been unified into a complete picture of hormone activity as we now know it. Next we shall consider the methods for the assay of these substances, treating them in sufficient detail for experimental use (Chapter III). Since most of these methods are founded upon cell elongation, and all other work has had its foundation thereon, it is natural to consider cell elongation in some detail first. The best demonstration of the effectiveness of these assay methods has been the working out of the chemical nature of the active substances (Chapter VII). The success of the chemical attack has made it possible for all the experiments described subsequently to be checked by use of the pure compounds ; this has had the effect of making the conclusions clear-cut and has avoided the difficulty of working with unknown extracts and mixtures. Parallel and simultaneous with work on the above lines, the role of the active substances in various aspects of plant growth has been elucidated (Chapter V), beginning with their formation (Chapter IV) and movement in plant tissue (Chapter VI). The latter phenomenon is of special interest, firstly because it offers an example of a naturally occurring substance whose movement can be followed quantitatively throughout the plant, and secondly because of the causal relation between the polarity of this transport and the wellknown polarity of plant structures. One of the most interesting aspects of the subject has been the attempt to analyze the various reactions interv^ening between the auxin and its final effect, —growth (Chapter VIII). In this, knowledge of the chemical nature of the substances has played an essential part. In close connection with problems of cell elonga- 1 References such as VIII G or III C refer to chapter (Roman numbers) and section (letters). INTRODUCTION 3 tion and transport come the tropisms, particularly the reactions of plants to gra\dty and light, insofar as they are caused by unequal growth (Chapter X). Further, these same active substances play an important role in a number of other correlations in plants, particularly in the formation of roots (Chapter XI), in bud inhibition (Chapter XII), and in the stimulation of cambial activity (Chapter XIII). There are also a number of other phenomena not so well understood. Finally the findings and general conclusions will be compared with those from other fields, bringing the work on plant hormones into the realm of general physiology (Chapter XIV). B. Definitions The definition of hormone which we propose to apply is this: a hormone is a substance which, being produced in any one part of the organism, is transferred to another part and there influences a specific physiological process. This is essentially the definition of Bayliss and Starling (1904): "the peculiarity of these substances (hormones) is that they are produced in one organ and carried by the blood current to another organ, on which their effect is manifested" (BayHss, 1927, p. 712). There is, of course, no blood stream in plants, but, as Bayliss emphasizes, ''these hormones are characterized by the property of serving as chemical messengers, by which the acti\'ity of certain organs is coordinated with that of others." ^ There is no strict necessity for the production of the hormone in speciaHzed organs, since even if all the cells of the plant should produce it, the phenomenon of polarity would bring about its specific distribution. In general, however, the points of production and response are spatially separated. To avoid the possibility of confusion with animal mechanisms the term phytohormones has been introduced for * The conception of hormones has recently been somewhat broadened (see Huxley, 1935). 4 PHYTOHORMONES such substances in plants. However, since in this book we shall deal only with the plant kingdom, the prefix can suitably be dropped. Thus our conception of hormone is essentially that of a correlation carrier, where correlation (as used in regard to plants) is defined as the influence exerted by one part of the plant upon another,—not in the sense of statistical correlation, but in the sense of causal relationship. In the beginning of the work in this field, the noncommittal terms growth substance, Wuchsstoff, growth regulator, and growth hormone were used, but as our knowledge developed, it became clear that the substances causing cell elongation must be regarded as a separate group. Since recent work indicates that this group is heterogeneous, the term auxins, first suggested by Kogl and Haagen Smit (1931), will be arbitrarily restricted to those substances which bring about the specific growth reaction which is conveniently measurable by the curvature of Avena coleoptiles. Whenever used in the physiological sense, the terms growth substance (g.s.) and growth hormone will be used throughout this book in the sense of auxins. The term Wuchsstoff in particular has been used for some of the growth substances of lower plants, such as Bios, but it cannot be too strongly emphasized that only those substances whose activity is determined on higher plants, preferably by the standard methods which are described in Chapter III of this book, can be termed auxins. C. Previous Reviews of the Field The rapid development of the field has resulted, as would be expected, in the publication of a number of reviews and summaries. Such reviews have rarely more than temporary interest, and many of these are already only of historical value (Babicka, 1934; Cholodny, 1935a; Kogl, 1932, 1932a, 1933, 1933a, 19336, 1933c; Laibach, 1934; Loewe, 1933; Malowan, 1934; Pisek, 1929; Snow, 1932; Soding, 1927, 1932; F. A. F. C. Went, 1927, 1930, 1931, 1932, 1932a, 19326, 1933, 1933a). INTRODUCTION 5 The newer and more extensive reviews are those of du Buy and Nuernbergk (1932, 1934, 1935), Erxleben (1935), von Guttenberg (1932, 1933, 1934, 1935, 1936), Heyn (1936), Jost (1935), Kogl (1935, 1935a, 1936), Haagen Smit (1935), Stiles (1935), Thimann (1935, 19366), Went i (19356, 1936a), and F. A. F. C. Went ^ (1934, 1935). The most complete account which has appeared up to now is that of Boysen Jensen (1935, translated and extended by Avery and Burkholder, 1936). We do not feel, however, that Boysen Jensen's publication makes our book superfluous, for several reasons. In the first place, it does not attempt to do more than review the past work, while in this book our aim is rather to analyze and integrate the material. In the second place, its scope is restricted largely to the role of hormones in growth and tropisms, while, as was stated above, the field has recently developed in quite different directions, which necessitates a revision and broadening of our ideas. Lastly, it appears from book reviews (Soding, 19356; Umrath, 1935) that one of the principal impressions which the book has made is in regard to the question of priority in the discovery of the auxins. We feel that the gradual unfolding of the current conceptions and the cooperation of different workers has made it impossible to credit any one person with such a discovery, and it is to be hoped that the reader of this book will gain the impression of a steady and collective advance rather than of individual contributions. > References to Went (without initials) refer to F. W. Went, the papers of F. A. F. C. Went being cited with initials. CHAPTER II DEVELOPMENT OF THE HORMONE CONCEPT A. Correlation and Formative Substances The idea that the phenomenon of correlation is brought about by substances or ''saps" is by no means new. No detailed consideration need be given to the very vague idea of Malpighi (1675) nor to the artistic conceptions of Agricola (1716) of a "materia ad radices promovendas." Careful experiments, however, were carried out by Duhamel du Monceau (1758), whose sound scientific reasoning led him to conceive of correlation as brought about by two saps, one moving downward, the other upward. The former was elaborated in the leaves and, after passing downward through the Fig. 1. The first published drawings of corcortex, waS USed for the relations in plants. Swellings occur above but -. •-• f +V, not below ring wounds, and in isolated pieces nutrition 01 tne rootS. of bark they are most marked when leaves or Jf however this downbuds are present. (From Duhamel du ' , . ' x^^^^ Monceau, 1758.) ^^^^ Stream were niLei cepted by ringing or other means, it caused the swellings, callus, and root formation which he observed above the point of interception (see 6 DEVELOPMENT OF THE HORMONE CONCEPT 7 Figure 1). So much stress was laid on the root formation that the sweUings and callus were considered as ''being much of the nature of roots" (Bk. IV, Ch. V). During the next hundred years the physiological concept of correlation seems to have been lost, the emphasis being placed on morphology, i.e. upon the inherent nature of the tissues themselves. The discovery of sieve tubes by Hartig and von Mohl led them to the opinion that there was indeed a downward-moving sap, and this was later proven by Hanstein (1860). The content of this sap, however, was studied from the \aewpoint of organic food materials rather than that of its correlating functions. At this time began the period of rapid development in plant physiology. The phenomenon of correlation was studied in greater detail and Sachs (1880, 1882, 1893) brought forward a complete theory, a modern version of Duhamel's views, which covered most of the known facts of morphogenesis and correlation, and can still be regarded as a modern treatment of the subject. Sachs' great achievement was that he applied the laws of causality to morphology. His starting point was the thesis that ''morphological differences between plant organs are due to corresponding differences in their material composition, which must be already present at the time of initiation, even though at this stage chemical reactions and other crude methods fail to show any differences." To account for these differences he assumed the existence of root-forming, flower-forming, and other substances, which move in different directions through the plant. For example, the former would be formed in leaves, and would move towards the base of stems. If a cut be made in a twig, this will be "an obstacle for further downward movement," and roots will be formed above the cut. Light and gravity were assumed to affect the distribution of these special substances. With only two assumptions: 1, the existence of organ-forming substances which, in minute amounts, direct development, and 2, polar distribution of these substances,—a distribution which may be modified by 8 PHYTOHORMONES external forces such as light and gravity,—correlations, normal development, galls and monstrosities were brought into one picture. In his very remarkable publications on galls Beijerinck (1888, 1897) elaborates the idea of ''growth-enzymes." Fig. 2. a, b, c, gall of Nematus capreae on leaf of Salix. a, egg deposited with some mucilage in mesophyll of young leaf; b, mature gall before hatching of larva; c, gall in which Ijy accident no egg has been deposited. The mucilage excreted by the gall-wasp has caused formation of an almost complete gall. This excretion is the first published example of an organ-forming substance. (From Beijerinck, 1888.) d, gall of Cecidomyia Poae on stem of Poa, showing excessive root formation. (From Beijerinck, 1885.) While he originally thought (1886) that ''it can not be doubted that nutritive stimuli must be considered as the primary cause" of root formation when parts of plants are cut off from the parent-plant, he afterwards modified his views in the direction of those of Sachs. In the case of the Capreae-gsdl on Salix (Beijerinck, 1888) he considers the development of the gall (see Figure 2) to be caused by "a protein, whose action differs from that of ordinary proteins, which only form an equivalent amount of protoplasm, and resembles that of an enzyme, whose effect is quantitatively of a different order of magnitude from the amount of active material." Thus we have to do here with a "material DEVELOPMENT OF THE HORMONE CONCEPT 9 stimulus" (or as we should say now, a stimulating material) (1888, p. 132). Beijerinck designates this protein as a "growth enzyme." Later (1897) he extended this \'iew to the development of organisms in general, and stated that "form is determined by liquid substances, which move freely through considerable numbers of cells in growing tissues" (1897, p. 203). Except for these observations of Beijerinck no direct evidence for the existence of such special substances as Sachs had postulated was obtained for nearly 40 years. On the other hand, at about the same period, the existence of polarity in correlation phenomena was proven, both for whole organs, and for each separate cell of a transplant, by Vochting (1878, 1884, 1892, 1908). His work, however, is primarily concerned with inherent morphological polarity, rather than with its physiological basis. After the time of Sachs and Vochting most of the studies on correlation laid emphasis on nutritional factors. Goebel (1908), for instance, in discussing quantitative correlations, says "of the numerous organ initials, many remain undeveloped because the building materials, which they need for their development, go to others which can ' attract ' these materials more powerfully." Similar \'iews, invoh-ing also the nitrogen content of the plant, i.e. "the carbon: nitrogen ratio," have been generally held by American workers (c/. e.g. Kraus and Kraybill, 1918). While their experiments show that there is a parallelism between a given carbon : nitrogen ratio and a given type of growth, no causal relation has been shown to exist. B. Tropisms About 1880 it began to be realized that tropisms were to be regarded as a special kind of correlation phenomenon. This aspect of tropisms was particularly emphasized by C. Darwin (1880). Both for roots and shoots he was able to show that the effects of light and gra\'ity are perceived by the tip, and that the stimulus is transmitted to the lower 10 PHYTOHORMONES regions, which then react. "We must, therefore, conclude 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 " (p. 474). In regard to geotropism of roots, he concludes ''that it is the tip alone which is acted on, and that this part transmits some influence to the adjoining parts, causing them to curve downwards" (p. 545). At first Darwin's statements met with much opposition, but Rothert (1894), working with phototropism of shoots, confirmed completely the separation between the zones which perceive and those which react. The connection between these processes was envisaged by Fitting (1907) as being due to a polarity set up by the light stimulus, which "spread out" from cell to cell. Fitting's work was closely followed by the experiments of Boysen Jensen (1910, 1911, 1913) which showed that a Fig. 3. First experimental demonstration of transmission of the phototropic stimulus across a wound gap. Five Avena coleoptiles were decapitated, and the tips replaced upon the three plants to the left, the wound being covered with cocoa-butter. Two plants to the right as controls. On illumination of the tips only, from the left, the plants with tips replaced show curvature in the base, the controls not. (From Boysen Jensen, Ber. d. hot. Ges. 3/ : 559-566, 1913; and Growth Hormones in Plants, tr. Avery and Burkholder, McGraw-Hill, 1936.) phototropic stimulus can be transmitted across a wound gap. Boysen Jensen cut off the tips of Avena coleoptiles and stuck them on again with gelatin (see Figure 3). He then illuminated the tip only and showed that curvature appeared not only in the tip but also in the base. From this he concluded that "the transmission of the irritation is of a material nature produced by concentration changes in the coleop- DEVELOPMENT OF THE HORMONE CONCEPT 11 tile tip." He was led to assume such concentration changes by comparison with Lehman's model of a nerve, in which the electromotive changes of a chain of zinc sulfate concentration cells were compared to the transmission of stimulus. The "material nature" of the transmission seemed not to be purely physical, but "on the other hand, various considerations make one think that the transmission is of a chemical nature." Under the influence of PfefTer and of the application of zoological concepts, it is clear that Boysen Jensen visualized the transmission of the phototropic stimulus as a complex chain of reactions. This is exemplified by his interpretation of it in terms of the transmission of irritation in a nerve. He assumes that the light causes a differentiation between the bright and dark sides of the coleoptile tip; this constitutes an "irritation" of the dark side, and this irritation "leaves the dark side of the tip to travel down the dark side of the coleoptile." Finally, this irritation "sets free," in the lower part of the coleoptile, an acceleration of growth. This separation into perception, transmission, and reaction made it impossible for him to conceive that all three were reahzed through the same agency. While his experiments prove that the transmission of the irritation is a transmission "of substance or of ions" he did not postulate that this was a special, growth-promoting substance, and it was left to Paal to show that the material nature of the transmission was due to a special substance which is active in promoting normal growth. It will be clear to the reader that the above analysis does not at all support Boysen Jensen's claim (1935, 1936) that "the existence of a growth substance in the Avena coleoptile during phototropic curvature was demonstrated" by his experiments. C. Birth of the Growth Hormone Concept Because of the importance of Boysen Jensen's experiment, it was repeated and extended by Paal (1914, 1919), who, after excluding the possibilities of the base being influ- 12 PHYTOHORMONES enced by scattered light, by contact stimulus, or by the asymmetrical weight of the bending tip, confirmed Boysen Jensen's finding. Varying the conditions of the experiment, he showed that the stimulus could cross a layer of gelatin, but not cocoa-butter, mica, or platinum foil. His next important step was to show that, even without light, curvatures could be induced in the base by the simple process of cutting off the tip and replacing it on one side of the stump. This makes it clear that ''the tip is the seat of a growthregulating center. In it a substance (or a mixture) is formed and internally secreted, and this substance, equally distributed over all sides, moves downwards through the living tissue. In the growing zone it causes symmetrical growth. If the movement of this correlation carrier is disturbed on one side, a growth decrease on that, side results, giving rise to a curvature of the organ" (Paal, 1919). This puts the whole problem on a new basis, namely the control of normal growth by a diffusible correlation carrier. Here, for the first time, the idea of a groivth hormone enters botanical literature. Paal then suggested that "this correlation carrier, which, under normal conditions continually moves downwards from the tip along all sides, is, upon illumination of the tip, either interfered with in its formation, photochemically inactivated, or inhibited in its downward movement, through some change in the protoplasm, these effects being greater on the lighted side." Paal thus established the theory that the growth of the coleoptile is controlled by the tip through the agency of a diffusible substance, and this was confirmed by the careful growth measurements of Soding (1923, 1925). Working not with curvatures but with straight growth, Soding proved that replacement of the cut tip would restore the greater part of the growth reduction which is caused by decapitation. Further, the success of Paal's work led to a search for a direct demonstration of the postulated growth-promoting substance. Stark (1921) introduced the method of applying small blocks of agar on one side of decapitated coleop- DEVELOPMENT OF THE HORMONE CONCEPT 13 tiles, the agar being mixed with various tissue extracts, but in no case did any of these promote growth, only inhibitions being observed. The same failure to extract the growth-promoting substance from coleoptile tips was experienced by Nielsen (1924) and by Seubert (1925). However, Seubert was able to prove that agar containing sali\'a, diastase, and malt extract caused a promotion of growth. This was the first evidence that growth-promoting substances exist outside the plant. Stark (1921), Stark and Drechsel (1922), Gradmann (1925), and Brauner (1922) attempted to explain tropisms in terms of special stimulus substances, or ''Tropohormones," but with little success, and shortly afterwards Cholodny (1924, 1926, 1927) and Went (1928) developed the view of Paal and attributed all tropisms to asymmetric distribution of the normal growth-promoting substance {cf. X B). D. Isolation of the Growth Hormone Success in obtaining the active substance from the coleoptile tip was finally achieved by Went (1926, 1928 ^). Using the findings of Paal and adapting the method of Stark (see III B), he placed coleoptile tips upon blocks of agar, and then placed the agar on one side of the stumps of decapitated coleoptiles. The result was a curvature away from the agar block (negative curvature). His fundamental improvement in technique was to measure this curvature, which was proven to be proportional, within limits, to the concentration of the active substance. This test, the '^ Avena test," was then used to determine some of the properties of the substance, which was shown to be thermo- and photostable, as well as readily diffusible. From the diffusion rate its approximate molecular weight was determined. Went interpreted the normal growth of the coleoptile in terms of the action of this growth substance in conjunction with other limiting factors. For the asymmetric growth involved > Published November, 1927. 14 PHYTOHORMONES in phototropism he obtained evidence that an asymmetric distribution of the growth substance occurs, this being the cause of the curvature. In recent years a large literature on this subject has developed. The later work, which will constitute the body of this book, need only be briefly referred to here. First of all must be mentioned the chemical investigation of the active substances, auxins, which, mainly in the skillful hands of Kogl, Haagen Smit, and Erxleben, led rapidly to the isolation in pure form of three highly active substances (this work will be discussed in Chapter VII). The occurrence and distribution of the auxins has been investigated by Soding, Thimann, Laibach, and others. In this connection the fact that the auxins have no specificity of action was first demonstrated by Cholodny, and confirmed abundantly by Nielsen, Soding, Uyldert, and others. The investigations of van der Weij have brought light to many remarkable facts concerning the transport of auxins in the coleoptile, particularly the strict polarity of its movement. Since the approach to knowledge of the auxins was through tropisms, it is natural that one of the main applications of the acquired knowledge of these substances should be in the explanation of tropisms. The investigations of Dolk, Cholodny, and Dijkman have shown that asymmetrical distribution of auxin under the influence of gravity quantitatively accounts for geotropic curvature. This rules out any need for assuming the action of "tropohormones." In phototropism the situation is somewhat more complicated, but the main Hues have been elucidated by Cholodny, Boysen Jensen, du Buy, and van Overbeek. In roots, the studies of Cholodny, Snow, Boysen Jensen, and others have made it clear that the effect of auxin is to inhibit, not to promote, elongation, and this makes it possible to explain geotropic behavior of roots along the same hues. Many attempts have been made to elucidate the mechanism of the growth-promoting action. Heyn and Soding succeeded in showing that one of the ultimate effects of DEVELOPMENT OF THE HORAIOXE CONCEPT 15 auxin is a change in the properties of the cell wall, which allows extension to take place. The intermediate stages between the entry of auxin and its final effect have been investigated by Thimann and Bonner. Following the discovery of the growth-promoting activity of the auxins, it was foimd that many well-known correlations in organ development are brought about by the same substances. As mentioned above, Sachs assumed that root formation is due to a special root-forming substance. Experimental e\4dence to support this \dew was brought forward by several investigators, particularly by van der Lek. Proof that a special substance is indeed concerned was obtained by Went and Bouillenne, and the isolation of this active substance by Thimann and Went led to its identification with the auxins, an identity which was independently confirmed by Laibach and others. Another phenomenon long known as a typical correlation is the inhibitory effect of the terminal bud of a shoot on the development of lateral buds (Goebel, Dostal, Reed, Snow); this effect was shown by Thimann and Skoog to be due to auxins, produced in the growing bud. Still other important correlative phenomena, such as cambial growth, swelling of the ovary of orchids after polHnation, petiole abscission, and callus formation have since been shown to be brought about by auxins (Snow, Laibach). The interrelations between the fields of organ formation, correlation, tropisms, and growth have been summarized in the chart. Table I, which shows how these four lines of approach have gradually come together, and how each investigator's contribution has been merged into a more and more complete system. Exceptions must be made for Beijerinck and Loeb, whose work, although of importance, had little influence on the development of this field. The soundness of their views, however, must now be acknowledged. The placing of an investigator's name between two vertical lines indicates that his work linked up these two lines ; thus Boysen Jensen showed that tropisms were a correlation 16 PHYTOHORMONES TABLE I Shows the Development of Four Apparently Independent Lines op Plant Physiology, and Their Gradual Synthesis through the Discovery OP THE Functions op the Auxins. (Heavy arrows indicate direct relationship between one piece of work and another, dotted arrows a PARTIAL relationship OF SOME KINd) Organ formation V I I Correlalion Vochting I (Klebs) (Goebel) I DuHamel Sachs \ Beijerinck I I I I Vochting I I I Tropisms Sachs i Growth Sachs Loeb^ Dostal K /Boysen Jensen Darwin \ Rothert I Pfeffer I Fitting Blaauw Paal \ Stkrk Soding T Cholodny 1 Went effer 1758 1870 1880 1890 1900 1910 1920 1925 )1UU 1 Vei I 1930 Kogl and Haagen Smit Bouillenne and Went \AThimann and Skoog /Thimann and Went I Recent developments in the auxin field 1936 DEVELOPMENT OF THE HORMONE CONCEPT 17 phenomenon, and Blaauw that they were a growth phenomenon. The vertical hne beginning with Paal is intended to be symmetrically placed between the three lines of correlation, tropisms, and growth. It goes without saying that such a chart is to a considerable extent arbitrary in its selection, and large numbers of valuable studies are of necessity omitted for one reason or another. An important point brought out by the chart is the true internationalism of these researches, the 27 investigators listed belonging to 10 nationalities. E. Other Hormones in Higher Plants The first investigator actually to work with hormones and active extracts in plants was Fitting (1909, 1910). He also was the first to introduce the word hormone into plant physiology, considering hormones merely as stimulative substances, without stressing the importance of their transport. He was able to show that the sweUing of the ovary, and other phenomena of post-floration in tropical orchids, are due to an active substance which could be extracted from the pollinia. As mentioned above, Laibach was able to identify this substance (with a high degree of probability) with auxin. An effect of perhaps similar nature has been shown by Swingle (1928) to be exerted in the development of the date fruit by the embryo ("meta-xenia"). Evidence for other types of hormones was obtained by Haberlandt and his pupils in a series of investigations, principally on cell division. Their fundamental experiment was the demonstration (Haberlandt, 1913) that in small pieces of the parenchymatous tissue of the potato tuber, cell division only occurs in the presence of a fragment of a vein containing phloem tissue. This influence was also shown to be exerted on the parenchjTna if the phloem was separated from it by a thin film of agar. In addition Haberlandt was able to show that cell division is promoted by the presence of crushed cells (1914). 18 PHYTOHORMONES In experiments with leaves of succulents and kohl-rabi, Haberlandt (1914, 1921) and Lamprecht (1918) also showed that cell divisions could be induced by spreading upon the cut surface some of the crushed tissue of other leaves. Haberlandt concluded (1913) that cell division was determined by two substances, one coming from the wound ("woundhormone"), and the other from the phloem tissue ('^leptohormone"). The latter was assumed to be present to a lesser extent in some other tissues. Subsequent investigators have tried to extract these hormones and to identify them with known substances (Reiche, 1924; Wehnelt, 1927). Their methods of testing were not specific enough to allow the drawing of any definite conclusions, but Jost (1935a) has confirmed and extended some of their results (c/. XIII A), and Umrath and Soltys (1936) have made a partial purification (see below) . Further, Bonner (1936), using Wehnelt's test not as a measure of cell division but simply of growth, has demonstrated that tissue extracts contain a growth-promoting factor not identical with auxin. By the addition of alcohol extracts of tissues to a culture medium he has obtained growth of undifferentiated parenchyma cells in vitro, which closely approached true plant tissue cultures. The experiments of Jacques Loeb (1917-1924) on correlations in Bryophyllum occupy a very isolated place in the literature. He studied regeneration, bud growth, and geotropic behavior in isolated leaves and stems. At first his explanations were based upon Sachs' ideas, as for instance: ''There must, therefore, be associated with the material which causes geotropic bending also something which favors the growth of roots, and this may be one of the hypothetical substances of Sachs" (1917a, p. 118). Subsequently he transferred the emphasis to more general factors of nutrition, controlled by simple mass action, as for instance: "These facts show that the regeneration of an isolated leaf of Bryophyllum is determined by the mass of material available or formed in the leaf during the experiment" (1923, DEVELOPMENT OF THE HORMONE CONCEPT 19 p. 852). The same views were developed with respect to geotropism and bud inhibition. The greater part of his results fit in very well with our present knowledge of the role of auxins and can be explained on that basis. The somewhat similar experiments of Appleman (1918) on the development of shoots in pieces of potato tubers led him to postulate both growth-promoting substances, present in the tuber, and growth-inhibiting substances formed in the growing buds. Another interesting case of correlation by the transmission of a substance was discovered by Ricca (1916). He found that the well-known transmission of excitation in the sensitive plant. Mimosa, takes place in the vessels by means of a substance, secreted into them upon stimulation and carried principally with the transpiration stream. Snow (1924, 1925) afterwards found that there are three types of stimulus involved, only one of which is due to a diffusible substance. Fitting (1930) investigated the action of pure substances of biological interest in stimulating Mimosa and found a number of compounds, especially a-aminoacids and anthraquinone derivatives, to be active. Their action, however, is not identical with that of the Mimosa leaf extract. The active substance in the leaf extract itself was shown to be heat-stable by Umrath (1927) and attempts to purify it have been made by Fitting (1936a) and by Soltys and Umrath (1936). The latter workers have succeeded in obtaining a highly purified extract, from the properties of w^hich they conclude that the active substance must be an oxy-acid with a molecular weight of about 500. They have also partly purified a similar oxy-acid active on the leaves of certain sensitive Papilionaceae (Umrath and Soltys, 1936), and this, it appears, may be identical with the substance active in the Wehnelt test. Although their hormonal nature in the strict sense is questionable, mention must be made here of those substances causing the onset of protoplasmic streaming in Vallisneria leaf cells. In the first place Fitting has shown 20 .PHYTOHORMONES that, just as is the case with auxins, a number of different substances can bring about the effect (Fitting, 1927, 1929, 1932, 1933, 19366). In the second place, as with the activity of auxins on Avena, some of them are active in extremely high dilution, and the activity of different substances varies widely. Thus, N-methyl-^histidine stimulates streaming at 1 part in 10^, or about 10^^ mol.; aspartic acid, asparagine, histidine, and some other amino-acids are active down to 10-^ mol.; alanine, serine, and phenyl-alanine down to 10~^mol., while glycine, proline, and glutamine have very low activity. Some, e.g. tryptophane, are completely inactive. A number of non-nitrogenous acids, including galacturonic acid, are active down to 10^^ to 10"^ mol. In the aminoacids activity decreases with increasing distance between the carboxyl and amino-groups. Neither peptides nor amines have much activity, so that, presumably, aminogroups only confer activity if they are accompanied by an acid radical. Small changes in the molecule greatly affect the activity; thus, N-methyl-/-histidine is five times as active as ^histidine itself. These differences may, of course, be caused by different rates of penetration into the cells, and not by true differences in ''sensitivity" of the protoplasm for these compounds, as is probably the case for the different activity of auxins (see VIII G). CHAPTER III THE TECHNIQUE OF AUXIN DETERMINATIONS Practically all of the facts known about plant hormones and most of the theoretical conclusions are based upon the Avena test, which in itself again is based upon growth in length of the Avena coleoptile. It will be well, therefore, to begin by considering this object. A. Morphology of the Avena Seedling Upon germination of the seed the primary root begins to grow out and is followed, within one day, by two lateral roots. jVIeanwhile the shoot also starts to elongate. It consists of the growing point, a very short stem with two partially coieoptiie — developed leaves, and a p^in^^ry leaf surrounding sheath, the s,mg„u^_ coleoptile (see Figure 4). Between the coleoptilar node and the insertion of Mesocotyi the SCUtellum there de- secondary root velops an internode, coleorhi Endosperm jza generally called the ' ' meso- Primary root — cotyl " or in older literature ^ ^ _ ., ,. , ^. ,, , , , Fig. 4. Longitudinal section through "hypocotyl, whose length ^^.g^ seedling after 30 hours' germinais dependent upon the tion, at the beginning of the period of ^ ,, 11 • rapid elongation. treatment of the seedlmg. If the coleoptile be considered as one cotyledon and the SCUtellum as the other, then this internode is truly the mesocotyi (= between cotyledons). If, on the other hand, the coleoptile be considered as the first true leaf, then this mesocotyi is simply the first internode (or epicotyl) (see 21 22 PHYTOHORMONES Avery, 1930, and Boyd and Avery, 1936). Since the term mesocotyl has been generally adopted in physiological literature, we shall retain its use. The growth of the coleoptile, which is a hollow cylinder with a solid conical top, takes place almost entirely in the Fig. 5. Cross section through Avena coleoptile at 5 mm. distance from the tip. The dorsal side faces the seed. longitudinal dimension. In its early growth, up to a length of 1 cm., cell divisions of the parenchyma accompany the elongation (Avery and Burkholder, 1936). The epidermal cells, however, cease dividing at a very early stage and grow only by extension. From a length of 1 cm. up to its final length (5-7 cm.), cell divisions are practically absent (see also Tetley and Priestley, 1927) and growth is entirely by cell elongation. On this account the coleoptile is a particularly suitable object for studies of growth uncomplicated by cell division, and whatever conclusions are drawn from it apply only to cell elongation. In transverse section, the coleoptile is elliptical, with the short axis in the plane of symmetry of the seedling. Two small vascular bundles run up on either side (see Figure 5). The cells at the tip of the coleoptile are morphologically distinguishable from the others by the fact that they do not elongate and are almost isodiametric (see Figure 6). The epidermal cells of the extreme tip stain somewhat more TECHNIQUE OF AUXIN DETERMINATIONS 23 —Pore heavily than the rest and are presumably richer in protoplasm. The region of isodiametric cells is hmited to the uppermost 0.5 mm. of the length of the coleoptile (du Buy and Nuernbergk, 1932). The growth of the primary leaf closely follows that of the coleoptile, so that under normal conditions the coleoptile is almost completely filled. At a length of 50-60 mm. the coleoptile is broken through, near the tip, by this leaf, which then starts to grow very rapidly (see Figure 27). At this time the growth of the cole- , ^ig. 6 Longitudinal section through mature . Arena coleoptile. Left, tip; right, middle zone, optile ceases. The which has greatly elongated. (From Avery whole period of growth ^"^ Burkholder, Bull. Torrey Bot. Club 63: lasts about 100 hours at 25° C, and the maximum growth rate, which is reached at an age of 70 hours, is approximately 1 mm. per hour (Figure 27). This mode of growth, beginning slowly, rising to a maximum and then decreasing again to zero, has been called, following Sachs, the ''grand period." Coleoptiles 25-35 mm. in length, which are growing at the maximum rate, are generally used for experimental purposes. During the growth of the coleoptile from about 20 mm. on, the uppermost 2 mm. scarcely elongate at all. The zone of maximum growth is first located near the base and migrates upwards so as to remain about 10 mm. below the tip. At about 40 mm. length, the growth of the most basal zones ceases altogether (see V A). 24 PHYTOHORMONES B. Evolution of the Avena Test Method By placing the cut tip asymmetrically on the stump, Paal (1919) showed that the growth of the lower zones is accelerated by a diffusible substance coming from the tip. His experiments were carried out with seedlings of Coix, and were afterwards confirmed on Avena by Nielsen (1924). This conclusion was confirmed by the careful measurements of growth made by Soding (1923, 1925). He compared first the growth of the stump, after decapitation, with the growth of the corresponding portion of the intact coleoptile. In the first 5 hours the growth was only about 40 per cent of that of the intact control. If, however, the tip were replaced, the growth of the stump was accelerated to 60-70 per cent of that of the intact control, thus proving that the decrease in growth after decapitation was not primarily due to any effect of the wound, such as the "wound substances" of Stark (1921). On the other hand the accelerating effect of the tip must be due to its secretion of growth-promoting substances. Soding states: "Since these substances certainly also influence growth in the intact plant, I propose to call them growth hormones." Cholodny (1924) carried out a similar experiment on Mais coleoptiles, in which the growth of coleoptile stumps on which the tips had been replaced was, in 3-4 hours, 144 per cent of that of the untreated stumps. When the growth measurements were continued beyond the first 5 hours, however, a new phenomenon appeared. In a representative experiment Soding obtained the following average growths in mm. (Table II). TABLE II TECHNIQUE OF AUXIN DETERMINATIONS 25 It will be seen that after the first 5-hour period the growth rate of the decapitated plants has increased again. If now the uppermost zone of these plants was cut ofT and placed upon freshly decapitated stumps, the growth of such stumps was accelerated, just as by tips. Other cylindrical zones of the coleoptile, similarly placed upon freshly decapitated stumps, had no such effect. Thus the uppermost zone of plants which have been decapitated for some time behaves Hke a tip in that from it diffuses a substance which accelerates the growth of the stump. This is the "regeneration of the physiological tip," and, as we shall see later, it explains the earlier observation of Rothert (1894) that tropistic sensitivity returns some hours after decapitation. The regeneration was studied in greater detail by Dolk (1926), from whose paper the curve of Figure 7 is taken. It 2 4 6 d 10 12 14 16 18 20 hrS. Fig. 7. Growth rate of Avena coleoptiles after decapitation. Ordinate, mm. elongation per hour; abscissa, time from decapitation in hours. (From Dolk, Proc. Kon. Akad. Wetensch., Amsterdam, 29: 1113-1117, 1926.) will be seen that immediately after decapitation the growth rate begins to fall, but after 2}/^ hours (at 21° C) a sudden break in the curve occurs and the growth rate rises to about 50 per cent of that of the intact plant. This sudden rise can be completely ehminated by a second decapitation 2 hours later, in which the uppermost 1 mm. of the stump is removed (Dolk, 1930). This proves that the rise is really due to regeneration of the production of growth hormone in the uppermost zone of the stump. It may be pointed out that this regeneration is not accompanied by any morphological change in the uppermost cells of the stump (Tetley and Priestley, 1927; Perry, 1932). 26 PHYTOHORMONES The above experiments provided evidence for the existence of the growth hormone in the coleoptile, and experiments of Beyer (1925) gave similar evidence in the case of Helianthus hypocotyls. Attempts were therefore made to prove the existence of this substance directly by extraction. Stark (1921) crushed up coleoptiles, mixed the extract with warm 5 per cent agar, and when the mixture had solidified, he divided it into small blocks and placed them one-sidedly upon the cut surface of decapitated coleoptiles. The decapitation was done by removing the tip without cutting it completely through, so that the primary leaf remained intact and could be used as a support for one side of the agar block. However, in all his experiments, embracing several hundred plants, only positive curvatures were obtained, that is, curvatures towards the applied block of agar. Extracts of stems or hypocotyls were similarly inactive. Hence no growth-promoting substance was extracted in this way; the explanation for the positive curvatures will be given later (III C 6). An important technical improvement was then introduced by Stark and Drechsel (1922), who pulled the primary leaf loose, breaking it at the base so that it did not grow further, and thus did not lift off the agar block. The differential growth rate of the leaf and coleoptile is caused by the decapitation, which only reduces the growth rate of the coleoptile itself. Nielsen (1924) and Seubert (1925) modified the experiment of Stark by crushing only the extreme tips of the coleoptiles, but, except for one experiment by Seubert, failed to obtain any growth-promoting extract. Seubert, however, was able to obtain growth promotion—that is, negative curvature—with agar blocks containing diastase, pepsin, malt extract, or saliva, and, in one experiment only, with concentrated press juice from coleoptiles. Starting from the facts that the growth hormone is able to diffuse through gelatin, and that only the tip actually produces it. Went (1926) ^ showed that the substance could 1 See footnote to page 5. TECHNIQUE OF AUXIN DETERMINATIONS 27 be "trapped" in the gelatin. He cut off a number of tips of coleoptiles, placed them for some time on gelatin, removed them, and placed the gelatin one-sidedly on the cut surface of freshly decapitated coleoptiles. For the preparation of the test coleoptiles he used essentially Stark's technique. However, as measure of the growth-promoting activity, he used, not the percentage of plants which curved, but the actual degree of curvature. The reason for this is that in any group of plants one or two will fail to curve due to experimental faults, and these should not be included in the mean. The percentage of plants which curve thus measures the efficiency of the technique rather than the growthpromoting activity. With this method he was able to show, amongst other things, that heating the gelatin for a short time at 90° C did not inactivate the substance, which cannot therefore be an enzyme. These fundamental experiments provided final proof of the material nature and stability of the growth-promoting substance of the coleoptile, and laid the foundation for further work. In a more detailed publication (Went, 1928), the Avena test method for determination of the growth hormone was worked out quantitatively. Since this method has been so extensively used as the basis of all phytohormone work it will be well to treat it here in detail. C. The Avena Method in Its Present Form 1. Dark Room and Equipment Although it is not strictly necessary to maintain darkness and constant conditions for carrying out qualitative work with Avena curvature, nevertheless for quantitative study the conditions outhned below must be strictly adhered to. Soding (1935) has used the Avena test in diffuse daylight with uncontrolled temperature, but under these conditions the sensitivity of the plants is very much less, and there is no strict proportionality between concentration of the active substances and curvature produced (see van Overbeek, 1933, 1936a). 28 PHYTOHORMONES For quantitative work, the temperature should be maintained constant: probably 25° C. has been the most used, but a somewhat lower temperature is more nearly optimal. The actual growth rate of the coleoptile, however, has its optimum close to 30° (Silberschmidt, 1928). Most of the data which we shall quote refer to 25° C. The maintenance of constant relative humidity is of great importance for a number of reasons: if the humidity is too high (above 90 per cent), guttation, i.e. exudation of water from the cut surface of decapitated coleoptiles, frequently occurs; this wets the whole cut surface, and may either wash off the agar block or may spread the substance which diffuses out of the block on to all sides of the plant. If, on the other hand, the humidity is too low, the agar dries out, which frequently leads to a failure of contact between block and cut surface. It is, of course, possible to maintain a suitable humidity in small chambers, inside a dark room, by merely lining them with wet filter paper. This is not, however, convenient for work on any considerable scale. Suitable thermostats and humidifiers have been described by Nuernbergk (1932). With the recent advances in the field of airconditioning a completely air-conditioned room is easily obtainable and is very suitable for hormone work. The effects of temperature, humidity, and some other factors have been worked out for a number of varieties of Avena saliva, as well as for some other plants, by Silberschmidt (1928). The Avena coleoptiles are exceedingly sensitive to light of the shorter wave-lengths ( < 550 m/u). This, if it falls on the plant from one side, causes phototropic bending, while, if symmetrically distributed, it causes a decrease of sensitivity to the applied auxin. Hence all manipulations must be carried out in orange or red light. Corning light filters 243 or 348, or Schott's O.G. 2, are very suitable to cut out the phototropically active wave-lengths from incandescent lamps. If no phototropic experiments have to be carried out in the dark room it is advisable to have the walls painted in a light shade. C. 2. Preparation of Test Plants Since most of the experiments in this field have been carried out with the genetically pure line of oats known as Victory oats (Segrehafer or Siegeshafer) it is advisable that TECHNIQUE OF AUXIN DETERMINATIONS 29 rack trough further work be continued with the same strain.^ Other lines have been used by various workers, perhaps the most notable being Gul Naesgaard in Denmark. The standard procedure for the preparation of the plants is as follows: The seeds are freed from their husks (glumes) and soaked in water for 2-3 hours. After this period they are laid out, groove downward, on wet filter paper in Petri dishes in the dark room. They are allowed to germinate in this way for about 30 hours, during which period it is ad\'isable to expose them to some red light, to suppress subsequent mesocotyl growth (Lange, 1927; du Buy and Nuernbergk, 1929). Originally the seeds were planted in a mixture of sand and leaf mould, but more recently, for the sake of uniformity and convenient handling, planting in water has been substituted. If the seeds are to be planted in such soil or in washed saw- p^^ § 4^^^^ seedling growing in dust, however, they should glass holder with roots in water coni . ,1 j.„»,„^„i^^f tained in the zinc trough. The adjustbe mserted at an angle of ^^^^^ ^^^^^^^^ ^^^ i^^i^^^^^ ^^^ ^,,„,,.g about 45° with the vertical, in order that the shoot may emerge straight. The sand or soil should be so wet that further watering is not necessary, as this will displace the seeds. If, on the other hand, it is too wet the sensitivity of the plants is reduced (Boysen Jensen, 1935). The depth of planting is also important and the shoot should be about 8 mm. below the surface ; if too shallow the seeds will be pushed out by the developing roots. For water culture convenient glass holders were devised by Went (1928) and a modification of his design is now in almost universal use (see Figure 8). The holders are coated thinly with paraffin, which prevents water from creeping into the guide and makes the fitting of the seeds into the > Obtainable from Sveriges Utsadesforening, Svalof, Sweden. 30 PHYTOHORMONES sockets somewhat easier. They are fixed in rows of 12 by means of brass chps which fit into grooves in a wooden rack; the clips are free to move, stiffly, in a vertical plane, while the holders can be rotated and thus any adjustment of the growing plant is possible. The germinated seeds are inserted into the sockets in such a way that the shoot comes under the center of the guide, and the roots point downwards. The whole rack, with its 12 seeds, is then placed with the roots at the surface of water contained in a zinc trough. The most usual dimensions are: rack 25 X 25 X 200 mm., trough 35 X 35 X 200 mm. About 48 hours after planting, the coleoptiles are 20-30 mm. long and ready for use. They are selected for straightness and uniformity, and Fig. 9. Row of 12 Avena seedlings as Figure 8. Left to right: 2 intact, 2 decapitated, 2 decapitated for the second time (primary leaf protruding), 2 from which primary leaf has been pulled loose, 2 with applied agar block, 2 showing curvature. since the holders are removable, rows of 12 good plants are assembled. It is advisable to bring all shoots into the strictly vertical position some 2 hours before use (see Figure 9). In the preparation of the test plants the first operation is to cut off the extreme tip with a razor (B, Figure 10). In the subsequent period a large amount of the growth hormone present in the stump is being used for its residual growth, and the test plants become more and more sensitive to any hormone which is applied (van der Weij, 1931). After 3 hours the topmost 4 mm. of the stump is cut off (C-E, Figure 10). This can be done by making an incision TECHNIQUE OF AUXIN DETERMINATIONS 31 on one side of the coleoptile without cutting the primary leaf; the top of the coleoptile is then bent so that it breaks at the incision and the topmost part is pulled off. Special decapitation scissors (Figures 11, 12) which cut the coleoptile on two sides without touching the leaf are very convenient for this, because with the closed scissors the cut-off m \l A u \/ B D H Fig. 10. Diagrammatic summary of procedure in the Avena test, showing the stages photographed in Figure 9. portion can be removed in the same movement. The primary leaf, which now protrudes for 5 mm. or more, is then pulled gently so that it breaks at the base and is partially drawn out (F, Figure 10); this can be conveniently done with cork-tipped forceps (Figure 11). If the cut surface is very wet it should be dried with filter paper. The small block of agar to be tested is then placed on one side of the cut surface, resting against the leaf, so that it is held in place by capillarity. The period elapsing between the second decapitation and the apphcation of the block does not usually exceed 20 minutes. To record the curvatures a piece of bromide paper is placed behind the plants; they are then illuminated from 32 PHYTOHORMONES Fig. 11. Convenient tools for the Avena test. 1, frame for dividing agar rectangles into 12 equal blocks with safety razor blade; 2, stamp for cutting the rectangles 8x11 mm. from agar disc or sheet 1-2 mm. thick; 3, spatula for application of agar block; 4, corktipped forceps for pulling primary leaf loose; 5, decapitation scissors (see Figure 12); 6, forceps for planting in glass holders. one side. The curvatures are measured on the resulting shadowgraph (see Figure 22) by means of a celluloid protractor with rotating arm (Figure 13). The angle measured is that between the tangent to the extreme curved tip and the straight base. Soding (1934) has described a method depending on the same principle, but measuring the curvature on the plant itself by placing it over the protractor. Fig. 12. Cutting end of decapitation ^^rdy (1921) haS^ develscissors. A, adjusting screw to regulate oped a method, which haS opening between knives; B, strips of been adopted by Boysen Jensaiety razor protrudmg 4 mm. beyond ^ » brass holders C; D, plant being decapi- Sen and by Nielsen, of meas- *^*^^uring the increased growth on . TECHNIQUE OF AUXIN DETERMINATIONS 33 the side to which the agar block has been apphed. Instead of measuring the angle (which is directly proportional to the difference in growth), the radius of curvature, r, thickness of the Fig. 13. Measurement of Avena curvatures. The vertical lines on the base of the transparent celluloid protractor are brought parallel to the uncurved base of the plant on photograph, B, while the lines on the moving arm, A, are brought parallel to the extreme curved tip. The angle (23J^°) is read directly. (From Went, Rec. trav. hot. need. 25: 1-116, 1928.) plant, t, and length of the curved zone, I, are measured (see Figure 14). Then the difference, d, between the growth on the two sides is expressed hy d = — Assuming a thickness of 1.5 mm., a rf- value of 0.1 mm. corresponds to 4° curvature, 0.5 mm. to IQ'', and 1.0 mm. to 38.5°. This procedure involves the making of three measurements, of which, however, t is taken as constant. Satisfactory measurement of the other two, r and I, depends upon the assumption that the curved 34 PHYTOHORMONES zone is a circular arc. It is, however, apparent from Dolk's (1930) measurements of plants curved under the influence of auxin that this is far from being the case, and that the radius of curvature decreases smoothly from the straight base to a region of maximum curvature some way below the tip (cf. X H). The values adopted for r and I are therefore of necessity arbitrary. If the test is carried out as above, the optimum time for photographing is about 90 minutes after application of the agar block {cf. Figure 20) ; after this time the curvature is no longer proportional to the concentration of hormone in the agar, because the curvatures produced by low concentrations decrease or remain stationary, while larger ones continue to increase. Fig. 14. Purdy's method of measuring the difference in growth between the two sides of a curved coleoptile. Explanation in text. (From Purdy, Kgl. Danske Videnskab. Selskab., Biol. Medd. 3: 3-29, 1921.) C. 3. Preparation of the Agar A good quality of agar is well washed and made up to a 3 per cent gel. To prepare the blocks containing auxin for the test, the agar may be either first cut into sheets and these soaked in the test solution or it may be melted and mixed with an equal volume of the test solution. The preparation of agar sheets of uniform thickness for the former method requires some precautions. The most satisfactory procedure is to cut a block of agar about 40 X 20 X 20 mm. and to surround this on 3 of its long sides with paraffin (see Figure 15). The agar is then cut in a microtome using a safety razor blade, and the resulting book of sheets (1 or 1.5 mm. thick) preserved in 60 per cent alcohol. Before use, these must be washed free of alcohol (1 hour). They are then cut into rectangles, with a special cutter, and these are placed in the test solution for at least 1 hour; sufficient solution must be present to ensure that the concentration is not changed appreciably by addition of the agar. For the second method, usually ]4, cc of the melted agar is mixed with 3^ cc. of the test solution, and }^cc. oi the resulting mixture quickly poured into a shallow circular mould with removable base whose dimensions, 10.3 mm. radius X 1.5 mm. deep, are such that it is TECHNIQUE OF AUXIN DETERMINATIONS 35 exactly filled by Yi cc. After cooling, the resulting disk of agar is cut into rectangles with the same cutter as above. With either method the rectangles are finally divided into 12 equal blocks by means of other special cutters (see Figure 11). The sizes of blocks adopted vary in different laboratories between 2 and 10 cmm. To avoid too much volume change by drying out, etc., it is advisable to use blocks of volume not less than 4 cmm., the volume changes being proportionately less the larger the block. It has been shown (Thimann and Bonner, 1932) that the curvature is primarily proportional to the concentration of auxin in the block and that the rate at which the auxin enters the plant is proportional to its f concentration in the block at any moment; since part of the auxin passes out of the Fig. 15. Method of cutting thin 1, 1 1 • ,1 ,, ,1 sheets of agar of uniform thickness. The block durmg the test, the agar, A, is embedded on 3 sides in parafchange of concentration in fin, B, and sliced on a microtome with a the block so caused is small '^^"^^ ^^^^'^ ^^^^^- ^^™"^ ^^'^^' ^^^s.) if the blocks are large. Thimann and Bonner (1932) have measured the amount of auxin which passes into the plant during 110 minutes' contact, and from the data have calculated the curvatures which would be produced by different sizes of blocks containing the same auxin concentration (see Table III). The calculations were found to agree closely with the curvatures observed. The discrepancy between Went's conclusion (1927), that with 0.9 cmm. blocks the curvatures are proportional to the absolute amount of auxin in the blocks, and the conclusions of van der Weij (1931) and Thimann and Bonner (1932) that the curvatures are proportional to the auxin concentration in the block, is directly explained by Table III. In Went's experiments 86 per cent of the auxin from the block entered the plant; with larger blocks only 15-30 per cent will pass into the plant. 36 PHYTOHORMONES TABLE III TECHNIQUE OF AUXIN DETERMINATIONS 37 found that curvature is produced. Apparently higher concentrations are needed than in the normal Avena test but the experiment provides an interesting demonstration that auxin may enter through the epidermis. The same fact has been utilized in a still further modification (Laibach, 1933a, 1935; Brecht, 1936) in which auxin is dissolved in 1 \ R n Fig. 16. De-seeding of Avena. A, normal seedling in glass holder; B, same with seed removed and coleoptile held in guide with cotton wool; C, decapitation; D, curvature. (From Skoog, J. gen. Physiol. 20: 311-334, 1937.) lanoline (Laibach's method), and this paste applied externally to intact or decapitated coleoptiles. If about 4 mg. of such paste be smeared over a length of 1 cm. of one side of the coleoptile, a curvature results which increases up to 24 hours, and is within limits proportional to the concentration of active substance in the paste. However, the lowest concentration which produces curvature appears to be about 10 times that which produces the same curvature when agar is used (see also Jost and Reiss, 1936). The method might be useful for the standardization of such pastes, but unfortunately it is highly non-specific, because acetic acid and other acids incorporated in the paste also give rise to curvatures. In the application of lanoline-auxin-paste to plants in general great care should 38 PHYTOHORMONES be taken to control the results with plain pastes, because Schilling (1915) has shown that treatment of twigs or leaves with vaseline, paraffin, cocoa-butter, etc., gives rise to various kinds of outgrowths, and even root formation may be induced in this way. Soding (1936) has found that seedlings of Cephalaria are very sensitive to low concentrations of auxin. His tests are carried out in diffuse light. If the concentration of hormone is very low, the Cephalaria test will show good curvatures where the (dayhght) Avena test shows none. cl\ 02"^S Fig. 17. Cephalaria test of Soding. A, decapitated hypocotyls with agar block applied; B, after 5 hours; curve C, relation between concentration of indole-acetic acid in mg. per liter and curvature of Cephalaria; curve D, the same for Avena. (From Soding, Jahrb. wiss. Bot. 82: 534-554, 1936.) The seedlings are decapitated, the stem being cut through very obliquely, and the block of agar is placed upon the lower half of the cut surface (see Figure 17). The curvature is measured after 5 hours. As may be seen, the sensitivity at low concentrations is much higher than with Avena but the maximum curvature obtainable is about the same. C. 5. Evaluation of Results The Avena coleoptiles, prepared in the way described, are of great physiological uniformity, as is shown by plotting the variability of the curvatures given by a large number of identically treated plants. Most of the experimental faults will tend to reduce rather than increase the curvature so that in a faulty test the variability curve will be skew towards the lower values (see Figure 18). If, in a test of 12 or more plants giving a fairly symmetrical distribution, TECHNIQUE OF AUXIN DETERMINATIONS 39 one or two values fall far off the distribution curve, they are generally neglected. If the values are too scattered, the experiment should be repeated with more uniform plants. As a rule, the mean of 12 good plants is taken. As measure of the variation among the test plants the standard deviation of the mean ^ is best used, and this I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 degrees Fig. 18. Distribution of individual curvatures in the Avena test. Abscissa, curvature groups; ordinate, number of plants in each group. Diagram A, perfect test, agreeing with the variability curve A'; diagram B, imperfect test, skew with relation to ideal variability curve B'. (After Went, 1928.) obviates the necessity of detailing the individual curva- tures. The possible causes for this variation between individual plants are many. Variations in thickness play no part (Went, 1928). Differences in actual age of the plants are of course excluded because all have been planted at the same time. Variations in the length of the cut tip do occur, but are of minor importance if they do not exceed a few millimeters. The following table shows the relative auxin curvatures of plants from which different lengths of tip have been cut off (Table IV). / '*' A"1 Standard deviation of the mean = A/ — , where A is the difference bey n{n — 1) tween the individual observation and the mean, 2 A^ the sum of the squares of these, and n is the number of observations. 40 PHYTOHORMONES TECHNIQUE OF AUXIN DETERMINATIONS 41 Figure 19). Below the upper limit, whose position differs for different active substances, the activity of a substance can therefore be quantitatively expressed. The amount of a substance necessary to produce a given curvature under standard conditions is taken as a unit. 04 f"^! Fig. 19. Relation between auxin concentration (in mg. indole-3-acetic acid per liter) and curvature. Two decapitations 3 hours apart, agar blocks 10 mm.\ 24° C, 85% relative humidity. The scale of the abscissae of the upper curve is 50 times that of the lower. Each point the mean of 30-50 plants. The "^rena-Einheit" (AE) of Kogl and Haagen Smit (1931) is that amount of substance which when applied in a 2 cmm. block of agar causes 10° curvature. The "plant unit" (p.u.) of Dolk and Thimann (1932) is that amount of substance which when applied in a 10 cmm. block causes 1° curvature. These workers also define as "1 unit per cc." that concentration of solution which when mixed with an equal volume of 3 per cent agar and made into 10 cmm. blocks gives 1° curvature. Under the conditions then used (40-60 minutes between decapitation and application of block) 1 AE = 2.5 p.u. How- 42 PHYTOHORMONES ever, under the conditions described in section C 2 (3 hours between decapitation and apphcation) 1 AE = about 1.5 p.u. The WuchsstoffEinheit (WAE) of Boysen Jensen (1933o) is defined as that amount of substance which, when dissolved in 50 cc. of water and mixed with an equal volume of 3 per cent agar, gives a d-value of 1 mm. Now that it has been shown that one of the auxins is indole-3-acetic acid, which is readily available in pure form, the importance of such units has largely disappeared, and all activities may be expressed in comparison with the effect of indole-acetic acid. However, for the better understanding of data given in the literature, the following comparisons are tabulated (Table V). It should be noted that, weight for weight, auxin a is about twice as active as indole- 3-acetic acid, or mol for mol about 3.75 times as active (Kogl, Haagen Smit, and Erxleben, 1934). C. 6. Positive Curvatures The curvature produced by auxin is a negative curvature, i.e. away from the side with the agar block, the active substance having caused an increase of growth on the side to which it is appHed. Stark (1921) and Nielsen (1924) never obtained such negative curvatures by the application of blocks containing plant extracts. Seubert (1925) found also that the majority of substances dissolved in agar caused positive curvatures. This was considered as evidence of the existence of growth-retarding substances which were identified by Stark with wound-substances. Seubert even found that the same substance appears to be stimulating or inhibiting according to its concentration; thus, high concentrations of saliva or malt extract caused negative, low concentrations positive curvatures. The absence of growth-promoting activity in plant extracts has been explained by Thimann (1934) ; it is due to the destruction of the auxin by enzymes set free on crushing the material. The production of positive curvatures, however, is something other than the mere absence of negative curvatures, and its mechanism has been elucidated by a O ; H ! n : o DO fi O'tT ^ S « Sm ^ ^ H O E W !s o w H !5 • g p H w H <; 05 CO o :^ « o E- O < K O <: S t) CO oCO •5 >> S SIS 3 S'=« m Q c oo Oo 2 <=> o o 03 o 2 o W H H <<*< < o3 O O 2 V > e o X w 00 K 140 PHYTOHORMONES herewith. This table (Table XII) is founded on those of Kogl and Kostermans (1935) and Haagen Smit and Went (1935), with additions from various sources. The conclusions to be drawn from this table are in some respects modified from those touched on in VII E. Tentatively we may say that: The primary growth-promoting activity is connected with the presence of: ^ the double bond, or aromatic unsaturation; 2, a carboxyl group, free, or if esterified, readily hydrolyzable; 3, a ring system, either 5-membered (auxin a and h), aromatic (naphthyl or phenyl), or a combination of both (indole, indene, etc.); 4, a minimum distance of at least one C atom between the carboxyl group and the ring; 5, a very definite steric structure, since in the one case studied the cis-compound is active, the trans-compound not. The chemical changes which do not directly influence the primary growth reaction, but modify the secondary properties of a growth-promoting substance, are: 1. length of the acid side-chain, 2. methyl substitution in the nucleus, 3. substitution in the side-chain, 4. the structure of the nucleus itself. A more detailed discussion and a hypothesis relating structure to activity has been given by Went, Koepfli, and Thimann (1937). Although this is only a beginning, we are already in a position to predict what substances should possess activity. Presumably an exact knowledge of the active grouping will help in determining the type of the reaction involved. CHAPTER IX THE GROWTH OF ROOTS In the preceding chapters reference has been made only to the relation of auxin to shoots. In roots the relations are entirely different, although in many respects analogous. A. The Effect of the Tip in Root Growth Unlike shoots, in which decapitation almost invariably reduces the growth rate, decapitation of roots does not greatly influence the growth. In the 70's and 80's of last century a flood of literature appeared deaUng with this subject. Every investigator used a different object, and opinions were about equally divided as to whether decapitation retarded growth or was without effect; Wiesner (1884), working with roots under water, even found an acceleration. Probably on account of the disagreement, the whole question was shelved for almost half a century. It was Cholodny who in 1924 and subsequent years took up the problem again and showed that in fact, if precautions were taken to ensure that the roots received sufficient water, there was an acceleration after decapitation (Cholodny, 1926). He worked first with Zea Mays and later confirmed the effect with Lupinus (1926). The differences were in all cases small (ca. 12 per cent), but real. Later Biinning (1928) showed that the acceleration was limited to a few mm. of the growing zone only, and that if sections of tip longer than 1 mm. were removed the acceleration did not take place. The acceleration is preceded by a temporary decrease in growth rate, which is probably due to a loss of turgidity of the cells resulting from the wounding (Janse, 1929). Biinning found this acceleration in Lupinus, Zea Mays, and Vicia Faba roots, but not in Pisum roots. Gorter (1932) also found that Pisum roots show no change in growth rate after 141 142 PHYTOHORMONES decapitation. It is thus clear that if the experiments are carried out carefully and not too much of the tip is removed, all of the roots studied—with the exception of those of Pisum—show a definite acceleration after decapitation (c/. also Cholodny, 1933). In an attempt to explain this phenomenon, Cholodny (1926, 1931a) replaced the tip on maize roots and found that their growth was again retarded. Coleoptile tips had previously been found to behave in the same way (1924). Keeble, Nelson, and Snow (1931a) confirmed these results. Further, when the tip is removed, the root stump loses its sensitivity to gravity, as has been known since Darwin (1880), but if root or coleoptile tips are replaced on the stump Cholodny (1924) found that its geotropic sensitivity is largely regained. This suggests, as Cholodny (1926) pointed out, that both the root tip and the coleoptile tip secrete a substance retarding root growth, and that this substance enables the root to give a tropistic response by becoming more concentrated on one side than the other (c/. X5). From its production by coleoptile tips it might be supposed that this growth-retarding substance is identical with auxin. B. The Effect of Auxin on Root Growth The above possibility was strongly borne out by the experiments of Nielsen (1930), Navez (1933), and Boysen Jensen (19336) (see also Keeble, Nelson, and Snow, 1930). They immersed roots of various plants in water and in difTerent concentrations of Rhizopus culture medium (which contains indole-3-acetic acid, cf. VII C). The growth of the roots was very greatly retarded by the active culture medium. That this retardation is really due to the auxin and not to the other substances present was shown by Kogl, Haagen Smit, and Erxleben (1934a) by immersing Arena roots in solutions of auxin a, auxin b, and indole-3-acetic acid, when they were strongly retarded. The extent of inhibition is proportional to the concentration of auxin, and THE GROWTH OF ROOTS 143 the lowest active concentration is about that which gives 5° curvature on the Avena coleoptile (Lane, 1936; Meesters, 1936). The inhibition is not due to pH, and is not comparable with that produced by toxic substances. Since indene-3-acetic acid exerts the same inhibiting effect (Thimann, 19356), it is presumably a general property of growth promoting substances to inhibit root elongation. Root inhibition can even be used as a method for the assay of auxin, o < IH O 5° o " Ul 100 80 60 40 20 0050 Hz^ Q020 0.004 0.0008 0.00016 .00003 2 CONCENTRATION IN MG. PER CC. I. IND0LE-3-ACETIC ACID 2. I ND0LE-3-PR0 PION IC ACID Fig. 46. Relation between auxin concentration and the inhibition of root elongation in Avena. The seeds were soaked 24 hours in water and then 24 hours in test solution. The concentration of indole-propionic acid necessary to cause a given inhibition is 25 times that of indole-acetic acid. (From Lane, Am. J. Bot. 23: 532-535, 1936.) for which it is both simple and sensitive, the inhibiting concentrations of auxin being much lower than those of ordinary toxic substances such as mercury and silver ions (Lane, 1936). The ratio of activities of indole-propionic and indole-acetic acids, which in Avena coleoptile curvature is 0.2 per cent, in Avena root inhibition is about 4 per cent, (see Figure 46). Extremely low concentrations of auxin may produce acceleration of root growth. Thus Amlong (1936) finds that if Vicia Faba roots are decapitated and 3 hours later, when their auxin content will have somewhat decreased, are treated with auxin solutions, then 10"^ mol. indole-acetic acid causes slight but definite growth promotion. Higher concentrations still inhibited growth. Fiedler (1936) obtained a similar result with isolated Zea Mays roots in culture medium without yeast extract ; their auxin content was 144 PHYTOHORMONES then practically nil, and 2.10"^ mol. indole-acetic acid (0.1 7 per liter) caused about 30 per cent acceleration in growth, while higher concentrations only inhibited. Thimann (1936a) obtained a slight acceleration of the growth of Pisum roots when auxin was applied to the base of the stem. He ascribed this acceleration to the very low concentrations of auxin which reach the root under these conditions, together with the relatively small amount of auxin present in these roots. Further, if Avena roots are inhibited in auxin solutions and then transferred to water, their growth is accelerated beyond that of water-treated controls; this, presumably, is due to the rapid disappearance of auxin from the root, so that its concentration eventually reaches the accelerating level (u). C. Auxin Production in the Root In order to complete the picture it is necessary to have proof that root tips really do produce auxin, and this has been given by a number of workers. First, Cholodny (1928) showed that placing a root tip of Zea Mays on decapitated Avena coleoptiles would increase their photo- and geotropic responses. Then Hawker (1932) in experiments primarily directed to the study of geotropism, placed root tips of Vicia Faba on gelatin blocks and showed that if subsequently applied to one side of decapitated root stumps the blocks caused the stump to curve towards them, i.e. the stump was inhibited on the side to which the gelatin was applied. However, Boysen Jensen (1933a), who similarly placed root tips on agar and then tested this agar on Avena coleoptiles, could obtain no auxin in this way from Zea Mays root tips. He found, however, that the tips of very young Zea Mays roots (not longer than 5 mm.) would cause curvature of Avena coleoptiles if placed one-sidedly upon them, but the tips of older roots would not do so. Believing that the reason for this failure was the need for nutrient, he used agar containing 10 per cent dextrose, and this brought immediate success. Root tips of Vicia Faba gave a small amount of auxin to plain agar (as Hawker had found), but five times THE GROWTH OF ROOTS 145 this amount with dextrose agar. With this method he showed that, in Zea "caragua,'' the concentration of auxin decreases rapidly from the tip of the root towards the base, and subsequently (19336) also confirmed Hawker's finding that more auxin diffuses out of the lower than the upper half of root tips which had been placed horizontally. The presence of auxin in Avena roots was proven in another way by Thimann (1934), who acidified the roots, extracted directly with chloroform, and subsequently tested the extract, freed from chloroform and dissolved in agar, upon coleoptiles. The concentration of auxin decreased from the tip towards the base, as may be seen from Figure 30, p. 68, which summarizes his results. Boysen Jensen (1936a) confirmed that auxin could be extracted from roots by chloroform and found that more was extracted from the lower half of horizontally placed roots than from the upper. The above experiments show that auxin is present in the root, and especially in the tip, but do not make it clear that it is actually produced there. Thimann (1934), using 10 mm. Avena root tips, compared the auxin obtainable by chloroform extraction with that obtainable by diffusion into dextrose agar, and stated that ''the only fair conclusion seems to be that growth substance is not produced in the root tip but merely accumulates there." However, Cholodny (1934) found that, if suppHed with dextrose gelatin, root tips of Zea Mays continue to yield auxin for 5-6 hours and Nagao (1936) obtained the same result with short (2 mm.) tips of Avena roots. The researches of Van Raalte (1936) go far towards clearing up this question. Using Vicia Faha roots, he found that addition of salts alone to the agar gives very good yields of auxin, but addition of dextrose isotonic with the salt is still more effective. Thus the dextrose acts in two ways—osmotically, by increasing the diffusion out of the root tip, and nutritively, by maintaining the root tip during the experiment. Boysen Jensen (1933a) had previously found that mannite was almost as effective as glucose and concluded from this that its action could not be 146 PHYTOHORMONES nutritive. Van Raalte further showed that on dextrose agar the production of auxin by the root tip is actually greater in the second hour than in the first, and if such tips were then extracted with ether the amount of auxin extractable had increased. There is therefore little doubt that auxin is produced for a short time in the isolated root tip, when it is fed with dextrose. Fiedler (1936), however, found that neither by diffusion into dextrose agar nor by extraction with chloroform could auxin be obtained from isolated root tips of Zea Mays and Vicia Faba in culture solutions after about 24 hours; even after 8 hours in the medium 80 to 90 per cent of the auxin originally present had disappeared. This disappearance of auxin could be largely prevented by coating the cut surface with gelatin or lanoline and is therefore probably due to enzymic oxidation (cf. IV A). Fiedler concludes that root tips in culture produce no auxin and adopts the view of Thimann (1934) that the auxin in root tips comes there from the seed or shoot. Since there are a number of chemically different auxins, which all have the same effect on root growth, it is of special interest to know which one is present in the root tip. This question has been studied by Heyn (1935), who found, by the determination of the diffusion coefficient, that the auxin of the root tip of Vicia Faba has a molecular weight of about 370, which is just the same as that for the Avena coleoptile tip ; it is therefore probably auxin a or b. The transport of auxin in the root has been the subject of conflicting experiments and views. Went (1932) suggested that the polarity of the shoot was continuous with that of the root, so that auxin transport would be towards the root tip. Gorter (1932) investigated transport directly by using Zea Mays root cyhnders in the same way as van der Weij with coleoptiles (Chapter VI C), and found that transport took place in both directions about equally (see also Heidt, 1931). The auxin appeared to be rapidly destroyed by the root. The same thing was found for Vicia Faba by Faber THE GROWTH OF ROOTS 147 (1936). The most basal zones of roots 60 mm. long or so, however, transported practically no auxin in either direction. The velocity of movement was about 4 mm. per hour. JMagao (1936) found in Vicia Faba, on the other hand, that the auxin diffusing out from cylinders taken from near the tip was greater from the basal than from the apical surface. In an experiment of Cholodny (1934), coleoptile tips were applied to the apical or the basal cut surface of decapitated roots; if these were then placed horizontally, the stumps with tips on their apical ends curved geotropically, the others not. He concluded that auxin moved only basipetally in the root, although Heidt's very similar experiment with Sinapis alba indicated movement in both directions. That some transport certainly takes place from base to apex is shown by the experiments of Thimann (1936a) on the formation of lateral roots of Pisum by auxin applied to the stump of the shoot (see XI F). He also found that auxin applied to the scutellum of Avena greatly inhibited the growth of the young roots, and if applied at the base of the shoot in older plants it here also—though to a lesser extent — inhibited the growth of the roots. Transport from base to apex in Avena roots therefore probably decreases with age. Such experiments on roots are made more difficult by the rapid destruction of auxin which takes place (Gorter, 1932; Fiedler, 1936), but there seems no doubt that transport in both directions occurs in Pisum, Zea, Avena, and Vicia, although doubtless the ratio of the transport intensities in the two directions varies. An interesting feature of the action of auxin on roots is its production of swellings. Cholodny (1931a) showed that when 3 maize coleoptile tips, instead of one, were applied to the root stump there was a marked swelling in the growing zone. This swelling was mostly confined to the cortical parenchyma. Bouillenne and Went (1933) noticed that cuttings on which leaves had been grafted, and which rooted \'igorously, frequently gave abnormal thickenings near the root tip, doubtless due to the excess of auxin coming from 148 PHYTOHORMONES the leaf; on removal of the leaf the roots resumed their normal growth. Kogl, Haagen Smit, and Erxleben (1934o) in showing that pure auxins inhibit growth of Avena roots, mention that the short roots were abnormally thick. Subsequently numerous workers have noticed the thickenings which are almost always produced by the action of auxin, particularly with high concentrations. In the shortened and thickened zone, there is radial elongation of the cells in the cortex, which are actually shortened in the longitudinal dimension (u). The thickening need not necessarily accompany inhibition of elongation, since Lane (1936) has shown that Avena roots may be almost completely inhibited in length without any appreciable thickening, by soaking the seeds in auxin solution. A special case of root thickening and lateral root formation is provided by the bacterial nodules on the roots of legumes. Thimann (1936) has shown that young growing nodules produce considerable amounts of auxin, which diffuses both from the apical and from the basal portion. He also showed that local application of auxin to a lateral root at its first appearance produced its complete inhibition, and thus deduced that nodules are due to the auxin formed by the bacteria in the tissues; this auxin induces a lateral root at the point of infection, but then inhibits its growth and causes it instead to swell. D. The Role of Auxin in the Growth of Roots The formation of and response to auxin in the root is so different from that of shoots, that the two-factor scheme, suggested to explain the distribution of growth in shoots, cannot be applied to roots. How then do roots grow? According to Czaja (1935) auxin in roots has fundamentally the same function as in shoots, that is, it causes elongation. He assumes two opposite streams of auxin, one coming from the stem and the other from the root tip, each of which by itself promotes growth, while the two in combination cause a growth retardation. This view, based on a few ex- THE GROWTH OF ROOTS 149 periments only, and theoretically impossible (see VI D), has been disproven both by Faber (1936) and by Thimann (1936). Czaja's experimentum crucis: negative geotropic curvatures in decapitated roots, which should have only one auxin "stream," could not be repeated. Further, auxin, when applied to the epicotyl stump of decapitated Pisum seedlings does not inhibit root growth, but even slightly accelerates it (Thimann, 1936), and the presence or absence of the root tip (i.e. the second auxin ''stream") does not affect the result. In Avena, in which the same treatment produces a slight inhibition, the presence or absence of the root tip is also without effect. One obvious suggestion as to how it is that auxin inhibits root elongation is that its action is exerted on the transverse walls and thus causes thickening instead of elongation (c/. e.g. F. A. F. C. Went, 1935). However, since inhibition appears to be independent of thickening, there must be another explanation. Another suggestion comes from Boysen Jensen (1936a). According to this, roots are much more sensitive to auxin than shoots; very low concentrations of auxin promote their growth, but the ordinary concentrations worked with are high enough to be above the optimum and thus cause inhibition. This is supported by the experiments of Amlong (1936a) and Fiedler (1936) on roots made very poor in auxin, whose growth is then slightly promoted by the lowest auxin concentrations (see IX B). A comparable inhibition of elongation is produced in stems, e.g. of pea and Helianthus, by very high concentrations of auxin. In stems, therefore, inhibition is caused only by the highest concentrations, while in roots it is caused by all but the very lowest. This raises the question as to why different roots react differently to auxin. Thus in Thimann's experiments (1936) Avena and Pisum behave in the opposite way to auxin applied basally; in Faber's experiments (1936) Avena and Vicia also react oppositely. Both have suggested that the difference is due to different concentrations of auxin already in the root, 150 PHYTOHORMONES roots already low in auxin giving an acceleration, roots high in auxin a retardation. This would correspond approximately with Boysen Jensen's suggestion. Jost and Reiss (1936) also tend to adopt this view. The other possibihty is that auxin is not necessary at all for the growth of roots. The growth of isolated roots in culture is known to be dependent on sugars, salts, and traces of special substances, which are present in yeast extract (White, 1933, 1934). These may perhaps include a special root-growth promoting substance, but in any event addition of auxin does not improve the growth, and such a medium suffices for the indefinite growth of the roots of wheat and tomato. The absence of detectable amounts of auxin from such isolated roots (Fiedler, 1936) would certainly indicate that it plays no part in their growth. The fact that in these cultures roots continue to branch, however, would indicate either that they produce traces of auxin or else that the small amount of auxin in the yeast extract or other constituent is sufficient to cause both growth and branching. Whether auxin plays any part in root elongation or not, the mechanism whereby it causes inhibition of root growth is, as stated above, not clear. Bonner (1935) has suggested that instead of diminishing the forces between the micelles of the cell wall, it increases them. However, Amlong (1936) has shown that the decapitation of Vicia Faba roots causes a decrease in their plasticity just as was shown by Heyn for coleoptiles (VIII D). Not only does this contradict Bonner's suggestion, but it raises again the question of whether the increased plasticity caused by auxin can be the direct cause of growth. In view of the incompleteness of our knowledge as to the mechanism of elongation, it is perhaps unreasonable to expect any better understanding of the mechanism of inhibition. Doubtless further advances in the analysis of both processes may be expected in the near future. CHAPTER X TROPISMS I. TROPISMS IN GENERAL The apparent lack of feeling or of responsiveness in plants was the basis of the old distinction between plants and animals: "Saxa crescunt, plantae crescunt et vivunt; animalia crescunt, vi\iint et sentiunt." However, in tropistic movements plants appear to exhibit a sort of intelligence; their movement is of subsequent advantage to them. In addition, tropisms are easily observable and lay themselves open to obvious experiments, so that it is not surprising that they have been a subject of investigation from the earliest times of plant physiology. This sensitivity, otherwise not noticeable in plants, explains why it is that in regard to tropistic responses the parallelism between plants and animals has been so much stressed. This had at first the drawback that many complex concepts of zoology were transferred to plants, but one of the great services which the discovery and study of the auxins has performed is that it has forced the development of new concepts independent of the zoological heritage—concepts not only new, but also more concrete and experimentally analyzable. It will be among the aims of this chapter to elucidate this change in thought, and with this in view, tropisms will be treated only so far as they are demonstrably due to differential growth and so far as their auxin relations have been investigated. The material in this chapter could also be regarded as an application of the knowledge of auxin which we have gained in the preceding pages. However, this would not be historically correct, because, as shown in Table I, p. 16, the early evidence for the existence and role of auxin came through the study of tropisms. In the recent development of the field, the center of interest has shifted away from the 151 152 PHYTOHORMONES explanation of tropisms to more general functions of auxin in correlative processes and to the growth process itself. A. Historical Introduction The early work on tropisms has been reviewed by Wiesner (1878, 1880), Pringsheim (1912), F. A. F. C. Went (1929, 1931), Rawitscher (1932), du Buy and Nuernbergk (1935) and need not be further discussed here. We can thus pass over the first period, consisting first of simple mechanical explanations, and then of their gradual replacement by more and more elaborate stimulus-concepts, none of which had a really concrete physical basis. This period of complexity of thought was accompanied by simpHcity of experimental approach, and work was of a purely qualitative nature. The experiments of Blaauw (1909, 1914, 1915, 1918) ushered in a new era,^ that of quantitative work; this was associated with greater simplicity of theories. He showed that, within limits, the response is proportional to the total energy received by the plant, and he envisaged tropisms simply as a phenomenon of differential growth. Thus: "Whenever light causes a growth reaction, unequal distribution of the light will cause unequal growth, which we call phototropism " (Blaauw, 1918, p. 171). Almost simultaneously with the first publication of Blaauw, the "stimulus" concept of tropisms received a blow from another direction. Boysen Jensen (1910, 1911, 1913) found that the transmission of the light stimulus does not involve any vital process, but can take place by simple diffusion. He concluded from his experiments, which have been discussed in H B, that the path of the phototropic stimulus is along the shaded side, that of the geotropic stimulus along the lower side, of the plant. The stimulus could cross a cut surface, but would not pass a sheet of mica; however, a slice of rattan inserted in the cut did not 1 The editors of the Zeitschrift fiir Botanik received Blaauw's paper "Licht und Wachstum, J" (1914), with the following unusual reservation: "We publish this paper although we do not agree in every respect with the author's theoretical con- ceptions." TROPISMS 153 interfere with its passage. The transmission of the stimulus is therefore due to a diffusible substance. Purdy (1921) confirmed his essential results. Soon after this first important step Paal's investigations (1914, 1919) brought another great advance. Paal found that not only after tropistic stimulation, but also in normal growth, the tip exerts an influence on the growth of the lower zones. Thus he developed the concept that the tip continuously forms growth-regulating substances. His great contribution to the field of tropisms was that he envisaged the response as due to an unequal supply of these growth regulators to the two sides (c/. II C). Considering phototropism in particular, he suggested three possible ways in which such an effect might be brought about : the regulator might ''disappear," its production might be reduced, or its transport might be interfered with, on the lighted side. The concepts of Stark (1921), Stark and Drechsel (1922), and Brauner (1922) represented a modification of Paal's theories, tinged with a return to earlier ideas. Stark showed that the traumatotropic stimulus, or the tendency of coleoptiles to curve towards a wound, could be transmitted, just as in phototropism, across a cut surface. Tips wounded on one side, e.g. with AgNOs, were replaced on stumps and induced curvatures, in the stump, towards the wounded ' side. If tissues were crushed and mixed with agar, then the agar, if applied one-sidedly to stumps, caused curvatures towards that side. Hence he assumed that there are special wound-hormones, which inhibit growth. Similarly he assumed special phototropo-hormones, (active in phototropism), which increase the growth rate (cf. footnote to p. 114). His evidence also indicated that these tropo-hormones had definite specificity. Brauner (1922) placed unilaterally-illuminated tips on unilluminated stumps, and unilluminated tips on unilaterallyilluminated bases; in both cases a curvature resulted, while the illumination of the stump alone caused no response. He explained these results in terms of an increased permeabil- 154 PHYTOHORMONES ity on the lighted side, which he afterwards (1924, 1935) was able to measure directly. The increased permeabiUty was assumed to allow increased transmission of growthinhibiting substances on the Ughted side. Seubert (1925) carried out experiments which can be regarded as an extension of those of Brauner. She illuminated coleoptile stumps unilaterally and found that if agar blocks containing sahva were then placed symmetrically upon them they curved towards the light; otherwise they did not. Brauner's explanation makes it clear on how slight an experimental basis the suggested relation between growth substances and phototropism was founded. There were 4 possible explanations, namely: 1 . Increased transmission of growth-promoting substance on the dark side (Boysen Jensen), 2. Decreased transmission of growth-promoting substance on the Ught side (Paal), 3. Increased transmission of growth-inhibiting substance on the light side (Brauner), 4. Decreased transmission of growth-inhibiting substance on the dark side. All but the fourth were held at different times by different workers, and it was some years before it was shown that in fact both 1 and 2 simultaneously occur (Cholodny, 1927; Went, 1928). B. The Cholodny-Went Theory In 1924 Cholodny published the first of a long series of valuable papers dealing with the relation between tropisms and growth hormones. Beginning with an investigation of roots he showed that decapitation removes the sensitivity of the stump to gravity, but that placing a coleoptile tip or a root tip upon the cut surface restores the geotropic sensitivity. In a somewhat similar way he later showed (1926), (c/. p. 75), that Lupinus hypocotyls from which the central cylinder had been bored out lose their geotropic sensitivity, but regain it if the tip of a Zea Mays coleoptile TROPISMS 155 be inserted into the cavity (see Figure 47). He thus arrived (1926) at a number of important conclusions. In logical order these are: (1) "growth hormones play an essential role in the mechanism of the geotropic reaction"; (2) ''in vertically placed stems and roots the growth-regulating substances are equally distributed on all sides"; (3) "as soon as these organs are placed in a horizontal position, Fig. 47. Geotropism of hypocotyl cylinders of Lupinus angustifolius, from which the central cylinder has been removed. Upper left, four boredout controls, no geotropic response; lower left, three intact controls, normal response. Right, six plants in the hollow of each of which a coleoptile tip of Zea Mays has been placed, as in diagram; geotropic sensitivity restored. (From Cholodny, Jahrb. vnss. Bot. 65: 447-459, 1926.) the normal diffusion of the growth hormones is disturbed; the upper and lower cortical cells now obtain different amounts of these substances." This unequal distribution is ascribed to a physiological polarity induced by gravity, a polarity which he had tentatively postulated in 1918; (4) the opposite signs of the reactions of roots and shoots fit in with the fact that they react in opposite ways to the growth hormones coming from their tips; (5) he concludes that "the problem of geotropism can be traced back to the much simpler problem of the chemical control of growth." This passage recalls Blaauw's (1918) statement that "the 156 PHYTOHORMONES problem of phototropism in itself has become void," phototropism being simply a phenomenon of growth. Simultaneously Went (1926) came to the same conclusions as Cholodny, from his experiments on the isolation of auxin from the coleoptile tip; ''geotropic perception is caused by a polar alteration in the coleoptile cells . . . instead of moving rectilinearly the growth regulators are more strongly conveyed towards that side which under geotropic stimulation was turned downwards." Consideration of the hterature on phototropism led Cholodny shortly after (1927) to the view that the apparently conflicting results of different workers could be brought into agreement by adapting his hormonal theory of geotropism as follows: "The cells of the coleoptile first become polarized under the influence of the unilateral illumination, and this causes the continuously produced growth hormones to diffuse from the light towards the light dark side more rapidly than in any other di- rection." No sooner had this theory of phototropism been formulated than it was independently Fig. 48. Col- stated, and this time with experimental proof, eoptiie tip, ar^y Went (1928). After having found that the the^auxfn from growth hormone of coleoptile tips (auxin) can the light and be determined quantitatively by the curvature tis iS? septest (see III C) and that without auxin no arate blocks, growth occurs, he coucludcd that a phenomeDegree of shad^^^ ^^ differential growth must be analyzed ing indicates »!.,.,. . , • i ,• tt relative in terms of differential auxin relations. Mavamounts of j^g found that auxin itself is not affected auxin CO ec e . ^^.^^^jy j^y hght, he looked for a difference in the production or distribution of auxin on the two sides of phototropically stimulated coleoptile tips. Coleoptiles were exposed unilaterally to hght of a suitable intensity, the tips then cut off and placed upon two blocks of agar separated by a razor-blade in such a way that the auxin from the two sides diffused into two different blocks TROPISMS 157 (see Figure 48). He found that more auxin diffuses out of the dark than out of the Hght side, and that the amount coming from the dark half was more than the amount coming from half an unilluminated tip. This shows that the principal effect of unilateral illumination is to cause the lateral transport of some auxin from the light to the dark side. On the basis of these experiments and concepts the general theory of plant tropisms, sometimes called the CholodnyWent theory, may be formulated as follows (see e.g. Cholodny, 1929) : Growth curvatures, whether induced by internal or by external factors, are due to an unequal distribution of auxin between the two sides of the curving organ. In the tropisms induced by light and gravity the unequal auxin distribution is brought about by a transverse polarization of the cells, which results in lateral transport of the auxin. ''These researches, which are grouped around a central idea, allow us to approach step by step to the solution of one of the most interesting problems of physiology. From the standpoint of the history of science, the present ^ state of the problem is of the greatest interest. Gradually there emerges from a chaos of facts the splendid form of a theory which promises to unite and to coordinate, in the very near future, the enormous mass of varied experimental data into a single principle" (Cholodny, 1927). This prophetic statement has to a large extent come true, and it will now be necessary to consider the ''enormous mass of experimental data" in more detail. In succession we shall treat geotropism, phototropism, and other tropisms so far as they are related to our subject. II. GEOTROPISM C. Geotropism of Shoots An interesting forerunner of the Cholodny-Went theory is Loeb's view of geotropism (1917). His experiments on » 1927. 158 PHYTOHORMONES the geotropism of Bryophyllum stems were tentatively interpreted on the basis of "specific geotropic substances or hormones . . . having the tendency to collect on the lower side of a horizontally suspended stem." His subsequent change of view is discussed in II E. Some experiments of Gradmann (1925) were at first thought to be in opposition to the above theory. He split stems of Lahiatae longitudinally and placed them horizontally : those with the cut surface uppermost curved normally and showed considerable growth, but those with the cut surface downward showed little growth or curvature. A large number of variations on this type of experiment gave similar results, and his general conclusion was that, in an intact organ, the influence of gra\dty produced geotropo-hormone only in the lower half and this then accelerated growth of this half. Isolated upper halves would thus have no such hormone. His interpretations have, however, been criticized both by Cholodny (1927, 1931) and Dolk (1930) on the ground that he completely neglected the effects of the wound, which are very great (see section J). It remained for Dolk (1929, 1929a, 1930, 1936) to show that the Cholodny-Went theory applies completely and in its simplest form to the case of geotropism in Avena. He first showed that neither upon rotating coleoptiles horizontally nor upon returning them to the vertical position was there any change in growth rate, i.e. there is no ''geogrowth reaction" (see also Cholodny, 1929a). This was afterwards confirmed by growth measurements made during the actual geotropic curvature by Navez and Robinson (1933). Correspondingly the total amount of auxin diffusing out of coleoptile tips is the same whether they have been placed horizontally or vertically. However—and this is the important point—the amount diffusing out of the lower half, when they have been placed horizontally, is 62.5 per cent, and that diffusing out of the upper half 37.5 per cent, of the total. Exactly the same distribution was found for Zea Mays coleoptile tips. (Navez and Robinson [1933] TROPISMS 159 subsequently found a somewhat greater difference, the lower side giving 73 per cent of the total auxin.) Hence gravity has no effect on the total auxin production but affects only its distribution between the two sides. Dolk found this difference in distribution to appear within 15 minutes after placing coleoptiles horizontally, and to disappear again within one hour after the horizontal exposure has ceased. In isolated coleoptile cylinders which have been symmetrically provided with auxin-agar and placed horizontally, the same distribution takes place and the same ratio, 38 to 62, was obtained. Polar distribution therefore may take place in any zone of the coleoptile. Dolk also measured the curvature of different zones of the Avena coleoptile as a function of time, and this brought to light a number of interesting points. Firstly, if the exposure to gravity is short, the curvature of the base is delayed until curvature of the upper zones is well under way, but if the exposure is 30 minutes or more all but the most basal zones begin to curve at about the same time. From this, and other experiments on bases provided with auxin and exposed to gravity, we may conclude that all zones are able to perceive gravity by redistributing their auxin, but that the redistribution in the basal zones only leads to curvature if the exposure has been 30 minutes or longer. In this respect the response is different from that to light (see XH). Secondly, as mentioned above, the unequal distribution does not last long after the stimulus is removed, and hence the curvatures do not go on increasing for more than 40 minutes after exposure. On the contrary, they begin to decrease from the top down, so that the zone of maximum curvature appears to migrate towards the base; this straightening Dolk explained in terms of the food factor (cf. YD). It thus appears that all the peculiarities of the geotropic reaction are explicable in terms of unequal auxin distribution {cf. also discussion in X H, p. 176). Dijkman (1933, 1934) has made a similar study of geotropism in Lupinus hypocotyls. Having first established 160 PHYTOHORMONES that gravity has no effect on the total production of auxin, he attempted to correlate quantitatively the unequal auxin distribution with the unequal growth distribution. The unequal auxin distribution begins half an hour after the plants are placed horizontally and the curvature begins at the same time. He showed that for these objects, as for Avena, straight growth is proportional to auxin applied, and he could thus calculate how much growth difference between the two sides of the geotropically curved organ would be expected from the observed auxin difference. He found that from 70 to 100 per cent of the growth difference may be accounted for by the auxin difference. It is possible that the pH difference (see below), if real, plays also a minor role in such curvatures, but on the whole the confirmation of the Cholodny-Went theory is excellent. Van der Laan (1934), in an investigation on the effect of ethylene, confirmed the older observation that in Vicia Faba seedlings exposure to ethylene causes downward bending of the shoots and found that this is correlated with an unequal auxin distribution such that the upper side has more than the lower. This, of course, is to be distinguished from the increase of auxin in the lower half of horizontally placed stems in pure air. The curious behavior of these seedlings in ethylene is thus explained. Boysen Jensen (1936a) has applied the chloroform extraction method of Thimann (see IV ^) to the direct determination of the auxin in the upper and lower halves of geotropically stimulated stems of Phaseolus and Vicia Faba. He finds that the distribution between the two sides is of the same order as that found by Dolk and Dijkman. However, the difference in auxin does not seem numerically great enough to account for the difference in growth. Boysen Jensen explains this by reference to a suggestion of Thimann (1934) that some of the auxin in the plant is present in a bound non-diffusible form, but that this is extracted by chloroform along with the free or active auxin (see discussion in VIII F). Hence the real difference between the free TROPISMS 161 auxin on the two sides is somewhat greater than that found. Another effect may play a role here. It has been found by Gundel (1933) and confirmed by Metzner (1934) that the pH of the lower side of horizontally placed stems is about 0.1 pH unit more acid than that of the upper side. According to Bonner (1934) this would increase the auxin which is in the active, free acid, form, by about 20 per cent, and thus the same total amount of auxin would give a greater growth on the lower side than on the upper. There are two apparent disagreements with the theory so far. One is the difficulty raised by Beyer (1932) that Taraxacum, Helianthus, and other stems and hypocotyls, just before or after they cease growing altogether, still give geotropic curvature. It might certainly be expected that if the auxin present were not enough to cause growth it would not be enough to cause curvature. Du Buy (1936) has shown, however, that in aging material a certain minimum concentration of auxin must be present before any growth can occur — a "threshold concentration." Beyer's material, just finishing its growth, was evidently in this state, and hence the increased concentrations of auxin on the lower side, produced by gravity, would be just sufficient to exceed this threshold and so cause growth to begin again. The other disagreement is the fact, discovered by Schmitz (1933) that mature nodes of grasses, which have ceased growing and which yield no auxin, begin to produce some auxin again if they are placed horizontally. This, of course, is connected with the well-known exceptional behavior of mature grass nodes which are still capable of geotropic response. It does not, however, agree with the CholodnyWent theory because there is here an apparent new formation of auxin upon stimulation. It must be left for the present as an unexplained curiosity. D. Geotropism of Roots The fact that auxin, as has been shown in IX B, inhibits the growth of roots (in all but the minutest concentrations), 162 PHYTOHORMONES makes it clear in what direction we must look for an explanation of the geotropism of roots. Unfortunately the facts are still partly in dispute. The experiment of Cholodny, mentioned above (1924), indicates that auxin is necessary for the response of the root to gravity (cf. Keeble, Nelson, and Snow, 1931; Keeble and Nelson, 1935). On the other hand, isolated roots in culture appear neither to contain nor to produce any auxin, and yet they are still able to curve geotropically (Fiedler, 1936). A possible explanation for this discrepancy may be sought in Fiedler's observation that his roots developed traces of chlorophyll when in agar media. This, as shown in IV A and B, would lead to the continuous production of traces of auxin, which might suffice for geotropic response, although not enough auxin would accumulate to be detectable by extraction methods. Since it is always difficult to prove a negative, i.e. the absence of auxin, we may for the present consider only the positive evidence. It is important that, although Keeble, Nelson, and Snow (1931) state the contrary, the growth rate of the root does not depend on its position in regard to gravity, i.e. there is no ''geo-growth reaction" (Cholodny, 1932; Navez, 1933). Hawker (1932) investigated the distribution of auxin in geotropically stimulated root tips. Zea Mays roots were placed horizontally and after 3 hours the tips were cut off and separated into upper and lower halves. These halves were then applied to half the cut surface of unstimulated root stumps, and the curvature of the stumps measured. The half of the tip which had been the lower during stimulation gave three times as big a curvature as that which had been the upper. Hence auxin passes to the lower side of the root under the influence of gravity and thus the auxin distribution in roots is the same as that in coleoptiles. Direct confirmation of this by the diffusion method was obtained soon after by Boysen Jensen (19336). He allowed the auxin from the upper and lower sides to diffuse into separate blocks of dextrose agar. These, when tested on TROPISMS 163 Avena, showed more auxin on the lower than on the upper side. Finally, the result was confirmed (Boysen Jensen, 1936a) by the chloroform extraction method, the auxin distribution found, 54 : 46, agreeing exactly with the ratio of the growth on the two sides during the action of gravity. This was not the case for stems, as mentioned above. It is of interest that roots which have been treated with eosin or erythrosin lose their geotropic sensitivity, as was shown by Boas and Merkenschlager (1925), and such roots were correspondingly found by Boysen Jensen (1934) to be almost devoid of auxin. Of a group of roots treated, some still gave positive geotropic curvature, some curved, but not in the vertical plane (''transversal") and some were completely ageotropic. The relative amounts of auxin obtained from these groups by diffusion into dextrose agar were 1.05, 0.49, and 0.08 respectively, against 1.26 for untreated controls. Geotropic response is thus proportional to the amount of auxin in the root. This action of eosin has been explained by Skoog (1935), who showed that traces of eosin cause rapid photodynamic inactivation of solutions of indole-acetic acid. No such inactivation occurs in the dark. The findings of Dolk can be used to explain the results of Keeble, Nelson, and Snow (1929), who carried out an experiment on roots comparable to that of Brauner (1922) on coleoptiles. They found that geotropically stimulated root tips of Zea Mays would induce curvature in unstimulated stumps. Correspondingly, unstimulated root tips would induce curvature in stimulated stumps, which alone do not curve. The transverse polarization of the root cells, leading to lateral transport of auxin, can thus take place in the lower zones as well as in the tip, but, in order that a markedly unequal distribution may be reached, an auxin supply from the tip must be provided. Altogether the relations between geotropism and auxin in the root are, except for the experiments on isolated roots, in complete agreement with the Cholodny-Went theory. 164 PHYTOHORMONES E. The Mechanism of Geotropic Perception It is interesting to recall that Czapek's theory of geotropism (1902) comes remarkably close to the auxin explanation in one particular respect. According to this theory, on geotropic (or phototropic) stimulation of plants, the reducible substances inside the cells increase, this being due mainly to the formation of homogentisic acid from tyrosine. The remarkable fact about this is that homogentisic acid, HOn -CHo COOH is closely related to phenyl-acetic acid, H —CH2 COOH which has definite growth-promoting activity ("primary activity") in the pea stem test. A number of attempts have been made to explain the way in which gravity may cause the unequal distribution of auxin. Two pieces of evidence indicate that the same basic principle underlies all geotropic reactions. In the first place, Navez (1929) has compared the temperature coefficient for the respiration of Vicia Faba seedlings with the temperature coefficients for the geotropic presentation and reaction times of the roots. Between about 5° and 21° all have the same value {fx = 16,200), from which he concludes that the geotropic response is governed by an oxidative or respiratory reaction. The role of a respiratory reaction in the response to auxin has been discussed in Chapter VIII. In the second place, the percentage of the total auxin which diffuses out of the lower half of horizontally placed shoots or roots of different plants varies within rather narrow limits, as Table XIII (ampHfied from that of Boysen Jensen, 1936a) shows. It may be concluded from the uniformity of the results with different plants that the basic factor inducing lateral auxin transport is the same throughout. A number of theories as to the mechanism of auxin dis- TROPISMS 165 tribution depend upon electrical changes. Cholodny (1926, 1927) suggested that the transverse movement of auxin is produced by an e.m.f., which is itself caused by the action of gravity. Such an e.m.f. had been observed first by Bose (1907) and was later studied by Brauner (1926, 1927, 1928). The latter found that if plant organs are placed horizontally a potential difference of the order of 5-10 mv. is established across them, the upper part always becoming negative to the lower (''geo-electric effect"). Roots and shoots behave in the same way. This behavior is not limited to living tissue, for even two electrolyte solutions separated by parchment paper show the same effect. The effect depends upon the charge of the membrane and the concentration of the electrolyte. Although Brauner and Amlong (1933) suggest a different interpretation, it seems probable that we have to do with a streaming potential between liquid and membrane. TABLE XIII Geotropicallt Stimulated Coleoptile tips of Avena (Dolk, 1930) " " " (Navez and Robinson, 1933) " " Zea Mays (Dolk, 1930) Root tips of Vicia Faha (Boysen Jensen, 19336) Root tips of Zea Mays (Hawker, 1932) Hypocotyls of Lupinus (Dijkman, 1934) Epicotyls of Vicia Faha (van der Laan, 1934) Per Cent of Auxin Diffusing from Upper Side 38 28 37 Lower Side 62 72 63 37 166 PHYTOHORMONES moved inside the plant by electric potentials, no direct evidence to prove this has ever been brought forward. In the experiments of Koch (1934) it appears that auxin is moved within Helianthus hypocotyls by the application of 10-100 mv. externally, but this deduction is founded only upon curvatures and not on direct auxin determinations. It is possible that these curvatures away from the + pole are due to the production of acid around this pole which would set free auxin (see VIII i^). As has been indicated on p. 103 the evidence that electric potentials play a part in auxin transport is as yet inconclusive. III. PHOTOTROPISM F. Auxin REDisTmsuTiON We have seen that in geotropism the Cholodny-Went theory quantitatively explains the observed facts. In phototropism, however, the situation is far more complicated, and it will therefore be desirable to differentiate clearly between the auxin distribution effect and the other phenomena. We shall therefore give up the historical order of treatment and consider first the simple auxin redistribution effect and afterwards the many complicating factors. As a beginning let us consider the Avena coleoptile. In the first place, the sensitivity of the extreme tip (the uppermost 0.25 mm.) to hght is about 1000 times as great as that of the lov/er zones (Sierp and Seybold, 1926; Lange, 1927). In the second place the curvature varies periodically with the amount of light (see curve. Figure 49). Small illuminations up to 4000 MCS ^ produce the so-called "first positive curvature"; the maximum curvature is reached at about 200 MCS. Above this and up to about 40,000 MCS the plant curves away from the light —''first negative curvature." With still more Hght there occurs a second and then a third positive curvature separated by a zone of indifference or of slight negative curvature. The third positive 1 MCS = meter-candle seconds, 25 meter-candles at 4360 A.U. being equal to 1 erg/cm^. TROPISMS 167 curvature is that which appears in ordinary dayhght (see: du Buy and Nuernbergk, 1934). All these curvatures may be brought about by illumination of the tip alone; illumination of the base gives only positive curvatures. That the curvature depends upon a gradient of light across the coleoptile was conidncingly shown by Buder (1920) and subsequently worked out for decapitated and regenerated coleoptile bases by du Buy (1934). The gradient can +75" 168 PHYTOHORMONES TABLE XIV Amount OF TROPISMS 169 effect, phototropic curvatures continue to increase for 6 hours or so after illumination (see X H) . The above considerations hold only for auxin redistribution in the tip. Since the extreme tip is sohd {cf. Figure 6, p. 23), it is clear that auxin redistribution can take place much more readily in it than in the lower, hollow zones. Thus, it was shown by Sierp and Seybold (1926) and by Lange (1927) that the minimum amount of light necessary to produce curvature is, if the upper 0.2 mm. are illuminated, 20 MCS; in the 0.2 mm. zone 0.8-1.0 mm. from the tip, 500 MCS; while if a 0.2 mm. zone 2 mm. below the tip is illuminated 20,000 MCS are required. We can now explain the experiment of Brauner (1922), mentioned above, in which unilluminated tips, replaced on pre\dously illuminated stumps, caused a curvature. This result is due to the persistence of the auxin-redistributing system after unilateral illumination has ceased. The fact that the illuminated stumps not provided with tips curved scarcely at all is due to the necessity of auxin for growth (and curvature). Instead of the tip a block of agar containing auxin is equally effective in allowing curvatures to take place in the stump (Seubert, 1925; Boysen Jensen, 1933; Reinders, 1934). The effect of the decapitation itself in temporarily inhibiting lateral transport (Nuernbergk, 1933) results, however, in a delay in the onset of such curvatures. The fact that phototropic curvature can only occur if sufficient free-mo\4ng auxin is present {i.e. curvature is dependent upon growth) is brought out very effectively by the work of Tsi Tsung Li (1934). A large number of coleoptiles were decapitated and then illuminated unilaterally at different time intervals after decapitation. With very short time intervals between decapitation and illumination they still gave a slight curvature, due doubtless to redistribution of the free-mo\'ing auxin still present, but afterwards the ability to curve became extremely small, i.e. the ''free" or redistributable auxin (see VIII F) almost disappears. Finally, as soon as auxin was regenerated the curvatures 170 PHYTOHORMONES increased markedly. The free or redistributable auxin disappears first from the upper zones and persists longest in the base (u). The response of tip and of stump thus both rest on the same phenomenon, namely the redistribution of free auxin. The two responses differ, however, (1) in the amount of light needed to bring them about, (2) in their spectral sensitivity, and (3) in their reaction times (Haig, 1935). Different phototropic sensitivity in different parts of the spectrum must be due to unequal absorption of light by the light-sensitive system in the coleoptile. Because of the role of auxin in the production of phototropic curvatures, it has been assumed that the greater sensitivity to blue and violet light is due to the absorption of these shorter wave-lengths by auxin. This, however, is not the case; the auxins have no pronounced absorption spectrum in the visible, while the phototropic sensitivity curve shows maxima at about 440 and 475 m/>t. Wald and du Buy (1936) have pointed out that the spectral sensitivity of Avena corresponds very well with the absorption spectrum of carotene, of which they showed small amounts to be present in etiolated coleoptiles. Castle (1935) has made a similar comparison for the phototropic sporangiophores of Phycomyces. The bulk of the curvature in the Avena coleoptile can thus be explained as due to the asymmetrical distribution of auxin, although more extensive data are very desirable. Just how the light can produce this distribution, and what role is played by the different pigments remains to be found out. Our knowledge of auxin redistribution under the influence of unilateral light is even scarcer in other objects than in Avena. Only in Raphanus has a complete set of data been collected (van Overbeek, 1933). These may be summarized here. The distribution of auxin diffusing from the apical 10 mm. of the hypocotyl in darkness was symmetrical; after 3 hours' continued illumination, however, the light side gave off only 15 per cent, the dark side 85 per cent of TROPISMS 171 the total. In hypocotyl cylinders supplied apically with auxin agar the same redistribution was observed; the length of these cylinders was only 6 mm. and thus one may calculate that the observed auxin difference (37 per cent on light and 63 per cent on dark side) corresponds quantitatively with the greater difference in the hj^pocotyl tips 10 mm. long. Van Overbeek also ascertained that the lateral auxin transport is induced only by wave-lengths less than 5460 A.U., i.e. by those causing phototropic curvature. The phototropic curvature of Phaseolus epicotyls is also accompanied by asymmetric distribution of auxin. Boysen Jensen (1936a) determined the auxin in the light and dark halves of the epicotyl by the chloroform method and found about one third of the total on the Ughted side and two thirds on the dark side. As already mentioned, the way in which unilateral light brings about this lateral transport is not understood. Light has no direct effect on the longitudinal transport of applied auxin through cylinders of A vena or Raphanus (see VI C), so that the explanation of du Buy (1933), based on decreased transport rate on the lighted side, cannot be valid. As with gravity, attempts have been made to ascribe the curvatures to electric potentials. Brauner (1927) found that unilateral illumination of Hordeum nodes caused the light side to become negative to the dark side. Waller (1900) and Bose (1907) had found similar effects. This potential would be in the right direction to produce an electrolytic transport of the anion of auxin, but there is no evidence that it does so. It is, however, a remarkable coincidence that both in geotropism and in phototropism the auxin moves to the side which becomes positive, as would be expected from its acid properties. G. Light-Growth Reactions As stated above, the asymmetric distribution of auxin is only one of the factors operating to produce phototropic curvatures. There are a number of other effects 172 PHYTOHORMONES of light on growth. We can imagine Hght having an effect on (1) auxin production, (2) auxin transport (already considered), (3) auxin destruction, or (4) on the reactivity of the plant to the auxin in it. Effects of light on permeability or on the protoplasm directly would appear, in most cases, under the last heading. As we have seen, the effect of light on lateral transport can only take place in an intact system or structure in which there is correlation between the different parts. In other words, the Cholodny-Went effect depends essentially on the behavior of the organ as a whole. This is shown very clearly by the experiments of Boysen Jensen and Nielsen (1926) and Boysen Jensen (1928), which show that direct contact between the light and dark sides of the coleoptile tip must be maintained in order to obtain good phototropic curvature. Insertion of platinum foil through the middle of the tip, perpendicular to the plane of the beam of light, practically prevents curvature. The other effects listed above, however, can take place at a single point, and each cell may react independently, its reaction being determined by the amount of light it receives. These reactions may be grouped together as light-growth reactions, and as such have been studied intensively by Blaauw (1914, 1915, 1918) and successors (Vogt, 1915; Sierp, 1918, 1921; Koningsberger, 1922; Van Dillewijn, 1927), and in Phycomyces by Castle (1930). In general, the originally constant growth rate of dark-adapted or dark-grown plants changes after illumination. This light-growth reaction in higher plants consists principally of a decrease in growth rate, with a maximal retardation M to 13^ hours after illumination, followed by an acceleration. When plants are unilaterally illuminated it is evident that the sides towards and away from the light will receive different amounts of light, and hence will give different light-growth reactions; this will in itself cause phototropic curvatures. However, the relative parts played in phototropic curvature by these light-growth reactions and by the auxin redistribution remains a point of controversy. TROPISMS 173 It would be out of place to review the publications dealing with the qualitative comparison of light-growth reaction and phototropic curvature; our concern is with the auxin side of the phenomenon. The simplest case is found in hypocotyls of Raphanus (van Overbeek, 1933), which probably are comparable in their behavior to those of Helianthus studied by Blaauw (1915). If Raphanus seedlings are exposed to strong continuous light (1000-2000 meter-candles) 20 15 10 (I 25 50 75 100 Fig. 50. Auxin curvatures of Raphanus hj^pocotyls. Ordinate, curvature in degrees; abscissa, concentration of auxin applied unilaterally. Curve D, in darkness; L, in light of about 2000 meter-candles. (From van Overbeek, Rec. trav. hot. neerl. SO: 537-626, 1933.) their growth is reduced by from 50 to 60 per cent. This reduction in growth rate is not due to decreased auxin production or transport, nor to increased destruction (see, however, pp. 88, 175) ; it must therefore be due to a decrease in the reacti\dty of the cells to auxin. The same effect may be seen from the effect of light on curvatures produced by one-sided application of auxin (see Figure 50). An exactly similar effect of light in reducing straight growth from applied auxin was found in Vicia Faba stems by Thimann and Skoog (1934). This reduced reacti\'ity of plant tissues in hght undoubtedly plays a part in phototropic curvatures. Van Overbeek calculated that about half the phototropic curvature of Raphanus hypocotyls is due to auxin redistribu- 174 PHYTOHORMONES tion (Cholodny-Went effect), and about half to this Ughtgrowth reaction (Blaauw effect). With Helianthus hypocotyls Blaauw measured lightgrowth reactions for different intensities and calculated the difference between the growth rates on the two sides, having previously found that the light side received 3^4 times as much light as the dark side. This difference accounted quantitatively for the curvature observed. Phototropism in Helianthus would thus appear to be largely accounted for by the Blaauw effect. In Avena, however, the Blaauw effect, as we have seen above, plays little or no part in phototropic curvatures at low light intensities. This is confirmed by experiments in which a light-growth reaction was observed without any accompanying phototropic curvature (Beyer, 1928) or a phototropic curvature without any light-growth reaction (Cholodny, 1932a, 1933a). The observed light-growth reaction of Avena at these intensities is apparently due to a reduction of about 20 per cent in the amount of auxin diffusing out of the tip within the first hour after illumination (Went, 1928). This corresponds well with the observation that this type of growth reaction (the '4ong reaction") occurs only after illumination has fallen on the tip of the coleoptile. A second type of light-growth reaction (the ''short reaction") reaching its maximum half an hour after illumination, occurs when the basal zones are illuminated. The response is not proportional to the total amount of light energy, as was shown by Burkhardt (1926), but appears to be more of the nature of a shock reaction or typical "stimulus"; it is ascribed by van Overbeek (1936a) to a transient decrease in sensitivity to auxin. It was shown by Cholodny (1930) that plants subjected to a gradual increase of unilateral illumination did not give this reaction, although their phototropic response was still normal. If, on the other hand, the light were applied to them suddenly, this lightgrowth reaction was very marked. In this connection an analysis of the effect of all-sided TROPISMS 175 illumination on auxin curvatures has been carried out byvan Overbeek (19366, 1936c). The effect depends upon the auxin used. If indole- acetic acid is applied, light causes a temporary decrease in rate of curvature, followed by an increase, so that the net effect is small, while if auxin a is used, light produces a considerable reduction in the curvature. On the other hand, if the plants are illuminated for 2 hours before the auxin is applied, the curvatures produced by indole-acetic acid and auxin a are both affected in the same way, i.e. they are increased. Thus we must conclude (1) that previous lighting increases the sensitivity of coleoptiles to both auxins, but that (2) lighting while the auxin is present decreases the response to auxin a only. The two effects could be interpreted as due to destruction of auxin a inside the coleoptile in the light. Thus, in (1), the increased sensitivity to applied auxin would be due to a reduced auxin content of the coleoptile, while in (2) the applied auxin a would be partially inactivated as soon as it enters. We saw in VI C that light has no effect on auxin transport, but on the other hand the auxin which is determined in transport experiments may be distinct from the auxin in the cell (c/. Table XI, p. 132). Hence light may inactivate only the auxin inside the cell, and not the transportable auxin. Another effect of light is to cause the formation of auxin in green parts, as discussed in Chapter IV A. Since the greening of etiolated plants is itself brought about by hght, this factor may also play a part in long-period illumina- tions. The so-called euphotometric movements, or bending of leaves out of the shade into the light (see e.g. Raydt, 1925), are growth reactions of the petioles. These are apparently due to a differential distribution of auxin in the petiole, caused by differential illumination of various parts of the leaf-blade {u). Laibach and Fischnich (1936a) have imitated the movements by applying dots of auxin paste on the leaf-blade of Coleus; if applied at one side the petiole 176 PHYTOHORMONES would bend away from the place of application. Our own experiments (u) have given similar results. H. Comparison of Phototropic and Geotropic Curvatures The similarities and differences between the different types of curvature in Avena coleoptiles are difficult to appreciate Geotropic Geotropic Decap, Phototropic Auxin Fig. 51. The course of curvatures in Avena coleoptiles. Ordinate: extent of curvature of each zone. Abscissae: towards right, time in minutes; towards background left, successive 2 mm. zones. At left of each diagram is a coleoptile corresponding with these zones, at the start of measurements. Top left: after 30 minutes horizontal; curvature already beginning in upper zones. Top right: the same, but decapitated immediately afterwards; autotropism delayed until after regeneration. Lower left: phototropic. Lower right: unilateral auxin application, the top two zones being removed. (After Dolk, 1930.) from the somewhat detailed discussion above. Dolk (1930, 1936) has, however, made a comparative study of the increase of curvatures with time and with distance from the tip, which we are now in a position to interpret. Figure 51 shows a three-dimensional composite picture in which the magnitude of curvature—represented by the ordinate—is plotted against time for each 2 mm. zone of the coleoptile. The plants were rotated on a clinostat throughout so that gravity could not counteract the curvatures. TROPISMS 177 The fourth diagram shows a curvature produced by unilateral application of auxin. After an interval of 35 minutes, in which no curvature takes place, curvature begins sharply in the upper zones 3 and 4 (zones 1 and 2 being removed by decapitation). It then spreads downwards from zone to zone at an average rate of 10-12 mm. per hour, the rate being somewhat faster in the upper zones. This rate agrees exactly with the transport velocity of auxin found by van der Weij (c/. VI C). At 110 minutes after application of the agar block (= 150 minutes after decapitation) regeneration begins, and immediately the curvatures in the uppermost zones decrease. The lower zones continue to increase in curvature so that the total curvature does not decrease but moves down the coleoptile. The final state of the plant, after 33^ hours, thus shows the curvature located mainly in the base. The third diagram (Figure 51) shows the curvature of an intact plant after one-sided illumination with 500 MCS. At first sight it resembles the auxin curvature. There is an interval of 40 minutes, after which curvature begins in several upper zones. The rate of movement of the initial curvature is somewhat faster than the above, about 16 mm. per hour; this agrees with Went's finding (1935) that in the intact plant auxin transport is slightly faster than when decapitated. The other characteristic of this diagram is that the curvatures do not decrease, but continue to increase in all zones for at least 3 hours. This agrees with the fact that the auxin redistribution caused by light persists for many hours (see p. 168). The final state of the plant therefore shows a curvature throughout. The first diagram shows geotropic curvature of plants placed horizontally for 15 minutes. The difference may be seen at a glance. The interval before curvature begins is only 20 minutes and the first five zones then all begin to curve simultaneously. This is due to the detection of gravity, with its resulting redistribution of auxin, not only by the tip but also by the zones below. In the sixth and lower zones the presentation time is 178 PHYTOHORMONES greater than 15 minutes and therefore curvature in these zones is dependent on transport of the redistributed auxin from above. From the fifth to the tenth zone the initial curvature therefore moves downwards at 12 mm. per hour, the rate of auxin transport. After only 30 minutes of curving the curvature begins to decrease very rapidly in the uppermost zones, so that they are straight within 2 hours ("autotropism"). This is due to the fact that the gravitational auxin redistribution does not persist and the autotropism is ascribed, as we have seen in V D, to the temporary exhaustion of the food factor at the point where growth has been rapid. The zone of maximum curvature is thus quite narrow and moves rapidly down the plant. The final state of the plant thus shows a very localized curvature in the base, the upper part being almost entirely straight. In decapitated plants this autotropic straightening is very much less marked, and is delayed until regeneration begins, 150 minutes after decapitation. This affords clear proof that autotropism is dependent upon a supply of auxin. Thus it may be seen that all the peculiarities of the curvatures in Avena may be satisfactorily explained in terms of what we know about auxin and its distribution. IV. OTHER TROPISTIC RESPONSES J. Traumatotropism The curvature of plants towards or away from a wound — traumatotropism—was first ascribed by Stark (1921) to the influence of special traumatotropic hormones which were produced in the damaged tissue. Beyer, however (1925), made it clear that all organs which curve towards a wound are those whose growth is retarded by removal of the tip. Correspondingly, if the tip be removed, such organs lose their ability to curve towards the wound. Hence he deduced, in agreement with Paal, that the curvatures are due to interference with the normally present growth-promoting substance, rather than to the production of a growthinhibiting substance. He also found that even in decapitated TROPISMS 179 Avena coleoptiles, curvatures could be induced if the wound was close to the base. This is doubtless due to interference with the upward-moving stream of substance from the seed (c/. YD). In general, all positive traumatotropic curvatures appearing above the incision (Stark, 1917; Beyer, 1925; Biinning, 1927) may probably be ascribed to interference with the upward-moving stream of food factor. Tendeloo (1927) has shown that while Avena normally gives positive traumatotropic curvatures, regeneration of auxin production at the lower cut surface of the incision may subsequently give rise to a negative curvature. The negative curvatures obtained by Weimann (1929) may also be due to this cause. Keeble and Nelson (1935) have explained the traumatotropic curvatures of roots by interference with the distribution of auxin, and there seems no reason to doubt that this is one of the principal factors operating in traumatrotropism. The effects of wounding, however, consist not only of interference with the transport, but also involve destruction of auxin by enzymes freed from the cut cells (Thimann, 1934). This phenomenon doubtless explains the results of Gradmann and Cholodny with split stems, discussed in X C. In Cholodny's experiments spHt halves of stems, placed horizontally with the cut surface downward, curve much less than those with the cut surface upward. We may tentatively explain this by saying that the auxin which accumulates under the influence of gravity on the lower side is, in the one case, partly inactivated by the wound enzjrnaes, in the other case not. The auxin inactivation may also play an important part in positive curvatures. In conclusion, it is evident that the whole phenomenon of traumatotropism needs to be reconsidered in the light of our present knowledge of auxins. K. Electrotropism Movements of shoots and roots towards electrodes have been known for a very long time; they are of interest to us 180 PHYTOHORMONES here on account of their possible connection with auxin movements. Since all known auxins are acids, their anions will move to the anode if solutions are electrolyzed. Such a movement in electrolysis through agar was shown to occur by Koch (1934). Electrolytic transport through agar also explains the results of Kogl, Haagen Smit, and Van Hulssen (1936) who found that the auxin curvature of decapitated Avena coleoptiles was increased if a small current was passed through the agar block containing auxin, the block being made negative to the plant. The question now arises, does this electrolytic auxin transport occur inside the plant, and if so, would it explain the observed electrotropisms? The phenomena of electrotropism are not altogether clear. Roots, when immersed in water through which a current is sent, curve towards the — pole at low current densities, and towards the + pole at high current densities or after long exposure to smaller currents. In air they curve towards the — pole. Coleoptiles and shoots in air curve towards the + pole (Brauner and Biinning, 1930; Hartmann, 1932; Amlong, 1933) and in water in the same direction (Koch, 1934), In regard to roots there is evidence that the curvature is due to accumulation of ions by electrolysis (Ewart and Bayliss, 1906; Navez, 1927) rather than to a direct effect of current on the root. Nevertheless, decapitation either of roots or of shoots prevents electrotropic curvature almost entirely (Amlong, 1933), and since decapitating roots does not retard their growth (cf. IX A) the effect cannot be due to influence on the growth rate alone. This last fact would indicate that electrolytic movement of auxin does play a part, and this is supported by an experiment of Koch (1934). He inserted electrodes on opposite sides of Helianthus hypocotyls, and applied 4 volts for 1 hour, when the plants curved towards the — pole. The hypocotyls were halved longitudinally and the halves applied one-sidedly to Pisum roots. The convex side produced more curvature than the concave. On the whole, it must be said that the analysis of electro- TROPISMS 181 tropic curvatures, so far as it has been carried, is suggestive rather than con\'incing. L. Chemotropism The only study of chemotropic curvature which is at all germane is that of Amlong (1933), who applied salt solutions of different concentrations to the two sides of Vicia Faba roots; the curvatures so caused were interpreted as due to the potential differences set up (concentration cell e.m.f.'s.) and thus to a kind of electrotropism. Under this heading we might also list the curvatures caused by the one-sided apphcation of acid (see III C4 and VIII F). M. Nastic Movements By these are understood movements caused by external forces, but whose direction is determined morphologically by internal structure. The only study of the role of auxin in these movements is that of Uyldert (1931). She showed that in Tradescantia the stems give their typical epinastic response only if supplied with sufficient auxin. When the plagiotropic lateral branches are in the vertical position auxin is transported along the dorsal side only, but when in the horizontal position auxin is also transported along the ventral side. We may therefore conclude that the epinastic curvature is due to the action of gravity in causing asymmetrical auxin transport. It is thus exactly comparable to geotropism except that the auxin accumulates, not on the lower side, but on the morphologically determined side. A geotropic accumulation of auxin on the lower side may take place at the same time ; an equilibrium position is then reached when the geotropic auxin distribution is equal and opposite to the plagiotropic auxin distribution. Crocker, Zimmerman, and Hitchcock (1932) studied the epinasty of tomato petioles produced by ethylene, and found it to be due to an acceleration of growth on the upper side, with or without some shortening of the lower side. The zone which reacts is the growing zone at the base of the petiole. 182 PHYTOHORMONES Their figures give no evidence that the ethylene produces any increase in the total growth rate. It is most probable, therefore, that the effect of the ethylene is to influence the distribution of auxin in the petiole. We have seen above that gravity may do the same thing, i.e. may cause unequal distribution of auxin. In a later paper (1935), Crocker, Hitchcock, and Zimmerman have argued that since application of auxin to the petiole can also produce unequal growth, the action of ethylene is the same as that of auxin. However, by the same argument, the action of gravity should be the same as that of auxin, i.e. gravity should be a growth hormone! This shows a confusion between the primary factor (auxin) and the forces which modify it, much as though the action of gasoline on an automobile were to be compared with the changing of the gears. Another unjustifiable comparison between ethylene and auxin is referred to in XI C. CHAPTER XI ROOT FORMATION A. Root Formation as a Correlation Phenomenon In Chapter II .4 it has been pointed out that our earUest knowledge of correlation was mainly based upon root formation. Beginning with Duhamel and Sachs, various investigators have explained root formation on cuttings by the accumulation of special root-forming substances near the basal cut surface. Beijerinck (1886) emphasized the importance of the leaves for root formation, although he was apparently thinking rather of a nutritional effect. Vochting (cf. VI B) studied both root formation and root development, but with particular reference to polarity, the formation of the roots being considered rather as an indicator of polarity than as a problem in itself. Among the factors investigated, Vochting stressed the importance of water, the inhibiting effect of hght, and the tendency of gra\'ity to induce root formation on the lower side of a horizontal cutting. Of the other investigations of the various factors controlling rooting, only the more relevant need be mentioned. In the first place numerous attempts, largely unsuccessful, have been made to correlate root formation or root gro\\i;h with nutritive factors, especially with the carbohydrate: nitrogen ratio (see for instance Goebel, 1902-1903; Reid, 1924; Carlson, 1929). However, it was emphasized by i\IacCallum (1905) that nutrient conditions are not the principal factors governing root formation. Morgan (1906) has shown the same thing for regeneration in animals; the rate of formation of new legs, after the removal of the original legs, was found, in Salamanders and other animals, to be the same when they were well-fed as when they were starved. In this connection Kupfer (1907) made the interesting 183 184 PHYTOHORMONES observation that in Commelina the yellow varieties do not root while green ones do, but the difference is not due to nutrition because addition of sugar or peptone does not affect it. In the second place the effects of various empirical treatments have been studied; Curtis (1918) found that permanganate promotes rooting of cuttings, while inorganic nutrient solutions have no effect, and a number of authors have shown that oxygen is necessary {e.g. Zimmerman, 1930, Graham, 1934). Treatment with carbon monoxide, ethylene, and other unsaturated gases stimulates root formation (c/. XI C). Graham and Stewart (1931) and Graham (1934, 1936) have studied the optimum experimental conditions for making cuttings of a large number of different plants. The most important factors are (1) time of year at which the cuttings are taken, which differs for different plants, (2) temperature relations of the cutting, and (3) ample watering without interference with aeration. They found that if these factors are considered, practically any plant can be induced to give 90 per cent rooting from stem or leaf cuttings. Loeb, in 1917, suggested that root formation in Bryophyllum is controlled by a special root forming substance or hormone. About this hormone he made some statements which in the light of our present knowledge seem quite remarkable. Thus, ''In Bryophyllum the hypothetical geotropic hormone is associated (or identical) with the rootforming hormone" (1917). Further, "these (bud-) inhibitory substances may be identical with or may accompany the root-forming hormones" (1917a). The inhibition of buds is, as will be shown in Chapter XII, also brought about by auxin. Loeb's subsequent change of view from special substances to mass action relations is discussed in II E. In a later study of Bryophyllum, F. A. F. C. Went (1930a) returned to the concept of root-forming substances, these substances being considered to be formed mainly in the older leaves. ROOT FORMATION 185 B. Hormones and Root Formation The first extensive study of root formation in which internal factors were taken into consideration was that of van der Lek (1925). He distinguished clearly between roots which develop from preexisting ''root germs" or initials, and those which are really formed anew. Since the bulk of the root germs are found in the apical part of any internode, isolated internodes do not show the usual polar distribution of roots. In longer cuttings, nevertheless, the number of root germs which develop is greater in the lower internodes, and hence a general polarity still persists. Of the cuttings he studied, Ribes nigrum and most species of Salix and Populus possessed numbers of root germs, while onl}^ four, Salix caprea, Salix aurita, Populus alba and Vitis vinifera, were free from them. In these latter species the polar distribution of roots is complete, even in single internodes. In all cases, the presence of a bud powerfully promotes root formation, especiall}^ if the bud is rapidly developing. Buds which are developing in the dark or which are enclosed in plaster of Paris also promote root formation, the latter only weakly, however. Removal of the buds stops root formation almost completely, especially in the species without root germs. If a portion of cortex below the bud is cut away down to the wood, root formation is reduced, showing that some influence travels through the cortex from the bud to the base of the cutting. To explain these results, van der Lek assumes that the developing bud forms one or more hormones, which are transported downwards through the phloem. These hormones he compares to the cell-divisionpromoting hormones postulated by Haberlandt. In a later study (1934) van der Lek found that in Populus cuttings taken in December or in January, the buds, which are now completely dormant, no longer promote root development, or even slightly inhibit it, but in the course of the next two months their favorable influence returns. A corresponding effect was found in Acalypha cuttings 186 PHYTOHORMONES by Went (1929); the buds strongly, and the leaves less strongly, promoted formation of roots. In these experiments distinction was made for the first time between number and elongation of the roots. Debudded and defoliated cuttings formed very few roots, or under some conditions none.^ If, however, the diffusate from Papaija leaves were mixed with agar and appHed to the cutting, an increase in the number of roots was observed. Still greater increases were subsequently obtained by the application of diastase (which was also active after being boiled) or of extract of rice polishings (Bouillenne and Went, 1933). However, Gouwentak and Hellinga (1935) were afterwards unable to obtain rooting by the application of diastase. Sugar solution was found to have no root-forming effect, so that the action is not one of nutrition. Bouillenne and Went (1933) found that the action of the extracts is exerted only at the base of the cutting, and inversion with respect to gravity did not alter this polarity; it was therefore deduced that "the polar locaUzation of new roots is caused by the polar transport" of the hormone. The formation of roots in seedlings is closely comparable; if the roots are removed from the base of Impatiens hypocotyls then the formation of new roots is greatly promoted by the presence of the cotyledons or, in the light, by leaves. The application of sugar to the hypocotyl base in these experiments increased the number of roots formed, but the cotyledons were necessary as well; there is therefore a differentiation between hormonal and nutritive factors, the influence of the cotyledons being explained as due to storage of root-forming hormone in them. It follows from these experiments that root-formation is due to a special substance or hormone (which Bouillenne and Went named "rhizocahne"); it is not itself a nutrient, is thermo-stable and is produced by leaves in the light. It is also stored up in cotyledons and buds, and its transport is basipetally polar. 1 Graham and Stewart (1931a) subsequently obtained good rooting on isolated Acalypha leaves. ROOT FORMATION 187 The production of a similar substance by bacteria would explain the results of Nemec (1930), who obtained formation of new roots on root cuttings of Cichorium intyhus by smearing the cut surface with a culture of Bacterium tumefaciens (cf. XIII C). In a later paper Nemec (1934) confirmed the fact that cotyledons and buds store not only food materials but also 188 PHYTOHORMONES node and, after washing, their bases are immersed in 0.05 per cent permanganate for four hours. This treatment disinfects the cuttings and improves their keeping quahty; it also, according to Curtis (1918), improves rooting. The terminal bud is then removed, and the apex of the stem, split longitudinally for 1-2 cm., is immersed for fifteen hours in 1 cc. of the test solution or extract (see Figure 52). Fig. 53. Root formation on etiolated pea stems 8 days after the treatment of Figure 52. Group 5, 10 mg. ; group 7, 100 mg. indole-3-acetic acid per liter; group 9, water. Note auxin curvatures of slit tops in 5 and 7. Ten plants per group. The test solution is applied at the apex because the substance is transported polarly from apex to base, as shown above. Only with very high concentrations of active substances can roots be formed by application at the base (see XI E). After the treatment, the split apex is rinsed. If the test solution contained auxin, the split halves will show the inward curvature described in III D. Finally the cuttings ROOT FORMATION 189 are placed for seven days with their bases in 2 per cent sucrose solution, followed by seven days in water. The number of roots, which by this time has reached its final value, is then a measure of the root-forming acti\'ity of the test solution (see Figure 53). The concentration necessary to produce one root under constant conditions is termed one root unit per cc. The usual procedure is to make serial dilutions of each solution to be tested, at least 10 plants being used for each dilution. To obtain reproducible results the sucrose should be purified by slightly acidifying and extracting with ether, since sucrose always contains a number of physiologically active substances. The ether extract so obtained is definitely toxic to pea cuttings (m). The buds of Pisum contain some of the root-forming substance, just as do the buds of Acahjpha mentioned above. This is shown by the fact that if all buds are removed, practically no roots are formed ; the number of roots formed—in plants not treated with any active solution—then depends quantitatively on the number of buds left on. The role of the buds is, however, dual, for in their absence roots are not formed even if the stem is treated with active solution; in order to carry out tests one bud must therefore be left on. If the one bud is left on for a short time part of its effect is exerted. Thus in one experiment pea cuttings were treated with a hormone preparation as described above, and in one group the bud was removed immediately after the treatment, in another group after 12 hours, while in the third group it was allowed to remain for the whole 15 days. The average number of roots per 10 plants was then 4, 12, and 22 respectively (u). This action of the bud cannot be replaced by any modification of the treatment, though it can be partially replaced by treatment with a water extract of pea cotyledons. Molisch (1935) also found that budless internodes of various plants root only weakly or not at all. He concludes not only that the buds contain a root-forming substance but also that they prolong the life of the internode. 190 PHYTOHORMONES C. Nature of the Root-Forming Substance The use of this method made it possible to investigate the chemical nature of the root-forming hormone (Thimann and Went, 1934). In the first place, tests on a number of pollens and other natural products showed that the rootforming hormone occurs almost always together with auxin (the latter being determined on Avena). In some cases there was even a good quantitative parallehsm between root-forming and growth-promoting acti\dty; the extract of Rhizopus medium, rich in auxin, was also very rich in rootforming hormone, and was therefore worked up. They found that the root-forming substance was extractable by organic solvents only from acid solutions and is therefore an acid; its dissociation constant, determined by shaking out from buffered solutions, was about 2.10-^. The distribution between different solvents was the same as that of auxin. The activity was readily destroyed by oxidizing agents and followed that of the auxin throughout the various stages of purification, even through vacuum distillations. It was therefore clear that the substance was either identical with, or very closely related to, auxin itself. It was then found that auxin b and, later, synthetic indole- 3-acetic acid (Thimann and Koepfli, 1935; Kogl, 1935) were as active in root formation as the purest Rhizopus preparation. This provides final proof that one, at any rate, of the hormones causing root formation is identical with auxin. The names ''rhizocaline" and "rhizogene," in so far as they really refer to the action of auxin, can therefore be dropped. The evidence against the identity of the root-forming substance with auxin was that the root-forming and growthpromoting activities of various natural preparations were not quantitatively parallel. The explanation for this must lie in the influence of secondary factors (c/. XI E). As to the path of transport of the hormone in cuttings, it seems that it moves through the living cells of the phloem, since Cooper (1936) showed that ringing, after appUcation of auxin at the top, prevents rooting at the base. The move- ROOT FORMATION 191 ment is longitudinal and not transverse, and in physiological concentrations is always polar. It is almost completely stopped by chilling to below 5° C. a 5 cm. section of the cutting. The promotion of root formation by auxins has been studied on a great number of different plants; Laibach, Muller, and Schiifer (1934) and Miiller (1935) found that Tradescantia internodes and Helianthus hypocotyls were induced to form roots by urine or orchid pollen applied in the form of lanohne paste; the effect was doubtless due to the auxin. Laibach (1935), Fischnich (1935), and Laibach and Fischnich (1935) subsequently obtained roots on intact plants of Coleus, Vicia Faba, and Solanum lycopersicum by appHcation of indole-acetic acid in lanoline. Hitchcock (1935, 1935a) and Zimmerman and Wilcoxon (1935), also working with intact plants, induced root formation on stems by local application of lanoline pastes containing indole-acetic acid and various other related substances. Crocker, Hitchcock, and Zimmerman (1935) then compared the long-known activity of ethylene, carbon monoxide, and other gases in promoting root formation with the action of the auxins. They concluded that ethylene may itself act as a hormone (c/. X M). However, the effect of ethylene on growth by elongation is to inhibit and not to promote it. Further, Michener (1935) has shown that in Pisum cuttings, which root vigorously in response to auxin, no roots are produced by ethylene, nor does ethylene increase the number of roots produced by a given auxin treatment. In Salix cuttings, ethylene alone has a small effect in increasing the number of roots, but if they are treated with auxin, its effect is greatly increased by ethylene treatment. Thus ethylene only seems to be effective in the presence of auxin. Since the experiments of Crocker, Hitchcock, and Zimmerman were carried out on green plants in the light, rich in auxin, it is highly probable that the action of ethylene which they observed was through its effect upon the auxin already in the plant. 192 PHYTOHORMONES The same authors have made some calculations which compare the minimum effective concentrations of ethylene and auxin {cf. X M). Such calculations are without any basis in the present state of our knowledge. In the first place it is impossible to compare an effect on Avena cell elongation with one on apple-twig intumescences. In the second place, we do not know whether the ethylene is really distributed between the air and apple-twig tissue as its equilibrium solubility in water would indicate, or whether it is completely absorbed. It is probably absorbed especially by those cells which react, which constitute only a very small fraction of the total tissue treated. Thirdly, the minimum effective concentration of auxin is not that which will produce 10° curvature as they assume, but that which will produce a just visible curvature, which, using de-seeded plants and automatic recording, is nearer to 0.1° than to 10°. (The value of one part of ethylene in 100,000,000 is recorded by Wallace for the smallest observable intumescence.) In addition there is an error of 10 times in the arithmetic as published, and a subsequent "erratum" distributed by Crocker in 1936 introduces a further factor of 1,000 times. Reliable data on the activity and penetration of ethylene will be needed before any comparative calculation of minimum effective concentrations is possible. It is of importance that all substances which act as auxins are, so far as they have been tested, also active in promoting root formation. Thimann (19356) showed that indene-3acetic and coumaryl-1-acetic acids, which, as discussed in VIII (t, are active in promoting growth but appear to be poorly transported, are also active in root formation. Their activity is, however, largely local, and is best exerted when applied at the base of the test cuttings. Phenyl-acetic acid, found to be active in root formation by Zimmerman and Wilcoxon (1935) was shown to act as an auxin by Haagen Smit and Went (1935). a-Naphthalene-acetic acid is also active for both functions (u). Indole-3-carboxylic acid, on the other hand, is inactive for both functions, while indole-3propionic acid has very low activity both for growth promotion and root formation (Thimann and Koepfli, 1935). D. Effect of Light on Root Formation The results concerning the effect of light on root formation have been very conflicting. Vochting showed that white light inhibited root formation of Salix cuttings. Some plants, ROOT FORMATION 193 e.g. Cinnamonum, root better if the twigs are etiolated ("blanched") for 2 weeks before taking cuttings (Blackie, Graham, and Stewart, 1926). On the other hand it is wellknown that leafy cuttings need light in order to root. A number of experiments have been carried out (Went, 1935a) on the effect of different colored lights on root formarools er ten planfs 60 1- 40 20 g ^ g violet white 400-460 400-700 1.2 X 100% color dark red red orange yellow green blue wavelengtil 610-700 580-700 520-700 480-700 480-570 400-520 energy o 93% 6|% 6i% 60% I.4X 22% Fig. 54. Root formation on pea stems exposed for 14 days to light of the color and intensity specified. Shaded columns, one leaf present; open columns, no leaf present, but treated with indole-acetic acid (20 mg. per liter for 20 hoiu's). 100% energy = about 100 erg/cm. ^/sec. tion. Etiolated pea cuttings, without leaves and treated with auxin in the standard way, were placed in a series of chambers illuminated through calibrated color filters for 15 days. The results (Figure 54) show that light of any wavelength reduces the number of roots formed below the number formed in darkness, white light having the greatest effect and blue the least. (The intensities of blue and green light used were, however, much lower than the others.) If, however, the leaves are left on and no auxin applied, the opposite result is obtained (Figure 54, shaded columns). Here the dark controls produce almost no roots, and the white light controls a maximum. Blue appears to be more active than 194 PHYTOHORMONES its intensity would indicate but the effects of the other Hghts are more or less proportional to their intensity. The white columns thus represent the effect of light on the effectiveness of the auxin in producing roots, while the shaded columns represent a combination of this with the action of light on auxin synthesis. The importance of light for the synthesis of auxin in green parts has been discussed in IV ^4. These experiments throw some light on the conflicting results mentioned above; in cuttings with an auxin storage (deciduous plants in fall and winter, cj. XI G), root formation will be best in darkness. In leafy cuttings without auxin storage, however, light will be required for auxin formation and therefore for rooting. E. Effects of Factors Other than Auxin Carbohydrate. The production of roots (or any other growth) by an etiolated cutting, deprived of the food reserves in its seed, requires carbohydrate. This was clearly shown by Bouillenne and Went (1933) with Impatiens; in one experiment they record, after 10 days in 1.5 per cent dextrose, an average of 7.25 roots per cutting, as against an average of only 1.0 in water. Sucrose behaved similarly. Leaving the cotyledons on, however, raised the average number to 15, which we can now explain as due to the combined effects of sugar and auxin, both coming from the cotyledons (c/. the analysis of the growth of hypocotyls in VD). In other etiolated material the same is true; cuttings of etiolated peas, for instance, gave 25 roots per 10 plants in 2 per cent sucrose, and only 9 when in water. The sugar must be applied very soon after the auxin treatment; if the plants are first placed in water for 2 days and then in sugar practically no roots are formed {u). The kind of sugar used is of considerable importance; sucrose and fructose give larger numbers of roots than dextrose under comparable conditions. The sugar also exerts an effect on the length of the roots produced, fructose giving the longest, and dextrose the shortest. Sucrose which has ROOT FOLIATION 195 been purified by ether extraction (c/. above) gives longer roots than if unpurified {u). Green plant parts, producing or containing ample carbohydrate, are of course not dependent on added sugar. Interrelations between auxins. It was mentioned above that the activity of various extracts was not always parallel to their auxin content. It has since been found {u) that the activity of auxin is increased by a number of other factors. One of the most interesting of these relationships is the effect of auxin a on the action of indole-acetic acid, of which Table XV {u) gives an example. In this experiment the cuttings were treated either with one auxin alone, or with indole-acetic acid in the highest concentration together with auxin a in varying concentrations. It will be seen that auxin a, even down to its lowest concentration (0.002 per cent = 2.10"^ mg. per cc), increases the number of roots formed in presence of excess indole-acetic acid by about one fourth. Urine, in concentrations which are not toxic, has a similar effect, which is undoubtedly due to its auxin a content, as is shown by the fact that its ether extract is also active in the same way {u). On the other hand, the reverse procedure, namely the addition of small amounts of indoleacetic acid when auxin a is in excess, has no such effect. TABLE XV Effect of Auxin a on the Root Form.\tion of Pea Seedlings by Indole- 3-AcETic Acid. Each Figure Represents the Number of Roots per 10 Pl.\nts and Is a Mean of 30-100 Plants Relative Concentration OF Auxin in Per Cent 196 PHYTOHORMONES The mechanism of this facihtating effect is unknown, but it is of considerable interest as indicating a physiological difference between the actions of the two auxins (cf. X G and Y F for another difference). These experiments with auxins were carried out by application at the apical end of the cutting. The transport of the root-forming hormone was earlier shown to be basipetal and this has been confirmed with pure auxin. Bios. There are other substances which are like the sugars in that they must be applied to cuttings from the base, but are unlike the sugars in that the amounts of them necessary to influence root formation are extremely small. In contrast to the sugars, addition to the cuttings of various amino-acids does not promote root formation. Tryptophane, however, is an exception; if applied at the base it gives rise to a large number of roots (u). This is doubtless due to its conversion by the plant to indole-acetic acid. The clearest example of a special substance, other than auxin, which is highly active when applied at the base, is that of the yeast-growth-promoting substance bios. Bios, which has been shown by Eastcott (1928) and subsequent workers to consist of a complex of at least 3 factors, is present in various sources including yeast extract itself. In a study of the effect of various additional substances on the formation of roots by pea cuttings it was found that yeast extract definitely increases the number of roots formed in presence of auxin (u). On this account and because ordinary aminoacids have no effect, an attempt was made to test the activity of the various constituents of bios. z-Inositol (Bios I) was inactive. Recently, however, one of the most important of the factors, biotin, has been isolated in a pure state by Kogl (1935, 1936) and a sample of this, from one of the last stages of purification, was tested in the same way (u). This work, which the authors carried out in cooperation with Professor Kogl, who supplied the biotin, showed a remarkable effect, one example of which is given in Figure 55 (cf. also Figure 62). Here the cuttings were treated apically with ROOT FORMATION 197 different concentrations of indole-3-acetic acid up to the maximum, which produced 102 roots per 10 plants; at this maximum concentration the biotin was subsequently apphed to the base, when the number of roots formed increased in proportion to the biotin concentration and reached a second maximum of 179 roots per 10 plants. Similar ISO 100 50 roots per ten plants -8 -7 -6 -5 -4 lo|.tnol. concentration of Indole acetic acid 0.4 2 10 50 250 1000 5000 S.E^^ units biotin Fig. 55. Root formation on etiolated pea stems with their bases in 2% sucrose solution. Left, indole-acetic acid alone applied to the apex (abscissa, log. molar concentration); right, optimal auxin concentration applied at apex, with biotin added to the sucrose solution at base (abscissa, Saccharomyces units per cc). (Kogl, Thimann, and Went, 1935, u.) application of biotin at the tip gave no effect. Without auxin, biotin, apphed either at tip or base, produces no roots, so that its effect is exerted only in presence of excess of auxin. This result is of special interest in \dew of the fact that up till recently biotin has only been known to exert its effect on growth of yeasts. However, Kogl and Haagen Smit (1936) have found that biotin also increases the growth in length of Pisum seedlings, from which the cotyledons have been cut off. It should be noted that the lowest concentration of biotin which definitely increases the number of roots is between 2 and 10 Saccharomyces units ^ per cc, i.e. a con- 1 1 mg. of biotin = 25 X 10^ Saccharomyces units. 198 PHYTOHORMONES centration which just exerts a detectable effect on yeast growth. Other factors. There is good evidence that pure theehn (female sex hormone or oestrin) also increases the number of roots produced by pure auxin, when it is appUed at the base (u). The effect is very much smaller than that of biotin, but like that of biotin it does not appear in the absence of auxin. Whether theelin plays any part in root formation in nature is doubtful, although it is frequently present in plant material. Bouillenne (1936) has stated that root formation in Impatiens seedlings is greatly hastened by carotene. The total number of roots produced was, however, not affected. The role of sugars, substances Uke biotin, and the auxins, provides an excellent example of an interlocking system of limiting factors (see Figure 62). By varying the conditions any one can become limiting; the activity of each can only be shown in the presence of sufficient of the other factors. Such a relationship is further borne out by the behavior of different races of peas; some, like Alaska and Gradus, give few roots unless supplied with auxin, while others, such as Dehcatesse, Dark Laxtonian and Perfection, give large numbers of roots on the controls, and auxin does not increase the number further (w). In the latter types auxin is evidently not a limiting factor. Others again give few roots even if supplied with auxin; in these evidently one of the other factors is limiting. Thus far the factors considered have been those which influence the formation of visible roots. In which stage of development each factor exerts its influence is unknown. To produce a visible root at least three processes must take place in succession: redifferentiation of pericycle cells into root initials, formation of a root primordium by these initial cells, and the outgrowth of the root primordium. From a standpoint of morphogenetics the first two processes are the most important; physiologically speaking they can be regarded as one process. There are several reasons for ROOT FORMATION 199 thinking that it is this process which is influenced by the factors discussed above; firstly, the length of the roots which have been formed is not materially influenced by the treatment, except by sugar; secondly, examination of the pea stems has not revealed any appreciable number of root primordia; thirdly, we know that the direct effect of auxin on roots is to inhibit their growth in length. Furthermore, the total length of roots formed per cutting is more or less constant, so that the more roots are formed the shorter they are. This indicates that the outgrowth of the roots is influenced not by the auxin treatment, but by an internal factor, which may become distributed over a large number of root primordia. This factor is probably the one contained in the yeast extract which White (1933, 1934) finds necessary for growth of excised root tips in synthetic media (see IX D) . There is, however, another factor of quite different type which apparently also takes part in the first stages of root formation. If a pea cutting is divided into a number of sections and each is placed in sugar solution and treated with high concentrations of auxin (in paste form), then the sum of the numbers of root primordia formed is about the same as if the intact cutting were so treated. The majority of these primordia are formed at the point of apphcation of the auxin. Only a small proportion of them grow out as roots, probably because of the high concentration of auxin, which inhibits growth in length (c/. IX B). In one experiment, there were 34 such primordia on the intact cutting; on 2 cuttings which were di\4ded into 4 the sum of the primordia produced at the 4 bases and apices was 27 and 33 ; on 2 cuttings similarly divided into 8 the sum of the primordia was 38 and 43 {u). The total number of primordia is thus nearly constant and must therefore be determined by an internal factor other than the auxin, which factor only becomes limiting when auxin is in excess. Further, the distribution of this factor inside the plant can be determined from the distribution of the primordia on the dissected cut- 200 PHYTOHORMONES tings; the bulk of the primordia are on those sections some distance from the apex, which therefore contain the most of this factor. However, when auxin was appUed to the intact cutting 30 primordia were formed at the top, but when apphed to the uppermost one eighth of a cutting, only about 7 were formed at the top: the auxin may, therefore, mobilize some of the other factor from the lower parts of -3 -4 ' -5 -6 -1 -« Fig. 56. Root formation on etiolated pea cuttings. Ordinate, average number of roots per plant formed near basal (crosses) and apical (circles) cut surface; abscissa, log. of the indole-acetic acid concentration in moles per liter. (From Went, "Allgemeine Betrachtungen fiber das Auxin-Problem," Biol. Zentralbl. 56: Fig. 1, P. 479, 1936; Verlag Georg Thieme, Leipzig.) the cutting. This suggests that an important function of the auxin is to control the movement of this other factor. This is supported by another fact, namely that with increasingly high auxin concentrations, applied at the tip, the number of roots at the base reaches a maximum and then decreases; at that concentration at which the decrease begins, roots begin to appear at the top of the cutting, i.e. at the point of apphcation of the auxin (see Figure 56). According to Went (1936) this means that the other factor is becoming mobilized at the tip by the very high auxin concentration and thus not enough is available for the base. ROOT FORMATION 201 If the number of roots at the base and the tip are added the total number per cutting increases smoothly with auxin concentration as shown in Figure 56. ^Yhen auxin is appUed at the apex, the lowest concentration needed to produce localized roots in this way is about 100 times that which will produce roots at the base. The concentrations needed to produce localized roots in the experiments of Hitchcock (1935a) are evidently those corresponding to these high values, since they too are of the order of 100 times the concentration which will, in our experiments, produce roots at the base. Experiments on intact plants in soil, of course, preclude observation of basal root formation. This ratio of 100 fits in very well with an observation of another sort. It was stated above that auxins must be applied at the apex to induce root formation at the base. An exception, of course, is given by those substances, such as indene-acetic and cumaryl-acetic acids, whose transport from apex to base is limited. These substances give excellent rooting when applied at the base, while if applied at the tip their activity is slight or zero (Thimann, 19356). Thus, on pea cuttings, indene-acetic acid applied at the tip gave, per 10 plants, 19 roots, while at the base the same concentration gave 98 roots. Root formation when true auxins are applied to the base can, however, also be obtained if very high concentrations are used. In one experiment basal root formation on Pisum cuttings was induced by 0.02 7 per cc. of indole-acetic acid when applied at the tip, but, when applied at the base, a concentration of 20 y per cc. was necessary. From this and other experiments the ratio of minimum effective concentrations applied at base and at tip is between 100 and 1000. Gouwentak and Hellinga (1935) report comparable data for Coleus cuttings. A dab of paste containing 0.01 y indole-acetic acid caused root formation at the basal cut surface only; with 20 y of the same compound in the paste, roots were formed at both apical and basal surfaces. When the 20 y was applied at the base it did not, of course, produce roots at the apex. These 202 PHYTOHORMONES results could be interpreted in terms of a local mobilization effect as described above. A conclusion of a similar kind may be drawn from the experiments of Cooper (1936) in which lemon cuttings were treated with auxin at the base. If subsequently the basal 10 mm. were cut off and the cuttings again treated with auxin no roots were formed, although controls from which the bases were not cut off rooted vigorously. This indicates that the second factor has already been accumulated at the base and was thus removed with the cut-off portion. It will be seen from this discussion that in the complex process of root formation many factors are involved. These include auxin, carbohydrates, a group of other substances such as biotin, and the internal factor discussed above. Much further study will be needed to elucidate their interactions. F. Root Formation on Other Organs Although we have dealt in this chapter only with root formation on stems and hypocotyls, roots will also be formed on the petioles of isolated leaves which have been treated Hke cuttings (for literature and for a list of leaves tested for root formation see Hagemann, 1932, and Graham, 1934). Since leaves form auxin and food materials in light, this is not surprising; it is also evident that addition of auxin will at best speed up root formation on petioles, but not materially change their rooting response in general. Root formation on roots might be considered as a special case, but Bouillenne and Went (1933) concluded that this was governed by the same factors as root formation on stems. They found, in Acalypha, that abundant hormone supply led to the formation either of a large number of sparsely branched roots, or of a few roots with numerous laterals, the controls forming only one or two roots without branches. This would indicate that if the excess hormone in the cutting is not used up in the formation of roots on the stem, it will move into the roots and cause the formation of numerous laterals on them. Zimmerman and Hitchcock ROOT FORMATION 203 (1935) described the formation of laterals on the aerial roots of Cissm, after apphcation of various auxins and auxin-like substances either as paste or in water solution to the growing zone. Application on the basal side of the elongating region was inefifective. The apphed auxin, however, inhibits the gro\\i;h of the main root, and to this they ascribed the branching; as soon as the main root recovered its original growth rate, the effect of the auxin paste on branching disappeared. The same phenomenon on a number of seedling roots was described by Faber (1936; see also Laibach, 1935), and by Thimann (1936). Faber obtained profuse branching of the roots at the place of application of the auxin paste. However, Thimann (1936) showed that in Pisum roots branching is independent of the growi^h of the main root. He applied aiLxin paste to the stump of the epicotyl, which produced no inhibition, but even a sHght acceleration of the main root; nevertheless it increased branching from in the controls to an average of 2.7 in the treated plants. On de-tipped roots the effect was still greater. This fact gives good e\'idence for acropetal auxin transport in the root (see IX C). Thimann also found that branching in both Avena and Pisum roots is inhibited by the presence of the root tip; since the root tip is a source of auxin this is probably due to factors other than auxin. Correspondingly, Katunskij (1935) reported that in Zea roots branching is inhibited by the apphcation of coleoptile tips. In Avena branching occurs very readily and is not increased by auxin application, so that auxin appears not to be the limiting factor for branching in these roots. If an incision be made on one side of Vicia Fdba roots the formation of laterals is prevented on the apical side of the incision for a considerable distance (de Haan and Petrick, 1935). This indicates that formation of laterals is controlled by something coming from the stem or cotyledons. All the above facts indicate that root branching is controlled both by auxin and by other factors. 204 PHYTOHORMONES G. Practical Applications Study of the use of auxin for rooting cuttings of commercially important plants has been begun by Cooper (1935). He obtained excellent root formation on cuttings of lemon, Acalypha, Lantana, and fig by apical application of auxin in lanoline. Subsequently the fact, mentioned above, that high concentrations of auxin cause root formation when applied at the base has been utilized successfully by Hitchcock and Zimmerman (1936) and by Cooper (1936) for cuttings of Ilex, Taxus, Hibiscus, Pachysandra, lemon, Chrysanthemum, and some other plants (see Figure 57). Private reports from a number of horticulturists have already extended this list considerably. In general the highest nontoxic concentration of indole-acetic acid, dissolved in water, will give the best results. This concentration varies for different plants, and is lowest for green cuttings. A treatment with 0.2 mg. per cc. for 12 to 24 hours can be recommended, but before large-scale applications are made the toxic limit for each species to be treated should be ascertained. Indolobutyric and naphthalene-acetic acids are also effective. For treatment by the lanoUne method a concentration of about 1 mg. indole-acetic acid per gram of lanoline is satisfactory. It is clear that some of the procedures used by gardeners to induce root formation have their foundation in the production and movement of auxin (see Bouillenne and Went, 1933). A curious example of this is found in the insertion, into the apical split end of cuttings, of a germinating wheat seed, as practised in parts of Holland and Scotland. As to more general principles, in non-deciduous plants leafy cuttings are always used, probably because no auxin is stored in their stems. In deciduous plants leafless cuttings are preferred because of the difficulty with water supply to the leaf, but here the bud acts as auxin supply. As one of numerous examples might be mentioned the holly cuttings of Zimmerman and Hitchcock (1929); leafless cuttings of the deciduous Ilex verticillata will root, but those of the evergreen varieties will not. ROOT FORMATION 205 Fig. 57. Rooting of lemon cuttings. Upper row, 8 hours in tap water; lower row, 8 hours in indole-3-acetic acid (highest non-toxic concentration, 500 mg. per liter). Photographed 17 days after treatment. (From Cooper, Plant Physiol. 10: 789-794, 1935.) 206 PHYTOHORMONES Layering probably depends for its success on the retarding influence on auxin transport exerted by high humidities {cj. XIII C), together with geotropic accumulation of auxin on the lower side of the stem. Rooting takes place usually at nodes, probably because there the transport of auxin is interfered with. {Clematis is an exception, rooting better at internodes.) The practice of ringing branches, either by cutting the cortex or by tying a tight wire round during growth, obviously operates in the same way. The optimum time of year to take cuttings varies from plant to plant (Graham, 1934) and depends upon a number of factors such as auxin production, storage, and destruction, as well as water supply and ease of wilting. Among the causes of failure of cuttings to root when treated with auxin, one of the most important is doubtless furnished by those plants in which not auxin, but one of the other factors, is limiting. Another cause is the loss of the applied auxin or other factors by exudation from the cut surface; cuttings which root with difficulty are frequently those from which much exudation takes place. Lastly, the inactivation of auxin at the cut surface, by enzymes set free in wounding, doubtless also plays a part. In conclusion, it may be pointed out that the role of auxins in root formation is a good example of a piece of research in pure physiology which has an immediate practical application. CHAPTER XII BUD INHIBITION A. Bud Inhibition as a Correlation Phenomenon It has been known from earliest times that lateral buds, low down on a stem, do not develop in presence of the terminal bud. If the terminal branch of a bud or shoot be removed, some of the laterals usually grow out at once; this is the basis of all pruning. Sachs' idea of bud-forming substances was abandoned by most workers, largely because the more obvious phenomenon is the inhibition, rather than the promotion, of bud development. However, Errera (1904) ascribed apical dominance and bud inhibition phenomena to "internal secretion," or, as we would say now, hormones. Goebel, in his earher works (e.g. 1903) favored Sachs' hypothesis, but later (1908) changed his views, he and his school interpreting bud inhibition as a nutrition phenomenon. Loeb carried out a number of experiments on shoot growth and inhibition in Bryophyllum (1915, 19176, 1918a, 1919, 1923, 1924), at the same time as his experiments on tropisms (see X C). He concluded that the growth of shoots was proportional to, and determined by, the amount of nutritive substances available. Thus, if one shoot of a plant is growing rapidly it deflects food substances away from other buds, which are therefore inhibited. However, as with root formation and geotropism, Loeb expressed different views at different times, and in 19176 attributed the phenomenon to the influence of special inhibiting substances formed in the leaf and transported basipetally in the stem. His experiments did not, however, pro\dde evidence in favor of either \dew, although his concept of bud-inhibiting substances was shown by Reed and Halma (1919) to explain satisfactorily their experiments on correlations in bud devel- opment. 207 208 PHYTOHORMONES A detailed study of bud inhibition was made by Dostal (1926). He placed isolated internodes of Scrophularia nodosa in water and found that if one of the pair of opposite leaves were removed, the bud in its axil began to develop, while the one in the opposite axil, with its leaf present, did not. The leaf, therefore, inhibits the bud in its own axil. A growing bud, however, he found to exert a greater inhibiting influence than a leaf, the effect of the leaf being merely to upset the balance between the pair of buds, so that one could get ahead of the other. Once ahead, this growing bud inhibited the other strongly. Dostal therefore made it clear that the balance between inhibition and growth is rather delicately poised. Like his predecessors, however, he interpreted his results in terms of nutrition and water relations, and his experiments were therefore not designed to throw any light on the role of special substances. Evidence for a special inhibiting substance was first brought by Snow (1925a), whose results were apparently not known to Dostal. Snow's essential experiment was to split the epicotyl of a Phaseolus seedling longitudinally from the roots up to 2 cm. above the cotyledons; another cut at an angle to the first then divided the plant into two parts, one of which, with one cotyledon, was completely isolated from the upper part, while the other, with the other cotyledon, was connected through the split stem with the upper part of the plant. The two halves were bound tightly together, and the growth of the buds in the axils of the cotyledons measured. The bud on the decapitated half was then found to grow out somewhat more slowly than that on a control decapitated and isolated split half. An inhibiting factor must therefore have come from the buds and leaves on the other half and crossed the cut surface. In later work (1929) he showed that the principal inhibiting effect was exerted by the very young leaves. A similar result was obtained by Weiskopf (1927). As to the path of movement of the inhibiting influence, Harvey (1920) found that if a stretch of the stem of Phaseolus BUD INHIBITION 209 was killed by steaming, axillary buds grew out below the dead zone although the part above remained alive ; transport downwards must therefore take place only through living tissue. A similar result was obtained by Child and Bellamy (1920), by chilUng a zone of the stem with a brine coil (compare also Cooper's experiment, p. 191.) Snow later (1931a) confirmed Harvey's finding, but also obtained some evidence that the inhibiting influence could—at least to some extent—travel up a lateral branch. Experiments of this kind also show that food relations play a subordinate part, since the axillary buds may develop while the top part is also developing. It appears, then, that the inhibition travels mainly from apex to base in living tissue, and is exerted principally by the young buds and leaves ; there is also some evidence that it may cross a cut surface and thus may be due to a diffusible substance. B. Bud Inhibition Caused by Auxin Thimann and Skoog (1933, 1934) pointed out that if inhibition were really due to a substance, then this substance would appear to behave like auxin in the plant. They determined the auxin production in Vicia Faba, using the diffusion method and the Avena test, and found that the terminal bud was the most active auxin-producing center (see Figure 28, p. 62). The leaves produce smaller amounts of auxin, but their production decreases with age (c/. Avery, 1935) ; the dormant axillary buds produce almost none, but as soon as they begin to develop they also begin to produce auxin. These results therefore closely parallel the inhibiting power of the different parts discussed above. Finally they removed the terminal bud and applied auxin, in agar, in its place, the auxin being renewed as soon as it had all diffused in from the agar, so as to duplicate the action of the terminal bud in providing a continuous stream. The laterals were then inhibited as completely as those on intact controls (see Figure 58). Their experiments were carried out with par- 210 PHYTOHORMONES tially purified auxin from Rhizop'us, but were later (Skoog and Thimann, 1934) confirmed with pure auxin b and indole-3-acetic acid. A clear picture of the effect of auxin in bud inhibition is given by Figure 59. The terminal bud was removed from etiolated Pisum seedlings, and instead pure lanoline or lanoline containing various amounts of indole-acetic acid was applied to the apical cut surface. The swellings produced by the auxin are negligible in comparison with the growth of the buds in the controls without auxin. As to the quantitative relations, Thimann and Skoog found it necessary, in order to obtain complete inhibition, to use an auxin concentration several times that which could be obtained directly from the terminal bud, but this difference is doubtless due to the inactivation of the applied auxin at the cut and the loss of auxin in non-transporting tissues. In any case the amount of auxin necessary for bud inhibition in Vicia Faba is considerably larger than that which causes maximal stem elongation, for, after decapitation, sufficient auxin for stem elongation, but not sufficient for bud inhibition, is produced in light by the remaining leaves. Thus only a growing bud, which is a very powerful source of auxin, can exert appreciable inhibition of lateral buds. I 2 3 4 3 6 7 a 9 10 II 12 days Fig. 58. Growth of axillary buds of Vicia Faba. Ordinate, length of bud in mm.; abscissa, time in days. A, plants decapitated, plain agar applied; B, plants intact; C, decapitated, 1600 plant units (=650 AE) of auxin applied in agar every 6 hours to apex — application stopped at arrow. (After Thimann and Skoog, Proc. Roy. Soc. B. 114: 317-339, 1934.) BUD INHIBITION 211 In this connection it is significant that many dwarfs, such as those of Pisum, Vicia, and Zea, have a bushy habit, i.e. the buds in their lower nodes develop extensively. If the dwarfing is due to a reduction in the amount of auxin present, as is true for the nana form of Zea Mays (van Fig. 59. Bud inhibition in etiolated Pisum seedlings. Nos. 1-5, decapitated below terminal bud; 6-9, decapitated below upper lateral bud; 10, intact controls. Auxin paste applied to apex immediately after decapitation. Concentrations: for 1 and 6, 10 mg.; for 2 and 7, 2.5 mg.; for 3 and 8, 0.6 mg.; for 4, 0.15 mg. indole-acetic acid per gram lanoline; for 5 and 9, plain lanoline. Overbeek, 1935), then the branching is doubtless due to the same cause. The above experiments leave no doubt that in Vicia and Pisum auxin is the factor inhibiting lateral bud growth. The same is true in other plants. Muller (1935) apphed lanoHne pastes containing urine, or orchid pollinia, to a number of decapitated plants other than those already mentioned. In Sinapis, Linum, Antirrhinum, Godetia, Zinnia, Helianthus, and Tradescantia, the pastes caused inhibition of lateral bud development, but in Polygonum and Tropaeo- 212 PHYTOHORMONES lum they were without effect. Both Miiller and Laibach (1933) also used, instead of lateral buds, the cotyledonary buds in legume seedlings, and these also were inhibited by appUcation of orchid poUinia to the decapitated stem. The orchid pollinia are, of course, rich in auxin. The treated stems frequently showed swelling or elongation or both, and this led Laibach and Miiller to postulate the mechanism of inhibition discussed below. If a leaf of Bryophyllum be cut off and replaced on its petiole, the bud in the axil continues to be inhibited, and the same result may be obtained by placing the leaf on agar, and then applying the agar block to the cut petiole (Uhrova, 1934). There is no reason to doubt that here, too, auxin is the active factor. In Solidago, the successive leaves of the rosette each inhibit the growth of the one following them for a time. Goodwin (1937) showed that the inhibiting power of the leaf ceases about the time that its auxin production falls off. Further, removal of one leaf accelerates the growth of the next, while application of pure auxin on the cut petiole inhibits the growth of the next. Apphcation of pure auxin inhibits bud development in the Aster. In this plant the much-branched habit of A. multiflorus is correlated with very small auxin production by the buds, while the almost unbranched A. novae-Angliae produces considerably more (Delisle, 1937). We may safely assume, therefore, that the inhibiting action of auxin on bud development is very general. Not only is the phenomenon common to a great many different plants, but it is also caused by a great many chemically different auxins. The natural auxin of the plant, pure auxin h, and indole-acetic acid all have the same effect. Further, indene-3-acetic and coumaryl-1-acetic acids, both of which have other properties of auxins, also strongly inhibit bud development of Pisum seedlings (Thimann, 19356). Hitchcock (1935a) mentions that indole-propionic and naphthalene-acetic acids had bud-inhibiting influence when applied in high concentrations to Nicotiana stems. BUD INHIBITION 213 C. Possible Mechanisms of Bud Inhibition The auxins thus possess not only growth-promoting and organ-forming ability, but under certain circumstances they may also inhibit growth. Leaving aside those inhibitions which are produced by unphysiologically high concentrations, the growth of lateral buds and of roots are both inhibited by auxin. This raises the question of how all these different actions may be brought about. It seems clear that the auxins control some master reaction in the cell (Thimann, 19356), which may then lead to different effects according to the age and position of the cell and the influence of other factors. However, the mechanism whereby any such action could lead to bud inhibition remains completely unexplained. Some hypotheses may be considered. In the first place, the production of auxin within the developing bud is probably from some precursor which is stored in it. Thus Went (1934a) found that the presence of buds on etiolated pea seedlings led to the formation of roots, presumably due to auxin storage in the buds. In Solidago the smallest leaf in the bud, weight for weight, produces the most auxin (Goodwin, 1937), and since these small leaves are completely enclosed in the bud their auxin production cannot be directly from photosynthesis (cf. IV B). Now Thimann and Skoog (1934) suggested that the relatively high concentration of auxin reaching the lateral bud from the tip shifts the equilibrium. Precursor ^ Auxin, towards the precursor, thus preventing the lateral buds from forming auxin themselves. Their diffusion experiments, however, showed that very little auxin diffuses out of the undeveloped lateral buds so that the amount actually in them cannot be very large. Laibach (1933) and Muller (1935), on the other hand, assumed that the swelling and growth, accompanying application of high concentrations of auxin to the cut surface, are the cause of the inhibition. According to this view, the inhibition is only a secondary result of the increased stem 214 PHYTOHORMONES growth produced by the auxin. However, this hypothesis was satisfactorily dismissed by Skoog and Thimann (1934) because in their experiments inhibition was complete without any noticeable growth increase at the top of the stem. This can be shown very clearly in peas by the inhibition of the bud in the axil of the cotyledon. Also, in Figure 59 the enormous bud development of the controls obviously represents more growth than the shght stem swelling in the auxin-treated plants, whose buds are completely inhibited. Le Fanu (1936) has brought to light some interesting new facts in connection with the inhibition. She found, firstly, that the application of dilute solutions of indole-acetic acid to the base of stem sections of Pisum inhibited their elongation ; if lanoline paste was used, the auxin paste decreased the growth when applied at the base, although (under somewhat different conditions) the same concentration increased the growth when apphed to the apex. These results resemble those of Faber (1936) and Thimann (1936a) on roots, in which the effect of auxin also depended on its point of application (c/. IX D), and they indicate that the effect of auxin on an organ may be determined by whether it has to move with or against the normal polarity. In the second place she showed that in plants with two nearly equal shoots, the shorter, which was being inhibited by the longer, yielded only traces of auxin to agar, while normal shoots under the same conditions yielded 26 units in 3 hours. Sections of the inhibited shoot also transport practically no auxin through them, whether it is applied apically or basally. Le Fanu concludes that this evidence is against the view that the shoot can be inhibited by the direct entry of auxin into it, and supports rather the concept of some indirect means of inhibition. However, it is clear that the inhibition of stem sections by auxin at the base seems to be comparable to the inhibition of one shoot or bud by another which supplied auxin to its base. In the hope of elucidating the mechanism of inhibition Went (1936) has attempted to assess the role of the other BUD INHIBITION 215 factors involved in bud growth. In etiolated Pisum seedlings the factor (or factors) necessary for growth in length of the stem or branch could be separated from factors governing leaf development and embryonic bud growth by cutting off different parts. The former comes mainly from the roots and to a less extent from the cotyledons, the latter mainly from the cotyledons. He suggested that the auxin coming from the terminal bud may influence the upward movement of the factor necessary for bud growth. Wherever an auxin production center is located, it would cause the bud-growth factor to move towards that place, preventing other, nonauxin producing, buds from obtaining this factor. This would be one way to explain the curious fact that the growth of laterals is inhibited by auxin coming from the terminal bud, but promoted by auxin produced in their own tissues. However, Le Fanu's (1936) observation of the inhibition due to basal application of auxin would also explain this. In XI E evidence has been given that auxin causes a redistribution of the specific root-forming factor, w^hich would be comparable to the action suggested above. This \dew is essentially a revival of the old hypothesis (see e.g. Goebel, 1903; Loeb, 1915) that the growing point is a ''center of attraction" for the material necessary for stem growth. The factors controlling bud growth are apparently subject to polar distribution in rhizomes, as Schwanitz (1935) has shown. If the rhizome is cut up immediately on removal from the plant, then each piece produces about the same number of buds, but if it is first placed in the ground for 16 days or so, and then cut up, the majority of the buds are formed in the apical portion. One of the factors for growth in length is apparently biotin, since Kogl and Haagen Smit (1936) have described how markedly it increases the growth in length of Pisum seedlings from which the cotyledons have been removed. Eastcott's finding (1928) that large quantities of bios are present in germinated malt seems significant in this connection. The influence of factors other than auxin may explain 216 PHYTOHORMONES why some buds have a greater tendency to develop than others. Thus in woody twigs after decapitation, it is frequently the first bud below the terminal which develops (c/. Reed and Halma, 1919), while in herbaceous plants the lowest bud often develops under these conditions. It is easy to see how this might be related to the distribution or storage of bud-growth promoting factors. Snow (1931) considered that, in Pisum, the inhibiting action of the terminal bud is greatest on the buds farthest from it, i.e. the inhibition increases with distance. The fact is, more probably, that the tendency to grow out is greatest in the basal buds, so that these buds show the greatest difference in growth between decapitated and intact plants. In this connection reference may again be made to the poorly-transported auxins, such as indene-3-acetic acid; Thimann (19356) has shown that the inhibition caused by this compound decreases very rapidly with increasing distance from the point of application. In callus formation, the activity even of readily transported auxins like auxin a and indole-acetic acid decreases very rapidly with distance (of. XIIIc). The mechanism of bud inhibition can probably not be understood until we know more about the fundamental mechanism of auxin action on the cell, and the role of other factors in bud growth. It is clear that many of the factors necessary for bud development, such as water, food, auxin, and the other substances, could, under the right conditions, become limiting factors in bud growth (cf. e.g. Denny, 1926; Moreland, 1934). However, the experiments in this chapter are concerned principally with the effects of auxin and it will be clear to the reader that the typical ''apical dominance" is an auxin phenomenon. CHAPTER XIII OTHER ACTIVITIES OF AUXINS A. Auxin and Cell Division It was mentioned in Chapter II that the work of Haberlandt and his students has shown that cell division in the parenchyma of a number of plants is controlled by diffusible substances, which are present in the phloem, and which are set free from other tissues by wounding. Such attempts as have been made to characterize these celldi\dsion substances chemically (Wehnelt, 1927; Jost, 1935a; Bonner, 1936 and u; Umrath and Soltys, 1936) give no reason to believe that they are in general identical with auxins. On the other hand, there are some conditions under which auxins certainly produce cell division. Thus, Jost (1935a) in studying the parenchyma of the pods of Phaseolus—the material used by Wehnelt—found that the cells could be induced to divide not only by bean juice and other preparations, but also by indole-acetic acid. However, this substance acted only at the very high concentration of 0.1 per cent in water, concentrations up to 100 times those present in the plant being completely inactive. Further, the bean extract, containing only 0.5 per cent dry matter, produced more divisions than the 0.1 per cent auxin solution. Since the physiological activities of indole-acetic acid and the auxin of the plant itself differ only in a very minor degree, it is clear that the powerful action of bean juice cannot possibly be ascribed to its content of auxin, but must be due to another special substance, probably active in high dilution. A somewhat lower concentration of auxin, namely 0.01 per cent in water, caused \'igorous cell division in the pith cells of the stem of Vicia Faha, and in the subepidermal parenchyma of the cotyledons of the white lupin. This concentration, however, is also 217 218 PHYTOHORMONES considerably higher than is Ukely to occur physiologically. The problem is further complicated by the fact, observed by Wilhelm (1930) and by Orth (1934), that the pith cells of Vicia Faba show growth and division on treatment with a variety of extracts and solutions, including sugar solution. Wehnelt (1927) and Jost (1935a) obtained cell divisions in Phaseolus pod parenchyma with 2 per cent levulose, 0.1 per cent NaCl, 0.01 per cent acetic acid, and 0.01 per cent citric acid. The apparent non-specificity of the reaction leads one to suspect that some of these materials may act indirectly by merely setting free the active substance in some way from the tissue, while others, such as the bean juice, really contain the active growth-promoting substance. To which type the action of auxins belongs cannot yet be definitely said. Thus both in the above work, and in that of Laibach and others on swellings (see XIII C), auxins bring about cell divisions in many kinds of tissue only when applied in high concentrations. The first stages in the initiation of new roots on stems have frequently been shown to be divisions in the pericycle, and it was shown in XI C that such root formation is brought about, and controlled, by auxin. This fact cannot, however, be taken as proof that auxin controls cell division in general, and since the phenomena of root formation are complex, it will be better to examine first the clearest case of the action of auxin on cell division. B. Cambial Growth The one type of cell division which appears to be really controlled by auxins under physiological conditions is the formation of, and division in, the cambium. This applies to the cambium of the stem, and to a large extent to both cambium and pericycle of the root. It was first shown by Jost (1891, 1893) that the activity of the stem cambium of dicotyledons is greatly stimulated immediately below the growing leaves. The effect is the same even if they are etiolated, and it is transmitted only in the morphologically OTHER ACTIVITIES OF AUXINS 219 downward direction, no effect being observable above the leaf. In 1910, when phytohormones were not yet recognized, Keeble ascribed this transmission of cambial activity to "chemical stimulators," while the first suggestion that the activation is due to a true hormone—produced in growing parts and transmitted in the morphologically downward direction—was made by Kastens (1924). Subsequently, in an extensive survey of cambial activity in tropical trees. Coster (1927, 1928) came to the conclusion that the young developing buds, and to a lesser extent the leaves, produce hormones which activate the cambium. These hormones may even be produced shortly before the first visible signs of bud development can be detected. In general it is observable that in trees the cambial acti\aty in the branches begins in spring at about the time the buds begin to develop. Snow (1932) mentions that growing inflorescences and fruits of Agrimonia, Spiraea, and Scrophularia stimulate the activity of cambium below them, and Priestley (1930) ascribes the same effect to the flowers, buds, and growing leaves of Fraxinus. Gill (1933) finds that the inflorescences of Populus serotina and Salix caprea, which are within the bud at the beginning of the spring, activate the cambium immediately below them as they expand in spring; trees whose catkins are exposed throughout the winter show no such effect in spring. Experimental evidence that cambial activation is due to a hormone was first brought by Snow in 1933. His experiments were apparently suggested by the work of Simon (1930) who found that, in grafting unrelated plants, vessel differentiation appeared to be stimulated in the neighborhood of the graft before the tissues had actually grown together. Snow therefore split longitudinally the stems of a Pisum and a Helianthus seedling for several cm., and severed half the Pisum stem at the lower end of the split, and half the Helianthus stem at the upper end. The downward-pointing half of the former was then brought into contact with the upward-pointing half of the latter, the two 220 PHYTOHOHMONES halves bound nrmly together, and after 22 days the region of contact was sectioned and examined. Many cambial layers had formed in and between the bundles in the Helianthus hypocotyl, although the tissues had not (with a few exceptions) grown together. Controls not in contact showed no such effect, so that the stimulus from the Pisum seedUng had passed across a protoplasmic discontinuity to act upon a plant of quite another family. In other experiments the upward- and downward-pointing portions were both from the same plant, Vicia Faba, but the stimulus passed across a piece of linen inserted in a cut in the stem. Snow and Le Fanu (1935) then found that the cambium in the Helianthus hypocotyl may be activated by applying urine, or the ether extract of urine, and finally Snow (1935, 1935a) obtained excellent activation by pure auxin a and indole-3-acetic acid. As stated above, in high concentrations auxin may cause cell division in a variety of tissues, but in this case it is of considerable importance that the activation is brought about by low concentrations of auxin comparable with those obtaining in the normal plant. Thus, comparing his results with the auxin determinations of Thimann and Skoog (1934), Snow found that moderate cambial activity is induced by an amount of auxin a about 2.5 times that produced hourly by the terminal bud of Vicia Faba. There can thus be no doubt that the auxin formed by buds and leaves is responsible for the cambial activation below them; the polar movement of this cambial stimulus is then due to the polar transmission of the auxin. In Snow's experiments cambium was activated for 2-3 cm. below the point of application. The exact tissue in which the auxin travels in such structures as stems has not yet been determined; Cooper's experiments (1936) indicate that it must be Hving tissue, possibly the cambium itself. The stimulus does not seem to travel very far, although the calculations and observations of Biisgen-Mlinch (1929) indicate that it takes several weeks to travel from the growing buds of trees down to the trunk, which would give OTHER ACTIVITIES OF AUXINS 221 it about the same velocity as that found for auxin in the coleoptile (Chapter VI). In trees the activation of cambial divisions by auxin has been studied by Soding (1936a), who showed that insertion of a crystal of indole-acetic acid into the cambium of woody twigs gives rise to a rapid growth of new secondary wood, which in the willow was up to 1 mm. wide; there was also some new production of cortex. The effect, which is due entirely to cambium activation, was only visible down to about 3 cm. below the point of application (cf. Rogenhofer, below). An interesting fact which has been described by various investigators, e.g. Coster (1927-1928) and Gouwentak and Hellinga (1935), is that wounding alone, without auxin addition, definitely produces some cambial stimulation; since, so far as we know, wounding will not produce any auxin, but will be more likely to destroy it (cf. lY A), this indicates that some other substance, probably of the type of the lepto-hormone of Haberlandt (see II E) is here involved. This fact, taken together with the relatively short distance of movement in Soding's and Snow's experiments, seems to raise the possibility that the cambial stimulus, which in nature travels very far, may not be entirely explained by the role of auxin. However, Avery, Burkholder, and Creighton (1937) have determined the distribution of auxin in twigs, following bud development, and find a parallelism between the downward spread of auxin and that of cambial activity. As was stated above, the action of auxin on roots also gives rise to cell division. The thickening of roots produced by moderate concentrations of auxin (cf. Chapter IX) is largely due to enlargement of the cortical cells, but it is usually accompanied by cell divisions either in the cambium (Jost, 1935a) or in the pericycle (Thimann, 1936) or both. Divisions in these layers give rise to lateral roots, which may be produced directly as a result of auxin application (cf. XI F), and at physiologically low concentrations. The induction of divisions in the stem or root pericycle is evi- 222 PHYTOHORMONES dently comparable with the formation of cambium in the stem; both must be ascribed to the ''sensitivity," or ''readiness to divide," of a certain layer of cells. Cells which have not this sensitivity can apparently not be stimulated to divide by auxin, except perhaps in unphysiologically high concentrations, and these cells therefore require the other factor or factors discussed above. Thus Snow (1935a) observed that an auxin concentration sufficient to produce marked activation of the cambium produced no divisions in the cortex or pith. We may perhaps conclude, though as yet without experimental support, that cell division results from the interaction of several factors, of which one is auxin; the distribution of these factors differs for different tissues, and those tissues, such as stem and root cambium and root pericycle, which divide readily on the application of auxin, do so because they already contain the other factors, and auxin is therefore limiting. Cells like those of the Phaseolus pericarp which do not respond to auxin must therefore be considered as having the other factors limiting. Finally, cells containing only traces of these other factors could respond to them if extremely high concentrations of auxin were added. The cambium or callus cultures of Gautheret (1934, 1935), which continued to proliferate on culture medium, presumably contained auxin and other factors in storage. C. Callus and Stem Swellings One of the most obvious results of the application of auxin to young tissues, especially if high concentrations are used, is the swelling of the tissue. This aspect of auxin activity has unfortunately not been studied very quantitatively, so that we cannot say just how the concentrations needed to produce swelling compare with those present in nature. The application of these findings to normal physiological processes is therefore difficult, and more exact work is needed, but it seems probable that they will have a bearing on pathology and teratology. In woody cuttings these OTHER ACTIVITIES OF AUXINS 223 swellings have their counterpart in the formation of callus at the basal cut surface. Such callus formation, which involves both cell enlargement and di\dsion, was observed in Acalypha cuttings by Bouillenne and Went (1933) after appUcation of diastase or extract of rice polishings. They concluded that the callus was caused by a special substance, analogous to, but probably not identical with the root-forming hormone. It was reported by Laibach (1933) that orchid pollinia, applied to decapitated epicotyls of Vicia Faba, cause a marked increase in thickness. The same result was obtained on other plants by Laibach, Mai, and Muller (1934), Mai (1934), and Miiller (1935), both with orchid pollinia and with ether extracts of these and of urine. ^ Later the same effect was obtained with pure indole-3-acetic acid in lanoUne by Laibach (1935) (see Figure 60), by Hitchcock (1935), the authors (u), and others, while similar results from the application of indole-but}Tic, naphthalene-acetic, and other acids have been mentioned by Zimmerman and Wilcoxon (1935). The only quantitative work so far carried out is that of Laibach and Fischnich (1935a) on swellings, and of Rogenhofer (1936) on callus proper. The former found that the extent of thickening of Vicia Faba epicotyls is proportional to the logarithm of the concentration of auxin applied, using lanoline paste. Rogenhofer determined the callus produced Fig. 60. Swelling and callus formation at apical cut surface of decapitated epicotyl of Vicia Faba, after treatment wdth indole-acetic acid paste. (From Laibach, Ber. d. hot. Ges. 53: 359-364, 1935.) 1 These workers proposed to call the active substance causing swellings " Meristine," but since it is identical with auxin the name can be dropped. 224 PHYTOHORMONES on sections of Populus twigs by separating and weighing it. The amount of callus formed by auxin decreases rapidly with distance from the point of application, approaching zero at about 3 cm. below it. If applied simultaneously at two points on the twig, the callus produced was equal to the sum of the amounts which would be produced by single applications at the same points; the method is thus satisfactory for quantitative work. Auxin a was somewhat less active in callus formation than indole-acetic acid. As to the histology of the thickening, it involves both cell enlargement and division, the enlargement, which is partly radial and partly isodiametric, being mostly confined to the cortical parenchyma and the pith cells (u). Laibach and Fischnich (1935) found that the proportion of divisions to enlargement was always about the same in Vicia Faba, and therefore use the total swelling as measure of the cell division; ^ they correspondingly speak of the swellings as ''callus." However, the term callus has usually been applied to the formation of undifferentiated and random-oriented cells at wound surfaces, whereas these swellings show at least in part the character of the stem; further, since enlargement of parenchyma accounts for a large part of the increase in volume, and in some cases (as claimed by Czaja, 19356 ^) for all of it, it seems better simply to use the term ''swellings." Within the swellings secondary lignification of the cell walls may often be observed, so that it seems possible that auxin controls or stimulates such secondary wall formation. Czaja (1935) has put forward the view that the effect of auxin depends largely on the direction of its application. If applied directly to the transversal cut surface of the Helianthus hypocotyl he finds increase in elongation to be the principal effect, but if applied externally, to the epidermis, the principal effect is swelling. His published measurements, 1 The controversy between Czaja and Laibach as to whether the auxin swellings are due to cell division or enlargement is easily explained: when auxin is applied, enlargement of the parenchyma cells takes place first, and cell divisions follow after a few daya (Went, 1936). OTHER ACTIVITIES OF AUXINS 225 however, do not bear this out, nor could Jost and Reiss (1936), nor the authors (u), confirm it. It is true that the place of apphcation of auxin is in some cases important for its efTect; this is shown in roots (Chapter IX) and also for the elongation or inhibition of stem growth (Le Fanu, p. 214). Czaja's theory, discussed in VI D, that the polarity of cells and tissues is caused by the direction of the prevailing auxin stream was based upon these experiments. Went (1936) has suggested a mechanism for the formation of swellings in terms of the interaction between auxin and other factors. The formation of callus at the base of a cutting is often, though not always, associated by gardeners with good rooting of the cutting. In the hght of the above, it is probable that a cutting which forms much callus does so because it is rich in auxin, and the root formation is then due to the auxin; the roots are not as a rule formed from the callus, but both result from the same internal cause, namely the auxin. The many exceptions to this rule pro\dde further evidence of the role of other, non-auxin, factors in both processes. The auxin swellings bear a close resemblance to the phenomena observed in some of the galls and other pathological outgrowths, and there is good evidence that auxin plays an important part in such growths. The root nodules of leguminous plants are active auxin-forming centers when still growing, and their initiation and growth are almost certainly due to the auxin produced by the invading bacteria (Thimann, 19366; cf. IXC). These can therefore be considered as root-galls, arising by pathological swelling of a lateral root initial. The crown-gall organism, Pseudomonas tumefaciens, also produces auxin, since Brown and Gardner (1936) have obtained typical swellings and outgrowth by extracts from cultures of this bacterium (see also Duyfjes, 1935). They have also produced large galls on Phaseolus by long-period application of pure indoleacetic acid at a cut surface (see Figure 61). The experiment of Nemec (p. 187) also indicates auxin production by this 226 PHYTOHORMONES bacterium. The apparently widespread ability of bacteria and fungi to produce indole-acetic acid from tryptophane, or possibly other auxins from other amino-acids (c/. IV E), is doubtless of great significance in this connection. Fig. 61. Gall, produced in 5 months on f^liaseolus stem by decapitating and smearing wound with indole-acetic acid paste (20 mg. per gram of lanoline). (From Brown and Gardner, Phytopath. 26: 708-713, 1936.) The intumescences on leaves of Populus grandidentata, which are not caused by microorganisms but arise under conditions of high humidity (La Rue, 1933) may be perfectly imitated by injection of indole-acetic acid in very low concentration ; if in some way the action of auxin in the leaf is intensified by high humidity this would explain them OTHER ACTIVITIES OF AUXINS 227 (La Rue, 1936). Probably many other pathological outgrowths are explicable in terms of auxin—a development which will open important fields in plant pathology. D. Miscellaneous Effects A number of other effects of auxin have been recorded. Laibach (1933) has described how tendrils of Cucurbita coil up when auxin paste is applied on one side, the reaction not being restricted to the place of application. This, however, is not generally true, for tendrils of Passiflora, similarly treated, show only local curvatures (u). The reactions of leaves are of some interest. Laibach (1934) found that when Phaseolus leaves are smeared over with auxin in lanoline, they bulge and curl, while Avery (1935) found that the midrib of the Nicotiana leaf curves readily away from auxin paste applied on one side. The authors' experiments {u) indicate that auxin causes elongation of the cells of the midrib and lateral veins, but not increase of surface of the mesophyll. This results in differential growth with bulging of the leaf-blade (particularly marked in Aristolochia) . In varieties like dejorrnis of tobacco (Honing, 1923) or wiry of tomato (Leslie, 1928), mesophyll growth is practically absent in the mature plant, so that the leaves are restricted to a midrib, with the lateral veins, which are still present, contracted against it. Thus the deformis and wiry genes affect only mesophyll growth. On the other hand, Aphids (see Maresquelle, 1935) and ''curlytop" \drus cause curling of the leaf, due to deficient vein growth, while the blade develops normally. Grieve (1936) has indicated that in ''spotted wilt" virus this curling may be due to lack of auxin. It is significant that it is usually associated with reduction in total growth. All these cases fit in with the view that auxin causes elongation of leaf veins, while the growth of mesophyll depends on other factors, such as that postulated by Gregory (1928). Certain other effects are not readily explicable on the basis of growth promotion. Thus if the leaf-blade of Coleus 228 PHYTOHORMONES be removed, the petiole subsequently falls off; but if urine or auxin paste be applied to the petiole it remains in position for a much longer time (Laibach, 1933a; Uhrova, 1934; La Rue, 1935). AppHcation of large amounts of indoleacetic, indole-butyric, or other related acids to the soil, 3 to 5 weeks before flowering was due, has been reported to hasten the formation of flower buds in Turkish tobacco (Hitchcock and Zimmerman, 1935). A hastening of flowering has been found to be produced in various plants by application to the soil of female sex hormones (see XIV B). Hastening of flowering has also been recorded in peas and beans as resulting from application of yeast extract (Virtanen and Hansen, 1933, 1934). Little can be said about the mechanism of such phenomena pending more careful study. Interesting, particularly from a historical standpoint, is the role which is apparently played by auxin in the phenomena of post-floration of some orchids. It was shown by Fitting in 1910 that the falling off of the flower and swelling of the gynostemium of some tropical orchids after flowering were brought about by the presence of the pollen grains, and could also be caused by water extracts of the pollinia (see II E) . The action was ascribed to a hormone in the pollen, and this was the first use of the term hormone in connection with plants. It was shown by Laibach (1932a) that this substance is extractable with ether, behaving like an acid in the extraction, and further that the extracts were active on Avena. Hibiscus pollen gave similar results. Laibach and Maschmann (1933) subsequently showed that ether extracts of urine and animal tissue behaved in the same way, and that action on the orchid gynostemium was parallel with auxin action on Avena, all extracts which had the one action having the other. The activity on the orchid was destroyed by H2O0. The sweUing was entirely due to cell enlargement. It was thus deduced that the effect is due to the auxin in the pollinia, so that Fitting's '^Pollenhormon," which may fairly be called the first plant hormone, is identical with auxin. Experiments have not OTHER ACTIVITIES OF AUXINS 229 been carried out with pure auxins, but there seems no reason to doubt the identity. In this case we have a combination of the growth-promoting effect with another effect resulting in flower fall, the mechanism of which is as yet unknown. We have seen that the auxins play a protean role in the development of plants, and influence a large number of processes, both normal and pathological. It is important to note that, as discussed in Chapters VIII and XII, in all cases investigated a substance which shows one of the effects of auxin shows them all. Its acti\dty may sometimes be concealed, as in those substances which cause no Avena curvature, or which produce root formation only locally, but it may be demonstrated by special experiments. Many of the apparently different effects are to be traced to different types of cell enlargement, but others, such as root formation, are more complex. It is difficult to avoid the conclusion that ''all these functions arise from one primary reaction in the cell; which physiological effect is produced depends upon the nature and position of the tissues affected. The actual primary reaction becomes, then, of greater interest than ever. It is a kind of master reaction governing the activities of the cell." So far as attempts have been made to analyze further the nature of this primary reaction, they have been summarized in Chapter VIII. It remains now to consider the role of auxin in comparison with some other aspects of growth and development. CHAPTER XIV GENERAL CONCLUSIONS In the preceding pages we have seen how from analyzing the complexities of correlation and of tropisms there has emerged a clear concept of hormones and of the role they play in the plant. It could hardly be expected, however, that, after only about 10 years of research this concept would have led us to a finished explanation of all the processes in which auxin is involved. Still less could we be expected to have progressed in our knowledge beyond this one group of hormones, the auxins. Our knowledge of the auxins has, as a matter of fact, laid open to experimental attack many problems concerned with other factors in the plant. However, it is fair to say that we already see the auxins and their properties as a continuous thread connecting most of the developmental and growth processes in the plant. Thus the auxins bring tropisms into that close relation with growth upon which Blaauw insisted in 1918; they bring growth, in the general sense of development and organ formation, into the same terms as growth in the special sense of cell elongation; and lastly, they bring the concept of correlation, which by its very name has previously defied causal analysis, into the realm of direct experimental attack. Perhaps the best example of this last phase is the meaning which is now given to the formerly elusive conception of polarity; polarity can now be expressed quantitatively as a function of the transport of a known substance in the tissues. Finally, few fields can have benefited so much from close interaction between the biological and chemical approaches, and the remarkable discoveries on the chemical side have made possible equally remarkable progress in the physiology. It has even been possible to inquire somewhat into the inner mechanism of the relation between auxins and their substrate. 230 GENERAL CONCLUSIONS 231 A. Quantitative Relations between Auxin and Growth The development of our knowledge of auxins has brought out the fact that their activity is quantitative as well as qualitative. The extent of growth, of root formation, etc., is directly dependent on the amount or concentration of auxin present. Since these quantitative relations have been proven for the simplified cases of direct experiment it is interesting to consider their bearing in general on growth and correlation. Thus absolute size, among individual plants of a species (within normal environmental conditions), remains fairly constant and is even used as a taxonomic character. The experiments described in Chapter VI allow us to ascribe this to genetically determined rates of auxin production. Ajnong relative size relationships, one of the most stressed is that between the above-ground and below-ground parts of the plant, the "shoot-root ratio." Such constancy as this ratio shows is probably due to a reciprocal control system in the plant. On the one hand the number and size of buds and leaves determines the rate of auxin production, and this auxin, reaching the roots, influences the development of the root system (c/. XI F) . On the other hand, the root system probably forms a factor or factors necessary for shoot growth (c/. XII C) and hence the increased root system increases the shoot. Another interesting relation is that between the place of auxin production and the length of the growing zone in a stem. In many stems {e.g. most seedlings and annuals — Pisum, Vicia, Nicotiana), the growing region is restricted to a few cm. below the terminal bud, and in these stems auxin production is mainly in the terminal bud and youngest leaves. Other plants exhibit growth over a longer region, and in these auxin production is probably vigorous in axillary buds or lower leaves as well as in the terminal bud. Examples are Asparagus (Oosterhuis, 1931), Polygonum (Fig- 232 PHYTOHORMONES ure 33), Tradescantia (Uyldert, 1931), and Bambusa (u); probably the shoots of all trees and shrubs with periodic growth behave in this way. The correlation between the amount of auxin which diffuses from buds of different plants and the rates of growth of the shoots below them (Zimmermann, 1936) may also be mentioned. All such observations support the view that quantitative relations between different parts of the plant are expressions of the quantitative relation between auxin and its growth effects. This generalization can now supersede the older view that such growth relations are determined by the amounts of food material present. B. Comparison with Animal Hormones The progress of the work on phytohormones compares very favorably with that which has been made in other fields. This may be attributed to the relative simplicity of the relation between the plant and its hormones, as contrasted with the apparent complexity in animals. The hormonal correlation between different parts of an organism will be the more complex the larger the number of parts; the plant, with few organs, is thus a relatively favorable organism for study. One important complexity in animals is that the action of hormones is so often indirect, so that the hormonal activity of one gland may be expressed through its effect on the activity of another gland. The pituitary exerts an effect on the gonads, vitamin D may act through the parathyroid, and so on. Where there are introduced in this way additional links in the chain of action the mechanism of the process is correspondingly harder to elucidate. This does not necessarily mean that in plants the fundamental process is itself any simpler, but it does mean that it is more open to attack, as is shown by the fact that it has been possible to separate the primary processes in which auxin takes part from the secondary ones which prepare for its action. GENERAL CONCLUSIONS 233 There are important contrasts between the hormones of the plant and animal kingdoms. In animals, of course, the foods and hormones all travel along the same path, namely that of the blood stream. This has the effect that all cells receive the same hormonal stimulus and the result will depend upon their ability to respond to it. Plants, on the other hand, have no true circulation and the movement of hormones in them is mainly unidirectional, independent of the mass movements of water and foods. Thus hormones and foods move by different paths and hence every cell is not in a position to respond; this results in local growth zones and phenomena such as apical growth. However, the fact that the term ''hormone," first coined for animal physiology, has been used for the auxins shows that there are important parallelisms. The fundamental function of hormones, namely that of chemical messengers, is the same in plants as in animals;—the phytohormones are true hormones. Further, the acti\'ity is in both cases exerted in concentrations too low to allow of their making up an appreciable part of the cell (cf. VHI E). Table XVI summarizes very approximately the minimal active doses per gram of fresh weight of the test organism, for some different hormones. These figures, however, are not really comparable, not only because the molecular weights are different, but also because we do not know the actual concentrations at the place of action. It is interesting to compare the specificity of the animal TABLE XVI Hormone 234 PHYTOHORMONES hormones with that of auxin. There is Uttle or no species specificity in either case, that is, one hormone performs the same function in large classes of organisms. On the other hand there is specificity of function, and this is far more marked in animals than in plants. Thus the auxins bring about a number of different effects, while the animal hormones in general have one special function. The latter are not as a rule merely growth-promoting, and their assay — except in the case of the pituitary hormone—is not based on growth measurements. An exception might be made for the sex hormones, which certainly act to produce a rapid growth of special tissues and which have actually been referred to as " Wuchsstoffe " (Butenandt, 1935, 1936). The specificity of function in animal hormones is strictly dependent on their molecular structure, so that in the sterols, for instance, small changes in the molecule completely alter certain functions of the substance: hydrogenation of one aromatic ring converts a female to a male sex hormone. In the auxins small changes merely alter the ciuantitative activity of the substance, but do not change the functions it performs. However, quantitative changes in activity with small changes in the molecule also occur in the sex hormones. Whether these changes are due to differences in secondary properties, such as penetration, inactivation, etc., as with the auxins (see VIII G), has not yet been ascertained. It is, however, very suggestive that the relative activity of two substances in two different tests may be quite different. Thus if the activity of androstandiol, a male sex hormone, is taken as 1 in both the capon and the rat test, then isoandrostandiol has an activity of 0.04 in the capon and 0.23 in the rat test (Butenandt, 1936). It seems justifiable, therefore, to suggest that the difference in relative activity between two such substances in the two tests is due to differences in their secondary properties. The effect of animal hormones on plant growth scarcely falls within the scope of this review, but may be mentioned briefly. There is evidence that oestrone in particular has GENERAL CONCLUSIONS 235 the effect of increasing the growth and development of a number of different plants. This is shown by increased production, dry weight, etc., in wheat, rye, barley, beans, and sugar-beets (Scharrer and Schrop, 1935) and tomatoes (Schoeller and Goebel, 1935). The effect can be ascribed to a stimulation of the earlier growth stages. Stimulation of flowering, either by an increase in the number of flowers or by the earlier opening of the first flowers, has also been reported (Schoeller and Goebel, 1935). This also seems to be correlated with better growth. The experimental conditions necessary to obtain positive results are of great importance, since various investigators were unable to confirm Schoeller and Goebel's earlier work (1931-1934) {e.g. Harder and Stormer, 1934; Virtanen, Hansen, and Saastamoinen, 1934). Indications that oestrone has an effect on root formation are mentioned in XI E. Comparison has recently been made between the phenomena of crown-gall disease and animal cancer (Levine, 1936). This comparison, which was first made 20 years ago by E. F. Smith, rests upon very superficial similarities and appears quite unjustified. Conversely, little comparison can be drawn between the auxins and the animal carcinogenic substances. These substances, which are hydrocarbons (see Fieser, 1936), are very slow in acting, and it seems probable that their effect is to induce normal cells to change into cancerous cells rather than to promote cell growth directly. C. Comparison with Growth Substances of Lower Plants In contrast to the above true hormones stand the growth promoting substances for fungi and microorganisms. While free movement of these substances may take place within the organism it is not an essential part of their acti\dty; this is particularly obvious for unicellular microorganisms, in which correlations as ordinarily understood cannot occur. While microorganisms, therefore, cannot, by definition, have 236 PHYTOHORMONES hormones, the question as to what name should be given to their growth-promoting substances remains in doubt. The term vitamin has the definite connotation of a food factor absorbed from the medium, while these growth substances are in some cases produced by the organisms themselves. On this account Kogl (1935) was led to term bios a phytohormone, because although yeasts require bios for growth, they also have the ability, though not usually in sufficient degree, to produce bios for themselves. According to Huxley's nomenclature (1935) we should name such substances local or intracellular activators. The reason why these growth substances have been mentioned here is because there are some interesting parallels between them and the phytohormones. The group includes bios, vitamin Bi, " Wuchsstoff B," and the substances active on bacteria and protozoa. Vitamin Bi is necessary for the growth of a few yeasts and numerous fungi. Wuchsstoff B is the term given by Nielsen and Hartelius (1932) to a substance, insoluble in ether, produced by Rhizopus cultures and active in promoting growth of Aspergillus. (The name should not be confused with auxin b.) Apparently both an organic growth substance and a group of accessory inorganic substances are involved (Nielsen and Hartelius, 1933). The term bios is specifically restricted to substances active in promoting the growth of yeasts. Bios is of particular interest here because it was the first example of a system of interlocking substances, the action of each of which is increased by the presence of the others. That any one bios produces an effect alone is probably to be ascribed to the presence within the cell of small amounts of the other factors. The work of the Toronto school has shown that bios consists of at least 3 fractions, of which bios I was shown by Eastcott (1928) to be z-inositol, and another, bios II, has recently been isolated, under the name of "biotin," by Kogl and Tonnis (1936). Like auxin, biotin is active at extremely high dilution, 1 part in 4 X 10^^ of solution having a detectable effect; however, in terms of GENERAL CONCLUSIONS 237 the weight of organism affected its acti\dty is not particularly high (see Table XVI). The following table from Kogl (1935) exemplifies clearly the interlocking action of the 3 fractions: TABLE XVII Additions to a Medium Containing 42 Grams of Yield per Gram of Dextrose and 23 Grams of Salts Inocdlom in 10 Hours Control 1-1.5 Bios I, 4 g. 1-1.5 Bios III, 2 g. 1-1.5 Bios I, 4 g. + Bios III, 2 g. 1-1.5 0.167 7 Biotin 4 1.67 7Biotin 7 1.67 7 Biotin + 4 g. Bios I 10 1.67 7 Biotin + 4 g. Bios I + 2 g. Bios III 14 The strain of yeast here used evidently contains none of the biotin, which thus acts as a limiting factor, but in the presence of a sufficient amount of biotin the other constituents become limiting and must be added to obtain maximal growth. Some yeasts, like that of Williams et al. (1933), need only one of the bios factors, presumably being able to synthesize sufficient of the others. This parallels closely the root formation of the different strains of Pisum discussed in XI E, where some varieties respond to auxin application and others not at all. In some of these, auxin is already in excess, as is shown by the large number of roots formed by untreated controls; in others some additional factor is lacking, for roots are not produced by any treatment. Root formation in Pisum provides a particularly good example of a system of limiting factors in higher plants, there being at least 3 substances known whose action interlocks. These 3 factors, sugar, auxin, and biotin are all available in the pure state and hence their interlocking action is easily studied (c/. XI E). A graphic representation of the interaction of these factors, drawn in a manner comparable to that of Blackman (1905), is shown in Figure 62. It will be seen that each factor reaches a concentration at which it is no longer limiting, and at this concentration root formation may be increased by adding the next factor, the 238 PHYTOHORMONES increase being proportional to the amount added. The activity of any one factor alone is zero, the action of sugar alone being ascribable to the presence of small amounts of auxin in the plant. We thus have a complete parallel to the interaction of bios I, II, and III on yeast growth, or that of the Wuchsstoff B and Co-Wuchsstoff on Aspergillus growth; or, to go still further, to the interaction of enzymes and coenzymes. Such interlocking systems of limiting factors are widely distributed in nature and probably will be encountered in any process which is sufficiently analyzed. While bios, a growth substance for fungi, plays a part in the growth of higher plants, there is no evidence that auxins have any action on fungi or other lower organisms. Boysen Jensen (1932), Nielsen and Hartelius (1932), Bonner (1932), and Biinning (1934) all found that the auxin produced by fungi has no effect upon their own growth. Ronsdorf (1935) failed to find any effect of the addition of auxin a to the culture medium of various fungi; the auxin, however, was apparently destroyed in the medium. Similar destruction of indole-acetic acid in Rhizopus cultures was also noted by Thimann and Dolk (1933) after the time of maximum auxin production had been passed. The above experiments make it highly improbable that the action of plant exudates on growth of Phytophthora can be due to auxins, as supposed by Leonian (1935). The report of Popoff (1933) that the excysting action of plant extracts on Euglena is due to auxin is probably equally unfounded. For the excystment of Colpoda Thimann and Barker (1934) found that at least two substances are involved; one is present in impure auxin preparations but is not identical with auxin, the other is completely unrelated to auxin. Pure indole-acetic acid and other auxins have no excysting action on Colpoda or some other protozoa (u). D. Regeneration We have made comparisons between the active substances of higher plants and those of lower plants and of animals. B rt S 239 240 PHYTOHORMONES We can now attempt some comparisons between the processes in which these substances are involved, in plants and animals. The analysis which we have made in the case of root formation enables us to consider organ formation in more general terms, and especially organ formation on isolated parts—the so-called ''regeneration." It is clear that the substances and processes acting to form roots are doing so continuously in the normal intact plant. In a cutting these factors will continue to operate in the same way as they would have done in the intact plant, the place of accumulation being now, of course, different. Thus the auxin which normally would have moved from the buds to the base of the shoot, or even into the roots, will now move merely to the base of the cutting. It follows that the roots so formed are not new formations (''regenerates"), nor is their formation in any way a response to the loss of the others. On the contrary it is merely the normal generation going on in an atypical—that is, an artificially conditioned—place, and thus has nothing to do with any "tendency towards completion (Ganzheit)." In animals, if a part is removed, there is a tendency to replace it, and this is very pronounced in the Coelenterates, which furnish the best comparison with root formation. These animals show apical growth, and, what is important from our point of view, they have no true circulation of food. Correspondingly, their growth is not diffuse, as in higher animals, but is typically polar. This must be due to the polar transport of one or more growth substances, which in higher animals would be carried in the blood stream instead. In view of the neglect of this subject in recent years, it would be of great interest now to transfer our knowledge of "regeneration" in plants back to such animals, with special reference to the role of growth substances and of their probable polar transport. A well-analyzed case of regeneration in a very interesting object is the work of Hammerling (1934, 1935, 1936) on the alga Acetabularia. This bears out all the points pre\4ously GENERAL CONCLUSIONS 241 discussed. In this large uninuclear siphonaceous cell, the apical end of any isolated middle section will regenerate apical organs; the basal end, on the other hand, will regenerate rhizoids. Sections containing the nucleus regenerate rapidly and completely, but without the nucleus regeneration is only complete if the section is fairly old and has therefore been under the influence of the nucleus for some time. Hammerling interprets his results as due to polar movement in opposite directions of two substances whose production is controlled by the nucleus, one apex-forming and the other base-forming. The percentage of regenerates, and the rate at which they form, are both proportional to the size of the isolated piece, and therefore, as he concludes, to the amounts of organ-forming substances present. The conclusions are supported by numerous additional experiments, but it must nevertheless be emphasized that as yet no direct e\adence for the postulated organ-forming substances has been brought forward. A comparable case is that of Griffithsia hornetiana. By passing an electric current through this alga, Schechter (1934) could induce rhizoids to form at the side facing the positive pole. The number of rhizoids was increased by increasing the current density. Schechter suggests that the formation of rhizoids is induced by some material inside the cell, which moves in an electric field. Centrifugal forces have a comparable effect (Schechter, 1935) ; shoots appear on the side where the hea\der matter collects. Similar electrical control of polarity was reported for the zygotes of Fucus by Lund (1923); the rhizoids grow out towards the positive pole. Thus, the inherent polarity in lower plants, or single cells, may be affected by electrophoresis of organ-forming materials resulting from applied potentials. This is not true for higher plants. To explain differences between types of regenerates, and between growth rates at different points, the concept of ''gradients" or ''fields" has been introduced. So long as the gradient is one of something intangible it is not open to 242 PHYTOHORMONES experimental attack, and is only a restatement of the observations in different terms. If, however, the term be interpreted as a gradient in the concentration of active substances it not only becomes experimentally analyzable but is in close agreement with the situation in plants. Plants have, in fact, a gradient of auxin concentration which is set up either by the apical auxin formation or by the polarity, the latter probably being the cause of the former. Heretofore polarity was only detected by its effect on regeneration or organ formation, and as so many other factors affect this process it was a doubtful criterion even for the existence of polarity. But now, by measuring the rate and capacity of polar auxin transport we can measure polarity quantitatively and this undoubtedly will lead to a better understanding of this remarkable property of living matter. Apex-to-base polarity provides an example of the simplest type of "field." E. Organization As we have seen, the factors operating in regeneration are those operating in normal development, and this suggests that the same principles also underlie development in the embryo. The effects of auxin in the plant compare very interestingly with embryonic development in the animal. Organization in the animal embryo, which may be defined as differentiation according to a definite pattern, is slightly different from Sachs' concept of organization referred to in VIII A. In the amphibian gastrula, any part of the ectoderm is apparently able to differentiate into a neural tube. That only one is formed is ascribed to the ''organizer," viz., the dorsal lip of the blastopore, which as Spemann and his school have shown will, after invagination, induce the formation of a neural tube in that part of the ectoderm against which it comes to lie. Recently attempts have been made to isolate chemically the active principle of the organizer (see the review of Weiss, 1935). As a result, certain substances have been found to have the power of causing GENERAL CONCLUSIONS 243 the differentiation of a neural tube. The chemical nature of these differentiating substances or ''evocators" (Waddington, 1934) does not appear to be highly specific. Fischer et al. (1935) have found that similar effects can be produced by highly purified oleic acid, hnolenic acid, and 12-octadecenic acid-1, as well as by muscle adenyUc acid. Saturated acids, including stearic acid, were not active. From these results they conclude that the induction can be regarded as a stimulation by acid ("Saure-Reiz"). Such a stimulation might be interpreted in the same way as the effect of acid on growth of plants, namely that the acids set free a certain amount of active substance, but have no activity themselves (cf. VIII F). The failure of the saturated acids to act may be due to their lower ability to enter the cell. The results of Waddington, Needham, et al. (1935) cannot be interpreted in this way, and the active sterols and hydrocarbons studied by them apparently possess evocator acti\aty of their own. It must be emphasized that such evocations are not to be confused with the action of the living organizer, which not only differentiates the neural tube but also controls all other differentiations, i.e. it imposes a pattern on the embryo. This pattern-formation seems to call for the assumption of some kind of "field," of the same type as the polarity of auxin transport in the plant. In organization in the plant, such as root formation, the ''field" is the polar transport of the active substances; the evocator itself can be compared with auxin. Although auxin is formed or present in many different tissues, organization takes place only in that special location which is determined by the polar accumulation of the auxin. Correspondingly, in animal embryos, the evocator may be present in other parts of the embryo, as Holtfreter (1935) showed; the lack of a "field," however, is probably the reason why it does not act there. There is thus a close parallelism between auxin and the evocator; both are present in many parts of the organism, but their effects are limited by an existing "field," i.e. by their polarized accumulation. As to the 244 PHYTOHORMONES organizer, it appears to furnish both field and evocator, and it must therefore be compared to the sum of auxin and polarity. F. The Stimulus Concept and the Nature of Auxin Action As has been shown in the latter half of this book, auxins bring about a number of different responses in plants. These are, specifically: growth by cell-elongation; the formation of roots—both on stems and on roots themselves; the inhibition of buds; the activation of the cambium; inhibition of root growth, and certain growth phenomena which involve cell enlargement and division together. We have seen already that a number of different substances can bring about the same growth response. It is also true that any one of the active substances can bring about all these different responses. This raises the question of the mechanism of these effects. There are two possibilities which may be considered; (1) the auxins bring about some master-reaction within the cell, the results of which will be determined by the presence and amount of other factors (''condition of the cell"); and by the conditions of the experiment; or (2) the auxins are stimulating substances setting free the energies stored up in the Uving protoplasm ("latente Reizbarkeiten"). This latter view has recently been emphasized by Fitting (1936); "I am convinced that we are right in including the hormones with the 'stimulus-substances' (Reizstoffe). By these are understood, following physiological usage, all those compounds which exert their physiological action through the intervention of the living substance, i.e. whose first point of attack is the living plasma. The typical physiological actions of stimulus substances show all the characteristics of stimulations. . . ." By the latter are meant latent time, presentation time, threshold concentration, excitation of the protoplasm, anti-reaction and recovery, etc. To make this somewhat old-fashioned idea clear to the modern reader it is necessary to picture the organism as con- GENERAL CONCLUSIONS 245 tinuously in touch with its surroundings: every quality of the surroundings, such as hght, gra\dty, or chemical change, acts as a stimulus on the organism, and the perception of this stimulus is followed by a response. These stimuh do not supply the organism with the energy which it uses in its response, any more than a Ughthouse supplies a ship with the energy to move out of its way. The stimulus is perceived by the helmsman and the response of the ship is achieved by the release of much larger amounts of energy. According to Fitting's definition the perception of the light stimulus must necessarily be through the hving protoplasm of the helmsman, although this is not entirely true because the helmsman can very well be replaced by a photoelectric cell. (A similar example has been given by Loeb [1918] in his "heliotropic machine.") However, it must be made clear that the responses of plants to auxin are by no means typical stimulus responses. Let us take as example the effect of light in causing phototropism. It was at first believed that the light acted by releasing the stored-up "bending tendency" in the plant. Blaauw and Froschel's ''Reizmengengesetz" dealt a serious blow to this concept by establishing a quantitative relation between the amount of energy applied (intensity X time) and the amount of response. Not only this, but the curvature of an Avena coleoptile is, as we now know, principally due to the redistribution of auxin between the light and dark sides. This redistribution requires far less energy than is suppUed by the light which causes it (Went, 1936). The ship is, in fact, being steered by the photoelectric cell itself. Thus one of the most typical properties of a stimulus, i.e. its action through the release of stored-up energy of the protoplasm, is here absent, and for phototropism, therefore, the stimulus concept is an unnecessary compUcation. The relation between auxin and growth also has nothing in common with stimulation. We have seen in the foregoing chapters, especially in III C and III D, that a given amount of auxin produces, if the conditions are constant, a 246 PHYTOHORMONES given amount of growth, the curve of proportionahty being a straight Une within well-defined limits. Under other conditions, of course (as for instance at other temperatures), the proportionality factor will be different. Old coleoptiles do not give the same amount of growth for a given auxin application as do young ones (du Buy, 1936) but in these, of course, it is the conditions which are not comparable. Further, there is no evidence for any threshold in the response of young Avena coleoptiles to auxin; the curve of auxin applied against growth produced passes directly through the origin (Figure 19, p. 41). As a matter of fact, even in phototropism the significance of the threshold has been made doubtful by the measurements of Arisz (1915), who showed that its value depended on the method of detection of the minimum curvature. Thus the use of the microscope reduced the apparent threshold illumination from about 5 MCS to 1.4 MCS. A final blow to the stimulus concept of auxin action is dealt by the stoichiometric relations in the pea test, in which the activity of a number of substances, at the lower limit of response, approaches the same value per mole (VIII G). This, together with the linear proportionality just mentioned, means that the auxins enter into a definite stoichiometric reaction with some constituent of the cell. The concept of stimulus, and all that it implies, has therefore no useful bearing on auxin problems. There remains the other possibility mentioned on pp. 229, 244, that of a master-reaction. According to this view auxin acts by taking part in some reaction in the cell, from which a chain of reactions leads to the observed response. The type of response then depends on the other factors, both internal and external, influencing the reaction-chain (Thimann, 19356). Thus it was shown in \ B that growth is controlled not only by auxin but also by another factor or group of factors; the most rapidly growing zone is that in which both are present in optimal concentrations. The auxin at the extreme tip of the coleoptile cannot cause growth because the other factors are limiting; the auxin at the ex- GENERAL CONCLUSIONS 247 treme base of the coleoptile cannot cause growth because the cells can no longer respond to it. Root formation furnishes a still more striking example. Here, as shown in XI E, the formation of roots is dependent upon the cooperation of a number of factors. The concentration of auxin determines not only the number of roots formed, but also the cells which will form them. In general, the response in organ formation is localized. To explain this localization of the action Went (1936) has suggested that in addition to its master-reaction effect, auxin acts by affecting the transport of the other factors, so that they become accumulated at the point of highest auxin concentration. However this may be, it is clear that when the influence of the other factors is taken into account, the apparently mysterious action of the auxins in bringing about so many responses no longer seems so obscm'e. It is true that we do not yet know the nature of the fundamental master-reaction, but there is good reason to hope that it is within our reach. G. Abnormal Growth If internal or external factors affect growth, they must do it through the growth-controlling system we have discussed. In some cases this will be through their effects on auxin. Numerous examples of this have been given. The effects of external factors, particularly radiation, are numerous, and a number of them have been analyzed in terms of auxin. In the case of visible light, these include phototropism (KE), auxin production (IV ^), and sensitivity to auxin (X B) ; in the case of x-rays, their general inhibiting effect on growth has been explained (V F). Of the effects of internal factors, only one group has yet been considered to any extent, namely the genes. The genes set up a chain of internal reactions which terminate in the observed effect ; but the last link in this chain, where genes affecting growth are concerned, is the effect on auxin. This has been partly analyzed in V E. Many of the infections to which plants are susceptible 248 PHYTOHORMONES become incorporated in the plant to such an extent that they may be regarded as internal factors. There are a number of diseases in which growth correlations are destroyed or new ones established, and in these auxin must play a part. Thus, in ''curly-top," one of the virus diseases, the growth of the shoot is greatly reduced, and Grieve (1936) has obtained evidence for a corresponding reduction in auxin content in the infected plant. Galls may be due to the opposite effect, namely production of growth hormones by the infective agent. Indeed, both Sachs (1882) and Beijerinck (1897) based their views on growth correlations partly on such pathological growths (c/. Figure 2). In a study of Erineum outgrowths on leaves (intumescences) La Rue (1935, 1936a) has shown that auxin will produce similar effects, while Brown and Gardner (1936) have shown the same thing for crown gall. In the case of root nodules there is good evidence that they are due to auxin produced by the infective agent, Rhizobium leguminosarum (see IX C). Comparable evidence for the production of auxin by the crown-gall organism has been given in XIII C. The galls due to gall-wasps {Cynipidae) and gall-midges (Cecidomyidae), which show elaborate differentiation, can probably not be considered in such simple terms. Another suggestive observation for the interpretation of abnormal growth is that of Laibach and Mai (1936), who have described malformations of leaves and buds caused by repeated application of concentrated auxin pastes to the growing point. H. Outlook This survey of the rapidly developing field of phytohormones shows that many problems have been solved and few really important points are still subjects of disagreement. This result has not been achieved without effort. In the first place emphasis has been laid from the very beginning on quantitative work. In the second place the development of the chemistry has made it possible to check GENERAL CONCLUSIONS 249 all findings with pure substances. However, the recent increasing use of these substances in unphysiologically high concentrations constitutes a danger, because the results do not necessarily have any bearing on the functions of auxin in the normal plant. Lastly, until quite recently it has been customary to investigate as far as possible every point bearing on a theory before the theory was enunciated, so that most of the views reached were well-founded and could be used as basis for further work. In the last year or two this procedure has not been so rigidly adhered to; examples of generahzations without sufficient experimental foundation are furnished by the comparison between the action of ethylene and auxin discussed in XI C, by the theory of the role of acids in producing growth directly (VIII F), by the interpretation of experiments on the movement of auxin in the transpiration stream (VI D), and by the two-stream theory of inhibition by auxin (VIZ), IX D). There is danger, in any rapidly developing field, of an accumulation of unclassified facts and unproven theories which makes further development much less certain. In the field of phytohormones this is particularly unjustifiable, because the experimental procedure is relatively simple and the equipment necessary is not too elaborate. If the criteria of high-class experimental work continue to be observed, then we may look forward in the next few years to the rapid solution of a great many of the interesting problems of growth and development. BIBLIOGRAPHY Agricola, G. a., 1716. Neu und nie erhorter doch in der Natur und Vernunft wohlgegrlindeter Versuch der Universal-Vermehrung aller Baume, Stauden und Blumen-Gewachse. Regensburg, 1716. Amlong, H. U., 1933. Untersuchungen liber die Beziehungen zwischen geoelektrischen Effekt und Geotropismus. Planta 21: 211-250. Amlong, H. U., 1936. Der Einfluss des Wuchsstoffes auf die Wanddehnbarkeit der Vicia-Faba-Wurzel. Ber. d. hot. Ges. 54: 271-275. Amlong, H. U., 1936a. Zur Frage der Wuchsstoffwirkung auf das Wurzelwachstum. Jahrb. wiss. Bot. 83: 773-780. Anderson, D. B., 1935. The structure of the walls of higher plants. Bot. Rev. 1: 52-76. Appleman, C. 0., 1918. Special growth-promoting substances and correlation. Science 48-' 319-320. Arisz, W. H., 1915. Untersuchungen iiber den Phototropismus. Rec. trav. bot. need. 12: 44-216. Avery Jr., G. S., 1930. Comparative anatomy and morphology of embryos and seedlings of maize, oats, and wheat. Bot. Gaz. 89: 1-39. Avery Jr., G. S., 1935. Differential distribution of a phytohormone in the developing leaf of Nicotiana, and its relation to polarized growth. Bull. Torrey Bot. Club 62: 313-330. Avery Jr., G. S. and P. R. Burkholder, 1936. Polarized growth and cell studies on the Avena coleoptile, phytohormone test object. Bull. Torrey Bot. Club 63: 1-15. Avery, G. S., P. R. Burkholder, and H. B. Creighton, 1937. Production and distribution of growth hormone in shoots of Aesculus and Mains, and its probable role in stimulating cambial activity. Am. J. Bot. 24: 51-58. Babicka, J., 1934. Die Wuchsstoffe. Beih. Bot. Centralbl. 52: 449-484. Bauguess, L. C, 1935. Plant responses to some indole derivatives. (Abstract). Am. J. Bot. 22: 910. Bayliss, W. M., 1927. Principles of general physiology. 4th Ed. London, 1927. Bayliss, W. M. and E. Starling, 1904. The chemical regulation of the secretory process. Proc. Roy. Sac. B 73: 310-322. 251 252 BIBLIOGRAPHY Beijerinck, M. W., 1885. Die Galle von Cecidomyia Poae an Poa nemoralis. Entstehung normaler Wurzeln in Folge der Wirkung eines Gallenthieres. Bot. Zeit. 43: 306-315, 320-331. Beijerinck, M. W., 1886. Beobachtungen und Betrachtungen iiber Nebenwurzeln. Verz. Geschr. II : 7-122. Beijerinck, M. W., 1888. tJber das Cecidium von Nematus Capreae auf Salix amygdalina. Bot. Zeit. 46: 1-11, 17-27. Beijerinck, M. W., 1897. Sur la cecidiogenese et la generation alternante chez le Cynips calicia. Verz. Geschr. Ill: 199-232. Beyer, A., 1925. Untersuchungen iiber den Traumatotropismus der Pflanzen. Biol Zentralhl. 45: 683-702, 746-768. Beyer, A., 1927. Zur Keimungsphysiologie von Awena saitVa. Ber. d. hot. Ges. 45: 179-187. Beyer, A.; 1928. Experimentelle Studien zur Blaauwschen Theorie. II. Planta 5: 478-519. Beyer, A., 1928fl. Beitrage zum Problem der Reizleitung. Z. /. hot. 20: 321-417. Beyer, A., 1932. Untersuchungen zur Theorie der pflanzlichen Tropismen. Planta 18: 509-524. Blaauw, a. H., 1909. Die Perzeption des Lichtes. Rec. trav. bot. neerl. 5: 209-372. Blaauw, A. H., 1914. Licht und Wachstum. I. Z. f. Bot. 6: 641-703. Blaauw, A. H., 1915. Licht und Wachstum. II. Z. f. Bot. 7: 465-532. Blaauw, A. H., 1918. Licht und Wachstum. III. Med. Landbouwhoogeschool 15: 89-204. Blackie, J. J., R. J. D. Graham, and L. B. Stewart, 1926. Propagation of camphor. Kew Bulletin, 380-381. Blackman, F. F., 1905. Optima and limiting factors. Ann. Bot. 19: 281-295. Boas, F. and F. Merkenschlager, 1925. Reizverlust, hervorgerufen durch Eosin. Ber. d. hot. Ges. 43: 381-390. Bonner, J., 1932. The production of growth substance by Rhizopus sidnus. Biol. Zentralhl. 52: 565-582. Bonner, J., 1933. The action of the plant growth hormone. J. gen. Physiol. 17: 63-76. Bonner, J., 1933a. Studies on the growth hormone of plants. IV. On the mechanism of the action. Proc. Nat. Acad. Sc. 19: 717-719. Bonner, J., 1934. The relation of hydrogen ions to the growth rate of the Avena coleoptile. Protoplasma 21: 406-423. BIBLIOGRAPHY 253 Bonner, J., 1934fl. Studies on the growth hormone of plants. V. The relation of cell elongation to cell wall formation. Proc. Nat. Acad. Sc. 20: 393-397. Bonner, J., 1935. Zum Mechanismus der Zellstreckung auf Grund der Micellarlehre. Jahrh. wiss. Bot. 82: 377-412. Bonner, J., 1936. Plant tissue cultures from a hormone point of view, Proc. Nat. Acad. Sc. 22: 426-430. Bonner, J., 1936a. The growth and respiration of the Avcna coleoptile. /. gen. Physiol. 20: 1-11. Bonner, J. and K. V. Thimann, 1935. Studies on the growth hormone of plants. VII. The fate of growth substance in the plant and the nature of the growth process. /. gen. Physiol. 18: 649-658. BosE, J. C, 1907. Comparative electro-physiology. London, 1907. BoTTELiER, H. p., 1934. Uber den Einfluss ausserer Faktoren auf die Protoplasmastromung in der Ayena-koleoptile. Rec. trav. bot. need. 31: 474-582. BoTTELiER, H. p., 1935. Oxygen as limiting factor of the protoplasmic streaming in Avena coleoptiles of different ages. Rec. trav. bot. neerl. 32: 287-292. Bouillenne, R. and F. W. Went, 1933. Recherches experimentales sur la neoformation des racines dans les plantules et les boutures des plantes superieures. Ann. Jard. bot. Buitenzorg 43: 25-202. Boyd, L. and G. S. Avery Jr., 1936. Grass seedling anatomy: the first internode of Avena and Triticuin. Bot. Gaz. 97: 765-779. Boysen Jensen, P., 1910. Uber die Leitung des phototropischen Reizes in At^ena-keimpflanzen. Ber. d. bot. Ges. 28: 118-120. Boysen Jensen, P., 1911. La transmission de 1' irritation phototropique dans VAvena. Bull. Acad. Roy. Danmark 1911, No. 1:3-24. Boysen Jensen, P., 1913. Uber die Leitung des phototropischen Reizes in der Afena-koleoptile. Ber. d. bot. Ges. 31: 559-566. Boysen Jensen, P., 1928. Die phototropische Induktion in der Spitze der Avena-koleoptile. Planta 5: 464-477. Boysen Jensen, P., 1931. Uber Wachstumsregulatoren bei Bakterien. Bioclicm. Zeits. 236: 205-210. Boysen Jensen, P., 1931a. Uber Bildung eines Wachstumsregulators durch Aspergillus niger. Biochem. Zeits. 239: 244-249. Boysen Jensen, P., 1932. Uber die Bildung und biologische Bedeutung des Wachstumsregulators bei Aspergillus niger. Biochem. Zeits. 250: 270-280. 254 BIBLIOGRAPHY BoYSEN Jensen, P., 1933. tJber die durch einseitige Lichtwirkung hervorgerufene transversale Leitung des Wuchsstoffes in der Avenakoleoptile. Planta 19: 335-344. BoYSEN Jensen, P., 1933a. Uber den Nachweis von Wuchsstoff in Wurzeln. Planta 19: 345-350. BoYSEN Jensen, P., 1933&. Die Bedeutung des Wuchsstoffes fiir das Wachstum und die geotropische Kriimmung der Wurzeln von Vida Faba. Planta 20: 688-698. BoYSEN Jensen, P., 1934. Uber Wuchsstoflf in Wurzeln, die mit Erythrosin vergiftet sind. Planta 22: 404-410. BoYSEN Jensen, P., 1935. Die Wuchsstofftheorie und ihre Bedeutung fiir die Analyse des Wachstums und der Wachstumsbewegungen der Pflanzen. Jena, 1935. BoYSEN Jensen, P., 1936. Growth hormones in plants. Translated and revised by G. S. Avery Jr., and P. R. Burkholder. New York, 1936. BoYSEN Jensen, P., 1936a. tJber die Verteilung des Wuchsstoffes in Keimstengeln und Wurzeln wahrend der phototropischen und geotropischen Kriimmung. Kgl. Danske Videnskab. Selskab., Biol. Med. 13: 1-31. BoYSEN Jensen, P. and N. Nielsen, 1926. Studien liber die hormonalen Beziehungen zwischen Spitze und Basis der Avena-koleoptile. Planta 1: 321-331. Brauner, L., 1922. Lichtkriimmung und Lichtwachstumsreaktion. Z. f. Bot. 14: 497-547. Brauner, L., 1924. Permeabilitat und Phototropismus. Z. f. Bot. 16: 113-132. Brauner, L., 1926. Uber das geo-elektrische Phanomen. Kolloidchem. Beihefte, Ambronn-Festschr., 23: 143-152. Brauner, L., 1927. Untersuchungen iiber das geoelektrische Phanomen. Jahrb. wiss. Bot. 66: 381-428. Brauner, L., 1928. Untersuchungen iiber das geoelektrische Phanomen. II. Membranstruktur und geoelektrischer Effekt. Jahrb. wiss. Bot. 68: 711-770. Brauner, L., 1935. Uber den Einfluss des Lichtes auf die Wasserpermeabilitat lebender Pflanzenzellen. Rev. Fac. So. Univ. d'Istanbul 1: 50-55. Brauner, L. and H. U. Amlong, 1933. Zur Theorie des geoelektrischen Effekts. Protoplasma 20: 279-292. Brauner, L. and E. BtJNNiNG, 1930. Geoelektrischer Effekt und Elektrotropismus. Ber. d. bot. Ges. 48: 470-476. BIBLIOGRAPHY 255 Brecht, F., 1936. Der Einfluss von Wuchsstoff- und Saurepasten auf das Wachstum von Avena- und Helianthus-keimYmgen und seine Abhangigkeit vom Sauerstoffgehalt der Luft. Jahrb. wiss. Bot. 82: 581-612. Brown, N. A. and F. E. Gardner, 1936. Galls produced by plant hormones, including a hormone extracted from Bacterium tumefaciens. Phytopath. 26: 708-713. BuDER, J., 1920. Neue phototropische Fundamentalversuche. Ber. d. hot. Ges. 38: 10-19. Buck, L., 1935. Dehnungsversuche an pflanzlichen Membranen. Beih. Bot. Centralbl. 53: 340-377. BuNNiNG, E., 1927. Untersuchungen iiber traumatische Reizung von Pflanzen, Z. f. Bot. 19: 433-476. BtJNNiNG, E., 1928. Zur Physiologie des Wachstums und der Reizbewegungen der Wurzeln. Planta 5: 635-659. BtJNNiNG, E., 1934. Wachstum und Stickstoffassimilation bei Aspergillus niger unter dem Einfluss von Wachstumsregulatoren und von Vitamin B. Ber. d. hot. Ges. 52: 423-444. BuRKHARDT, H., 1926. Untcrsuchung iiber die Gultigkeit des Reizmengengesetzes fiir die Lichtkriimmung der Awna-koleoptile. Z.f. Bot. 18: 273-317. BuRKOM, J. H, VAN, 1913. Het verband tusschen den bladstand en de verdeeling van de groeisnelheid over den stengel. Diss. Utrecht, 1-188. BtJSGEN, M. and E. Munch, 1929. Structure and life of forest trees. Trans, by T. Thomson. New York, Wiley. BuTENANDT, A., 1935. Vhev die stoffliche Charakterisierung der Keimdriisenhormone : ihre Konstitutionsermittlung und kunstlicne Herstellung. Deutsche Med. Wochenschrift 781. BuTENANDT, A., 1936. Ergebnisse und Probleme in der biochemischen Erforschung der Keimdriisenhormone. Naturwissensch. 24: 529-536 and 545-552. Buy, H. G. du, 1931. Uber die Bedingungen, welche die Wuchsstoffproduktion beeinflussen. Proc. Kon. Akad. Wetensch. Amsterdam 34: 277-288. Buy, H. G. du, 1933. Uber Wachstum und Phototropismus von Avena sativa. Rec. trav. bot. neerl. 30: 798-925. Buy, H. G. du, 1934. Der Phototropismus der Avena Koleoptile und die Lichtabfallstheorie. Ber. d. bot. Ges. 52: 531-559. Buy, H. G. du, 1936. The change in the response of Avena coleoptiles to growiih regulators produced by aging. Proc. Nat. Acad. Sc. 22: 272-275. 256 BIBLIOGRAPHY Buy, H. G. du and E. Nuernbergk, 1929. tJber das Wachstum der Koleoptile und des Mesokotyls von Avena saliva unter verschiedenen Aussenbedingungen. Proc. Kon. Akad. Wetensch. Amsterdam 32: 614-624. Buy, H. G. du and E. Nuernbergk, 1929a. Weitere Untersuchungen liber den Einfluss des Lichtes auf das Wachstum von Koleoptile und Mesokotyl bei Avena saliva. II. Proc. Kon. Akad. Wetensch. Amsterdam 32: 808-817. Buy, H. G. du and E. Nuernbergk, 1930. tJber das Wachstum der Koleoptile und des Mesokotyls von Avena saliva unter verschiedenen Bedingungen (III). Proc. Kon. Akad. Wetensch. Amsterdam 33: 542-556. Buy, H. G. du and E. Nuernbergk, 1932. Phototropismus und Wachstum der Pflanzen. Ergeb. Biol. 9: 358-544. Buy, H. G. du and E. Nuernbergk, 1934. Phototropismus und Wachstum der Pflanzen. II. Ergeh. Biol. 10: 207-322. Buy, H. G. du and E. Nuernbergk, 1935. Phototropismus und Wachstum der Pflanzen. Ill, Ergeh. Biol. 12: 325-543. Carlson, M. C., 1929. Microchemical studies of rooting and non-rooting rose cuttings. Bol. Gaz. 87: 64-80. Castle, E. S., 1930. Phototropism and the light-sensitive system of Phycomyces. J. gen. Physiol. 13: 421-435. Castle, E. S., 1935. Photic excitation and phototropism in single plant cells. Cold Spring Harbor Symposia 3: 224-229. Child, C. M. and A. W. Bellamy, 1920. Physiological isolation by low temperature in Bryophyllum. Bol. Gaz. 70: 249-267. Cholodny, N., 1918. tJber den Einfluss der MetaUionen auf die Reizerscheinungen bei den Pflanzen. Schriften d. Univers. Kiew 1918: 1-33. Cholodny, N., 1924. tJber die hormonale Wirkung der Organspitze bei der geotropischen Krummung. Ber. d. bol. Ges. 1^2: 356-362. Cholodny, N., 1926. Beitrage zur Analyse der geotropischen Reaktion. Jahrb. wiss. Bol. 65: 447-459. Cholodny, N., 1927. Wuchshormone und Tropismen bei den Pflanzen. Biol. Zentralbl. 4.7: 604-626. Cholodny, N., 1928. Beitrage zur hormonalen Theorie von Tropismen. Planta 6: 118-134. Cholodny, N., 1929. Einige Bemerkungen zum Problem der Tropismen. Planta 7: 461-481. BIBLIOGRAPHY 257 Cholodny, N., 1929a. Uber das Wachstum des vertikal und horizontal orientierten Stengels in Zusammenhang mit der Frage nach der hormonalen Natur der Tropismen. Planta 7: 702-719. Cholodny, N., 1930. Mikropotometrische Untersuchungen iiber das Wachstum und die Tropismen der Koleoptile von Avena saliva. Jahrb. wiss. Bot. 73: 720-758. Cholodny, N., 1931. Verwundung, Wachstum und Tropismen. Planta 13: 665-694. Cholodny, N., 1931a. Zur Physiologie des pflanzUchen Wuchshormons. Planta 14: 207-216. Cholodny, N., 1932. 1st die Wachstumsgeschwindigkeit der Wurzel von deren Lage abhangig? Planta 17: 794-800. Cholodny, N., 1932a. Lichtwachstumsreaktion und Phototropismus. II. Ber. d. bot. Ges. 50: 317-320. Cholodny, N., 1933. Zum Problem der Bildung und physiologischen Wirkung des Wuchshormons bei den Wurzeln. Ber. d. hot. Ges. 51: 85-98. Cholodny, N., 1933a. Beitrage zur Kritik der Blaauwschen Theorie des Phototropismus. Planta 20: 549-576. Cholodny, N., 1934. tJber die Bildung und Leitung des Wuchshormons bei den Wurzeln. Planta 21: 517-530. Cholodny, N., 1935. tJber das Keimungshormon von Gramineen. Planta 23: 289-312. Cholodny, N. G., 1935a. Investigations on the growth hormone of plants in U.S.S.R. Herbage Rev. 3: 210-214. Claek, W. G., 1935. Note on the effect of light on the bioelectric potentials in the Avena coleoptile. Proc. Nat. Acad. Sc. 21: 681-684. Cooper, W. C, 1935. Hormones in relation to root formation on stem cuttings. Plant Physiol. 10: 789-794. Cooper, W. C, 1936. Transport of root-forming hormone in woody cuttings. Plant Physiol. 11: 779-793. Coster, C, 1927, 1928. Zur Anatomie und Physiologie der Zuwachszonen und Jahresringbildung in den Tropen. Ann. Jard. Bot. Buitenzorg 37: 49-160; 38: 1-114. Crocker, W., P. W. Zimmer\l4.n, and A. E. Hitchcock, 1932. Ethyleneinduced epinasty of leaves and the relation of gravity to it. Contrib. Boyce Thompson hist. 4' 177-218. Crocker, W., A. E. Hitchcock, and P. W. Zimmerman, 1935. Similarities in the effects of ethylene and the plant auxins. Contrib. Boyce Thompson Inst. 7: 231-248. 258 BIBLIOGRAPHY Curtis, 0. F., 1918. Stimulation of root growth in cuttings by treatment with chemical compounds. Cornell Univ. Agr. Expt. Sta. Mem. 14CzAJA, A. Th., 1931. Der Einfluss von Korrelationen auf Restitution und Polaritat von Wurzel- und Sprossstecklingen. Ber. d. hot. Ges. 49: (67)-(71). CzAJA, A. Th., 1934. Der Nachweis des Wuchsstoffes bei Holzpflanzen. Ber. d. hot. Ges. 52: 267-271. CzAJA, A. Th., 1935. Polaritat und Wuchsstoff. Ber. d. hot. Ges. 53: 197-220. CzAJA, A. Th., 1935a. Wurzelwachstum, Wuchsstoff und die Theorie der Wuchsstoffwirkung. Ber. d. hot. Ges. 53: 221-245. CzAjA, A. Th., 19356. Die Wirkung des Wuchsstoffes in parallelotropen Pflanzenorganen (Eine Entgegnung). Ber. d. hot. Ges. 53: 478-490. CzAPEK, F., 1902. Stoffwechselprocesse in der geotropisch gereizten Wurzelspitze und in phototropisch sensiblen Organen. Ber. d. hot. Ges. 20: 464-470. Darwin, C, 1880. The power of movement in plants. London, 1880. Delisle, a. F., 1937. The influence of auxin on secondary branching in two species of Aster. Am. J. Bot. 24: 159-167. Denham, W. S. and T. Lonsdale, 1928. Testing instruments for yarns and fibres. J. Set. Instr. 5: 348-354. Denny, F. E., 1926. Effect of thiourea upon bud inhibition and apical dominance of potato. Bot. Gaz. 81: 297-311. Dijkman, M. J., 1933. A quantitative analysis of the geotropical curvature in Dicotyledons. Proc. Kon. Akad. Wetensch. Amsterdam 36: 749-758. Dijkman, M. J., 1934. Wuchsstoff und geotropische Krummung bei Lwpinus. Rec. trav. hot. neerl. 31: 391-450. DiLLEWiJN, C. VAN, 1927. Die Lichtwachstumsreaktionen von Avena. Rec. trav. hot. neerl. 24: 307-581. DoLK, H. E., 1926. Concerning the sensibility of decapitated coleoptiles of Avena sativa for light and gravitation. Proc. Kon. Akad. Wetensch. Amsterdam 29: 1113-1117. DoLK, H. E., 1929. tJber die Wirkung der Schwerkraft auf Koleoptilen von Avena saliva. Proc. Kon. Akad. Wetensch. Amsterdam 32: 40-47. DoLK, H. E., 1929a. tJber die Wirkung der Schwerkraft auf Koleoptilen von Avena sativa. IL Proc. Kon. Akad. Wetensch. Amsterdam 32: 1127-1140. BIBLIOGRAPHY 259 DoLK, H. E., 1930. Geotropie en groeistof. Diss. Utrecht, 1930. English translation in Rec. trav. hot. need. 33: 509-585, 1936. DoLK, H. E. and K. V. Thimann, 1932. Studies on the growth hormone of plants. I. Proc. Nat. Acad. Sc. 18: S0-4Q. DoLLFUs, H., 1936. Wuchsstoffstudien. Planta 25: 1-21. DosTAL, R., 1926. tjber die wachstumsregulierende Wirkung des Laubblattes. Acta Soc. Sci. Nat. Mwavicae 3: 83-209. DuGGAR, B. M., 1936. Biological effects of radiation. New York, 1936. 1343 pp. DuHAMEL Du MoNCEAU, 1758. La Physique des arbres. Paris, 1758. DuYFjES, H. G. P., 1935. Het probleem der actieve immunisatie van planten tegen Pseudomonas tumefaciens Smith en Town. Diss. Utrecht, 1935. 100 pp. Eastcott, E. v., 1928. Wildiers' Bios. The isolation and identification of "Bios I." /. physical Chem. 32: 1094-1111. Errera, L., 1904. Conflits de preseance et excitations inhibitoires chez les vegetaux. Bull. Soc. Roy. Bot. Belgique 1^2: 27. Erxleben, H., 1935. Uber die Chemie und Physiologie der Auxine. Ergeb. Physiol, exp. Pharm. 37: 186-209. Ewart, a. J. and J. S. Bayliss, 1906. On the nature of the galvanotropic irritability of roots. Proc. Roy. Soc. B 77: 63-66. Faber, E. R., 1936. Wuchsstoffversuche an Keimwurzeln. Jahrb. wiss. Bot. 83: 439-469. Fiedler, H., 1936. Entwicklungs- und reiz-physiologische Untersuchungen an Kulturen isolierter Wurzelspitzen. Z. f. Bot. 30: 385-436. Fieser, L. F., 1936. The chemistry of natural products related to phenanthrene. A.C.S. Monograph, New York, 1936. Fischer, F. G., E. Wehmeier, H. Lehmann, L. Juhling, and K. HuLTZscH, 1935. Zur Kenntnis der Induktionsmittel in der Embryonal-Entwicklung. Ber. d. chem. Ges. 68: 1196-1199. FiscHNicH, 0., 1935. tJber den Einfluss von /S-indoIylessigsaure auf die Blattbewegungen und die Adventivwurzelbildung von Coleus. Planta 24: 552-583. Fitting, H., 1907. Die Leitung tropistischer Reize in parallelotropen Pflanzenteilen. Jahrb. wiss. Bot. 44: 177-253. Fitting, H., 1909. Die Beeinflussung der Orchideenbluten durch die Bestaubung und durch andere Umstande. Z.f. Bot. 1: 1-86. Fitting, H., 1910. Weitere entwicklungsphysiologische Untersuchungea an Orchideenbluten. Z. f. Bot. 2: 225-267. 260 BIBLIOGRAPHY Fitting, H., 1927. Untersuchungen iiber Chemodinese bei Vallisneria. Jahrb. wiss. Bot. 67: 427-596. Fitting, H., 1929. tjber die Auslosung von Plasmastromung durch optisch-aktive Aminosauren. Jahrb. wiss. Bot. 70: 1-25. Fitting, H., 1930. Untersuchungen iiber endogene Chemonastie bei Mimosa pudica. Jahrb. wiss. Bot. 72: 700-775. Fitting, H., 1932. Untersuchungen iiber die Empfindlichkeit und das Unterscheidungsvermogen der FaMisnen'a-protoplasten fiir verschiedene a-Aminosauren. Jahrb. wiss. Bot. 77: 1-103. Fitting, H., 1933. Untersuchungen iiber den Plasmastromung auslosenden Reizstoff in den Blattextrakten von Vallisneria. Jahrb. wiss. Bot. 78: 319-398. Fitting, H., 1936. Die Hormone als physiologische Reizstoffe. Biol. Zentralbl. 56: 69-86. Fitting, H., 1936a. Untersuchungen iiber die chemischen Eigenschaften des Reizstoffes von Mimosa pudica. Jahrb. wiss. Bot. 83: 270-314. Fitting, H., 19366. tJber Auslosung von Protoplasmastromung bei Vallisneria durch einige Histidinverbindungen. Jahrb. wiss. Bot. 82: 613-624. Fliry, M., 1932. Zur Wirkung der Endknospe auf die Hypokotylstreckung des Dikotylenkeimlings. Jahrb. wiss. Bot. 77: 150-184. Frey-Wyssling, a., 1935. Die Stoffausscheidung der hoheren Pflanzen. Monogr. ges. Physiol. Pflanzen Tiere 32: 378 pp. Berlin, Springer. Frey-Wyssling, A., 1936. Der Aufbau der pflanzlichen Zellwande. Protoplasma 25: 261-300. Frieber, W., 1921. Beitrage zur Frage der Indolbildung und der Indolreaktionen sowie zur Kenntniss des Verhaltens indolnegativer Bakterien. Zentralbl. f. Bakt., I Abt. 87: 254-277. Friedrich, G., 1936. Untersuchungen iiber die Wirkung des natiirlichen Wuchsstoffes und der /S-Indolyl-Essigsaure auf den Stoffwechsel der Pfianze. Planta 25: 607-647. Garbarini, G., 1909. Reinigung von Ather (abstract). Chem. Zentralbl. 80: 1126. Gautheret, R. J., 1934. Culture du tissu cambial. Compt. rend. Acad. Sci. 198: 2195. Gautheret, R. J., 1935. Recherches sur la culture des tissus vegetaux. These. Paris. Gill, N., 1933. The relation of flowering and cambial activity. New Phytol. 32: 1-12. BIBLIOGRAPHY 261 Glover, J., 1936. Skatole as a growth-promoting substance. Nature 137: 320-321. GoEBEL, K., 1903. Regeneration in plants. Bull. Torrey Bot. Club 30: 197-205. GoEBEL, K., 1905. AUgemeineRegenerationsprobleme. F?ora 55; 384-411. GoEBEL, K., 1908. Einleitung in die experimentelle Morphologic der Pflanzen. Leipzig, 1908. Goodwin, R. H., 1937. The role of auxin in leaf development in Solidago species. Am. J. Bot. 2J^: 43-51. GoRTER, C. J., 1927. On the occurrence of growth-accelerating and growth-retarding substances. Proc. Ron. Akad. Wetensch. Amsterdam 30: 728-733. GoRTER, C. J., 1932. Groeistofproblemen bij Wortels. Diss. Utrecht, 1932. GouwENTAK, C. A. and G. Hellinga, 1935. Beobachtungen iiber Wurzelbildung. Med. Landbouwhoogeschool Wageni}ige7i 39: 1-6. Gradmann, H., 1925. Untersuchungen iiber geotropische Reizstoffe. Jahrb. wiss. Bot. 64: 201-248. Gradmann, H., 1928. Die Lateralwirkung bei den Windepflanzen. Jahrb. wiss. Bot. 68: 46-78. Graham, R. J. D., 1934. The work of Laurence Baxter Stewart. Trans. Bot. Soc. Edin. 31: 450-459. Graham, R. J. D., 1936. Laurence Baxter Stewart's methods of vegetative propagation at Edinburgh. Sci. Hort. 4- 97-113. Graham, R. J. D. and L. B. Stewart, 1931. Special methods of practical utility in vegetative propagation of plants. Proc. Xlth Int. Horticultural Congress, 1930, 159-164. Graze, H. and G. Schlenker, 1936. Versuche zur Klarung der reziproken Verschiedenheiten von Epilobium-BastaTden. III. Vergleichende Untersuchungen iiber den WuchsstofTgehalt bei verschiedenen Biotypen von Epilobium hirsutxim. Jahrb. wiss. Bot. 82: 687-695. Gregory, F. G., 1928. Studies in the energy relations of plants. II. The effect of temperature on increase in area of leaf surface and in dry weight of Cucumis sativus. Ann. Bot. 4^' 469-507. Grieve, B. J., 1936. Spotted Wilt virus and the hormone heteroauxin. Nature 138: 128. Gundel, W., 1933. Chemische und physikalisch-chemische Vorgange bei geischer Induktion. Jahrb. wiss. Bot. 78: 623-664. Guttenberg, H. von, 1913. tJber akropetale heliotropische Reizleitung. Jahrb. wiss. Bot. 52: 333-350. 262 BIBLIOGRAPHY GuTTENBERG, H. VON, 1933. Reizperzeption und Wuchsstoffwirkung. Planta 20: 230-232. GuTTENBERG, H. VON, 1932, 1933, 1934, 1935, 1936. Wachstum und Bewegung. in: Fortschritte der Botanik I, II, III, IV, V. Berlin, 1932, 1933, 1934, 1935, 1936. Haagen Smit, a. J., 1935. Over auxinen. Chem. Weekblad 32: 398-403. Haagen Smit, A. J. and F. W. Went, 1935. A physiological analysis of the growth substance. Proc. Kon. Akad. Wetensch. Amsterdam 38: 852-857. Haan, I. DE and L. Petrick, 1935. Polaire wortelvorming. Natuurw. Tijdsch. 17: 117-127. Haas, R. Horreus de, 1929. On the connection between the geotropic curving and elasticity of the ceU-wall. Proc. Kon. Akad. Wetensch. Amsterdam 32: 371-373. Haberlandt, G., 1913. Zur Physiologie der Zellteilung. Sitz. her. k. preuss. Akad. Wiss. 1913: 318-345. Haberlandt, G., 1914. Zur Physiologie der Zellteilung. Sitz. her. k. preuss. Akad. Wiss. 1914: 1096-1111. Haberlandt, G., 1921. Wundhormone als Erreger von Zellteilungen. Beitr. allg. Bot. 2: 1-53. Hagemann, a., 1932. Untersuchungen an Blattstecklingen. Gartenhauwiss. 6: 69-195. Haig, C., 1935. The phototropic responses of Aveiia in relation to intensity and wave-length. Biol. Bull. 69: 305-324. Hammerling, J., 1934. tJber formbildende Substanzen bei Acetahularia viediterranea, ihre raumhche und zeitHche Verteihmg und ihre Herkunft. Arch. Entwicklungsmech. 131: 1-81. Hammerling, J., 1935. tJber Genomwirkungen und Formbildungsfahigkeit bei Acetahularia. Arch. Entwicklungsmech. 132: 424-462. Hammerling, J., 1936. Studien zum Polaritatsproblem. I-III. Zool. Jahrb. 56: 440-486. Hanstein, J., 1860. Versuche iiber die Leitung des Saftes durch die Rinde und Folgerungen daraus. Jahrb. wiss. Bot. 2: 392-467. Harder, R. and I. Stormer, 1934. Uber den Einfiuss des Follikelhormons auf das Bliihen von Pflanzen. Jahrb. wiss. Bot. 80: 1-19. Hartmann, H., 1931. Reaktionen von Koleoptilen und Wurzeln im elektrischen Feld. Beitr. Biol. Pfl. 19: 287-333. Harvey, E. N., 1920. An experiment on regulation in plants. Amer. Nat. 54: 362-367. BIBLIOGRAPHY 263 Hawker, L. E., 1932. Experiments on the perception of gravity by roots. New Phytol. 31: 321-328. Heidt, K., 1931. tiber das Verhalten von Explantaten der Wurzelspitze in nahrstofffreien Kulturen. Arch. Exp. Zellforsch. 11: 693-724. Hess, K., C. Trogus, and W. Wergin, 1936. Untersuchungen iiber die Bildung der pflanzlichen Zellwand. Planta 25: 419-437. Heyn, a. N. J., 1930. On the relation between growth and extensibihty of the cell wall, Proc. Kon. Akad. WeteTisch. Amsterdam 33: 1045- 1058. Heyn, A. N. J., 1931. Further experiments on the mechanism of growth. Proc. Kon. Akad. Wetensch. Amsterdam 34: 474-484. Heyn, A. N. J., 1931a. Der Mechanismus der Zellstreckung. Rec. trav. hot. neerl. 28: 113-244. Heyn, A. N. J., 1932. Recherches sur les relations de la plasticite des membranes cellulaires et la croissance des vegetaux. Compt. rend. Acad. Sci. 194: 1848-1850. Heyn, A. N. J., 1932a. Sur la methode de determination de plasticity des membranes cellulaires. Compt. rend. Acad. Sci. 195: 494-496. Heyn, A. N. J., 1933. Further investigations on the mechanism of cell elongation and the properties of the cell wall in connection with elongation. I. The load extension relationship. Protoplasma 19: 78-96. Heyn, A. N. J., 1933o. X-ray investigations of the cellulose in the wall of young epidermic cells. Proc. Kon. Akad. Wetensch. Amsterdam 36: 560-565. Heyn, A. N. J., 1934. Die Plastizitat der Zellmembran unter Einfiuss von Wuchsstoff. Proc. Kon. Akad. Wetensch. Amsterdam 37: 180- 182. Heyn, A. N. J., 1934a. Weitere Untersuchungen iiber den Mechanismus der Zellstreckung und die Eigenschaften der Zellmembran. H. Das Rontgendiagramm von jungen wachsenden Zellwanden und parenchymatischen Geweben. Protoplasma 21: 299-305. Heyn, A. N. J., 19346. Weitere Untersuchungen iiber den Mechanismus der Zellstreckung und die Eigenschaften der Zellmembran. HI. Die Anderungen der Plastizitat der Zellwand bei verschiedenen Organen. Jahrh. wiss. Boi. 79: 753-789. Heyn, A. N. J., 1935. The chemical nature of some growth hormones as determined by the diffusion method. Proc. Kon. Akad. Wetensch. Amsterdam SS; 1074-1081. Heyn, A. N. J., 1936. Auxine. Handb. biol. Arbeitsmethoden 5: 823-861. 264 BIBLIOGRAPHY Heyn, a. N. J. and J. van Overbeek, 1931. Weiteres Versuchsmaterial zur plastischen und elastischen Dehnbarkeit der Zellmembran. Proc. Kon. Akad. Wetensch. Amsterdam 34-' 1190-1195. HiNDERER, G., 1936. Versuche zur Klarung der reziproken Verschiedenheiten von Epilobium-Bastarden. II. Wuchsstoff und Wachstum bei reziprok verschiedenen Epilobium-Bastarden. Jahrb. wiss. Bot. 82: 669-686. Hitchcock, A. E., 1935. Indole-3-n-propionic acid as a growth hormone and the quantitative measurement. Contrib. Boyce Thompson Inst. 7: 87-95. Hitchcock, A. E., 1935a. Tobacco as a test plant for comparing the effectiveness of preparations containing growth substances. Contrib. Boyce Thompson Inst. 7: 349-364. Hitchcock, A. E. and P. W. Zimmerman, 1935. Absorption and movement of synthetic growth substances from soil as indicated by the responses of aerial parts. Contrib. Boyce Thompson Inst. 7: 447-476. Hitchcock, A. E. and P. W. Zimmerman, 1936. Effect of growth substances on the rooting response of cuttings. Contrib. Boyce Thompson Inst. 8: 63-79. Holtfreter, J., 1935. Der Einfluss thermischer, mechanischer und chemischer Eingriffe auf die Induzierfahigkeit von Triton-Keimteilen. Arch. Entwicklungsmech. 132: 225-306. Honert, T. H. van den, 1932. On the mechanism of the transport of organic materials in plants. Proc. Kon. Akad. Wetensch. Amsterdam 35: 1104-1111. Honing, J. A., 1923. Nicotiana deformis n. sp. und die Enzymtheorie der Erblichkeit. Genetica 5: 455-476. Huxley, J. S., 1935. Chemical regulation and the hormone concept. Biol. Rev. 10: 427-441. Janse, J. M., 1926. On new phenomena caused by irritation of roots. Proc. Kon. Akad. Wetensch. Amsterdam 29: 834-842. JosT, L., 1891. Uber Dickenwachsthum und Jahresringbildung. Bot. Zdt. J^: 485-630. JosT, L., 1893. tJber Beziehungen zwischen der Blattentwicklung und der Gefassbildung in der Pflanze. Bot. Zeit. 51: 89-138. JosT, L., 1935. tJber Wuchsstoffe. Z.f. Bot. 28: 260-274. JosT, L., 1935a. Wuchsstoff und Zellteilung, Ber. d. bot. Ges. 53: 733-750. JosT, L. and E. Reiss, 1936. Zur Physiologie der Wuchsstoffe. II. Einfluss des Heteroauxins auf Langen- und Dickenwachstum. Z. f. Bot. 30: 335-376. BIBLIOGRAPHY 265 JuEL, I., 1936. tJber die Genauigkeit der Wuchsstoffbestimmungsmethode. Planta 25: 307-310. Kastens, E., 1924. Beitriige zur Kenntnis der Funktion der Siebrohren. Mitt. Inst. Allg. Bot. Hamburg 6: 33-70. Katunskij, V. M., 1935. Growth promoting substance as a factor in the formation of plant organism. Compt. rend. Acad. Sci. U.R.S.S. 1: 665-667. Keeble, F., 1910. Plant-Animals. Camh. Univ. Press, 163 pp. Keeble, F. and M. G. Nelson, 1935. The integration of plant behaviour. V. Growth substance and traumatic curvature of the root. Proc. Roy.Soc.B 117:92-119. Keeble, F., M. G. Nelson, and R. Snow, 1929. The Integration of Plant Behavior. I. Separate Geotropic Stimulations of Tip and Stump in Roots. Proc. Roy. Soc. B 105: 493-498. Keeble, F., M. G. Nelson, and R. Snow, 1930. A wound substance retarding growth in roots. New Phytol. 29: 289-293. Keeble, F., M. G. Nelson, and R. Snow, 1931. The Integration of Plant Behavior. III. The Effect of Gravity on the Growth of Roots. Proc. Roy. Soc. B 108: 360-365. Keeble, F., M. G. Nelson, and R. Snow, 1931o. Integration of Plant Behavior. IV. Geotropism and Growth-Substance. Proc. Roy. Soc. B 108: 537-545. Kisser, J., 1931. Die stofflichen Grundlagen pflanzlicher Reizkriimmungen. Verh. Zool. Bot. Ges. Wien. 81: (34)-(38). Koch, K., 1934. Untersuchungen iiber den Quer- und Langstransport des Wuchsstoffes in Pflanzenorganen. Planta 22: 1-33. t KoGL, F., 1932. tJber die Chemie des Auxins, eines pflanzlichen Wuchsstoffs. Chem. Weekblad 29: 317-318. KoGL, F., 1932fl. tiber die Chemie des Auxins und sein Vorkommen im Pflanzen- und Tierreich. Forsch. und Fortschr. 8: 409-410. KoGL, F., 1933. iiber Auxine. Z. Angew. Chem. 46: 469-484. KoGL, F., 1933fl. Die Chemie des Auxins und sein Vorkommen im Pflanzen- und Tierreich. Naturwiss. 21: 17-21. KoGL, F., 19336. On plant growth hormones (Auxin A and auxin B) Rep. British Assoc. 600-609. KoGL, F., 1933c. Chemische und physiologische Untersuchungen iiber Auxin, einen Wuchsstoff der Pflanzen. Angew. Chemie 46- 166-167. KoGL, F., 1935. tiber Wuchsstoffe der Auxin- und der Bios-Gruppe. Ber. d. chem. Ges. 68: 16-28. 266 BIBLIOGRAPHY KoGL, F., 1935a. Untersuchungen iiber pflanzliche Wuchsstofife. Naturwiss. 23: 839-843. KoGL, F., 1936. Untersuchungen iiber pflanzliche Wuchsstoffe. Proc. 6th Int. Bot. Congress. 1: 97-107. KoGL, F., 1936a. tjber pflanzUche Wuchshormone. Svensk Kem. Tidskr. 48: 145-155. KoGL, F. and H. Erxleben, 1934. tJber die Konstitution der Auxine a und b. X. Mitteihmg iiber pflanzliche Wachstumsstoffe. Z. physiol. Chem. 227: 51-73. KoGL, F. and H. Erxleben, 1935. Synthese der " Auxin-glutarsaure " und einiger Isonierer. XV. Mitteilung. Z. physiol. Chem. 235: 181-200. KoGL, F., H. Erxleben, and A. J. Haagen Smit, 1933. Uber ein Phytohormon der Zellstreckung. Zur Chemie des krystallisierten Auxins. V. Mitteilung. Z. physiol. Chem. 216: 31-44. Kogl, F., H. Erxleben, and A. J. Haagen Smit, 1934. Uber die Isolierung der Auxine a und b aus pflanzlichen Materialen. IX. Mitteilung. Z. physiol. Chem. 225: 215-229. Kogl, F. and A. J. Haagen Smit, 1931. tJber die Chemie des Wuchsstoffs. Proc. Kon. Akad. Wetensch. Amsterdam 34: 1411-1416. Kogl, F. and A. J. Haagen Smit, 1936. Biotin und Aneurin als Phytohormone. Ein Beitrag zur Physiologic der Keimung. XXIII. Mitteilung. Z. physiol. Chem. 243: 209-226. Kogl, F., A. J. Haagen Smit, and H. Erxleben, 1933. tJber ein Phytohormon der Zellstreckung. Reindarstellung des Auxins aus menschlichem Harn. IV. Mitteilung. Z. phijsiol. Chem. 214: 241-261. Kogl, F., A. J. Haagen Smit, and H. Erxleben, 1933a. Studien iiber das Vorkommen von Auxinen im menschlichen und im tierischen Organismus. VII. Mitteilung. Z. physiol. Chem. 220: 137-161. Kogl, F., A. J. Haagen Smit, and H. Erxleben, 1934. Uber ein neues Auxin ("Heteroauxin") aus Harn. XI. Mitteilung. Z. physiol. Chem. 228: 90-103. Kogl, F., A. J. Haagen Smit, and H. Erxleben, 1934a. tJber den Einfluss der Auxine auf das Wurzelwachstum und iiber die chemische Natur des Auxins der Graskoleoptilen. XII. Mitteilung. Z. physiol. Chem. 228: 104-112. Kogl, F., A. J. Haagen Smit, and C. J. van Hulssen, 1936. Uber den Einfluss unbekannter ausserer Faktoren bei Versuchen mit Avena saliva. XIX. Mitteilung. Z. physiol. Chem. 241 •' 17-33. Kogl, F., A. J. Haagen Smit, and B. Tonnis, 1933. Uber das Vorkommen von Auxinen und von Wachstumsstofifen der " Bios "-Gruppe in Carcinomen. VIII. Mitteilung. Z. physiol. Chem. 220: 162-172. BIBLIOGRAPHY 267 KoGL, F. and D. G. F. R. Kostermans, 1934. Hetero-auxin als Stoffwechselprodukt niederer pflanzlicher Organismen. Isolierung aus Hefe. XIII. Mitteilung. Z. physiol. Chem. 228: 113-121. KoGL, F. and D. G. F. R. Kostermans, 1935. tJber die KonstitutionsSpezifitat des Hetero-auxins. XVI. Mitteilung. Z. phijsiol. Chem. 235: 201-216. KoGL, F. and B. Tonnis, 1936. tJber das Bios-Problem. Darstellung von krystallisiertem Biotin aus Eigelb. Z. physiol. Chem. 21^.2: 43-73. KoK, A. C. A., 1931. tJber den Einfluss der Plasmarotation auf den Stofftransport. Proc. Kon. Akad. Wetensch. Amsterdam 34: 918-929. KoK, A. C. A., 1932. Uber den Transport von Kaffein und LiNOs durch parenchymatisches Gewebe. Proc. Kon. Akad. Wetensch. Amsterdam 35: 241-250. KoNiNG, H. C, 1933. Het winden der slingerplanten. Diss. Utrecht, 1933. KoNiNGSBERGER, C., 1936. De auto-inactiveering der Auxinen. Diss. Utrecht, 1936. KoNiNGSBERGER, V. J., 1922. Tropismus und Wachstum. Rec. trav. hot. neerl. 19: 1-136. KoRNMANN, P., 1935. Die Aufhebung der Wuchsstoffwirkung durch lebende Pflanzenteile. Ber. d. hot. Ges. 53: 523-527. Kraus, E. J. and H. R. Kraybill, 1918. Vegetation and reproduction with special reference to the tomato. Ore. Agr. Expt. Sta. Bidl. 149. Kropp, B. and W. J. Crozier, 1934. The production of the crustacean chromatophore activator. Proc. Nat. Acad. Sc. 20: 453-456. Kupfer, E., 1907. Studies in plant regeneration. Mem. Torrey Bot. Cluh 12. Laan, p. a. van der, 1934. Der Einfluss von Aethylen auf die Wuchsstoffbildung bei Avena und Vicia. Rec. trav. bot. neerl. 31: 691-742. Laibach, F., 1932. Interferometrische Untersuchungen an Pflanzen. II. Die Verwendbarkeit des Interferometers in der Pflanzenphysiologie. Jahrh. wiss. Bot. 76: 218-282. Laibach, F., 1932a. Pollenhormon und Wuchsstoff. Ber. d. hot. Ges. 50: 383-390. Laibach, F., 1933. Wuchsstoffversuche mit lebenden OrchideenpoUinien. Ber. d. hot. Ges. 51: 336-340. Laibach, F., 1933a. Versuche mit Wuchsstoffpaste. Ber. d. hot. Ges. 51: 386-392. Laibach, F., 1934. Zum Wuchsstoffproblem. Der Ziichter 6: 49-53. Laibach, F., 1935. tJber die Auslosung von Kallus- und Wurzelbildung durch /3-Indolylessigsaure. Ber. d, hot. Ges. 53: 359-364. 268 BIBLIOGRAPHY Laibach, F. and 0. Fischnich, 1935. Kiinstliche Wurzelneubildung mittels Wuchsstoffpaste. Ber. d. hot. Ges. 53: 528-539. Laibach, F. and 0. Fischnich, 1935a. tJber eine Testmethode zur Priifung der kallusbildenden Wirkung von Wuchsstoffpasten. Ber. d. hot. Ges. S3: 469-477. Laibach, F. and 0. Fischnich, 1936. Die Wuchsstoffleitung in der Pflanze. I. Planta 25: 648-659. Laibach, F. and O. Fischnich, 1936o. tJber Blattbewegungen unter dem EinfluvSS von kiinstlich zugefiihrtem Wuchsstoff. Biol. Zentralbl. 56: 62-68. Laibach, F. and P. Kornmann, 1933. Zur Methodik der Wuchsstoffversuche. Plaiita 19: 482-484. Laibach, F. and P. Kornmann, 1933a. Zur Frage des Wuchsstofftransportes in der Haferkoleoptile. Planta 21: 396-418. Laibach, F. and G. Mai, 1936. tJber die kiinstliche Erzeugung von Bildungsabweichungen bei Pflanzen. Arch. Entwickhmgsmech. 134: 200-206. Laibach, F., G. Mai, and A. Muller, 1934. Uber ein Zellteilungshormon. Naturwiss. 22: 288. Laibach, F. and E. Maschmann, 1933. Uber den Wuchsstoff der OrchideenpoUinien. Jahrb. wiss. Bot. 78: 399-430. Laibach, F. and F. Meyer, 1935. tJber die Schwankungen des Auxingehaltes bei Zea Mays und Helianthus annuus im Verlauf der Ontogenese. Senckenbergiana 17: 73-86. Laibach, F., A. Mijller, and W. Schafer, 1934. ttber wurzelbildende Stoffe. Naturwiss. 22: 588-589. Lamprecht, W., 1918. tJber die Kultur und transplantation kleiner Blattstiicken. Beitr. allg. Bot. 1: 353-398. Lane, R. H., 1936. The inhibition of roots by growth hormone. A?/i. J. Bot. 23: 532-535. Lange, S., 1927. Die Verteilung der Lichtempfindhchkeit in der Spitze der Haferkoleoptile. Jahrb. iviss. Bot. 67: 1-51. Larsen, p., 1936. liber einen wuchsstoffinaktivierenden Stoff aus Phaseolus-Keimpflanzen. Planta 25: 311-314. La Rue, C. D., 1933. Intumescences on poplar leaves. I and II. Am. J. Bot. 20: 1-17 and 159-175. La Rue, C. D., 1935. The role of auxin in the development of intumescences on poplar leaves; in the production of cell outgrowths in the tunnels of leaf-miners; in the leaf-fall in Coleus. Am. J. Bot. 22: 908. BIBLIOGRAPHY 269 La Rue, C. D., 1936. Tissue cultures of spermatophytes. Proc. Nat. Acad. Sc. 22: 201-209. La Rue, C. D., 1936fl. Intumescences on poplar leaves. III. The role of plant growth hormones in their production. Am. J. Bot. 23: 520-524. Le Fanu, B., 1936. Auxin and correlative inhibition. New Phytol. 35: 20&-220. Lehmann, E., 1936. Versuche zur Klarung der reziproken Verschiedenheiten von Epilobium-Bastarden. I. Der Tatbestand und die Moglichkeit seiner Klarung durch differente Wuchsstoffbildung. Jahrh. miss. Bot. 82: 657-668. Lek, H. a. a. van der, 1925. Over de wortelvorming van houtige stekken. (With summary: Root development in woody cuttings). Diss. Utrecht, 1925. Lek, H. a. A. van der, 1934. Over den invloed der knoppen op de wortelvorming der stekken. (With summary: On the influence of the buds on root-development in cuttings). Meded. Landbouwhoogeschool Wageningen 38 (2): 1-95. Leonian, L. H., 1935. The effect of auxins upon Phytophthora cactorum. J. Agr. Res. 51: 277-286. Leslie, J. W. and M. M., 1928. The "wiry" tomato. /. Hered. 19: 337-344. Levine, M., 1936. Plant tumors and their relation to cancer. Bot. Rev. 2: 439-455. Li, T.-T., 1930. The appearance of the new physiological tip of the decapitated coleoptiles of Avena saliva. Proc. Kon. Akad. Wetensch. Amsterdam 53; 1201-1205. Li, T.-T., 1934. Phototropism of decapitated coleoptile of Avena sativa. Rep. Nat. Tsinghua Univ. 2: 1-10. LoEB, J., 1915. Rules and mechanism of inhibitions and correlation in the regeneration of Bryophyllum calycinum. Bot. Gaz. 60: 249-276. LoEB, J., 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. LoEB, J., 1917a. The chemical basis of regeneration and geotropism. Science 46: 115-118. LoEB, J., 19176. The chemical basis of axial polarity in regeneration. Science 46: 547-551. LoEB, J., 1918. Forced movements, tropisms, and animal conduct. Philadelphia, 1918. 270 BIBLIOGRAPHY LoEB, J., 1918a. Chemical basis of correlation. I. Production of equal masses of shoots by equal masses of sister leaves in Bryophyllum calycinum. Bot. Gaz. 65: 150-174. LoEB, J., 1919. The physiological basis of morphological polarity in regeneration. I. /. gen. Physiol. 1: 337-362. LoEB, J., 1923. Theory of regeneration based on mass action. J. gen. Physiol. 5: 831-852. LoEB, J., 1924. Regeneration from a physicochemical viewpoint. New York, 1924. LoEHWiNG, W. F. and L. C. Bauguess, 1936. Plant growth effects of hetero-auxin applied to soil and plants. Science 84-' 46-47. LoEWE, S., 1933. Analyse der Pfianzenhormone. Handb. Pflanzenanalyse 4: 1005-1041. Lund, E. J., 1923. Electrical control of organic polarity in the eggs of Fucus. Bot. Gaz. 76: 288-301. MacCallum, W. B., 1905. Regeneration in plants. I and 11. Bot. Gaz. 40: 97-120 and 241-263. Mai, G., 1934. Korrelationsuntersuchungen an entspreiteten Blattstielen mittels lebender OrchideenpoUinien als Wuchsstoffquelle. Jahrh. wiss. Bot. 79: 681-713. Malowan, S. L., 1934. Wuchsstoffe und Pflanzenwachstum. Protoplasma 21: 306-322. Malpighi, M., 1675. Anatome Plantarum. (Die Anatomie der Pflanzen.) Ostwald's Klassiker 120. Leipzig, 1901. Mangham, S., 1917. On the mechanism of translocation in plant tissues. An hypothesis, with special reference to sugar conduction in sievetubes. Ann.Bot.31:2^2,-Zn. Manske, R. H. F. and L. C. Leitch, 1936. The synthesis of 5- (3indolyl)-valeric acid and the effects of some indol acids in plants. Can. J. Res. B I4: 1-5. Maresquelle, H. J., 1935. Defaut d'allongement et depolarisation de la croissance dans les morphoses parasitaires. Rev. gen. Bot. ^7; 129-143, 193-214, 273-293. Maschmann, E., 1932. Der WuchsstofT bosartiger Geschwiilste. Naturwiss. 20: 721-722. Maschmann, E. and F. Laibach, 1932. Uber Wuchsstoffe. Biochem. Z. 255: 446-452. Maschmann, E., and F. Laibach, 1933. Das Vorkommen von Wuchsstoff in tierischem und pflanzlichem Material. Naturwiss. 21: 517. BIBLIOGRAPHY 271 Meesters, a., 1936. The influence of heteroauxin on the growth of root hairs and roots of Agrostemma Githago L. Proc. Kon. Akad. Wetensch. Amsterdam 39: 91-97. Meissner, K. W., 1932. Interferometrische Untersuchungen an Pfianzen. I. liber ein handliches Prazisions- Instrument zur Messung von Dimensionsanderungen auf Grund des interferometrischen Messprinzips. Jahrb. iciss. Bot. 76: 208-217. Metzner, p., 1928. Das Mikroskop. Leipzig, 1928. Metzner, p., 1934. Zur Kenntnis der Stoffwechselanderungen bei geotropisch gereizten Keimpflanzen. Ber. d. hot. Ges. 52: 506-522. Meyer, F., 1936. Uber die Verteilung des Wuchsstoffes in der Pflanze wahrend ihrer Entwicklung. Diss. Frankfurt, 1936. MiCHENER, H. D., 1935. Effects of ethylene on plant growth hormone. Science 82: 551. MoissEJEWA, M., 1928. (Zur Frage nach den Wuchshormonen des Getreides.) Visnik, Kiivsk Bot. {Bull. Jard. Bot. Kieff), 7/8: 1-16 (Ukrainian with German summary). MoLiscH, H., 1935. Das knospenlose Internodium als Steckling behandelt. Ber. d. bot. Ges. 53: 575-586. MoRELAND, C. F., 1934. Factors afTecting the development of the cotyledonary buds of the common bean, Phaseolus vulgaris. Cornell Univ. Agr. Expt. Sta. Mem. 167: 3-28. Morgan, T. H., 1906. The physiology of regeneration. J. exp. Zool. 3: 457-500. MiJLLER, A. M., 1935. Uber den Einfluss von Wuchsstoff auf das Austreiben der Seitenknospen und auf die Wurzelbildung. Jahrb. wiss. Bot. 81: 497-540. Nagag, M., 1936. Studies on the growth hormones of plants. I. The production of growth substance in root tips. Rep. Tohoku Imp. Univ. 10: 721-731. Navez, a. E., 1927. "Galvanotropism" of roots. /. gen. Physiol. 10: 551-558. Navez, A. E., 1929. Respiration and geotropism in Vida Faba. J. gen. Physiol. 12: 641-667. Navez, A. E., 1933. Geo-growth reaction of roots of lupinus. Bot. Gaz. 94: 616-618. Navez, A. E., 1933. "Growth-promoting substance" and elongation of roots. /. gen. Physiol. 16: 733-739. Navez, A. E., 1933a. Growth-promoting substance and illumination. Proc. Nat. Acad. Sc. 19: 636-638. 272 BIBLIOGRAPHY Navez, a. E. and B. Kropp, 1934. The growth-promoting action of crustacean eye-stalk extract. Biol. Bull. 67: 250-258. Navez, A. E. and T. W. Robinson, 1933. Geotropic curvature of Avena coleoptiles. /. gen. Physiol. 16: 133-145. Nemec, B., 1930. Bakterielle Wuchsstoffe. Ber. d. hot. Ges. 48: 72-74. Nemec, B., 1934. Ernahrung, Organogene und Regeneration. Vest. Krai. Ces. Spol. Nauk. Ir. II: 1-34. Nielsen, N., 1924. Studies on the transmission of stimuH in the coleoptile of Avena. Dansk. Bot. Arkiv. 4 (8). Nielsen, N., 1928. Untersuchungen iiber Stoffe, die das Wachstum der Avenacoleoptile beschleunigen. Planta 6: 376-378. Nielsen, N., 1930. Untersuchungen iiber einen neuen wachstumsreguherenden Stoff: Rhizopin. Jahrb. wiss. Bot. 73: 125-191. Nielsen, N., 1931. tlber Wuchsstoffe der Hefe. Biochem. Z. 237: 244-246. Nielsen, N., 1931a. The effect of rhizopin on the production of matter of Aspergillus niger. Compt. Rend. Lab. Carlsberg 19 (5) : 1-10. Nielsen, N., 1932. tjber das Vorkommen von Wuchsstoff bei Boletus edulis. Biochem. Z. 249: 196-198. Nielsen, N. and V. Hartelius, 1932. The separation of growthpromoting substances. Compt. Rend. Lab. Carlsberg 19 (8): 1-17. NuERNBERGK, E., 1932. PhysikaHschc Methoden der pflanzlichen Lichtphysiologie. Handb. biol. Arbeitsmethoden 4 (5) : 739-950. NuERNBERGK, E., 1933. tJber den Auxin-Quertransport und den Geotropismus der A?;ena-koleoptile: Einfluss der Dekapitation. Flora 128: 99-110. NuERNBERGK, E. and H. G. du Buy, 1930. Uber Methoden zur Analyse von Wachstumserscheinungen. Rec. trav. bot. neerl. 27: 417-520. NuERNBERGK, E. and H. G. du Buy, 1932. Die Analyse von pflanzlichen Wachstumsvorgangen. Handb. biol. Arbeitsmethoden 9 (4) : 951-1014. Oholm, L. W., 1912. Die freie Diffusion der Nichtelektrolyte. Meddel. Vet.-Akad. Nobelinst. 2, No. 23. OosTERHUis, J., 1931. Der Einfluss der Knospen auf das Stengelwachstum von Asparagus plumosus und A. Sprengeri. Rec. trav. bot. neerl. 28: 20-74. Orth, H., 1934. Die Wirkung des Follikelhormons auf die Entwicklung der Pflanze. Z. f. Bot. 27: 565-607. OvERBECK, F., 1934. Beitrage zur Kenntnis der Zellstreckung. (Untersuchungen am Sporogonstiel von Pellia epiphylla.) Z. f. Bot. 27: 129-170. BIBLIOGRAPHY 273 OvERBEEK, J. VAN, 1932. An analysis of phototropism in dicotyledons. Proc. Kon. Akad. Wetensch. Amsterdam 35: 1325-1335. OvERBEEK, J. VAN, 1933. Wuchsstoff, Lichtwachstumsreaktion und Phototropismus bei Raphanus. Rec. trav. hot. need. 30: 537-626. OvERBEEK, J. VAN, 1935. The growth hormone and the dwarf type of growth in corn. Proc. Nat. Acad. Sc. 21: 292-299. OvERBEEK, J. VAN, 1936. Growth honnone and mesocotyl growth. Rec. trav. hot. neerl. 33: 333-340. OvERBEEK, J. VAN, 1936a. Light growth response and auxin curvatures of Avena. Proc. Nat. Acad. Sc. 22: 421^25. OvERBEEK, J. VAN, 19366. Different action of auxin-o and of heteroauxin (Preliminary note). Proc. Nat. Acad. Sc. 22: 187-190. OvERBEEK, J. VAN, 1936c. Growth substance curvatures of Averia in light and dark. /. gen. Physiol. 20: 283-309. Paal, a., 1914. tJber phototropische Reizleitungen. Ber. d. hot. Ges. 32: 499-502. Paal, A., 1919. tJber phototropische Reizleitung. Jahrb. wiss. Bot. 58: 406-458. Perry, J. L., 1932. A possible hormone-secreting region in the grass coleoptile. Science 76: 215-216. Pfaeltzer, J. W., 1934. Lengtekracht, groeistof en groei bij het coleoptiel van Avena saliva. Diss. Utrecht. 121 pp. PiSEK, A., 1929. Wuchsstoff und Tropismen. Osterr. Bot. Z. 78: 168-186. PoHL, R., 1935. tJber den Endospermwuchsstoff und die Wuchsstoffproduktion der Koleoptilspitze. Planta 24: 523-526. PoHL, R., 1936. Die Abhangigkeit des Wachstums der Avena-Koleoptile und ihrer sogenannten Wuchsstoff-produktion von Auxingehalt des Endosperms. Planta 25: 720-750. PopoFF, M., 1933. Uber die pfianzlichen Auxine und ihre Wirkung auf Einzellige. Biol. Zentralhl. 53: 661-668. Priestley, J. H., 1930. Studies in the physiology of cambial activity. III. The seasonal activity of the cambium. New Phijtol. 29: 316-354. Pringsheim, E. G., 1912. Die Reizbewegungen der Pflanzen. Berlin, 1912. PuRDY, H. A., 1921. Studies on the path of transmission of phototropic and geotropic stimuli in the coleoptile of Avena. Kgl. Danske Videnskah. Selskah., Biol. Medd. 3: 3-29. Raalte, M. H. van, 1936. On the influence of glucose on auxin production by the root tip of Vicia Faba. Proc. Kon. Akad. Wetensch. Amsterdam 39: 261-265. 274 BIBLIOGRAPHY Ramshorn, K., 1934. Experimentelle Beitrage zur elektrophysiologischen Wachstumstheorie. Planta 22: 737-766. Rawitscher, F., 1932. Der Geotropismus der Pflanzen. Jena, 1932. Raydt, G., 1925. tJber die Bewegungen euphotometrischer Blatter. Jahrh. wiss. Bot. 64: 731-769. Reed, H. S. and F. F. Halma, 1919. On the existence of a growth inhibiting substance in the Chinese lemon. Univ. Calif. PxM. Agr. Sci. 4' No. 3, 99-112. Reiche, H., 1924. Uber Auslosung von Zellteilungen durch Injektion von Gewebesaften und Zelltriimmern. Z. f. Bot. 16: 241-278. Reid, M. E., 1924. Quantitative relations of carbohydrates to nitrogen in determining growth responses in tomato cuttings. Bot. Gaz. 77: 404-418. Reinders, D. E., 1934. The sensibility for light of the base of normal and decapitated coleoptiles of Avena. Proc. Kon. Akad. Wetensch. Amsterdam 37: 308-314. RiccA, U., 1916. Solution d'un probleme de physiologic: la propagation de stimulus dans la Sensitive (resume). Arch. ital. Biol. 65: 219-232. Robinson, T. W. and G. L. Woodside, 1937. Auxin in the chick embryo. I. /. Cell. Comp. Physiol. 9: 241-260. Ronsdorf, L., 1935. Vergleichende Untersuchungen liber die Wirkung verschiedener Wuchsstoffe auf das Wachstum einiger Pilze. Arch. Mikrobiol. 6: 309-325. RoTHERT, W., 1894. Uber Heliotropismus. Cohn's Beitr. Biol. Pfl. 7: 1-212. Sachs, J., 1880, 1882. Stoff und Form der Pflanzenorgane. I and II. Arb. Bot. Lnst. WUrzburg 2: 452-488 and 689-718. Sachs, J., 1893. Ueber Wachsthumsperioden und Bildungsreize. Physiologische Notizen VI. Marburg. ScHARRER, K. and W. Schropp, 1935. Die Wirkung von FollikelhormonKristallisaten auf das Wachstum einiger Kulturpflanzen. Biochem. Z. 281: 314-328. ScHECHTER, V., 1934. Electrical control of rhizoid formation in the red alga, Griffithsia bornetiana. J. gen. Physiol. 18: 1-21. ScHECHTER, V., 1935. The effect of centrifuging on the polarity of an alga, Griffithsia bornetiana. Biol. Bull. 68: 172-179. Schilling, E., 1915. Vhev hypertrophische und hyperplastische Gewebewucherungen an Sprossachsen, verursacht durch Paraffine. Jahrb. wiss. Bot. 55: 177-258. BIBLIOGRAPHY 275 ScHLENKER, G. and G. Mittmann, 1936. Versuche zur Klarung der reziproken Verschiedenheiten von Epilobium-Bast&Tden. IV. Internodienwachstum und Zellstreckung bei Epilohium hirsuhim unter dem Einfluss synthetischer |8-Indolylessigsaure. Jahrh. wiss. Bot. 83: 315-323. ScHMiTZ, H., 1933. tJber Wuchsstoff und Geotropismus bei Grasern. Planta 19: 614-635. ScHOELLER, W. and H. Goebel, 1931. Die Wirkung des Follikelhormons auf Pflanzen. Biochem. Z. 340: 1-11. ScHOELLER, W. and H. Goebel, 1932. Die Wirkung des Follikelhormons auf Pflanzen. II. IJber den Einfluss des kristallinischen /3-Follikelhormons. Biochem. Z. 251: 223-228. ScHOELLER, W. and H. Goebel, 1934. Die Wirkung des Follikelhormons auf Pflanzen. III. Biochem. Z. 272: 215-221. ScHOELLER, W. and H. Goebel, 1935. Die Einwirkung ostrogener Substanzen auf Pflanzen. IV. Biochem. Z. 278: 298-311. Schumacher, W., 1936. Untersuchungen iiber die Wanderung des Fluoresceins in den Haaren von Cucurbita Pepo. Jahrh. wiss. Bot. 82: 507-533. ScHWANiTZ, F., 1935. Beitrage zur Analyse der pflanzlichen Polaritat. Beih. Bot. Centralhl. 54 A: 520-530. Seubert, E., 1925. Uber Wachstumsregulatoren in der Koleoptile von Avena. Z. f. Bot. 17: A9-S8. Sierp, H., 1918. Ein Beitrag zur Kenntniss des Einflusses des Lichtes auf das Wachstum der Koleoptile von Avena saliva. Z. f. Bot. 10: 641-729. Sierp, H., 1921. Untersuchungen uber die durch Licht und Dunkelheit hervorgerufenen Wachstumsreaktion bei der Koleoptile von Avena saliva und ihr Zusammenhang mit den phototropischen Krummungen. Z.J. Bot. 13: 113-172. Sierp, H. and A. Seybold, 1926. Untersuchungen iiber die Lichtempfindlichkeit der Spitze and des Stumpfes in der Koleoptile von Avena saliva. Jahrh. wiss. Bot., 65: 592-610. SiLBERSCHMiDT, K., 1928. Uutersuchungeu uber die Abhangigkeit des pflanzlichen Wachstumsverlaufes und der erreichten Endlange von konstanten Temperaturgraden. Bihliotheca Botanica 97, Stuttgart. Simon, S. V., 1930. Transplantationsversuche zwischen Solanum melongena und Iresine Lindeni. Jahrh. wiss. Bot. 72: 137-160. Skoog, F., 1934. The effect of x-rays on growth substance and plant growth. Science 79: 256. 276 BIBLIOGRAPHY Skoog, F., 1935. The effect of x-irradiation on auxin and plant growth. /. Cell. Comp. Physiol. 7: 227-270. Skoog, F., 1937. A deseeded Aveyia test method for small amounts of auxin and auxin precursors. /. gen. Physiol. 20: 311-334. Skoog, F. and K. V. Thimann, 1934. Further experiments on the inhibition of the development of lateral buds by growth hormone. Proc. Nat. Acad. Sc. 20: 480-485. Snow, R., 1924. Conduction of excitation in stem and leaf of Mimosa pudica. Proc. Roy. Soc. B 96: 349-374. Snow, R., 1925. Conduction of excitation in the leaf of Mimosa Spegazzinii. Proc. Roy. Soc. B 98: 188-201. Snow, R., 19250. The correlative inhibition of the growth of axillary buds. Ann. Bot. 39: 841-859. Snow, R., 1929. The young leaf as the inhibiting organ. New Phytol. 28: 345-358. Snow, R., 1931. Experiments on growth and inhibition. I. The increase of inhibition with distance. Proc. Roy. Soc. B 108: 209-223. Snow, R., 1931fl. Experiments on growth and inhibition. II. New phenomena of inhibition. Proc. Roy. Soc. B 108: 305-316. Snow, R., 1932. Growth-regulators in plants. New Phytol. 31: 336-353. Snow, R., 1933. The nature of cambial stimulus. New Phytol. 32: 288-296. Snow, R., 1935. Activation of cambial growth by pure hormones. Nature 135: 876. Snow, R., 1935a. Activation of cambial growth by pure hormones. New Phytol. 34: 347-360. Snow, R., 1936. Upward effects of auxin in coleoptiles and stems. New Phytol. 35: 292-304. Snow, R. and B. Le Fanu, 1935. Activation of cambial growth. Nature 135: 149. Soding, H., 1923. Werden von der Spitze der Haferkoleoptile Wuchshormone gebildet? Ber. d. hot. Ges. 4I: 396-400. Soding, H., 1925. Zur Kenntnis der Wuchshormone in der Haferkoleoptile. Jahrh. wiss. Bot. 64: 587-603. Soding, H., 1926. tJber den Einfluss der jungen Infloreszenz auf das Wachstum ihres Schaftes. Jahrh. wiss. Bot. 65: 611-635. Soding, H., 1927. Uber Wuchshormone. Zellstimulationsforsch. 2: 381- 392. Soding, H., 1929. Weitere Untersuchungen liber die Wuchshormone der Haferkoleoptile. Jahrh. wiss. Bot. 71: 184-213. BIBLIOGRAPHY 277 SoDiNG, H., 1931. Wachstum und Wanddehnbarkeit bei der Haferkoleoptile. Jahrh. wiss. Bot. 74-- 127-151. SoDiNG, H., 1932. Hormone und Pflanzenwachstum. Beih. Bot. Centralbl. 69: 469-481. SoDiNG, H., 1932a. Uber das Streckungswachstum der Zellwand. Ber. d. hot. Ges. 50: 117-123. SoDiNG, H., 19326. liber das Wachstum der Infloreszenzschafte. Jahrb. wiss. Bot. 77: 627-656. SoDiNG, H., 1934. tJber die Wachstumsmechanik der Haferkoleoptile. Jahrb. wiss. Bot. 79: 231-255. SoDiNG, H., 1935. Die Ausfiihrung des Wentschen Auxintestes am Tageslicht. Ber. d. hot. Ges. 53: 331-334. SoDiNG, H., 1935a. tJber den Wuchsstoff in der Basis der Haferkoleoptile. Ber. d. hot. Ges. 53: 843-846. SoDiNG, H., 19356. Review of: Boysen Jensen, Die Wuchsstofftheorie etc. Z. f. Bot. 28: 466-467. SoDiNG, H., 1936. Wirkt der Wuchsstoff artspezifisch? Jahrh. wiss. Bot. 82: 534-554. SoDiNG, H., 1936a, tlber den Einfluss von Wuchsstoff auf das Dickenwachstum der Baume. Ber. d. hot. Ges. 54: 291-304. SoLLNER, K., 1933. Zur Aufklarung einiger Membranvorgange. KolloidZ. 62: 31-37. Soltys, A. and K. Umrath, 1936. tJber die Erregungssubstanz der Mimosoideen. Biochem. Z. 284: 247-255. Stanley, W. M., 1936. The isolation from diseased Turkish tobacco plants of a crystalline protein possessing the properties of tobacco mosaic virus. Phytopath. 26: 305-320. Stark, P., 1917. Beitrage zur Kenntnis des Traumatotropisraus. Jahrb. wiss. Bot. 57: 461-552. Stark, P., 1921. Studien iiber traumatotrope und haptotrope Reizleitungsvorgange mit besonderer Beriicksichtigung der Reiziibertragung auf fremde Arten und Gattungen. Jahrh. wiss. Bot. 60: 67-134. Stark, P., 1927. Das Reizleitungsproblem bei den Pflanzen im Lichte neuerer Erfahrungen. Ergeb. Biol. 2: 1-94. Stark, P. and 0. Drechsel, 1922. Phototropische Reizleitungsvorgange bei Unterbrechung des organischen Zusammenhangs. Jahrh. vriss. Bot. 61: 339-371. Stiles, W., 1935. Recent advances in science;—plant physiology. Sci. Progress, 30: 313-317. 278 BIBLIOGRAPHY Strugger, S., 1932. Die Beeinflussung des Wachstums und des Geotropismus durch die Wasserstoffionen. Ber. d. hot. Ges. 50: (77)-(92). Strugger, S., 1933. tJber das Wachstum dekapitierter Keimpflanzen. Ber. d. hot. Ges. 51: 193-209. Strugger, S., 1934. Beitrage zur Physiologie des Wachstums. I. Zur protoplasma-physiologischen Kausalanalyse des Streckungswachstums. Jahrh. wiss. Bot. 79: 406-471. Swingle, W. T., 1928. Metaxenia in the Date Palm, J. Hered. 19: 257-268. Tammes, p. M. L., 1931. tJber den Verlauf der geotropischen Krlimmung bei kiinstlich tordierten Koleoptilen von Avena. Rec. trav. hot. neerl. 28: 75-81. Tendeloo, N., 1927. Onderzoekingen over zoogenaamde traumatotropische krommingen bij kiemplanten van Avena sativa. Proc. Kon. Akad. Wetensch. Amsterdam 36: 661-666. 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. /. gen. Physiol. 18: 23-34. Thimann, K. V., 1935. Growth substances in plants. Ann. Rev. Biochem. 4: 545-568. Thimann, K. V., 1935a. On the plant growth hormone produced by Rhizopus suinus. J. Biol. Chem. 109: 279-291. Thimann, K. V., 19356. On an analysis of the activity of two growthpromoting substances on plant tissues. Proc. Kon. Akad. Wetensch. Amsterdam 38: 896-912. Thimann, K. V., 1936. On the physiology of the formation of nodules on legume roots. Proc. Nat. Acad. Sc. 22: 511-514. Thimann, K. V., 1936a. Auxins and the growth of roots. Am. J. Bot. 23: 561-569. Thimann, K. V., 19366. The physiology and chemistry of the plant hormones. Current Science 4- 716-721. Thimann, K, V. and H. A. Barker, 1934. Studies on the excystment of Colpoda cucullus. II. The action of the excystment inducing substance. /. exp. Zool. 69: 39-57. Thimann, K. V. and J. Bonner, 1932. Studies on the growth hormone of plants. II. The entry of growth substance into the plant. Proc. Nat. Acad. Sc. 18: 692-701. BIBLIOGRAPHY 279 Thimann, K. V. and J. Bonner, 1933. The mechanism of the action of the growth substance of plants. Proc. Roy. Soc. B 113: 126-149. Thimann, K. V. and H. E. Dolk, 1933. Conditions governing the production of the plant growth hormone by Rhizopus cultures. Biol. Zentralhl. 63: 49-66. Thimann, K. V. and J. B. Koepfli, 1935. Identity of the growthpromoting and root-forming substances of plants. Nature 135: 101. Thimann, K. V. 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. Sc. 19: 714-716. Thimann, K. V. and F. Skoog, 1934. On the inhibition of bud development and other functions of growth substance in Vicia Faba. Proc. Roy. Soc. B 114: 317-339. Thimann, K. V. and F. W. Went, 1934. On the chemical nature of the root-forming honnone. Proc. Kon. Akad. Wetensch. Amsterdam 37: 456-459. Uhrova, a., 1934. Uber die hormonale Natur der Hemmungswirkung der Blatter bei Bryophyllum crenaium. Planta 22: 411-427. Umrath, K., 1927. tJber die Erregungssubstanz der Mimosoideen. Planta 4: 812-817. Umrath, K., 1935. Review of: Boysen Jensen, Die Wuchsstofftheorie etc. Protoplasnia 23: 143-144. Umrath, K. and A. Soltys, 1936. Uber die Erregungssubstanz der Papilionaceen und ihre zellteilungsauslosende Wirkung. Jahrb. miss. Bot. 84: 276-289. Ursprung, a. and G. Blum, 1924. Eine Methode zur Messung des Wand- und Turgordruckes der Zelle, nebst Anwendungen. Jahrb. wiss. Bot. 63: 1-110. Uyldert, I. E., 1927. The influence of the growth-promoting substances on decapitated flower-stalks of Bellis perennis. Proc. Kon. Akad. Wetensch. Amsterdam 31: 59-61. Uyldert, I. E., 1931. De invloed van groeistof op planten met intercalaire groei. Diss. Utrecht, 1931. Virtanen, a. I. and S. von Hausen, 1933, 1934. Effect of yeast extract on the growth of plants. Nature 132: 408-409; 133: 383. Virtanen, A. I., S. von Hausen, and S. Saastamoinen, 1934. Die Einwirkung des Follikelhormons auf das Bluhen der Pflanzen. Biochem. Z. 272: 32-35. VocHTiNG, H., 1878 and 1884. tJber Organbildung im Pflanzenreich. I and II. Bonn, 1878, 1-258; 1884, 1-200. 280 BIBLIOGRAPHY VocHTiNG, H., 1892. liber Transplantation am Pflanzenkorper. Tubingen, 1892, 1-162. VocHTiNG, H., 1908. Untersuchungen zur experimentellen Anatomie und Pathologic des Pflanzenkorpers. Tubingen, 1908, 1-318. VoGT, E., 1915. tJber den Einfluss des Lichts auf das Wachstum der Koleoptile von Avena sativa. Z. f. Bol. 7: 193-270. Vries, H. de, 1885. Uber die Bedeutung der Circulation und der Rotation des Protoplasma fiir den Stofftransport in der Pflanze. Bot. Zeit. JfS: 1-6 and 17-26. Waddington, C. H., 1934. Experiments on embryonic induction. I, II, and III. J. exp. Biol. 11: 211-227. Waddington, C. H. and D. M. Needham, 1935. Studies on the nature of the amphibian organization centre. II. Induction by synthetic polycyclic hydrocarbons. Proc. Roy. Soc. B 117: SlO-317. Waddington, C. H., J. Needham, W. W. Nowinski, and R. Lemberg, 1935. Studies on the nature of the amphibian organization centre. I. Chemical properties of the evocator. Proc. Roy. Soc. B 117: 289-310. Wald, G. and H. G. du Buy, 1936. Pigments of the oat coleoptile. Science 84.: 247. Waller, A. D., 1900. The electrical effects of light upon green leaves. Proc. Roy. Soc. B 67: 129-137. Warner, T., 1928. tJber den Einfluss der geotropischen Reizung auf den Zucker- und Sauregehalt von Sprossen. Jahrb. iviss. Bot. 68: 431-498. Weber, U., 1931. Wachstum und Kriimmung 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. Weij, H. G. van der, 1931. Die quantitative Arbeitsmethode mit Wuchsstoff. Proc. Kon. Akad. Wetensch. Amsterdam 34: 875-892. Weij, H. G. van der, 1932. Der Mechanismus des Wuchsstofftransportes. Rec. trav. bot. neerl. 29: 379-496. Weij, H. G. van der, 1933. On the occurrence of growth substance in marine algae. Proc. Kon. Akad. Wetensch. Amsterdam 36: 759-760. Weij, H. G. van der, 1933a. Uber Wuchsstoff bei Elaeagmis angustifalius. Proc. Kon. Akad. Wetensch. Amsterdam 36: 760-761. Weij, H. G. van der, 1933&. Uber das Vorkommen von Wuchsstoff bei Meeresalgen. Pubbl. Staz. Zool. Napoli 13: 172-179. BIBLIOGRAPHY 281 Weij, H. G. van der, 1934. Der Mechanismus des Wuchsstofftran&portes II. Rec. trav. hot. need. 31: 810-857. Weimann, R., 1929. Untersuchungen uber den Traumatotropismus der .4iiena-koleoptile. Jahrb. wiss. Bol. 71: 269-323. Weiskopf, B., 1927. Sur les conditions correlatives de la croissance en longueur des bourgeons chez quelques Papilionac^es en voie de germination (resume). Publ. biol. ecole veter. Brno 6: 67-103. Weiss, P., 1935. The so-called organizer and the problem of organization in amphibian development. Physiol. Rev. 15: 639-674. Went, F. A. F. C, 1927. Groeistoffen. Jaarh. Kon. Akad. Wetensch. Amsterdam. 1927. Went, F. A. F. C., 1929. Plant movements. Proc. Int. Congr. Plant Set. Ithaca. Went, F. A. F. C, 1930. Les conceptions nouvelles sur les tropismes des plantes. Rev. gen. des Sci. 41- 631-643. Went, F. A. F. C., 1930a. tJber wurzelbildende Substanzen bei Brijophyllum calycinum Salisb. Z. f. Bot. 23: 19-26. Went, F. A. F. C, 1931. In: Kostytchew: Pflanzenphysiologie 11. Berlin, 1931. Went, F. A. F. C, 1932. Pflanzenwachstum und Wuchsstoff (Auxin). Forsch. und Fortschr. 8: 371-372. Went, F. A. F. C., 1932a. Over groeistoffen bij planten. Chem. Weekblad 29: 316-317. Went, F. A. F. C., 1932&. Wuchsstoff—Auxin—bei Pflanzen. ChemikerZeit. 56: 782-783. Went, F. A. F. C, 1933. Die Bedeutung des Wuchsstoffes (Auxin) fur Wachstum, photo- und geotropische Kriimmungen. Naturwiss. 21: 1-7. Went, F. A. F. C., 1933a. Growth substance (auxin) in plants. Nature 133: 452-453. Went, F. A. F. C, 1934. Hormone bei Pflanzen. Verh. Schweiz. Naturf. Ges. 1934: 220-240. Went, F. A. F. C., 1935. The investigations on growth and tropisms carried on in the Botanical Laboratory of the University of Utrecht during the last decade. Biol. Rev. 10: 187-207. Went, F. W., 1926. On growth-accelerating substances in the coleoptile of Ave7ia saliva. Proc. Kon. Akad. Wetensch. Amsterdam 30: 10-19. Went, F. W., 1928. Wuchsstoff und Wachstum. Rec. trav. hot. neerl. 25: 1-116. 282 BIBLIOGRAPHY Went, F. W., 1928a. Die Erklarung des phototropischen Krummungsverlaufs. Rec. trav. hot. neerl. 25a: 483-489. Went, F. W., 1929. On a substance causing root formation. Proc. Kon. Akad. Wetensch. Amsterdam 32: 35-39. Went, F. W., 1932. Eine botanische Polaritatstheorie. Jahrb. wiss. Bot. 76: 528-557. Went, F. W., 1934. On the pea test method for auxin, the plant growth hormone. Proc. Kon. Akad. Wetensch. Amsterdam 37: 547-555. Went, F. W., 1934a. A test method for rhizocaline, the root-forming substance. Proc. Kon. Akad. Wetensch. Amsterdam 37: 445-455. Went, F. W., 1935. Coleoptile growth as affected by auxin, aging and food. Proc. Kon. Akad. Wetensch. Amsterdam 38: 752-767. Went, F. W., 1935a. Hormones involved in root formation. Proc. 6th Int. Bot. Congr. 2: 267-269. Went, F. W., 19356. Auxin, the plant growth-hormone. Bot. Rev. 1: 162-182. Went, F. W., 1936. Allgemeine Betrachtungen iiber das Auxin-Problem. Biol. Zentralbl. 56: 449-463. Went, F. W., 1936a. The growth hormone in plants. Proc. 25th Ann. Celebration, Univ. So. Calif., 223-228. White, P. R., 1933. Concentrations of inorganic ions as related to growth of excised root-tips of wheat seedlings. Plant Physiol. 8: 489- 508. White, P. R., 1934. Potentially unlimited growth of excised tomato root tips in a liquid medium. Plant Physiol. 9: 585-600. WiESNER, J., 1878, 1880. Die heliotropischen Erscheinungen im Pfianzenreiche. I, II. Denkschr. A-. Akad. Wiss. Wien 39: 143-209; 43: 1- 92. WiESNER, J., 1884. Note iiber die angebliche Function der Wurzelspitze. Ber. d. bot. Ges. 2: 72-78. WiLHELM, A. F., 1930. Untersuchungen iiber das Chromogen in Vicia Faba. Jahrb. wiss. Bot. 72: 203-253. Williams, R. J., C. M. Lyman, G. H. Goodyear, J. H. Truesdail, and D. HoLADAY, 1933. "Pantothenic acid," a growth determinant of universal biological occurrence. J. Am. Chem. Soc. 55: 2912-2927. WoLK, P. C. VAN DER, 1911. Oudcrzoekingen over de geleiding van lichtprikkels bij kiemplantjes van Avena. Versl. Kon. Akad. Wetensch. Amsterdam 1911 : 258-273. Zimmerman, P. W., 1930. Oxygen requirements for root growth of cuttings in water. Am. J. Bot. 17: 842-861. BIBLIOGRAPHY 283 Zimmerman, P. W., W. Crocker, and A. E. Hitchcock, 1933. Initiation and stimulation of roots from exposure of plants to carbon monoxide gas. Contrib. Boyce Thom-pson Inst. 5: 1-17. Zimmerman, P. W. and A. E. Hitchcock, 1929. Vegetative propagation of holly. Am. J. Bot. 16: 556-570. Zimmerman, P. W. and A. E. Hitchcock, 1935. Response of roots to "root-forming" substances. Conlnh. Boyce Thompson Inst. 7: 439- 445. Zimmerman, P. W., A. E. Hitchcock, and F. Wilcoxon, 1936. Several esters as plant hormones. Contrib. Boyce Thompson Inst. 8: 105-112. Zimmerman, P. W. and F. Wilcoxon, 1935. Several chemical growth substances which cause initiation of roots and other responses in plants. Contrib. Boyce Thompson Inst. 7: 209-229. ZiMMERMANN, W. A., 1936. Untcrsuchungen iiber die raumliche und zeitliche Verteilung des Wuchsstoffes bei Baumen. Z. f. Bot. 30: 209-252. ZoLLiKOFER, C, 1928. Ubcr Dorsiventralitatskriimmungen bei Keimlingen von Panicurn und Sorghum und den Einfluss der Koleoptile auf das Mesokotylwachstum. Rec. trav. hot. neerl. 2oa: 490-504. ZoLLiKOFER, C, 1935. Zur Rolle der Membrandehnbarkeit bei der floralen Bewegung. Ber. d. bot. Ges. 53: 152-177. ADDENDA Clark, W. G., 1937. Electrical polarity and auxin transport. Plant Physiol. 12: 409-440. KoEPFLi, J. B., K. V. Thimann, and F. W. Went, 1937. Plant Hormones: Structure and physiological activity. I. In press. Rogenhofer, G., 1936. Wirkung von Wuchsstoffen auf die Kallusbildung bei Holzstecklingen. I. Sitzungsber. Akad. Wie^i I, 14S: 81-99. AUTHOR INDEX References to pp. 251-283 are to the Bibliography. Agricola, 6, 251 Amlong, 123, 143, 149, 150, 165, 180, 181,251,254 Anderson, 120, 251 Appleman, 19, 251 Arisz, 246, 251 Asana, 167, 168 Avery, 5, 22, 62, 69, 91, 97, 100, 209, 221, 227, 251, 253, 254 Babicka, 4, 251 Barker, 238, 278 Bauguess, 81, 139, 251 BayHss, 3, 180, 251, 259 Beijerinck, 8, 9, 15, 16, 183, 248, 252 Bellamy, 209, 256 Burkhardt, 174, 255 Burkholder, 5, 22, 62, 221, 251, 254 Burkom, van, 77, 255 Busgen-Miinch, 220, 255 Butenandt, 234, 255 Buy, du, 5, 14, 23, 29, 40, 46, 51, 59, 60, 75, 76, 83-85, 98, 152, 161, 167- 171, 246, 255, 256, 272, 280 Carlson, 183, 256 Castle, 170, 172, 256 Child, 209, 256 Cholodny, 4, 13, 14, 16, 24, 53, 58, 63, 65, 75, 80, 100, 141-147, 154- 158, 162, 165, 174, 179, 256, 257 Beyer, 26, 58, 60, 64, 75, 83, 93, 94, Clark, 104, 257, 283 161, 174, 178, 179, 252 Cooper, 91, 190, 202, 204, 205, 209, Blaauw, 16, 17, 152, 155, 172-174, 220,257 230, 245, 252 Coster, 219, 221, 257 Blackie, 193, 252 Creighton, 62, 221, 251 Blackman, 45, 237, 252 Crocker, 181, 182, 191, 192, 257, 282, Blum, 120, 279 283 Boas, 163, 252 Crozier, 71, 267 Bonner, 15, 18, 35, 45, 52, 53, 72, 75, Curtis, 184, 188, 258 86, 105, 123, 125, 127, 128, 130, 131, Czaja, 45, 61, 98, 120, 148, 149, 224, 150, 161, 217, 238, 252, 253, 278, 225, 258 279 Bose, 171, 253 Bottelier, 101, 253 Bouillenne, 15, 16, 147, 186, 194, 198, 202, 204, 223, 253 Boyd, 22, 253 Boysen Jensen, 5, 10-16, 29, 32, 42, 43, 58, 61, 68, 71, 72, 93, 94, 98, 112, 131, 142, 144, 145, 149, 150, 152, 154, 160-165, 168-172, 238, 253, 254 Brauner, 13, 101, 153, 154, 163, 165, 169, 171, 180, 254 Brecht, 36, 37, 254 Brown, 225, 248, 255 Buck, 121, 255 Buder, 167, 255 Banning, 81, 141, 179, 180, 238, 254, 255 Czapek, 164, 258 Darwin, 9, 16, 92, 142, 258 Delisle, 212, 258 Denham, 124, 258 Denny, 216, 258 Dijkman, 14, 60, 69, 73, 84, 97, 159, 160, 165, 258 Dillewijn, van, 172, 258 Dolk, 14, 25, 34, 40, 42, 60, 71, 72, 74, 85, 105, 111, 112, 158-160, 163, 165, 176, 238, 258, 259, 279 DoUfuss, 68, 259 Dostdl, 15, 16, 208, 259 Drechsel, 13, 26, 153, 277 Duggar, 88, 259 Duhamel du Monceau, 6, 7, 16, 183, 259 Duyfjes, 225, 259 285 286 AUTHOR INDEX Eastcott, 196, 215, 236, 259 Errera, 207, 259 Erxleben, 5, 14, 42, 63, 65, 70, 106- 110, 113, 116, 117, 142, 148, 259, 266 Ewart, 180, 259 Faber, 146, 149, 203, 214, 259 Fiedler, 57, 143, 146-150, 162, 259 Fieser, 235, 259 Fischer, Emil, 118 Fischer, F. G., 243, 259 Fischnich, 100, 175, 191, 223, 224, 259, 268 Fitting, 10, 16-20, 74, 228, 244, 259, 260 FHry, 60, 85, 260 Frey-WyssHng, 120, 125-127, 260 Frieber, 112, 260 Friedrich, 124, 260 Froschel, 245 Garbarini, 58, 260 Gardner, 225, 248, 255 Gautheret, 222, 260 Gill, 219, 260 Glover, 116, 261 Goebel, H., 235, 275 Goebel, K., 9, 15, 16, 183, 207, 215, 261 Goodwin, 67, 212, 213, 261 Gorter, 44, 105, 141, 146, 147, 261 Gouwentak, 97, 186, 201, 221, 261 Gradmann, 13, 58, 158, 179, 261 Graham, 184, 186, 193, 202, 206, 252, 261 Graze, 87, 261 Gregory, 66, 227, 261 Grieve, 227, 248, 261 Gundel, 161, 261 Guttenberg, von, 5, 93, 261, 262 Haagen Smit, 4, 5, 14, 16, 40-43, 49, 50, 53, 63, 65, 70, 71, 106-110, 113, 116, 117, 128, 140, 142, 148, 180, 192, 197, 215, 262, 266 Haan, de, 203, 262 Haas, Horreus de, 121, 262 Haberlandt, 17, 18, 185, 221, 262 Hagemann, 202, 262 Haig, 170, 262 Halma, 207, 216, 274 Hammerling, 240, 262 Hanstein, 7, 262 Harder, 235, 262 Hartelius, 113, 236, 238, 272 Hartig, 7 Hartmann, 180, 262 Harvey, 208, 209, 262 Hausen, von, 228, 279 Hawker, 61, 144, 145, 162, 165, 263 Heidt, 146, 147, 263 Hellinga, 97, 186, 201, 221, 261 Hess, 126, 263 Heyn, 5, 14, 36, 113, 121-125, 146, 150, 263, 264 Hinderer, 87, 264 Hitchcock, 51, 56, 91, 99, 116, 139, 181, 182, 191, 201, 202, 204, 212, 223, 228, 257, 264, 282, 283 Holtfreter, 243, 264 Honert, van den, 102, 103, 264 Honing, 227, 264 Hulssen, van, 49, 50, 128, 180, 266 Huxley, 236, 264 Janse, 141, 264 Jost, 5, 18, 37, 40, 53, 54, 84, 150, 217, 218, 221, 225, 264 Juel, 43, 49, 265 Kastens, 219, 265 Katunskij, 203, 265 Keeble, 142, 162, 163, 179, 219, 265 Kisser, 265 Klebs, 16 Koch, 166, 180, 265 Koepfli, 140, 190, 192, 279, 283 Kogl, 4, 5, 14, 16, 40-43, 49, 50, 63, 65, 70, 71, 106-118, 128, 140, 142, 148, 180, 190, 196, 197, 215, 236, 237, 265-267 Kok, 102, 267 Koning, 58, 62, 267 Koningsberger, C., 88, 109, 267 Koningsbergtr, V. J., 52, 172, 267 Kornmann, 36, 46, 47, 52, 57, 80, 90, 91, 97, 267, 268 Kostermans, 111, 113, 114, 140, 267 Kraus, 9, 267 Kraybill, 9, 267 Kropp, 71, 267, 272 Kupfer, 183, 267 Laan, van der, 160, 165, 267 Laibach, 4, 14, 15, 17, 36, 37, 46, 47, AUTHOR INDEX 287 52, 56, 62, 63, 70, 80, 88, 90, 91, 97, 100, 175, 190, 203, 212, 213, 218, 223, 224, 227, 228, 248, 267, 268, 270 Lamprecht, 18, 268 Lane, 143, 148, 268 Lange, 29, 166, 169, 268 Larsen, 57, 268 La Rue, 226-228, 248, 268, 269 Le Fanu, 214, 215, 220, 225, 269, 276 Lehmann, 87, 269 Leitch, 116, 270 Lek, van der, 15, 16, 92, 185, 269 Leonian, 238, 269 Leslie, 227, 269 Levine, 235, 269 Li, 44, 169, 269 Loeb, 15, 16, 18, 157, 184, 207, 215, 245, 269, 270 Loehwing, 81, 270 Loewe, 4, 270 Lonsdale, 124, 258 Lund, 241, 270 MacCallum, 183, 270 Mai, 97, 223, 248, 268, 270 Malowan, 4, 270 Malpighi, 6, 270 Mangham, 102, 270 Manske, 117, 270 Maresquelle, 227, 270 Maschmann, 70, 88, 228, 268, 270 Meesters, 143, 270 Meissner, 51, 270 Merkenschlager, 163, 252 Metzner, 51, 124, 161, 271 Meyer, 45, 62, 63, 268, 271 Michener, 97, 191, 271 Mittmann, 81, 87, 275 Mohl, von, 7 Moissejewa, 75, 271 Molisch, 189, 271 Moreland, 216, 271 Morgan, 183, 271 Muller, 191, 211, 212, 213, 223, 268, 271 Nagao, 59, 145, 147, 271 Navez, 60, 71, 142, 158, 162, 164, 165, 180, 271, 272 Needham, 243, 280 Nelson, 142, 162, 163, 179, 265 Nemec, 187, 225, 272 Nielsen, 13, 14, 24, 26, 32, 40, 42, 52, 58, 68, 71, 105, 112, 113, 142, 172, 236, 238, 254, 272 Nuernbergk, 5, 23, 28, 29, 46, 51, 83-85, 152, 167-169, 256, 272 Oholm, 113, 272 Oosterhuis, 60, 231, 272 Orth, 218, 272 Overbeck, 272 Overbeek, van, 14, 27, 57, 59, 60, 73, 81, 83, 86-88, 97, 98, 122, 136, 167, 168, 170-175, 211, 264, 273 Padl, 11-13, 16, 17, 24, 58, 92, 152, 154, 178, 273 Perrv, 25, 273 Petrick, 203, 262 Pfaeltzer, 60, 98, 273 Pfeffer, 11, 16 Pisek, 4, 273 Pohl, 63, 64, 65, 100, 273 Popoff, 238, 273 Priestley, 22, 25, 219, 273, 278 Pringsheim, 152, 273 Purdy, 32, 153, 273 Raalte, van, 145, 146, 273 Ramshorn, 104, 274 Rawitscher, 152, 274 Raydt, 175, 274 Reed, 15, 207, 216, 274 Reiche, 18, 274 Reid, 183, 274 Reinders, 169, 274 Reiss, 37, 40, 54, 84, 150, 225, 264 Ricca, 19, 274 Robinson, 71, 72, 158, 165, 272, 274 Rogenhofer, 139, 221, 223, 283 Ronsdorf, 238, 274 Rothert, 10, 16, 25, 77, 92, 274 Saastamoinen, 235, 279 Sachs, 7, 9, 15, 16, 18, 23, 92, 119, 183, 207, 242, 248, 274 Salkowski, 111 Schiifer, 191, 268 Scharrer, 235, 274 Schechter, 241, 274 Schilling, 38, 274 Schlenker, 81, 87, 261, 275 Schmitz, 161, 275 Schoeller, 235, 275 288 AUTHOR INDEX Schropp, 235, 274 Schumacher, 102, 275 Schwanitz, 92, 215, 275 Seubert, 13, 26, 42, 70, 105, 154, 169, 275 Seybold, 166, 169, 275 Sierp, 166, 169, 172, 275 Silberschmidt, 28, 275 Simon, 219, 275 Skoog, 15, 16, 36, 45, 61, 64, 66, 68, 88, 89, 97, 100, 115, 163, 173, 209, 210, 213, 214, 220, 275, 276, 279 Smith, 235 Snow, 4, 14-16, 19, 90, 97, 99, 142, 162, 163, 208, 209, 216, 219-222, 265, 276 Soding, 4, 5, 12, 14, 16, 24, 27, 32, 36, 38, 58, 67-69, 121-123, 126, 221, 276, 277 SoUner, 102, 277 Soltys, 18, 19, 217, 277, 279 Stanley, 69, 277 Stark, 12, 13, 16, 24, 26, 27, 42, 67, 153, 178, 179, 277 Starling, 3, 251 Stewart, 184, 186, 193, 252, 261 Stiles, 5, 277 Stormer, 235, 262 Strugger, 130, 131, 278 Swingle, 17, 278 Tammes, 91, 278 Tendeloo, 179, 278 Tetley, 22, 25, 278 Thimann, 5, 14-16, 35, 40, 42, 43, 52, 53, 57, 58, 61, 63, 67, 68, 71, 72, 75, 78, 86, 88, 89, 97, 100, 105, 111, 112, 116, 125, 127, 128, 131, 132, 135, 136, 139, 140, 143-149, 160, 173, 179, 190, 192, 197, 201, 203, 209-214, 216, 220, 221, 225, 238, 246, 253, 259, 276, 278, 279, 283 Tonnis, 70, 71, 236, 266, 267 Trogus, 126, 263 IJhrova, 212, 228, 279 IJmrath, 5, 18, 19, 217, 277, 279 Ursprung, 120, 279 Uyldert, 14, 56, 58, 60, 181, 232, 279 Virtanen, 228, 279 Vochting, 9, 16, 92, 183, 192, 279, 280 Vogt, 172, 280 Vries, de, 101, 280 Waddington, 243, 280 Wald, 170, 280 Wallace, 192 Waller, 171, 280 Warner, 124, 280 Weber, 86, 280 Wehnelt, 18, 19, 217, 218, 280 Weij, van der, 14, 30, 35, 36, 57, 59, 61, 90, 94-97, 99, 101, 146, 177, 280, 281 Weimann, 179, 281 Weiskopf, 208, 281 Weiss, 242, 281 Went, F. A. F. C, 4, 149, 152, 184, 281 Went, F. W., 5, 13, 15, 16, 26, 29, 35, 53, 54, 57, 59, 60, 63, 74, 76-81, 92, 98, 101, 103, 105, 113, 116, 135, 136, 140, 146, 147, 154, 156, 157, 167, 168, 174, 177, 186, 187, 190, 192-194, 197, 200, 202, 204, 213, 214, 223-225, 245, 247, 253, 262, 279, 281, 283 Wergin, 126, 263 White, 150, 199, 282 Wiesner, 141, 152, 282 Wilcoxon, 51, 116, 139, 191, 192, 223, 283 Wilhelm, 218, 282 Williams, 237, 282 Wolk, van der, 92, 282 Woodside, 71, 274 Zimmerman, P. W., 51, 91, 99, 116, 139, 181, 182, 184, 191, 192, 202, 204, 223, 228, 257, 264, 282, 283 Zimmermann, W. A., 61, 70, 232, 283 ZoIIikofer, 44, 123, 283 SUBJECT INDEX Page numbers in italics refer to extended or systematic treatment of the subject. Absidia ramosa, 105 Absorption spectrum of auxin, 109 of carotene, 170 Acalypha, 185-189, 202, 223 Acetabularia, 240, 241 Acid curvatures, 37, 130, 131, 181 AE (Avena Einheit), 41, 43 Aesculus, 61, 62 Agar, 12, 13, 34-36 Age of coleoptile for auxin test, 39, 40 Ageotropic, 163 Aging, 75-77 Agrimonia, 219 Alaska peas, 54, 198 Amino acids, 19, 20, 40, 72, 112, 196 Animal hormones, 232-235 Animals, auxin in, 70-71 Antirrhinum, 211 Aphids, 227 Apical dominance, 216 Arachis oil, 116 Aristolochia, 227 Asparagus, 60, 231 Aspergillus, 112, 113, 236, 238 Aster, 212 Autotropism, 86, 178 Auxanometer, 52 Auxenolonic acid, see Auxin h Auxentriolic acid, see Auxin a Auxin, definition, 4 Auxin a and h chemistry, 106-110, 113, 114 esters of, 65, 116, 117 physiological activity, 42, 65, 142, 146, 175, 190, 195, 210, 220, 224 Auxin-glutaric acid, 109 Avena, 4, 10, 21-53, 58, 61, 63, 64, 73, 82, 84, 88, 113, 142-149, 156- 172, 203 Avena test, 13, 21, 27-51 Axillary buds, 60, 207-216 Bacteria, 71, 106, 112, 113, 116, 148, 187, 225, 226 Bacterium tumefaciens, 187, 225 Bambusa, 232 Bios, 4, 196, 215, 236-238 Biotin, 196, 197, 215, 236, 237 Blaauw effect, 174 Blastanin, 63 Bound auxin, 68, 131, 132, 160 Bryophyllum, 18, 158, 184, 212 Bud formation, 92 Bud growth, 18, 207-216 Bud growth substances, 215, 216 Bud inhibition, 3, 19, 56, 184, 207-216 Calibrating plants, 51 Callus, 6, 216, 222-227 Cambial growth, 3, 97, 218-222 Cancer, see Carcinoma Capacity of auxin transport, 95-97, 102, 103 Carbon-nitrogen ratio, 9, 183 Carcinogenic substances, 235 Carcinoma, 70, 71, 235 Cardamine, 58 Carotene, 170, 198 Cecidomyia Poae, 8 Cecidomyidae, 248 Cell division, 17, 18, 22, 217, 218, 221, 223, 224 Cell elongation, 2, 4, 22, 119-127 Cell enlargement, 223, 224, 228, 229 Cellulose, 124-127 Cell wall, 119-127 Center of attraction, 215 Cephalaria, 36, 38, 58, 68 Chemotropism, 181 Chloroform extraction, 58, 67, 68, 128, 145, 146, 160, 163 Cholodny-Went theory, 154-17 ^ Chromatophore activator, 71 Chrysanthemum, 204 Cichorium, 187 Cinnamonum, 193 289 290 SUBJECT INDEX Cissus, 203 Citrus, 91, 202, 204, 205 Clematis, 206 Cocos fat, 117 Coelenterates, 240 Coix, 24, 58 Coleoptile, 21 ff. Coleoptile, length for auxin test, 39, 40 Coleoptile, thickness, 39 Coleus, 97, 175, 191, 227 Colpoda, 238 Commelina, 184 Convolvulus, 58 Copenhagen, 49 Corn germ oil, 63, 106, 116 Corn meal, 63 Correlation, 2, 4, 6, 7, 9, 16, 91, 104, 183, 207-209, 230, 231, 248 Correlation carrier, 4, 12 Cotton-hair, 126 Cotyledon, 21, 60, 84, 186, 194, 208, 215, 217 Co-Wuchsstoff, 238 Crown gall, 225, 235, 248 Crustaceae, 71 Cucurbita, 102, 227 Curly-top, 227, 248 Cuttings, 183-187, 192-206, 222, 239, 240 Cynipidae, 248 Dark room, 27, 28 Date (Phoenix), 17 Decapitation, 10, 12, 24 ff., 128, 141, 154, 167, 169 Decapitation scissors, 31 Deformis tobacco, 227 De-seeded test, 86, 64, 65, 68, 100 Dextrose agar, 61, 144-146, 162, 163 Diastase, auxin in, 13, 26, 105, 186, 223 Didymoplexis, 67 Differentiation, 119, 198, 242, 243 Diffusion of auxin, 90, 95-97, 101, 113, 145, 146 Diffusion method, 57 Dihydro-auxin a, 108, 115 Diseases, plant, 72, 248 Dissociation constant, 106, 130, 131 Distribution of auxin, 67-70, 86, 99, 156-182, 245 Distribution of growth, 51, 77-86, 148 Dog, 70 D-value, 33 Dwarfs, 81, 86-88, 211 Elasticity, 121-124 Eleagnus, 61, 97 Electrical field, 180, 241 Electric potential, 11, 103, 104, 165, 166, 171, 181 Electro-tropism, 165, 179-181 Embryo, 17, 71, 242, 243 e.m.f., see Electric potential Eosin, 163 Epicotyl, 21, 82, 85 Epilobium, 87 Epinasty, 56, 116, 181-182 Erineum, 248 Esters of auxins, 65, 114-117, 139 Ether narcotization, 96, 97 Ethylene, 160, 181, 182, 184, 191, 192 Euglena, 238 Euphotometric movements, 175 Evocator, 243, 244 Excitation, 244 Excystment, 238 Extraction of auxin, see Chloroform Eye stalk, 71 Falling of petiole, 228 Fiber-like structure, 125, 126 Field, 241-244 First negative curvature, 166 First positive curvature, 166 Flower formation, 7, 228, 235 Flower stalk, 58, 122, 123 Fluorescein, 102 Food factor, 78-80, 82, 84-86, 159, 178, 179 Formation of auxin, 57-67, 69, 194 Formation of roots, see Root Formation of shoots, 92 Formative substances, 6 Fraxinus, 219 Free auxin, 68, 130-132, 160, 161, 169, 170 Fucus, 241 Fungi, 57, 71, 72, 105, 112, 113, 116, 235-238 Galls, 8, 225, 248 Ganzheit, 240 Gastrodia, 67 Gelatin, 12 Gene, 86-88, 247 SUBJECT INDEX 291 Genetics, 87 Geo-electric efifect, 165 Geo-growth reaction, 158, 162 Geotonus, 97, 98 Geotropic perception, 164 Geotropic sensitivity, 154, 163 Geotropism, 10, 18, 46, 61, 86, 91, 97, 124, 142, 144, 145, 147, 152, 165- 168, 171, 181, 207 Geotropo-hormone, 158, 184 Germination and auxin content, 63 Glass holder, 29, 30 Godetia, 211 Gradient, auxin, 96-99, 242 Gradient, metabolic, 92, 241, 242 Grafting, 219 Grand period, 23 Grass nodes, 161 Gravity, 60, 92, 97, 98, 142, 154-166, 183, 186, 245 Griffithsia, 241 Growth, definition of, 119 Growth enzymes, 8, 9 Growth hormone, definition, 4 Growth-inhibiting substances, 19, 42, 45, 128, 129, 143, 154, 178, 207, 208, 213 Growth of roots, 56, 85, Ul-lSO Growth promoting substance, 12, 13, 19, 24, 26, 42, 45, 53, 105, 154, 213 Growth regulator, 4, 12, 153, 155 g.s. (growth substance), 4 Guttation, 28 Gynostemium, 228 Heat treatment, 83, 84, 87 Hehanthus, 26, 58, 60, 62, 64, 82, 84, 85, 88, 124, 149, 161, 174, 180, 187, 191, 211, 219,220, 224 Heliotropic machine, 245 Hemicellulose, 124, 127 Hen, 70, 71 Hetero-auxin, 110 Hibiscus, 204, 228 Hicoria, 63 Homogentisic acid, 164 Hordeum, 63, 86 Horizontal microscope, 51 Hormone, definition, 3 Humidity, 28 Hydration of protoplasm, 130 Hypocotyl, 21 Ilex, 204 Impatiens, 186, 194, 198 Inactivation of auxin, 42, 57, 58, 65, 72, 86-89, 105, 112, 128-130, 136, 146, 147, 163, 172, 175, 179, 206, 210 Inactivation, spontaneous, 109 IndoIe-3-acetic acid, 42, 51, 72, 110- 112, 142, 175, 190, 195, 210, 220, 221, 224, 225 Indole compounds, 112-116, 132-140 Inositol, 196, 236 Interface, 102 Interferometer, 51 Internal factor for root formation, 199-202 Intracellular activators, 236 Intumescences, 192, 226 Intussusception, 120, 123, 126 Ionization of the air, 50 Ipomoea, 58, 62 Irritation, 10, 11 Isoelectric point of protoplasm, 130 Isolated roots, 146, 150, 162 Key, simile of, 118, 134, 135 Kohl-rabi, 18 Labiatae, 158 Lactone of auxin a, 106, 1 10 Lanoline paste, 37, 191, 204, 210, 214, 223 Latente Reizbarkeiten, 244 Latent time, 244 Lateral transport, 46, 91, 98, 136, 157, 163, 169, 171 Layering, 206 Leaf, auxin production in, 61, 62, 69, 148, 203, 213, 219 Leaf growth, 66, 227 Lemon, see Citrus Lepidium, 60 Lepto-hormone, 18, 221 Light, effects of, 60, 66, 88, 89, 98, 166-178, 192-194, 245 Light-growth reaction, 101, 104, ni- ne Light-sensitive system, 170 Lignification, 224 Limiting factor, 45, 198, 199, 236-239 Linum, 211 Load-extension apparatus, 124 Long reaction to light, 174 Lupinus, 60, 66, 69, 73, 75, 84, 97, 141, 154, 159, 165, 187 292 SUBJECT INDEX Malt, 13, 26, 42, 105 Malus, 62 Malva, 91 Master reaction, 229, 244, 246, 247 Maturation, 119 Maximum angle, 45-49 MCS (meter-candle seconds), 166 Meristine, 223 Mesocotyl, 21, 29, 82-84, 86, 87, 100 Mesophyll, 66, 100, 227 Messengers, chemical, 3 Meta-xenia, 17 Micelles, 125-127, 150 Microorganisms, 71, 72, 235-238 Micropotometer, 53 Midrib, 227 Mimosa, 19 Molecular weight, 112, 113 Monstrosities, 8 Morphogenesis, 7, 119 Mouse, 70, 233 Moving picture camera, 51 Nana, 86, 87, 211 Narcosis, 97, 99 Nastic movements, 181-182 Native growth hormone, 113, 114, 146 Negative curvature, 42, 44 Negative osmosis, 120 Nematus capreae, 8 Nerve, 10 Nicotiana, 56, 62, 69, 91, 97, 212, 227, 228, 231 Nodes of grasses, 161 Nodules, see root nodules Nutrition, 9, 183, 207, 208 Oestrin 1 Oestriol \ see Theelin Oestrone J Orchids, 17, 228 Organ formation, 2, 8, 16, 230 Organization, 119, 242-2U Organizer, 242-244 Osmotic pressure, 120 Ovary, 17, 67, 228 Oxidase, 86 Oxidation of auxin, 58, 86, 88, 106, 146, see also Inactivation Pachysandra, 204 Papaver, 123 Papaya, 186 Papihonaceae, 19 Partition coefficient, 106 Pasadena, 49, 50 Passiflora, 227 Paste, see Lanoline Pathology, 222, 227 Pea test, 40, 53-56, 133-139 Pectin, 124, 127 Pepsin, 70, 105 Peptone, 111, 112, 184 Perception, 11, 164-166, 171 Pericycle, 198, 218, 221, 222 Permeability, 120, 128, 136, 153, 154, 172 Peroxide, 58, 105 Petiole, 175, 202, 212, 228 pH, 40, 106, 130-134, 143, 160, 161 Phaseolus, 160, 165, 171, 208, 217, 218, 222, 225, 226, 227 Phloem, 91, 185, 190, 217 Photosynthesis and auxin formation, 66, 213 Phototropic hormones, 114, 153 Phototropism, 10, 14, 28, 53, 91-94, 152-156, 166-178, 245, 247 Phycomyces, 113, 170, 172 Phytohormones, 3, 232, 233, 236, 248, 249 Phytophthora, 238 Pisum, 54-56, 82, 85, 88, 97, 141- 149, 180, 187-189, 191, 197-201, 203, 210-220, 231, 237 pK, 106, 130, 131 Plagiotropic, 181 Plant unit, 41-43 Plasmoptysis, 120 Plasticity, 120-124, 150 Plumula, 60, 84 Poa, 8 Polarity, 2, 3, 9, 90-10^, 146, 147, 155, 157, 183, 185, 186, 214, 225, 230, 242 Polarizing microscope, 125 Polar transport of hormone, 92-104, 156, 159, 186, 188, 191, 215, 220, 240, 241 Pollen, 63, 190, 191, 211, 212, 223, 228 Pollen-hormone, 228 Pollinia, 17, 62, 63, 223, 228 Polygonum, 77, 211, 231 Populus, 61, 185, 219, 224, 226 Positive curvatures, 26, 42-^5 SUBJECT INDEX 293 Post-floration, 17, 228 Potato, 17, 19 Potential gradient, 103 Precursor of auxin, 59, 6^-67, 70, 100, 116, 117, 213 Presentation time, 177, 244 Primary activity, 132-140 Primary leaf, 23, 31, 68 Primordium, organ, 119, 198, 199 Proportionality between concentration and curvature, 27, 33, 35, 40, 41, 54-56 Proportionality of growth, 52, 53, 73, 86, 128 Protoplasmic streaming, 12, 19, 101, 102 Protractor, 32, 33, 55 Pruning, 207 Pseudo-auxin, 109 Pseudomonas, see Bacterium p.u. (plant unit), 41-43 Purification of auxin, 105-112 Quercus, 61 Radiation, 88, 89 Raphanus, 60, 66, 73, 88, 89, 97, 98, 170, 171 Recovery, 244 Regeneration, 18, 92, 183, 238-242 Regeneration of the physiological tip, 25, 36, 44-47, 64, 75, 76, 84, 121, 167 Reizmengengesetz, 245 Rejuvenation, 76, 77 Residual growth, 51, 53 Respiration, 128, 164 Reviews, 4, 5 Rhizobium, 248 Rhizocaline, 186, 190 Rhizogenes, 187, 190 Rhizoid, 241 Rhizome, 92, 215 Rhizopin, 111 Rhizopus, 72, 105, 106, 111, 112, 113, 142, 190, 210, 236, 238 Ribes, 185 Rice polishings, 63, 186 Ringing, 6, 206 Ring structure, 126 Roots, auxin formation in, 61, 144- 148 Root formation, 3, 6-8, 56, 92, 97, 100, 183-206, 218, 225, 229, 237, 246 Root forming substance, 183, 184, 186, 189-192, 215, 223 Root germs, 185 Root inhibition, 141-144 Root nodules, 148, 225 Root tip, 61, 141, 142, 199 Root unit, 189 Saccharomyces unit, 197 Salamanders, 183 Saliva, 13, 26, 42, 70, 105, 154 Salix, 8, 61, 92, 97, 185, 192, 219 Salts, 40, 145 Sap, 6, 7 Scrophularia, 208, 219 Scutellum, 21, 100, 147 Secale, 81 Secondary activity or properties, 132- 140 Sections of coleoptile, 52, 53, 75 Seeds, auxin in, 63 Sensitive plant, 19 Sensitivity of Avena, 27-29, 36, 40, 64, 75-77 Sequoia, 63 Setaria, 84 Sex hormones, 109, 198, 228, 234, 235 Shadowgraph of curvatures, 32, 34, 48, 55 Shock reaction, 174 Short reaction to light, 174 Sinapis alba, 147, 211 Size of agar blocks, 35, 43 Size of plant, limitation of, 80 Size relationships, 231, 232 Solidago, 212, 213 Sorghum, 68 Specificity of auxins, 114 Spiraea, 219 Spotted wilt, 227 Standard deviation, 39 Stimulus, 152, 174, 244-247, see also Transmission Stimulus substances, 244 Stoichiometric relationship, 128, 136, 246 Straight growth and auxin, 51-53, 231, 232 Succulents, 18 Suction force, 120 Sugars, 40, 85, 123, 124, 144, 145, 184, 186, 189, 194, 195, 199, 218, 237, 239 294 SUBJECT INDEX Supramaximal angle, 47 Swelling of roots, 147-149 Swelling of stems, 6, 56, 81, 210, 213, 218, 222-227, 228 Tannic acid, 45 Taraxacum, 161 Taxus, 204 Temperature, 28, 60, 246 Temperature coefficient, 95, 164 Tendril, 227 Teratology, 222 Terminal bud, 60-62, 207-209 Theelin, 109, 198, 233-235 Theelol, 109 Threshold, 56, 161, 244, 246 Tissue cultures, 71 Tissue tension, 54 Tobacco, see Nicotiana Tomato, 181, 191 Toxic compounds, 40, 44, 45, 55, 143, 189 Tradescantia, 60, 181, 191, 211, 232 Transmission of irritation or stimulus, 10, 11, 19, 92, 94, 97, 101, 152, 153 Transpiration stream, 91, 97, 99 Transplant, 9 Transport of hormone, 90-104, 136, 146, 147, 171, 172, 186, 190, 209 Transverse polarization, 157, 163 Traumatotropism, 153, 178-179 Tropaeolum, 211 Tropisms, 2, 3, 9, 16, 151-182, 230 Tropohormones, 13, 153 Tryptamine, 45, 115, 116 Tryptophane, 20, 72, 111, 112, 115, 116, 196 Tube structure, 125, 126 Tussilago, 123 u (unpublished), 1, 36, 40, 45-47, 49, 55, 59, 67, 70-72, 75, 80, 81, 87, 90, 91, 97, 100, 115, 116, 132, 135, 144, 148, 170, 175, 176, 189, 192, 194- 199, 217, 223-225, 227, 232, 238 Ultra-violet light, 88, 110 Urine, 70, 71, 106, 107, 110, 111, 117, 191, 195,211,220,223,228 Utrecht, 49 Vallisneria, 19 Valonia, 57 Variability of test, 38, 39, 49-51, 101 Vascular bundle, 90, 91 Veins, 66, 69, 91, 100, 227 Velocity of auxin transport, 94-96, 101-103, 147, 177, 221 Vicia Faba, 61, 62, 88, 89, 97, 113, 141, 143-147, 149, 150, 160, 164, 165, 173, 181, 191, 203, 209-211, 217, 218, 220, 223, 224, 231 Virus, 69, 227, 248 Viscosity of protoplasm, 130 Vitamin B, 236 Vitis, 185 WAE (Wuchsstoff-Avena-Einheit), 42,43 Wheat germ oil, 63 Wiry tomato, 227 Wound hormone, 18, 153, 178 Wound substances, 24, 42 Wuchsstoff, 4, 234 Wuchsstoff B, 113, 236 Wuchsstoff Einheit, 42 X-ray photography, 125, 126 X-rays, 88 Yeast, 72, 106, 111-113, 143, 150, 196, 199, 228, 236-238 Zea Mays, 24, 58, 62, 63, 75, 81-83, 86, 87, 113, 141, 143-147, 154, 158, 162, 163, 165, 203, 211 Zinc trough, 30 Zinnia, 211 Zones, growth of, 51, 76, 77, 80, 81 Zoology, 11, 151