jE. ; ,! >! r \' M W: o > C J3 o c c3 O 3 03 CI o t/3 a S o H aj o o P O ' / > i / GROWTH - r( of PLANTS T'wenty Years' Researcli at Boyce Tkompson Institute by WILLIAM CROCKER Managing Director, Boyce Thompson Institute for Plant Research, Inc., Yonkers, N. Y. REINHOLD PUBLISHING CORPORATION 330 West 42nd St., New York 18, U. S. A. 1948 Copyright, 1948, by REINHOLD PUBLISHING CORPORATION All rights reserved Printed in U.S.A. by NORWOOD PRESS, NORWOOD, MASS. Contents Page Introduction 1 The Founder of the Boyce Thompson Institute • Aims and Scope of Work of the Institute • Organization and Purpose of Book • Acknowledgments Chapter 1. Early Problems 10 Yellows and Virus Diseases of Plants • Duck Food Problem • Stiidy of Aquatic Plants 2. Life Span of Seeds 28 Seeds of Long Life Span • Seeds of Short Life Span • Life Span of Seeds in Soil • Dormancy and Delayed Germination in Seeds of Wild Plants • Why Seeds Remain Dormant in a Germinator or in Soil • Hard Seeds Best Adapted for Long Life Span • Storage of Seeds • Why Seeds Degenerate with Age 3. Dormancy in Seeds 67 Significance of Dormancy in Seeds • Categories of Dormancy: Hard Coats; Light as a Factor in Dormancy; Oxygen Deficiency and Dormancy; Moist Low-Temperature Stratification • Two-Year Seeds: Seeds with Resistant Coats and Dormant Embryos; Dormant Epicotyls; Seeds Re- quiring Two Low-Temperature Exposures • Period of Dry Storage After-Ripens Many Seeds • Temperature a Factor in Overcoming Dor- mancy • Quick Vitality Tests for Dormant Seeds • Chemicals as Forc- ing Agents far Dormant Seeds • Summary /> Physiological Effects op Ethylene and Other Unsat- urated Carbon-Containing Gases 139 Early Experiments • Responses Induced by Gases Containing Un- saturated Carbon: Epinasty of Leaves; Proliferation of Tissues; Abscis- sion of Leaves, Flowers, and Fruits; Other Metabolic Changes in Living Plant Tissues; Root and Root-Hair Initiation; Color and Ripening of Fruits; Other Physiological Effects • Respiring Plant Tissue Produces Ethylene • Summary Effect of Certain Lethal Gases Upon Plants and Animals 172 Injury to Trees and Shrubs from Leaks of Artificial Illuminating Gas • Injury to Plants in a Greenhouse by Mercury Vapor • Effect of Sulphur Dioxide on Plants • Sulphur Dioxide of Atmosphere as a Sxdphur Source for Plant Nutrition • Effect of Sulphur Dioxide on Animals • Effect of Hydrogen Sulphide on Plants • Effect of Chlorine Gas and Chlorinated Water on Plants and Aniynals • Comparative Effect of Ammonia, Chlorine, Hijdrogen Cyanide, Hydrogen Sulphide, Sulphur Dioxide on Plants and Animals • Summary «' 62169 iv CONTENTS Chapter Page 6. Plant Hormones by P. W. Zimmerman 204 Definition of Hormone • Brief Historical Picture and Recent Experi- ments • Physiological Activities in Substances: Methods and Applica- tions; Induction of Adventitious Roots; Propagation of Plants; Pre- harvest Apple Drop; Inhibition of Growth; Fruit Set and Seedless Fruit; Formative Influence of Growth Substa7ices • Other Subjects of Interest Involving Plant Hormones: Absorption and Movement of Growth Sub- stances; Light and Dark Effects; Tropic Curvatures; Anesthesia; Activa- tion of Cinnamic Acid with Ultraviolet Light; Natural Influences; Comparative Effectiveness of Adds, Esters, and Salts 7. Dormancy in Buds 230 Dormancy in Potato Buds • Dormancy of Gladiolus Corms and Cormels • Forcing Dormant Buds of Deciduous Trees and Shrubs • Metabolic Changes Induced by Chemicals That Force Dormant Potato Buds • Metabolic Changes Induced by Ethylene Chlorhydrin Compared with Effects of Other Chemicals Including Other Bud Forcers • Summary 8. Plant Cell Membranes by Wanda K. Farr 260 General Studies of Cotton Fiber Groivth • Broadening of the Experi- mental Approach • Formation of the Cellulose Particles • Summary 9. Plants Grown Under Controlled Environmental Con- ditions 285 Plants Grown Entirely in Artificial Light • Growing Plants Under a Combination of Sunlight and Artificial Light • The Carbohydrate Nitrogen Ratio in Plants • Spectral Greenhouses • Some Effects of Ultraviolet Rays on Plants • Miniynum Light Intensity for the Survival of Green Plants • Light Measurement • Effect of Radiation on Trans- piration • Environmental Conditions and the Development of Chlorophyll Pigments • Environmental Conditions Modify the Microchemistry and Anatomy of Plants • Some Lcnv-Temperature Effects • Summary 10. Research on Insecticides by Albert Hartzell 343 Efficiency and Mode of Action of Contact Insecticides • Fumigants • Search for New Insecticides • Some Local Problems in Insect Control • Summary 11. Fungicide Investigations by S. E. A. McCallan 362 Fungicidal Action of Sulfur • Fungicidal Action of Copper • The Laboratory Slide-Germination Method of Evaluating Fungicides • Greenhouse Methods of Evaluating Fungicides • Cumulative Error Terms • Correlations Between Laboratory and Greenhouse Methods of Testing Fungicides • New Fungicides • Past and Future 12. Miscellaneous 386 Factors for Color in the Production of Potato Chips • Physiological and Biochemical Effects of Carbon Dioxide • Diurnal and Autumn Changes in Leaves of Deciduous Plants • Concerning the Determination of the Isoelectric Poirit of Protoplasm • Growth Substances arid Vitamin Bi for Seed Treatment • Importance of the Mother-Tuber in the Growth of the CONTENTS V Chapter Page Potato Plant • Flower Color of Hydrangea macrophylla • Soil Studies • Studies on Lilium, Gladiolus, and Dahlia • Propagation of Trailing Arhxdus and Lycopodiimi • Two Studies on Physiology and Cytology of Fungi • Effect oj Nitrogenous and Carbohydrate Reserves on Growth of Seedlings • Analytical Methods • Experimental Planning and Statistics Author Index 445 Subject Index 449 Figure 2. Colonel William Boyce Thompson. Introduction The Founder of the Boyce Thompson Institute Boyce Thompson Institute for Plant Research, Inc., was founded and en- dowed by Colonel William Boyce Thompson of Yonkers, New York. His total gift to the Institute in property and endowment amounted to some- what more than ten million dollars. The Institute was formally dedicated on September 24, 1924.2 The writer is often asked, "Who was Colonel Thompson and why was he so much interested in research on plants?" Colonel Thompson * was born in Alder Gulch, near Virginia City, Montana, in 1869, and spent the first seventeen years of his life in the wild frontier towns of Virginia City, Glendale, and Butte, Montana. It is not strange that his main interest was in mining, for that was the industry of the region; besides, his father formed an mvestment company that dealt in mining properties. After a period at Phillips Exeter Academy and Columbia School of Mines, Colonel Thompson returned to Montana and engaged in various mining operations there until 1899. At this date he moved to the East; operating in New York and Boston, he began dealing in mining stocks and developing mines. His first success was the development of the Shannon Mine at Clifton, Arizona, 1899 to 1903. This netted enough to pay off all indebtedness of the Thompson Investment Company with an additional net personal asset of $70,000. The stockholders profited similarly. This was only pocket change compared with what was to come later. In the development of this property, efficiency of operation was the keynote of success, as was the case in his future opera- tions: for example, when copper prices were low, the cost of production must be reduced to meet the price. Later the Colonel developed or aided in developing a series of mines that were highly profitable: Nipissing, Ely, and Mason Valley, Nevada, Bingham Canyon, Utah, and Inspiration and Magma in Arizona. The last two were models in efficiency, and the efficiency grew out of careful and ex- tensive researches preliminary to carrying out the plan of operation and building the concentrators and refineries. The former especially because of its size gave great returns in dividends during World War I. If the Colonel's savings at this point reached the fifty million mark, they were to be greatly increased by his part in the development of Texas Gulf Sulphur and by the organization of the Newmont Mining Corporation, the latter an investment corporation handling mining stocks. * The data for the brief biography of Colonel Thompson are taken mainly from Hermann Hagedorn's book, "The Magnate: William Boyce Thompson and His Time, 186^1930." » The author includes some statements based on his personal contacts, 1 2 GROWTH OF PLANTS While the Colonel's biggest philanthropies were the financing of Boyoe Thompson Institute and numerous big gifts to Phillips Exeter Academy, other sizable philanthropies could be named by the score: Belgian relief, Roosevelt Memorial, financing the Red Cross Commission to Russia, 1917, very large relief gifts to Russia during the sojourn of the Red Cross Commis- sion there, and many contributions to worthy charities during and after World War I. One of the very interesting things about Colonel Thompson was his friendships. His close personal friends ranged from ditch diggers to able scientists, statesmen, businessmen, and rulers. These friendships were generally determined by positive worthy characteristics of the friend, especially insight, integrity, and sincerity. The friendship with Doby Tom, a Slovak ex-sailor, working in Boyce Thompson Southwestern Arboretum, was a matter of mutual entertainment. Tom had definite ideas about his own rights and began answering fanciful criticism of the Colonel. Later their arguments often took on the appearance of bitter personal quarrels, to the great amusement of the Colonel as well as of Tom. The friendship with Johnny Schaller, a stone-mason, was unique. Jolmny was for nineteen years, up to his death, superintendent of "Alders," Colonel Thompson's estate at Yonkers, N. Y. The friendship and the employment of Johnny as superintendent was an outgrowth of the Colonel's admiration of Johnny's excellence as an artisan and approval of sincere and sturdy German charac- ter in Avhich realism far outweighed imagination. The Colonel's friendship with Dr. Robert Kennedy Duncan, organizer and first Director of Mellon Institute, started with the reading of Duncan's books, "New Knowledge" (1905) and "The Chemistry of Commerce" (1907), which gave a preview of the coming importance of chemistry in the industries. This friendship was deepened by frequent visits while the Colonel was having research done with Duncan on leaching and flotation of copper ore and on certain sulfur problems. These copper researches aided in making Inspiration and Magma efficient and profitable mines, and the research on sulfur assisted in building up a fine technical organization under Walter Aid ridge that made Texas Gulf Sulphur technically so nearly perfect and financially so highly profitable. Dr. Duncan's books were not only excellent expositions of the subjects discussed, but they were simple, direct, and highly inspirational. Such passages as: "the romance of untrodden ways, the romance of unguessed to-morrow" and "man can really live only when he has the chance to live. There is but one way of lifting man to a higher moral and spiritual plane, and that is by lifting to a higher plane the condition of his material sur- roundings" evidently impressed the Colonel. The effect of association with Duncan through his books and personally was to convince Colonel Thomp- son of the great contribution that research could make to human progress. It also convinced him that one of the best investments one could make for society was properly chosen and directed research. Colonel Thompson had INTRODUCTION 3 practically decided to finance Dr. Duncan's research in industrial chemistry when A. W. Mellon made his gift of the Mellon Institute. If Mr. Mellon had not anticipated the Colonel in financing Dr. Duncan's researches, it is almost certain that there would not have been a Boyce Thompson Institute nor a Mellon Institute under that name. After the death of Dr. Duncan, Dr. Raymond Foss Bacon, the next Director of Mellon Institute, assumed the place of Dr. Duncan as a friend and scientific counsellor to Colonel Thompson. It was with Dr. Bacon's advice and assistance that plans for Boyce Thompson Institute developed. It w^as also through Dr. Bacon's suggestion that Prof. John Merle Coulter was called in to aid in developing plans for the Institute. One of the Colonel's most interesting friendships was that with Colonel Raymond Robins, a well-knowTi social worker. Neither had met before being appointed to the Russian Red Cross Commission in 1917, and both appar- ently avoided meeting as long as possible after going on the Commission. Colonel Robins was said to have asked, "\\Tiy was that several-sorts of a plutocrat, Thompson, appointed on the Commission?" Thompson was sup- posed to have asked a like question about that several-sorts of a socialist. WTien they did meet and exchange views, they found themselves in agree- ment on most matters, even on social views, and especially on the social needs of Russia at that time. As one reads the operation of this Commission as reported in "The IMagnate,"^ he is inclined to believe that if the ad- vice of Thompson and Robins had been followed by the Allies, including our o^^^l country, Russia would have been kept active on the Eastern front and the future history of Russia would have been very different. The Thompson-Robins recommendation that the Allies authorize Kerensky to parcel out the land of Russia immediately to the peasants and let the remuneration for the land rest for later adjustment, brought a loud howl of disapproval from the Allies. Also, the recommendation that Russia be given adequate material help of all kinds fell on deaf ears. Even when Kerensky lost out to the Soviets and Lenin, largely because of his failure to distribute the land to the peasants, Thompson and Robins still recom- mended full cooperation mth Russia. This was still more bitterly opposed by the Allies, and both the magnate and the socialist, now mutually called "Chief" and "Panther," were considered dangerous radicals. A friendship which started and grew under the trying conditions of the Red Cross Com- mission in Russia was intensified with time and association only to be ended by the death of Colonel Thompson in 1930. As a boy and throughout life, the Colonel seemed to have two deep-seated longings: a desire to understand the logical order behind life and the uni- verse, and a longing for beauty. Neither of these longings received much satisfaction in the rough, helter-skelter, sulfur dioxide-scorched Butte of that day. The Colonel was very fond of his cultured, sympathetic mother, who also had a keen sense for beauty. His father was stern, with a strict Methodist interpretation of conduct and with little understanding of the 4 GROWTH OF PLANTS aspirations of a growing boy. To this boy the Methodist religion represented a vengeful rather than a just and sympathetic God. Arthur Cotton Newell, a classical student from Baliol College, Oxford, and a high-school teacher at Butte, seems to have given the boy the first start in proper orientation Avdth the world, both by personal contact and by placing in his hands Olive Schreiner's "African Farm" and Herbert Spencer's "Data of Ethics." The logical orientation Avith the world was furthered still more during his happy days at Exeter. Here he came under the influence of able teachers and outstanding characters: George Wentworth, teacher of mathematics and author of textbooks on the subject; Professor Tufts, English; George Lyman Kittredge, Latin ; and Bradbury Cilley, Greek. After two very busy decades in developing mining interests, Colonel Thompson at last found some leisure time to indulge his two natural bents: the desire for beauty and the desire to understand and contribute to the logical order of society. Furthermore, he had acquired the wherewithal to gratify these mterests. His fine home on North Broadway, Yonkers, mth its beautiful landscaping, largely planned and developed by himself, is an expression of his sense of beauty. With the aid of the artistic touch of Dr. Fred J. Pope, a mining and chemical engineer in the Colonel's employ, he collected and arranged in the basement of his home two valuable and beau- tiful exhibits: one of minerals and another of carved jades. While Dr. Duncan had impressed the Colonel with the social value of research, the Colonel had to decide the area in which such research was most needed. In planting his estate, he learned from consulting experts that there was much still unkno\\Ti about factors affecting plant develop- ment, especially about controlling insect pests and plant diseases. His ex- periences in starving and freezing Russia emphasized the significance of plants to man as the ultimate source of all his food, nearly all his clothing, and much of his shelter. Statements of the Colonel quoted from "The Magnate" (pp. 290-291) show how his mind was working. "'When I have enough money,' he said one day, 'I am going to build a laboratory to study some of the fundamental things. I want to do something to get at the bot- tom of the phenomena of life processes and I think a good place to study them would be in the realm of plants. Any principles concerning the nature of life that you can establish for plants will help you to understand man, in health and in disease. So, by helping men to study plants, I may perhaps be able to contribute something to the future of mankind.' "The thought made contact in his mind with other thoughts rising out of his Russian experience, his impatience with the ineffectiveness and unreality of the political approach to national problems, the waste and stupidity of politics. The phrase, 'when there are two hundred million of us' came again and again to his lips. He saw hope only in an order based on economics, illuminated and disciplined by science. He sent his imagination playing along the highways of tomorrow. 'What you are doing in politics and social welfare is all right, Panther,' he said to Robins one day as they stood to- INTRODUCTION 6 gether in the sunken garden. 'But there will be two hundred million people in this country pretty soon. It's going to be a question of bread, of a primary food supply. That question is going to be beyond politicians and sociologists. I think I'll work out some institution to deal mth plant physiology, to help protect the basic needs of the two hundred million. Not an uplift foundation but a scientific institution dealing with definite things, with germination, parasites, plant diseases, plant potentialities. I can understand a thing of that sort. I could do something with it.'" In 1919 the Farm and Research Corporation was formed, but no plans had been developed as to what it was to do or how it was to be done. Evi- dently the Colonel assumed that Dr. Pope would plan and build the labora- tory. Pope was innocent of any considerable knowledge about plants, and nobody knew better his innocence of such knowledge than Pope himself. The Colonel advised Pope that if he didn't know about such work, he'd better pack his grip and go where he could get sound advice. Pope visited various universities and agricultural colleges, among them Cornell, Univer- sity of Wisconsin, and University of Illinois, with the question, "What should the proposed institution undertake, mainly research or mainly ex- tension?" Eugene Davenport, Dean of Agriculture of the University of Illinois, gave the deciding answer when he said in effect, "Agricultural ex- tension is well organized and cared for in the United States, but if we do not have more fundamental knowledge, we may soon have nothing more to extend." Basic research on plants became the function of the corpora- tion. Then followed a series of conferences A\ath plant scientists in several institutions as to the best organization for such an institution and the most significant problems that could be undertaken. The early suggestions were not satisfactory to Colonel Thompson, so Dr. Bacon suggested that Prof. John M. Coulter, Professor of Botany at the University of Chicago, be called in as an adviser. Prof. Coulter visited Colonel Thompson at his home in Yonkers in the fall of 1920 and presented a proposed outline for the organization of the Institute. This was accepted by the Colonel and the selection of a Director to plan and build the Institute was taken up immediately. Prof. Ezra J. Kraus of the University of Wisconsin was offered the position, but after due consideration decided not to accept. Later, the author, then Associate Professor of Plant Physiology at the University of Chicago, was offered the position and accepted. On February 1, 1921, the Director and Mr. John M. Arthur, an assistant and graduate student in plant physiology at the Uni- versity of Chicago, began planning the Institute. Their duties at Chicago re- quired one half of their time until August last of that year. The other half was used in visiting research laboratories in the United States, in searching for and buying books for the new library, and in considering pro- jects that ought to be investigated, along with the type of equipment and building and personnel needed to develop the projects effectively. The Director spent the fall of 1921 and a portion of the winter of 1922 in 6 GROWTH OF PLANTS Europe, visiting biological laboratories and experiment stations and con- sulting mth prominent men in biology. During this trip the nucleus of the present library was purchased. The purchases consisted mainly of complete bound sets of German, English, and French periodicals in botany, biology, and chemistry, including a complete set of Justus Liebig's Annalen and several thousand German dissertations. The German publications and some publications in other languages that were for sale in Germany were pur- chased on very favorable terms because of the highly inflated German currency. Mr. Arthur and the writer spent the rest of 1922, 1923, and some of 1924 in working mth The J. G. White Engineering Corporation, the builders of the laboratories, in designing and equipping the laboratories. Because of the great amount of control equipment that was installed for the first time in biological laboratories this proved to be a rather arduous task. A refrig- eration room was built to run at a regulated temperature considerably be- low freezing in which several thermostatically controlled chambers were placed to give the constant temperatures needed for plant studies. A scrub- ber system was installed to scrub the flue gas from the boilers as a source of carbon dioxide for the greenhouses. The greatest amount of control apparatus was developed for the proposed studies on light. Much of this is described and illustrated in Chapter IX, "Plants Grown under Controlled Environmental Conditions." During the period of building, much attention was given to selecting the scientific staff for the Institute. We were anxious to get research started at an early date. Dr. L. O. Kunkel came to Yonkers in the spring of 1923 and started his work on the yellows disease of plants. This work is described later in the first chapter. Mr. Arthur was working on the Botrytis disease of tulips under the direction of Prof. H. H. Whetzel of Cornell, and on the fireholding qualities of tobaccos treated with various salts, and the writer was studying germination problems. Research did not start in full force, however, until the building was finished and a bigger staff was assem- bled in the fall of 1924. Aims and Scope of Work of the Institute From our studies of biological laboratories in the United States and Europe, we came to six conclusions concerning the aims, organization, and scope of the work to be undertaken at the Institute that would make it most serviceable in plant science at the time. (1) It should do basic research, as had been determined by the founder, but basic research should not be contrasted mth applied research as pure and applied research had been contrasted in some European laboratories, with harm, as we believed, to best progress. We felt that any project or problem tackled should be studied in all its relations, including its meaning in nature, in agriculture, and in the industries. Our later experience has INTRODUCTION 7 shown that much is added to knowledge in applying findings in the labora- tory to behavior in nature and to practice. (2) We had found too many botantists working on problems as individ- uals, hence with inadequate techniques for proper solution of the problems, likewise with inadequate equipment. We decided that we would not organize as departments based on techniques, but would organize to attack projects focusing enough different techniques on the projects to bring evidence on them from many, if not from all, angles. We believe this has minimized department jealousies such as sometimes appear in universities where, be- cause of the necessity of teaching, departments must be organized on the basis of technique. It has also led to excellent cooperation between workers. This, of course, called for a staff with a great range of techniques and knowledge of the subject; not only must we have personnel covering every phase of botany needed for the projects, but we must have several kinds of chemists, physicists, entomologists, et al. (3) We decided that botanical laboratories were generally inadequately equipped for effective work and that botanists should be supplied vnth many accurate instruments and controls to make their researches thoroughly reliable. The Institute was, as origmally organized, almost a model of equip- ment for the projects m line for solution, and it has always given great attention to equipment since, so far as finances made this possible. Some- times this ideal has brought embarrassment. One worker on light effects asked for a quartz prism that cost $1800.00 and two quartz lenses costing $300.00 each. He got the three desired gems. (4) We found many botanists spending their time in doing work that they were not trained to do and doing it poorly or only moderately well. So we early installed an excellent photographic and illustration division, a H- brary force that furnished citations of all literature bearing on the several projects, an engineering and mechanical division that kept all controls in operation and built any apparatus that was not available on the market, and finally a trained greenhouse and garden force that could carry through many of the larger scale experiments under the guidance of the investiga- tors. The investigators were also provided with all needed laboratory helpers. These were generally high-school graduates who were encouraged to take college work while working for the Institute. A number of the labora- tory helpers have since received college degrees, some Master's degrees, and one a Doctor's degree. (5) Prompt publication of the researches was also considered desirable. Consequently at first extra space was purchased in the Botanical Gazette and the American Journal of Botany and finished researches published promptly. It is interesting to note that at least one botanist protested to these journals against this practice as unfair in that it gave the scientists of the Institute an unfair advantage over other scientists, namely, opportunity for prompt publication of their results. The Institute merely held that prompt publica- tion is a desirable part of research and that all research institutions should 8 GROWTH OF PLANTS consider it as such and provide for it as an integral part of research. Later, the quarterly journal, Contributions from Boyce Thompson Institute, was started, as well as a series of Professional Papers dealing with practical applications of researches at the Institute. The latter consist mainly of separates published in trade journals or other professional periodicals. Several members of the staff have published one-chapter monographs in treatises on various phases of biological science or in review periodicals. (6) Finally it was decided not to duplicate botanical research that was already being adequately covered in the northeastern United States. This was especially true of systematic botany and genetics. This left fundamental researches in plant physiology, pathology, and biochemistry as the main fields of activity for the Institute. Organization and Purpose of the Book The first eleven chapters of this book describe the researches on twelve larger projects with their many scientific and practical ramifications. Most of these projects have been in operation throughout the life of the Institute and some phases of all of these are still under study except for the Duck Food and Plant Cell Membranes projects, the first of which was finished and the second discontinued. Chapter XII presents fourteen shorter projects which, in the main, have been finished or discontinued. This book is a critical summary of the researches carried on at Boyce Thompson Institute. Only such outside researches are discussed as are nec- essary to orient the work at the Institute generally in the whole field of plant science. A critical discussion of all the literature on every subject treated would expand the work far beyond a moderate sized volume. Acknowledgments The author expresses his gratitude to each member of the scientific staff of Boyce Thompson Institute for critically editing the portions of the man- uscript covering his researches. Gratitude is also due the Publication Com- mittee (F. E. Denny, A. E. Hitchcock, N. E. Pfeiffer, L. P. Miller, S. E. A. McCallan, Z. Troy, B. M. Brooks) of the Institute for editing all the manu- script for the book. The author gives special recognition to the several members of the Institute who wrote chapters or sections of the book. The follomng is a list of co-authors with the portions written by each: Dr. P. W. Zimmerman, Chapter VI — "Plant Hormones"; Mrs. Wanda K. Farr, Chapter VIII — "Plant Cell Membranes"; Dr. Albert Hartzell, Chapter X — "Research on Insecticides"; Dr. S. E. A. McCallan, Chapter XI — "Fungicide Investigations"; and Dr. W. J. Youden, last two topics of Section 13 and all of Section 14 in Chapter XII — "Miscellaneous Chap- ter." Special gratitude is due Mrs. Bettie M. Brooks for directing the typing and looking after the details of arrangmg chapters, sections, and figures INTRODUCTION 9 for the book, to Dr. L. P. Miller for a critical reading of all chapters, to Mrs. Lillian Teller for indexing the volume, and to Mr. L. P. Flory for the curves and most of the photographs used. Literature Cited 1. Hagedorn, Hermann, "The Magnate: William Boyce Thompson and His Time, 1869-1930," 343 pp., John Day Company, New York, 1935. 2. "Organization — equipment — dedication," C. B. T. I.* 1 : 1-58 (1925). * C. B.T.I . \\\\\ be used throughout the citations in this volume to indicate Contributions from Boyce Thompson Institute. CHAPTER 1 Early Problems Yellows and Virus Diseases of Plants At the time Dr. L. O. Kiinkel started his researches at the Institute, "yellows" were very obscure plant diseases. Peach yellows was described in 1791 by Judge Richard Peters of Philadelphia. There were violent outbreaks of it; some of. them destroying whole orchards, in 1791, 1806-07, 1817-21, 1845-58, 1874-78, 1886-88, and 1920. The symptoms of the disease are as follows: the fruits ripen early mth deep red color of both skin and flesh, and the fiesh is bitter; the leaves are yellowed, rolled, and drooping; the new shoots are thin and wiry, growing upright and bearing narrow yellow leaves; the buds that remain dormant on healthy trees grow prematurely on diseased trees, producing witches' broom effect; the diseased tree be- comes worthless and dies in two to six years. The disease is limited mainly to southeastern Canada and northeastern United States. Some outbreaks have occurred as far south as Texas and as far west as Arkansas and Ne- braska. The disease had been fairly extensively studied by able pathologists, including Penhallow (1882-83) and Erwin F. Smith (1888-94), but neither the causative agent nor the method of transmission had been learned. Dr. Ermn F. Smith described aster yellows in 1902 and suggested that it might go to other composites closely related to the China aster. The causa- tive agent and method of transmission of this disease was likewise unkno\vn when Dr. Kunkel started his work on yellows diseases. He had recently re- turned from his research on sugar mosaic, the insect vector for which he had discovered. Dr. Kunkel did not seem enthusiastic about the problem, al- though his later discoveries in the field did much to enhance his already high standing as a plant pathologist. Recently the author once accused him of showing little en husiasm for this new undertaking. His answer was that he never liked to undertake a new problem. Anyone who knows Dr. Kunkel, able and a bit phlegmatic as he is, would know that his lack of enthusiasm did not augur lack of future accomplishment in the problem. Prof. L. R. Jones, a member of the Board of Directors of the Institute, had some doubts of the wisdom of having a scientist undertake such an obscure prob- lem. He might spend many years on it without success. Dr. Kunkel started on aster yellows searching for possible insect vectors by the methods he used in discovering the insect vector of sugar cane mosaic. About six weeks after he got the insect isolation cages, he announced that of the several insects feeding commonly on asters, one and only one 10 ■*1F >> » ^ i ^g o o o ^ OJ +J cc t^ "o .'^'^ a^ 3 "H. -O T3 fl o =3 ^ /- o C/2 --H ca o &>H o ^ «u -o O '"' -3^ V !=^ fl iH OJ OJ ^ CO t-. to bC o3 > QJ- bC bC fl olia you -T^- ellowed f Healthy ^ O >" ^ CQ ^ a . o O T3 bD 03 -ki ^^-H O ill O 33 -i-i 3 J C -73 -»-:> _bp 1- <« 'G o — a, -is -^^ 3 eg fi S3 _2 -T3 a; CO o 2^ -^ in (/2 r^ '% Qd o z < o a *n >- o V > < Q < N I L. O u Z u O I tf Q u. O o z u CB ■2 < N Isl 5 z u u § O t- u X H 3 O b. z -1 u. u o z < a H z u 3 -1 o u. *" o s s -t T3 -^ cS C tc « § § C o cc & h o -So •^^.^ g &R !=! £ o .^i -tJ CO to C " « (B ?i w "^ „ (B "3 ^ o3 cj 20 GROWTH OF PLANTS main source of the fluctuating salt content of these two bodies of water. The decline in duck feeds in these waters from 1918 to 1926 turned out to result from the removal of the sea-level lock mentioned above. Figure 9. Submerged plants covered with colonial growths of the brackish-water hydroid, Cordylophora lacustris AUman. Offhand, one might be inclined to assume that flow of water from the canal injured the plants in Currituck Sound and Back Bay by increasing the salt concentration. Apparently Bourn was early inclined to this view. Culture of the main duck food plants of this region in various dilutions of sea water disproved this. Potamogeton pedinatus and P. perfoliatus grew better in dilute sea water than in fresh water; 20 per cent sea water proved optimum and 36 per cent showed retardation but not complete inhibition. P. foliosus withstood 36 per cent of sea water. Vallisneria spiralis could not be grown successfully in concentration above 12 per cent sea water, but did well in 8 to 12 per cent. Ruppia maritima thrived in any concentration of sea water from 0 to 80 per cent and lived and stayed healthy in 150 per cent sea water. These facts eliminate salt concentration as any considerable factor of injury to the main duck food plants in these waters. The only place that salt concentration might act as an injurious factor to the dominant plants of the region was in the northerly part of Currituck Sound. One of the big causes of injury to the plants was a hydroid, Cordylophora lacustris Allman, carried do\xn Avith the brackish polluted waters of the canal EARLY PROBLEMS 21 and favored in its growth by these waters. The profusion of growth of this hydroid on leaves and stems of aquatic plants is shown in Fig. 9. Fig. 10 shows the hydroid growing on a leaf of Potamogeton, and Fig. 11 shows other stages in the development of the hydroid and accompanying organisms. This hydroid is not a parasite on the plants but feeds instead on the abundance Figure 10. Photomicrograph (about 50 X) of Cordylophora lacustris AUman entwining a leaf of Potamogeton pedinatus L. of plankton in the polluted water. The hydroid forms a gelatinous sheath about the stems and leaves of the plants and the main injury occurs after the hydroid dies. This gelatinous sheath is also a good cultural medium for other organisms such as larvae, worms, diatoms, rotifers, fungi, and bacteria, which add to the injury. As a result of the smothering activity of all these organisms the plants were partly killed, and were broken from their moorings, after which they sank to the bottom and added to the pollution and turbidity of the water or were carried to the shore by the wind. Bourn indicates that the biggest factor in denuding these waters of plants was the great turbidity of the water. He made many measurements of the light intensity at different depths in these waters during the growing season. He also determined in cultures the light intensity needed for the growth of the various duck food plants. On the basis of these two sets of measure- ments he came to the conclusion that during the growing season in many of these waters there was not enough light penetrating three feet below the surface to support plant growth. Although the minimum light require- ments for the two important bottom-cover plants, Chara and Nitella, were not determined, it is probable that light was deficient in the deeper waters for these also. The lack of light was especially destructive to Potamogeton 22 GROWTH OF PLANTS pedinatus and Vallisneria spiralis. The former thrives in clear water up to a depth of 8 feet and the latter up to a depth of 12 feet. Recent work ^^ indicates that in clear water the latter and perhaps the former may grow at two or three times the depths mentioned. There were two sources of par- ticles of matter causing the turbidity — organic and inorganic. The water Figure 11. Photomicrographs (about 50 X) of Cordylophora lacustris Allman. A, Typical hydranth. B, Stalk of a colony with its gelatinous secretions. C, Embryos. D, Diatoms growing in the gelatinous secretions left by hydroid colonies on a leaf of an aquatic plant. flowing down the canal had many sewage particles in suspension. This and plants killed by the hydroid formed a sludge on the bottom that was being continually stirred up by wave motion. Inorganic clay particles were kept in suspension by wave action because of the lack of a bottom-cover of Chara and Nitella. McAtee ^ reports that at the time of his study of these waters in 1909 there was an almost complete bottom-cover of Chara in Currituck Sound. In 1926 to 1930 there were only patches here and there of Chara and Nitella as bottom-covers. Where they did appear the water Avas clear; also there EARLY PROBLEMS 23 was a good growth of Potamogetons. Potamogeions, especially P. pectinatus, require special emphasis in connection with this project, for they are prime duck food plants, furnishing as they do both seeds and tubers; in the days of thriving gro^\i;h they constituted the greater part of the plants of these waters. McAtee says that in 1909 P. pectinatus alone comprised 60 per cent of the abundant duck feed in Currituck Sound. Bourn found the oxygen content of these waters low and the carbon diox- ide content high. This was especially true of the water of the northern part of Currituck Sound and of the canal. He thinks neither of these, as such, sig- nificantly limited the growth of green aquatic plants and that both would have changed if other growth conditions had been favorable. The situation was different with fish. The oxygen content of the water was so low that only low-oxygen-requiring herbivorous fish were present in any abundance and these were minor destructive agents to plants. The high-oxygen-requir- ing, plankton-consuming fish were scarce and consequentl}^ did not compete \\ath the hydroid for plankton food. During his studies in the region. Bourn cut off several coves from the main body of water by means of bulkheads and watched the natural recovery of vegetation in these protected coves or the development of re-set plants or sown seeds in them. In these protected areas, provided the water was not too shallow, the water plants thrived and finally gave a good stand of vege- tation. Also the turbidity of the waters in these coves disappeared soon after the construction of the protecting bulkheads. This was true in spite of the fact that the bulkheads were not water-tight but allowed some movement of water, as was shown by the fact that the change of water level in the en- closed areas always accompanied that in the main body of water. The District Army Engineer stated in 1922 ^- p-^^: "the salinity of Currituck Sound is caused not by accession of salt from the north or through the Albemarle and Chesapeake Canal, but from salt water washing over the beach into Currituck Sound at times of storm, and from Oregon Inlet." The Oregon Inlet through the sand bar opens into the north part of Albe- marle Sound. When the evidence was all in, the Army Engineers reversed themselves on this point and expressed themselves as in agreement with the conclusions of the Institute. This is showm by a quotation from the re- port in 1929 of Major General H. Taylor, U. S. A., Retired, formerly Chief of Engineers. He says ^' p-^^: "The Division Engineer seems to agree with this view, for he says: 'Con- sidering the possible reasons for the present salinity in these waters, the salt water from Oregon Inlet, the sea washing over the beach, the seepage through narrow parts of the beach and from a salt water table can be disre- garded, as there is nothing to show that there has been any increase in salin- ity from these sources. In fact, there should be a decrease, as Oregon Inlet is said to be smaller than in former years and the beaches have been con- siderably improved by sand-fence construction.' "Among the papers submitted in connection with this case is a report sub- 24 GROWTH OF PLANTS mitted by the Boyce Thompson Institute and filed mth the River and Har- bor Board under date of February 23, 1929. This report describes present conditions existing in Currituck Sound and particularly refers to the effect of the contamination of these waters by sewage passing through the Albe- marle and Chesapeake Canal into them. This report, in my opinion, is a very convincing report as to the detrimental effects caused in Currituck Sound and Back Bay by the removal of the lock. It will be noted that this report was filed in February 1929, while the report of the District Engineer was dated October 11, 1927, the report of the Division Engineer was dated October 20, 1927, and the report of the Board of Engineers for Rivers and Harbors was dated April 3, 1928. The pollution of the waters of Currituck Sound by sewage coming from Norfolk Harbor was discussed by representa- tives of the Boyce Thompson Institute at the hearing held by the Board of Engineers for Rivers and Harbors, December 14, 1927, but the statements made at that hearing as shown by the record are much less impressive than the carefully prepared paper which was filed after the Board made its report and which was based on studies which were continued after the date of the hearing as well as what had been learned before that date. "An examination of the Boyce Thompson report shows conclusively that the sewage which is brought through the canal from Norfolk Harbor into Currituck Sound is in itself sufficient to cause destruction of the duck food plants. Whatever difference of opinion there may be as to the possibility of salt water coming into Currituck Sound in other ways than through the canal there can be no difference of opinion as to the sewage, for that can only come from Norfolk Harbor through the canal." He also says ^' p-'^: "I can come to no conclusion except that the removal of the lock has been the principal cause of the destruction of the duck food plants in Back Bay and Currituck Sound and that it has been detrimental to navigation and to the fishing industry. The history of the Albemarle and Chesapeake Canal shows that the injury to the hunting and fishing interests of Currituck Sound and Back Bay caused by the infiltration of contami- nated waters into these waters has been progressive. Every year that the contaminated water from Norfolk Harbor is allowed to freely flow south the damage is increased and the longer the construction of a new lock is postponed the greater the damage will be." Mr. Bourn and members of the Institute feared that it would take years after the locks were replaced for the natural restoration of the plants in these waters if the process was not accelerated by artificial planting. These fears were based on two facts. (1) There were relatively few seeds, bulbs, and tubers left in the soil of the water bed as a source of new plants. (2) The bottom-cover of Chara and Nitella had largely disappeared, and with it the best preventive against turbidity. The following quotations from Bourn in the report referred to by General Taylor^' p-^ summarize the conclusions reached by the Institute on the basis of this research : EARLY PROBLEMS 25 "The damage is not necessarily confined to the direct loss of plants, and the reduction of fish and wild fowl. While the productivity of the region was at its best, hundreds of sportsmen were attracted to the region. More than 40 hunting clubs were established on the shores of Back Bay and Cur- rituck Sound. More than $5,000,000 were invested in marsh lands and prop- erties suited to the sport of gunning, and suited to nothing else. Generous sportsmen contributed sums running into hundreds of thousands toward the erection and maintenance of modern schools, for the construction of roads, and for the general welfare of the community. Fine bus lines have been established and operated at private expense for the conveyance of children to and from the schools, and appreciable sums have been expended in the welfare of these children. Furthermore, there accrues from these investments to the public an annual sum of approximately $500,000 in the way of taxes, licenses, purchases, and other items in connection with the sport of fowling. In addition it is estimated that 5,000 people depend almost wholly for their livelihood and an equal number are partially dependent upon the sport of gunning and fishing. This means of livelihood and the returns from these investments are seriously endangered, for there is now little gunning and fishing and the property values have depreciated greatly in value, some as much as three-fourths." Bourn also states ^- p--^-^^: "In view of our findings and in view of the fact that the opening of the Albemarle and Chesapeake Canal has been the sole disturbance in the natural conditions of this region we are forced to the logical conclusion that the restoration of the guard-lock in the canal is the only remedy for the present conditions in Back Bay and Currituck Sound and the only remedy that will restore the sole natural resource to this vast region. When we consider the extent of the damage to the region, the economic losses involved, the loss of the sole, large winter-feeding ground on the Atlantic Coast for wild fowl, the number of people and interests concerned, the reduction and threatened extinction of our wild fowl and our fresh water fishes, and our moral obligation to posterity and our treaty obligations to Great Britain for the preservation and protection of our rapidly vanishing wild life, we are further forced to conclude that the application of this remedy should not longer be delayed." In a letter to the author under date of June 25, 1943, Dr. W. S. Bourn, now of the United States Fish and Wildlife Service, makes the following statements about the recovery of duck feeds after the restoral of the locks: ''You undoubtedly will be delighted to know that the waters in question have returned to a maximum productivity of duck food plants. Last year the peak was reached and sago pondweed seed was washed ashore in wind- rows. The history of the return of sago pondweed is very interesting. As you will remember, you and myself predicted that first Chara and Nitella would carpet the bottom. It took about two years for these growths to be- come appreciable in extent. In the third year these plants covered the bot- tom and the water became crystal clear. Then by the fifth year sago pond- 26 GROWTH OF PLANTS weed {Potamogeton pectinatus L.), redhead grass (P. perfoliatus L.), and wild celery (Vallisneria spiralis L.) returned beyond expectation. So by last season the old residents maintained that these plants had returned to abundances that existed before the locks in the canal had been removed. It is regrettable that some scientific agency could not have continued the study of the progressive changes in the flora. About all the opportunity I have had to observe these has been on short periodic visits once or twice a season, but I had no time to make various tests. "As to the seeding of the depleted areas, this may be attributed to dis- semination by waterfowl. On the beach strip between the ocean and Pam- lico Sound a few years ago the Fish and Wildlife Service made a large arti- ficial pond by dikes on the sand. This is on what is known as Pea Island, just below Oregon Inlet towards Cape Hatteras, on the sand spit of barrier beach between the two bodies of saltwater. The impoundment filled to a de- sirable depth with rainwater, as there the normal rainfall exceeds evapora- tion. Without any seeding except through the agency of waterfowl a good stand of sago pond weed became established the first season. This could have happened in Back Bay and Currituck Sound, but then we knew nothing about the probable continued presence of dormant seeds in the mud of these waters." In a letter under date of August 9, 1943, Bourn further states: "It may interest you further to learn that with the recovery of the plant life in Back Bay and Currituck Sound the black bass, the important game fish, also returned. Now on 'blue bird' days during the duck shooting season when the birds are reluctant to fly without a stimulating wind, sportsmen are accustomed to cast from their shooting blinds and catch a limit of these game fish, the commercial seining of which is no longer permitted in Back Bay and Currituck Sound. So the area may again be thought of as a 'sports- man's paradise,' brought about by the restoration of the locks in the Intra- coastal Waterway." Literature Cited 1. Beale, Helen Purdy, and Mary E. Lojkin, "Quantitative studies on the precipitin reaction of the tobacco-mosaic virus-antiserum system," C. B. T. I., 13 (1944): 385-410 (1945). 2. , and Beatrice Carrier Seegal, " N^ormal-tobacco-plant protein and tobacco- mosaic-virus protein as anaphylactogens and precipitinogens in the guinea pig," C. B. T. I., 11 : 441-454 (1941). 3. Bourn, W. S., "Ecological and physiological studies on certain aquatic angiosperms/' C. B. T. I., 4 : 425-496 (1932). 4. Denny, F. E., "Field method for determining the saltiness of brackish water," Ecology, 8 : 106-112 (1927); also in B.T.I. Prof. Pap., 1 : 20-26 (1927). 5. "Documentary proof of immediately imperative necessity for restoration of lock in Albemarle and Chesapeake Canal," 39 pp. (1929), 6. Hildebrand, E. M., C. H. Berkeley, and D. Cation, "Handbook of virus diseases of stone fruits in North America," 76 pp. Misc. Publ. Michigan Agric. Exp. Sta. (May, 1942). EARLY PROBLEMS 27 7. Kunkel, L. O., "Studies on aster yellows," Atner. J. BoL, 13:646-705 (1926); also in C. B. T. /., 1 : 181-240 (1926). 8. , "Studies on aster yellows in some new host plants," C. B. T. I., 3 : 85-123 (1931). 9. , "Heat treatments for the cure of yellows and other virus diseases of peach," Phytopath., 26 : 809-830 (1936). 10. Lojkin, Mary E., and Helen Purdy Bealc, "A colorimetric method for the quantita- tive determination of minute amounts of tobacco-mosaic virus and for the differ- entiation between some of its strains," C. B. T. I., 13 : 337-354 (1944). 11. Manns, T. F., "Peach yellows and httle peach," Delaware Agric. Exp, Sta. Bull. 236, 50 pp. (1942). 12. Meyer, Bernard S., Frank H. Bell, Lawrence C. Thompson, and Edythe I. Clay, "Effect of depth of immersion on apparent photosynthesis in submersed vascular aquatics," Ecology, 24 : 393-399 (1943). CHAPTER 2 Life Span of Seeds How long do seeds live? This is a very complex question to answer, for there are many species of seeds varying greatly from one to the other in life span under any one condition of storage. ^^ The condition of storage modifies the life span of seeds tremendously. A given storage condition may lengthen the life span of one species and shorten that of another. In his well known and excellent book, "On the Longevity of Seeds," published in 1908, Alfred J. Ewart^^ gives a rather pessimistic view of the accuracy of the knowledge in this field. He says: ''Probably few sections of human knowledge contain a larger percentage of contradictory, incor- rect and misleading observations than prevail in the works dealing Avith this subject, and, although such fables as the supposed germination of mummy wheat have long since been exploded, equally erroneous records are still current in botanical physiology. In addition, there are considerable dif- ferences of opinion as to the causes which determine the longevity of seeds in the soil or air. The works of de Candolle, Duvel, and Becquerel are the most accurate and comprehensive dealing -with the question, and, in addi- tion, Vilmorin has published very useful data in regard to the seeds of culi- nary vegetables. The subject is still, however, in an incomplete and fragmen- tary condition." Since Ewart's classic work was written, many new data and much evi- dence have been accumulated in this field, and definite advances have been made in several phases of the subject, including the nature of the changes involved in the degeneration of seeds with age and the effect of storage con- ditions on their rate of degeneration. Many new records have appeared on life span of seeds of mid plants in herbaria and seed cupboards and of seeds of cultivated plants in storage. We also now have available a good deal of reliable data on the life span of seeds in soil. On the basis of their life span under optimum conditions, Ewart divides seeds into three biological classes: (a) microbiotic, whose life span does not exceed 3 years; (b) mesobiotic, whose life span ranges from 3 to 15 years; and (c) macrobiotic, whose life duration ranges from 15 to more than 100 years. As we shall see later, we do not have final information on the opti- mum storage conditions of many kinds of seeds, and in spite of the great amount of research that has been done on seed storage in recent years it is questionable whether anyone can give optimum conditions for the storage of any sort of seed, although one can give good conditions that will greatly 28 LIFE SPAN OF SEEDS 29 lengthen the formerly assumed life span. Until we possess such information, these terms do not have very definite meaning. As we learn of better and better storage conditions for a given species of seed, it may jump from the microbiotic to the mesobiotic or even to the macrobiotic class. Seeds of Long Life Span Let us look at the percentage germination of certain old macrobiotic seeds that have been taken from seed cupboards or herbaria. Becquerel ^~ gives a very interesting record. He had access to a batch of old seeds in a storage room in the National Museum of Paris. The time of collection of these seeds varied from 1819 to 1853. He ran germination tests on these seeds in 1906 and again in 1934. For the 1934 test, Humbert and Metman furnished him about 20 seeds of Cassia multijuga which were collected in 1776. These seeds were all hard-coated, so they demanded special treatment. They were sterilized, the coats broken, and put to germinate in tubes under sterile conditions at 28° C. The seed stock was considered so precious that only ten of each sort were used for the test. Of Cassia multijuga only two seeds were used. Table 1 shows the results obtained for the 13 kinds, sho\\ing germination in either the 1906 or the 1934 test. In the last column Becquerel estimates the probable life span of several of the seeds, based on the data for the two tests. Table 1. "Becquerel's Record of Old Seeds Macrobiotic species Date collected Seeds ing ir grow- 1 1906 Seeds ing ir grow- i 1934 Determined longevity, yrs. Probable longevity, yrs. Mimosa glomeraia Forsk. 1853 5 out of 10 5 out of 10 81 221 Melilotus lutea Gueld 1851 3 " " 10 0 " " 10 55 Astragalus nmssiliensis Lam. 1848 0 " " 10 1 " " 10 86 100 Cytisus austriacus Linn. 1843 1 " " 10 0 " " 10 63 Lavatera pseiido-olbia Desf. 1842 2 " " 10 0 " " 10 64 Dioclea pauciflora Rusby 1841 1 " " 10 2 " " 10 93 121 Ervum Lens Linn. 1841 1 " " 10 0 " " 10 65 Trifolium arvense Linn. 1838 2 " " 10 0 " " 10 68 Leucaena leucocephala Linn. 1835 2 " " 10 3 " " 10 99 155 Stachys nepetifolia Desf. 1829 1 " " 10 0 " " 10 77 — Cytisus biflorus L'Herit. 1822 2 " " 10 0 " " 10 84 Cassia bicapsularis Linn. 1819 3 " " 10 4 " " 10 115 199 Cassia multijuga Rich. 1776 2 " '< 2 158 All these seeds are of the Leguminosae, except those of Lavatera (Mal- vaceae) and Stachys (Labiatae). The seeds of Cassia multijuga germinated after 158 years of storage. This exceeds the records of Robert Brown for Nelumbium speciosum ^^ from the British Museum, which were 150 years; also the records of Ewart for Goodia lotifolia and Hovea heterophylla, which 30 GROWTH OF PLANTS were 105 years. Becquerel believes the long life span in all these seeds is made possible by impermeability of the coats, which prevents any exchange of gases or water between the embryo and endosperm and the outside atmosphere, and by the high degree of desiccation, 2 to 5 per cent mois- ture, and absence of oxygen in which the embryos exist within the hard coats. Late work shows that hard seeds of Alhizzia julibrissin in the British Museum ^^ were alive after 149 years, and seeds of Nelumhium (Robert Brown's collection) after 250 years of storage. Table 2. Turner's Record of Old Seeds Species Age, yrs. Germination Anthyllis Vtdneraria 90 4 per cent Trifolium striatum 90 14.1 " " Trifolium prateme 81 2.6 " " Lotus uliginosus 81 9.6 " " Melilotus alba 81 163 seeds, 1 germinated Cytisus scoparius 81 636 " 4 Medieago orbiculans > 78 22 per cent Ipomoea sp. 43 6 " " Turner," of Kew Botanical Garden, tested the vitality of old seeds from several sources. All the viable seeds were hard-coated and were treated with sulfuric acid to render the coats permeable. Table 2 gives data on the seeds he found viable. Schjelderup-Ebbe •'•'^ tested the vitality of 1254 batches and nearly as many species of seeds stored in bottles or paper bags for 34 to 1 1 2 years. The oldest living sort found by this author is that of Astragalus utriger, 82 years old, with 6 per cent germination. Some kinds that were not so old showed relatively high percentages of germination. The following families are represented by these macrobiotic seeds: Cannaceae, Leguminosae, Euphor- biaceae, Malvaceae, Thymelaeaceae, Convolvulaceae, Solanaceae, and Com- positae. Of more than 1400 sorts (including species and varieties) of old seeds tested, Ewart ^^ found 49 that retained their vitality after more than 50 years of storage: 37 Leguminosae, genera Acacia, Alhizzia, Canavalia, Cytisus, Eutaxia, Galega, Gompholobiiim, Goodia, Hardenhergia, Hovea, Indigofera, Jacksonia, Kennedya, Melilotus, Mimosa, Oxylobium, Psoralea, and Pultenaea; 4 Malvaceae, genera Ahutilon, Hibiscus, Modiola, and Sida; 1 Tiliaceae, genus Entelea; 2 Euphorbiaceae, genera Euphorbia and Pseu- danthus; 1 Labiatae, genus Stachys; 1 Iridaceae, genus Watsotiia; 1 Ster- culiaceae, genus Hermannia; and 1 Polygaleae, genus Comesperma. Amongst these were Goodia lotifolia, 105 j'^ears old with 7.7 per cent germination, and Hovea linearis, 105 years old with 17 per cent germination. Seeds of several species show relatively high germination in spite of great age: Cytisus LIFE SPAN OF SEEDS 31 albus, 51 years old, 78 per cent germination; Entelea arhorescens, 51 years old, 47.3 per cent; Indigofera cytisoides, 51 years old, 51.2 per cent; and Melilotus gracilis, 58 years old, 28.8 per cent. Seeds of Short Life Span Some seeds lose their vitality in a very short time if they are kept in open air after harvest. Until recently this rapid loss of vitality was supposed to be due mainly or solely to the drying effects of the air, that is, the proto- plasm of the embryo was killed by partial desiccation. No doubt this is the case with some short-lived seeds, but other factors determine life span in other short-lived seeds. A consideration of some of the later more critical work on various short-lived seeds will show the significance of several factors in the loss of vitality. According to Jones,'^ the seeds of the river maple (Acer saccharinum) are killed by relatively slight drying. When they fall from the tree in June they bear about 58 per cent water. Regardless of the temperature of expo- sure (0° to 35° C [32° to 95° F]), they were killed when the moisture content reached 30 to 34 per cent. In his experiments it required six days at 35° C (95° F) and 92 days at 0° C (32° F) to reach this water content, or the death point. When these seeds were stored in a closed vessel over water at the freezing point and provision made for preventing carbon dioxide accumulation, they retained full vitality for 102 days, which was the limit of the test. The low temperature prevented germination and reduced the rate of metabolism. The latter is an important consideration, for these seeds are fleshy and have rapid respiration at higher temperatures. They should be sowed as soon as they fall. If this is impossible, because of the necessity of shipping or for any other reason, they should be kept near the freezing point, and water loss prevented. River maple seeds are not dor- mant but begin germination in nature as soon as they reach the moist ground. The seeds of the fall-fruiting sugar maple {A. saccharum) showed very different behavior. They endure complete air-drying and respond to several weeks' low temperature stratification for eliminating dormancy. Duvel " finds that wild rice (Zizania aquatica) seeds lose their vitality if they are allowed to dry in the air for even a few days, but that they retain their vitality perfectly until spring if stored in water at 0° to 1° C (32° to 36° F). In the spring they must be transferred from storage water to the water in which they are to grow, without being allowed to dry. Seeds of wild rice are dormant when mature, and storage in water near the freezing point after-ripens them as well as maintains their vitality. According to Barton,^" various citrus seeds endure only partial drying in the air. Grapefmit and sweet orange seeds are injured by drying to 52 and 25 per cent moisture content, dry weight basis, respectively, at labora- tory temperatures but grapefruit seeds retain their full vitality for more than a year when stored in the open at 5° C (41° F), where the moisture falls 32 GROWTH OF PLANTS to 17 per cent. Rate of drying as well as degree may be important, or it is possible that the temperature at which the drying occurs is a determining factor. Sour orange and rough lemon seeds also retain their vitality well when stored open at 5° C (41° F). They also endure more drying than the other two sorts of citrus seeds. Storage at — 5° C (23° F) was injurious to all four sorts of citrus seeds because of their necessarily high water content. Other seeds of the temperate zone that lose their vitality readily when stored in open air are oaks, beeches, horse chestnuts, walnuts, hickories, and chestnuts. These are generally stratified Avith moisture at a low tem- perature in the fall, which prevents germination until spring and after- ripens such as are dormant. Delavan,^^ working on seeds of three species each of hickories and of the white oak and the black oak groups, found that the seeds kept well until the following spring in an ice box and in a pit out- side, but lost their vitality in a few months in dry storage. They also gradu- ally after-ripened at the low temperatures, as shown by quicker germina- tion with the lengthening of the period of storage. Barton " found low- temperature stratification necessary for the after-ripening of seeds of hickory, walnut, and butternut. Seeds of willows also lose their vitality quickly when exposed to the air. This has been assumed to be due to excessive drying, but Nakajima's work disproves this assumption. He found ^^ that seeds of Salix opaca, S. japon- ica, and S. Reinii retained their vitality much better in closed tubes over a solution of 50 per cent by volume of H2SO4 in water than they did in open air. In later work he found that seeds of Salix Pierotii and S. japonica in open air lost their vitality completely within a week, but when stored over the sulfuric acid solution mentioned above and kept in an ice chest they still gave 53 per cent germination after 360 days of storage. Such a solution gives a relative humidity of only 13 per cent, which is much lower than the average humidity of the atmosphere at ripening time of the seeds. Evidently the injury in open air is not caused by excessive drying. Valuable informa- tion might have been obtained if he had also tried low oxygen pressure and absence of oxygen, as Busse did for aspen seeds. It is a well known fact that poplar seeds lose their vitality within a few weeks when left in the air. Busse " believes this is due to the injurious action of oxygen, also that higher temperatures hasten the degeneration. Storage of these seeds in a vacuum in a cellar preserved 90 per cent viable after 22 months. Seeds of the English elm lose their vitality almost completely within six months of open storage and seeds of the American elm keep little better." Barton ^ finds that sealed storage of American elm seeds at low tempera- tures, 5° and — 5° C (41° and 23° F), prolongs the life of these seeds greatly. There seems to be little difference whether the moisture is 2, 3, or 7 per cent. In sealed low-temperature storage these seeds retained full vitality for five years, with the experiment still running. Sugar-cane seeds degenerate rapidly when stored in open air. This makes LIFE SPAN OF SEEDS 33 it impossible to ship them A\ath assurance from one sugar region of the world to distant regions where seedlings are desired for breeding. Verret " found that vitality could be lengthened materially by taking the seeds from the thoroughly air-dried heads, placing them m cans with 9 grams of CaCb to 1 liter of space, displacing the air wnth carbon dioxide, hermetically sealing, and storing at the freezing point. In these seeds, low and perhaps constant water content and absence of oxygen seem to be necessary for retention of vitality. It is possible also that carbon dioxide plays a positive role rather than merely displacing oxygen. Kidd " finds that the life span of seeds of Hevea brasiliensis can be greatly lengthened by sealing them in 40 to 45 per cent carbon dioxide. He assumes that this gas acts as a narcotic and that it induces dormancy. There are many other seeds that retain their vitality for only a few weeks or less than a year when stored in the au*. For most of these there is httle information on the effect of the several factors involved in atmospheric stor- age. It is generally assumed, however, that the seeds are injured by drying, but the possible error m such a conclusion is made evident by the discussion above. Among the seeds of tropical plants that have a short life span, Hevea and sugar cane have been mentioned already. Others are Boea, Thea, Co- cos, Oreodoxa, Sabal, Attalea, Mauritia, Thrinax, and Acrocomia. De Candolle " says seeds of most species of the families Rubiaceae, Myrtaceae, and Lauraceae lose the germinative capacity soon after being detached from the mother plants. There are, however, exceptions in the first and second families. Coffea (Rubiaceae) seeds are used for planting up to three years. Ewart ^ reports several Myrtaceae that retain their vitality for considerable periods. Nearly all of 35 species of Eucalyptus reported upon show some vitality in the seeds after 10 years of storage. E. calophylla seeds 10 years old gave 96 per cent germmation, and those 32 years old, 5 per cent. Leptospermum scoparium 10 years old gave 8.2 per cent, and those 16 years old, 2.4 per cent. Callistemon lanceolatus seeds 16 years old gave 75 per cent germination, and C. rigidus seeds 22 years old, 2.8 per cent. A thorough study would probably show many other excep- tions to de Candolle's statement. The seeds mentioned by de Candolle as short-lived generally are sown soon after harvest. While the studies mentioned above have thro\Mi much light on the factors determining the life span of short-lived seeds, our knowledge would be much fuller and more conclusive if the life span of each seed had been studied under a wide range of intensity of each of the effective atmospheric variables, singly and in combination. Life Span of Seeds in Soil In the literature there are hundreds of records of seeds that are supposed to have lain in the soil for decades, still dormant but capable of germination. These determinations are based on the appearance of plants, not common 34 GROWTH OF PLANTS in the region, on recently excavated soils, or similar phenomena on recently- plowed meadows or pastures of long standing. Peter " studied the seed content of soils of the forest that had been planted on meadow, swamps, or pastures for known periods and kept free from open land plants by deep shading. In general, as the age of the forests increased, seeds of field plants became more scarce and those of forest plants more abundant in the soils of the forest. He found seeds of the following in deeper layers of soils of forests 100 years old: Hypericum humifusum, Stellaria media, and Juncus hujonius. In soils of forests 20 to 46 years old, he found seeds of a large number of open-land plants belonging to various genera, such as Thlaspi, Plantago, Sinapis, Juncus, Stellaria, Stachys, Anagallis, Polygonum, Cheno- podium, etc. Peter concludes that seeds of some meadow and swamp plants may lie in the soil more than fifty years, still capable of germination. Ewart 27. p. 182-183 has the following to say about the reliability of Peter's conclusions: "Peter's observations are good evidence of the readiness of dispersal of certain seeds, but as evidence of their longevity are quite un- trustworthy. They contain a grain of truth buried in a mass of inaccuracy. The same applies to all similar records of supposed old seed in soil or under water being germinable, from the classical case of Mummy Wheat down- wards. Here and there long-lived seed has accidentally been hit upon, but in the great majority of cases the records are incorrect." Later accurate records of Beal and the United States Department of Agriculture on the life span of seeds buried in the soil lead one to conclude that the claims of Peter, and others mentioned above, may be true in the main and that Ewart's criticism of their claims is far too severe. Ewart was especially concerned because the seeds that Peter claimed had long life in the soil were mainly small. The buried seed results, as we shall see later, show that very small seeds as well as larger seeds may live for long periods in the soil. There is little doubt of Ohga's "^ claim of great age of the Nelumbo nucifera seeds he excavated from a naturally drained lake bed in Manchuria. The seeds were buried about 1.5 meters deep in a layer of gray mud covered in turn by a layer of peat and a layer of loess. The eroding river which drained the lake has now cut a channel through the lake bed about 13 meters deep. Since there were no Nelumbo plants growing in the region, and the seeds were buried so deep, Ohga concludes that the seeds were from plants growing in the lake before it was drained. Judging from the rate at which the river is eroding its bed, the age of the trees growing on the land since drainage of the lake, and the record of a family that has been farming the drained lake bed for several generations, Ohga concludes that the seeds have been buried for at least 120 years and more likely for 200 to 400 years. They still give perfect germination after treatment of coats. United States Department of Agriculture buried seed project. The most extensive buried seed project on which considerable data have already accumulated is that of the Seed Testing Laboratory " of the United States Department of Agriculture, designated hereafter as U.S.D.A. These seeds LIFE SPAN OF SEEDS 35 were buried bj'' Duvel in 1902. Thirty-two sets of 107 species of wild and cultivated plants were placed in sterile soil in flower pots covered with porous clay lids and buried outside at three different depths, 8, 22, and 42 inches. Records of the tests for 1, 3, 6, 10, 16, and 20 years are now available. The tests for the 30-year period were made in 1933, but unfor- tunately there has been great delay in publication. Seeds of a number of sorts of wild plants showed little or no germination after shorter burial periods, but considerable to excellent germination after longer burial periods. This may have resulted either from the use of less favorable germination conditions in the earlier tests, or from the gradual after-ripening of the seeds in the soil. At any rate, the conditions used for the later tests Avere fairly favorable for the germination of seeds of wild plants on the surface of flats of sterilized soil in a greenhouse. This provided light, fluctuating temperatures, and the stimulative effect of the soil. It is regrettable that samples of the seeds were not placed in dry storage under a variety of conditions, including sealed storage in the soil. This would have answered the interesting question of the relative life span of the seeds in several dry storage conditions and in the moist soil. After 20 years' burial, some seeds of 51 of the 107 species were still alive. The folloAving gives the families, genera, and the highest germination for each genus of those seeds still alive after 20 years. Family Genus Per cent germ. Family Genus Per cent germ. Gramineae Chaetochloa 26 Malvaceae Abutilo7i 57 i( Phalaris 11..5 (< Hibiscus 57.5 It Phleum 12.5 Onagraceae Oenothera 87.5 Robinia 31 '^- them back at the same temperature induced 83 per cent germina- tion; partial desiccation and fluctuating temperatures are also effective. Denny ^^ finds that dormant corms of gladiolus that have remained in moist soil at room temperature for a year or more can be thrown out of their dormancy and caused to grow by only a few hours of exposure to 5° C (41° F), whereas freshly harvested corms require several weeks at 5° C (41° F) to after-ripen. Dormancy and Delayed Germination in Seeds OF Wild Plants In connection with the statements about buried seeds it has been men- tioned that seeds of wild plants are much more commonly and more per- sistently dormant than those of cultivated plants, and consequently their germination is much more delayed. There are many records of long- delayed germination in seeds of wild plants. Nobbe and Hanlein *^ placed 400 seeds each of 31 species of wild plants, mainly weeds, in Petri dishes as germinators at room temperature and kept records on the germination for 1173 days. All the species of seeds used in this experiment were said to be soft-coated, that is, absorbed water. Table 8 shows the number of germinations of the different kinds of seeds after various numbers of days in the germinator, also the total germination and per cent germination for each after 1173 days. It will be noted that there was no germination, also no decay, of the Phyteuma spicatum, Primula elatior, and Verbascum nigrum seeds. Lack of decay indicates that the seeds were alive, and the failure to germinate indicates that room temperature in the germinator failed either to furnish the conditions for after-ripening or for germination. The evidence in the next chapter will indicate that it was the former. Five other sorts of seeds gave less than 1 per cent germination; likewise, these showed no decay, and the low germination is undoubtedly explained on the same basis as the three mentioned above. In several of the seeds that showed moderate or considerable germination, the germination is 42 GROWTH OF PLANTS »ooom»cicmiomoooiooiciooooQic»oiraoiraioo»coioo ^'l0oc^^-(^^^^t^^-oo^0(NOC^^^-_pooo(NI>;^^oc<^^>pl>ot>;p OT^^Gdooo5o6o6;deooo•>*05coo«o'eO"$'^-oooMOlra^»0'--^o>o^ I— ( iC (N'-iTt0Oir5>-HO500 00 '^ OS 1—1 .—I b» iC (N Tt< 05 MoO(Ni-«iot^(MCOoot^'<^'^r^c^coooO' b lO PO i-H t^ 0> 00 C5 ^ lO 0> -^ ■* »-i fC 00 ^ (N '-H T-HcoeoN eoeo ec coco iCO»CO^HT-HOt^OCOCD rti m O CO 00 00O5 CO(N —I CO CI 00 o CO a. o -3 "<*< o in IN CO CO i-H(M OOO eo(N ^H OS'-H 1—1 CO COi-KN 1-100 00 CO I I C0C0t^.. o3 COO'-i |l I o3 c3 (N *3 +i -_ to O! CO Q^ Q; 1 1 CO(M CO I --H I "-I I CO»-i Tt* I I I (N ^ 1^ I s?^ I I n 12 »— I I— I CD .-1 (M OSTtcoiMiM m I > .^ ^ «-^ iC CO ■* >— I CO CO I I N.^ CO(M OOO I I ■>* COi-H CO (N I 00 CI iC CO l*^ Mi2 I 08" cot>-_ ^ ^ >— I >> — CO I O I"! "O ■■u " ID CO o CO "^2 ■* I r 1 I I I I I t>.CO 00 (N lO-* t>.co coos ■* (M I l>t^ 1 r~» i>- TfiO I >C »o OOCO I co^ CO I loeo I I (N (N I ^ CO •" I ri IM CO CO CD 00 a. m ^ o e c3 o g.=3 ^ 'S 8ec^ 8 2^ s b~ ""J "** ^o ^« I^ ^4e4eo■^lOc6^-lo6ojo-HC^■eoTJ^lOcd^^o6ojd•-j^"co;^lOO^^ 1— ir-ii— ii— iT-ii— (I— li— ii— (1— (dddOdC^C^C^ddCOCO LIFE SPAN OF SEEDS 43 distributed over much of the 1173-day period: Campanula rotundifolia, C. persicifolia, Chenopodium album, Capsella Biirsa-pastoris, and several others. Delayed and time-distributed germination of seeds of wild plants is striking in the results reported in this table. Nobbe and Hanlein used room temperature with relatively little fluctuation in temperature. In the soil out-of-doors in Germany there would be much greater fluctuation in temperature during more than three years of the experiment. Table 9. Germination Experiments with Seeds of Various Wild-Growing Plants (From Dorph-Petersen) Species Seed har- vested Put into germinator Per cent germination after years 1 1 0 6 14 1 73 96 12 35 35 29 0 15 2 2 0 15 32 0 4 11 12 11 10 0 20 3 34 54 47 2 9 1 15 10 16 5 0 2 4 19 15 23 3 7 0 18 22 13 7 0 12 5 3 2 39 1 16 26 1 11 3 52 12 6 26 1 0 3 2 2 10 3 7 2 29 1 4 10 13 9 8 36 0 22 7 9 4 1 3 10 2 11 0 12 1 Total Stellaria nemormn Sisymbrium sophia Sisymbrium officinale Thlaspi arvense It K (( l( Sinapis arvensis II II Geranium molle Malva vulgaris Cynoglossum officinale Ballota nigra July 5 '97 Sept. 20 '98 Aug. 24 '96 Sept. 20 '98 July 27 '96 Aug. 28 '97 Aug. '04 Aug. 11 '99 Sept. 21 '03 July 31 '96 Aug. 24 '96 Aug. 10 '99 Aug. 8 '96 Aug. 16 '97 Oct. 8 '98 Sept. 11 '96 Oct. 8 '98 Aug. 22 '96 Sept. 1'97 Sept. 9 '05 Sept. 2 '99 Oct. 5 '03 Aug. 22 '97 Sept. 11 '96 Sept. 4 '99 Aug. 27 '96 87 69 93 91 87 94 96 86 80 93 94 82 73 Dorph-Petersen -* carried on similar experiments with weed seeds at Copenhagen, using the Jacobsen germinator, but ran them in duplicate for a period of 11 years. One set of germinators was kept in the laboratory and the other on an open porch without heat. In one set of these experi- ments he got much earlier and much higher percentage of germination in the outdoor germinators for Anthriscus silvestris, Primula elatior, and Galeopsis tetrahit, and somewhat earlier and higher for Potamogeton natans, while Datura stramonium gave 99 per cent germmation after eight years (98 per cent during the eighth year) inside and only 2 per cent after 11 years outside. In another set of experiments he showed that in the germi- nator on the porch with the wide fluctuations in temperature, there was marked delay and distribution in the germination of weed seeds (Table 9) . Why Do Seeds Remain Dormant in a Germinator OR IN THE Soil? Considering both the indoor and outdoor germinators, Dorph-Petersen had a range of temperatures that cared for one of the very important factors in the after-ripening and germination of dormant wild seeds. As we shall see in later chapters, many seeds require a period in a germina- 44 GROWTH OF PLANTS tion medium just above freezing in order to after-ripen the seeds prepara- tory to germination. Some seeds require low temperatures for germination, while others germinate only at high temperatures, and finally others germi- nate well if the temperature fluctuates rather ^^^dely from day to night. There are other factors active in the soil that might either stimulate the germination of dormant seeds or prolong their dormancy. Nitrates and other nitrogen compounds generally present in the soil promote the germi- nation of certain seeds. This is especially true of many of the light- stimulated seeds.^^ Deep burial in the soil excludes light. Light-stimulated seeds, such as celery, tobacco, and timothy, have been mentioned above as remaining in the soil for several years in the dormant condition. Why do seeds of so many wild plants lie in a germinator or in the soil in conditions that are favorable for germination of seeds of our cultural plants and fail to germinate even after years? The answer to this question is very complex and involved. The next chapter, which deals in large part with types of dormancy in seeds, will throw much light upon it. At this point we shall discuss only two factors that may play a part in keeping seeds of wild plants dormant in the soil: (1) inhibiting chemicals in the fruits, seed coats, or seeds themselves, and (2) unfavorable germination conditions in the soil that throw seeds into secondary dormancy. Many investigations ^^' ^^' ^^' ^^- ^^- ^^' ^^' ^^ show that fleshy and dry fruits, seed coats, and even the living parts of seeds contain chemicals which inhibit germination of seeds. For instance, the fleshy fruits of the tomato, cucumber, pawpaw {Carica papaya), and many other plants con- tain chemicals that inhibit the germination of the seeds while they are within the fruits, and this mthout any permanent injury to the seeds. The woody material of the seed balls of beets and the coats of lettuce seeds contain chemicals that inhibit germination. This becomes very noticeable if batches of either are germinated repeatedly on the same moist filter papers so that the inhibiting chemicals become concentrated. The em- bryos and endosperms of some seeds also contain inhibiting chemicals. Mostly these chemicals are not specific in their action but inhibit other species of seeds as well as the species in which the chemicals develop. In some cases the chemicals are more effective as inhibitors on seeds of other species than on those of the species producing the chemicals. Some of these chemicals are volatile and others are not. They are mostly heat- stable, although some workers claimed to find inhibitors that are destroyed at the boihng point of water. Some of the substances that have been found in fruits and seeds that act as germination inhibitors are ammonia, hydrocyanic acid (from amygdalin of rosaceous seeds), essential oils, alka- loids, glycosides, and an unidentified substance, "Blastokolin." "^ These inhibitors are more effective in ordinary germinators than in the soil, for the soil moisture allows them to diffuse away from the seeds; also soil, like animal charcoal, adsorbs the inhibitors and removes them from action on the seeds. LIFE SPAN OF SEEDS 45 For such inhibiting chemicals to be effective in holding seeds dormant in the soil for years, two conditions \\ill have to prevail: (1) the inhibiting chemicals must be very stable in the soaked-up seeds, and (2) they must be prevented from diffusing out of the seeds by semipermeable membranes of either the living protoplasm or of the non-li\'ing seed coats. Both these conditions may be met by alkaloids and glycosides. On the whole, how- ever, much more investigation will be needed before we can attribute to inhibiting chemicals an important role in holding many seeds of wild plants dormant in the soil for years. We have learned already that many of them do stay dormant for years in the soil in the imbibed condition. Secondary dormancy. It has long been kno^^'n that unfavorable germi- nation conditions often throw seeds into dormancy so they will not germi- nate when shifted to a favorable condition. Kinzel ^^ showed that seeds of Nigella sativa, which are prevented from germinating by light, if placed in an illuminated germinator, soon change so they will not later germinate in darkness. They become "light-hard." This holds for other light- inhibited seeds. Many seeds that need light for germination when placed in a dark germinator become "dark-hard," so they will not germinate later in light. Kidd ^^ has sho^^^l that high partial pressures of carbon dioxide, 24 per cent more or less, will inhibit germination of certain seeds and in time throw them into dormancy. Reduced pressures of oxygen render the carbon dioxide effective in lower concentrations. There are many other cases of bad conditions in a germinator that throw seeds into dormancy. Most of these conditions are active in the soil and may, in part, account for the long rest period of seeds in nature. In 1916 the author ^° spoke of dormancy in seeds induced by bad conditions in a germinator as "second- ary dormancy." B C D Figure 12. Ambrosia trifida fruits and seeds: A, the fruit; B, the seed; C, the embryo; D, section of the two-layered seed coat. 46 GROWTH OF PLANTS The most thorough study in inducing dormancy m seeds is that of W. E. Davis "• ^o on seeds of Ambrosia trifida and XantUum. This was a joint contribution of this Institute and the Department of Botany and Plant Pathology of Kansas State Agricultural College. Ambrosia seeds Figure 13. Germination of embryos of Ambrosia trifida taken from fruits that had been stored for three months as follows: A, dry; B, in a germinator m a refrigerator; and C, in a germinator at 27° to 30° C (80° to 86° F). consist of an embryo covered with a paper-thin transparent seed coat. The seed is enclosed in a thick, woody, indehiscent fruit coat (Fig. 12). In controllmg germination and dormancy the seed coat and not the fruit coat is important. Ambrosia seeds (embryos) are dormant and they after- ripen slowly and incompletely after several months of dry storage and LIFE SPAN OF SEEDS 47 completely after three months in a germinator at 1° to 10° C (34° to 50° F). After the seeds are after-ripened they will germinate slowly but nearly completely in a germinator at 20° C (68° F) and more rapidly but more sparingly in a germinator at 30° C (86° F) . At the higher temperature many of the seeds go back into the dormant condition. The secondary dormancy is induced by the seed coats restricting the oxygen supply to the embryo in a germinator at a high temperature. Fig. 12 pictures the fruit, seed, embryo, and seed coat structure of Am- brosia. Fig. 13 shows the behavior of isolated embryos of Ambrosia when / / A ^ Figure 14. A, seeds of cocklebur in clay in a high temperature germinator to produce dormancy; B, imbedded in agar for the same purpose. placed in a germinator for three to four days at room temperature after three months' storage of fruits m three different conditions. Note that the embryo of intact fruits in a germinator at low temperature, 1° to 10° C (34° to 50° F), after-ripened so that they germinated promptly when the fruit and seed coats were removed and the embryos were placed in a germinator at room temperature. Those which had been kept in a germinator at a low temperature gave prompt, complete germination; those in dry storage a small percentage germination; and those in a high-temperature germi- nator no germination. The embryos of the latter were in deep dormancy. The embryos of the cocklebur -^ are also enclosed in very thin seed coats that interfere with oxygen diffusion to the embryos. They differ from Ambrosia in three ways: the involucral fruit coat (bur) encloses two seeds, an upper and lower; the embryos are not dormant in the mature seeds, but the thin seed coats hinder germination by limiting oxygen supply to the embryos at certain temperatures, that is, below 20° C (68° F) for lower 48 GROWTH OF PLANTS seeds and below 30° C (86° F) for upper seeds. We shall describe more fully the behavior of the two cocklebur seeds in the next chapter. To throw the embryos of cockleburs into the dormant condition, the intact seeds were placed in a germinator at a high temperature, 28° C (82° F) or above, with greatly reduced oxygen pressure. The reduced oxygen pres- sure can be obtained by displacing some of the oxygen in the air with a gas such as nitrogen or hydrogen, or by imbedding the whole seed in agar jelly or the radical end in modelling clay in a germinator as shown in Fig. 14. With the intact seeds in a high-temperature germinator at re- duced oxygen pressure the embryos become dormant in from two to several months. Thornton ^^ confirms Davis' work on the cocklebur. The dor- mant seeds can be thro\vn out of dormancy by placing the seeds in a germi- nator at 5° C (41° F). While the intact seed in secondary dormancy will not germinate, the embryo can be made to germinate if the seed coat is removed, but the growth is very slow and sluggish and the seedling is dwarfish mth abnormal leaves. Fig. 15 shows that the non-dormant em- bryo gives a much bigger plant after 14 days' growth than the embryo from the dormant seed gives after 32 days' growth. Likewise, the non- dormant embryo gives much more growth after 21 days of growth than the embryo from the dormant seed gives after 47 days. As we shall see later, so-called dormant embryos are not generally incapable of growth if the coats are removed, but they have so little vigor of growth that they cannot overcome the slight resistance of even so thin a coat as the cockle- bur seed bears. In general, also, dormant embryos, if forced to grow, pro- duce dwarfish plants so far as the above-ground portion of the plant is concerned. The root system is not dwarfish but is large in comparison with the top. In the next chapter we shall have much more to say about the effect of oxygen pressure on germination and dormancy induction, about the importance of seed and fruit coats in dormancy of seeds, and about dormant embryos. Davis was able to throw seeds (embryos) into and out of dormancy repeatedly and at will. Nature induces dormancy in embryos of some species of plants when they mature. Many seeds after-ripen in nature in the soil during the cool weather of fall, winter, and spring. If Davis and nature use the same methods in inducing and overcoming embryo dor- mancy, embryos in nature are thrown into dormancy as the seeds mature because of the relatively high temperature during maturing and because of limited oxygen supply to the embryo caused by seed coats and other tissues about embryos. As we shall see in the next chapter, many seeds in the temperate zone after-ripen in soil at temperatures slightly above freezing. Do many seeds in the soil after-ripen in nature in the cool weather of fall, winter, and spring, which gives an abundance of germination in early spring? Does the hot weather of summer throw many of the seeds in the soil back into the dormant condition to be after-ripened again the LIFE SPAN OF SEEDS 49 Figure 15. Embryos of lower seed of cocklebur. A, Imbibed embryo of non-dormant seed; B, same 14 days after planting; C, same 21 days after planting. D, Embryo of dormant seeds after 18 days in germinator; E, same as D after 18 days in germinator, plus 14 days in soil, or 32 days in germinating conditions; F, same after 18 days in germinator, plus 29 days in soil, or 47 days in germinating conditions. follo^^^ng mnter? In any case, Davis has thrown much hght upon the dormancy and after-ripening rhythm of seeds in nature. Hard Seeds Best Adapted for Long Life Span We have been discussing the Hfe span of soft seeds (seeds that absorb water) in the soil and find that some of them stay aUve for 60 years or 50 GROWTH OF PLANTS more, but the seeds best adapted for long life in the soil are certain hard seeds, i.e., seeds that do not absorb water. This is true only if the hard coats offer great resistance against softening in the soil. The most striking example of this is the old seeds of the East Indian lotus (Nelumho nucifera) m — ii NEW OLD mm: B Figure 16. A, Relative rate of growth of freshly-harvested and century-old seeds. Coats must be broken to permit water absorption and germination. B, Structure of coats showing water-resistant layers. The water-resistant layers are (a) epidermis, and (6) outer end of palisade layer, down to (m) the "light line." C, 30-60 ft. bank left by the cut of the river. Seeds located in strata 3-6 ft. from top of this precipice. D, Plant grown from one of the old seeds. that Ohga ^^ dug from a naturally drained lake bed in Manchuria. He offers evidence that these seeds, many of which were still hard and 100 per cent viable, had been in the soil for two centuries or more. Fig. 16 shows the vigor with which the old seeds germinate when the coat is made per- meable by sulfuric acid treatment, also the several layers of the seed coat, indicating the parts that prevent water absorption. In the old seeds that had been in the soil the epidermis and much of the outer end of the pali- LIFE SPAN OF SEEDS 51 sade cells had been eaten away by bacteria and fungi during the long period in the soil. No doubt Ohga recovered only a small remnant of the seeds originally buried. During the centuries the bacteria and fungi had eaten the coats of most of them down to the "light line," the seeds had swollen and germinated, and the seedlings were killed due to deep burial. Many leguminous seeds are hard-coated, and no doubt many of them remain alive in the soil for years because of this fact. Goss ^'^ found seeds of several species of clovers viable after 20 years of burial. Allers ^ claims that hard yellow lupine seeds lay in the soil 40 years unswoUen and fully viable. Nobbe found that some seeds of various species of Papilionaceae, Mimo- saceae, Cannaceae, and Ranunculacae remain hard and viable after being soaked in water for years. Different crops of seeds, red clover from vari- ous parts of Europe, varied greatly in percentage of seeds remaining hard after 10 days' soaking at room temperature as well as in percentage remain- ing hard after 12 years of soaking; one sample had 5.33 per cent still hard after 12 years in water and a sample of very small seeds gave 53.33 per cent ■'^ hard after 37 years of soaking. After 9 years in water, ''^ white sweet clover had 48.93 per cent hard, Vicia cracca 43.36 per cent, Labur- num vulgare 94.5 per cent. Two samples of black locust showed 18.5 per cent and 5.5 per cent hard after 153^ years of soaking. Davis ^i found that some velvet-leaf {Ahutilon Theophrasti) seeds remained hard after 20 years in water. Kondo **^ found some seeds of Astragalus sinicus still hard and alive after soaking in water for 21 years and ^- 5.50 per cent of black locust seeds were hard and alive after soaking 14 years. Rees ^'•' found that seeds of Alhizzia lophantha that had been in the soil at least 23 years were still hard and viable. The conditions in the soil with varia- tions in temperature, moisture supply, and presence of organisms would probably decrease the resistance to swelling. Yet it is likely that very hard leguminous seeds such as palo verde," Kentucky coffee tree,^* and certain Albizzias, Acacias, and Cassias may lie in the soil under favorable conditions hard and viable for as long periods as the East Indian lotus seeds. The only chance in the main for leguminous seeds to remain in the soil alive for a long time is their failure to swell. Once they swell, the coats in most species split and swell up into massive gelatinous tissue very subject to attack by pectin bacteria ^- and the embryos are left unprotected and subject also to bacterial attack unless good germination conditions pre- vail. Bier '' indicates that some yellow lupine seeds bear substances that offer considerable anti-bacterial action. The same thing is true of many other hard seeds. The seeds that lie in the soil viable and swollen for years have coats that do not rupture or otherwise decompose markedly. They furnish good protection to the embryo and endosperm. In the next chapter, under types of dormancy, we shall have occasion to discuss the anatomical, environmental, and genetical factors that deter- 52 GROWTH OF PLANTS mine the hardness of leguminous seed coats as well as factors that over- come hardness. Storage of Seeds Conditions for storing seeds so that they will maintain their full vitality for a considerable period are not only of academic interest but of great economic importance. Some plants give big crops of seeds only on alter- nate years, or in some cases only on occasional years, with several years of poor crops between. The latter is true of red pine. Such seeds must be stored so that nurseries may have an ample supply of good seeds to plant every year. No doubt many seeds could be produced more cheaply if a larger acreage were grown only alternate years or even less frequently. Seed producers must plant sufficient acreage to supply the need for the follo\ving year even when the season proves poor for production. Conse- quently, in good cropping years there is a great excess of production over the needs for the following year. We have been asked to determine good storage conditions for Cinchona seeds so that the greater part of seeds of various selections and crosses can be held viable until samples are gro\vn and the quinine yield of the resulting plants determined. We have already discussed seeds that are ordinarily short-lived because they \vill endure only slight drying in the air. Most seeds ^vill endure complete drying in the air and considerable additional desiccation. What are the best storage conditions for such seeds? We have noticed that the seeds that live longest in seed cupboards and in the soil are of the hard- coated type. Nature may give us a hint at best storage conditions for seeds that stand moderate to considerable drying.- Hard coats prevent any exchange of moisture and air between the outside atmosphere and the living parts of the seeds. The hard coats hermetically seal the embryos individually, Becquerel ^^ has determined that the percentage of water is low, 2 to 5 per cent, in hard-coated seeds of legumes and of course aerobic respiration is prevented except for oxygen within the coats. The storage of such seeds can be made almost perfect by placing them at low tempera- tures. Perhaps it might prolong the life of these hard seeds if two impos- sible changes could be made in them, namely, withdrawal of the last trace of oxygen and most or all of the water from A\dthin the coats. The recipe for prolonging the life of seeds that endure drying is to remove as much water from them as possible mthout injury, and seal them so as to hold the moisture low and constant in absence of oxygen at a low temperature. Let us examine the effects of these three storage factors — moisture con- tent, temperature, and oxygen. Seeds mth soft coats stored in the air fluctuate in water content ^vith the relative humidity of the air. Barton * determined the water content of tomato, pine, and lettuce seeds stored open in the laboratory at Yonkers eight different months of the year. Fig. 17 shows the results. During the LIFE SPAN OF SEEDS 53 winter months in the heated laboratory the water content was low. During the moist, hot weather of summer the water content was high, reaching a maximum in August when the water content was about twice that of the winter months. It is also evident that different kinds of seeds vary con- siderably in their water content when stored under identical atmospheric conditions. Seeds in which the storage substances are mainly fats absorb Lettuce FEB. 1939 MAY JULY AUG. SEPT. NOV. DEC. JAN. 1940 Figure 17. Moisture contents at various times of the year of seeds stored open in the laboratory. Moisture expressed as percentage of dry weight of seeds. ess water than those in which the storage substances are mainly carbol- hydrates. Peanut and pine seeds are high in fats, tomato seeds low, and flax seeds intermediate. No complete analyses for onion and lettuce seeds are available, but microchemical tests show them rich in fats. None of these six kinds of seeds bear carbohydrates as the main storage material. All store their foods mainly in the form of proteins and fats. The nature and thickness and chemical nature of the coats and other factors modify the amount of water absorbed. The pine seeds absorb a higher percentage of water than lettuce, although both are fatty seeds. Barton ^ has shown not only that is high moisture content injurious to the keeping quality of seeds but that fluctuation in moisture content is also detrimental. Fig. 17 shows the mde variation in water content in open storage. This is avoided by sealed storage or by hard-coated seeds. Another interesting observation made by Barton ^ is the fact that with different temperatures under the same relative humidities the amount of moisture held by the seeds varies. Fig. 18 shows this variation. This is especially marked at the higher humidities, 76 per cent and 55 per cent, 54 GROWTH OF PLANTS 20 16 14 12 10 u tr 16 t- (J) O 14 2 ijj o cr liJ 12 10 - 8 10 10 20 TEMPERATURE " C. Onion Lettuce Peanut Figure 18. Moisture content of seeds after 43 days of storage at relative humidities of 76, 55, and 35 per cent. LIFE SPAN OF SEEDS 55 where the maximum amount of water is held at 10° C (50° F) with less at 5°, 20°, and 30° C (41°, 68°, and 86° F). It is less marked at 35 per cent relative humidity. The explanation for the difference is not known. Per- haps the colloidal condition, and consequently the water-holding power of the seeds, varies with the temperature. Why seeds have maximum water- retaining or absorbing power at 10° C (50° F), especially at high humidities, is an interesting academic question for an investigator to answer in the future. It is of little significance in practical seed storage. The curves in Fig. 18 show again the great difference in the amount of water that differ- ent kinds of seeds will hold when they are in equilibrium with the atmos- phere at various humidities and temperatures. Here again the fatty seeds, peanut, lettuce, flax, and pine, are relatively low in water content at all temperatures and humidities as compared with tomato and onion seeds, which are also fatty. If low water content is required for storage of seeds, how low should it be? Probably the lower the better, provided the drying itself does not injure the seeds. We have already seen that some seeds endure little dry- ing and also, in the case of citrus, that seeds will endure more drying at low temperatures with slow drying than at high temperatures with fast drying. It would be well if we had data for all commercial seeds that can be stored dry, on the best conditions for drying, as well as the degree to which they can be dried without injury. We do not have such data, but we do have enough to conclude that complete removal of water is injuri- ous to most seeds and that the degree of drying endured without injury varies with different seeds. Ewart -^ states that seeds will not endure dry- ing below 2 to 3 per cent moisture. Waggoner ^^ dried Icicle radish seeds to 0.4 per cent moisture, and Joseph ^s. se dried parsnip seeds to 0.4 per cent moisture and paper birch seeds to 0.6 per cent moisture without injury. Kiesselbach ^^ dried maize grains to 5 per cent moisture without injury but did not find that the maximum drying endured without injury. Gray birch seeds ^^ were injured by drying to 5.2 per cent moisture and some pine seeds ■* are injured by over-drying. If we are interested in the water content necessary to keep seeds per- fectly for a few years in sealed storage using other good storage conditions, the answer is easy. Fatty seeds should be reduced to 4 to 5 per cent mois- ture and starchy seeds to 5 to 6 per cent moisture before sealing. The second important storage condition affecting the life span of seeds is temperature. In the case of seeds which contain so much water that they soon perish at 20° or 30° C (68° or 86° F), lowering the temperature to near the freezing point will prolong the life markedly. With such seeds, lowering the temperature far below the freezing point may prove injurious due to freezing. As the moisture is reduced, lower and lower temperatures can be used, until in seeds having very low moisture the temperature of liquid air is not injurious." Guillaumin " found that soybeans stored in air lost their vitality com- 56 GROWTH OF PLANTS pletely in six years, while those stored in nitrogen gas or in a vacuum retained their full vitality for the same period. Some other fatty seeds seem especially sensitive to oxygen during storage; flax seeds, on the other hand, seem to endure air storage very well. Dillman and Toole " found that the latter stored in the dry air of Mandan, North Dakota, still showed 58 per cent germination after 18 years of storage. We have mentioned other cases above where short-lived seeds were benefited by being stored in absence of oxygen. In seeds with high moisture the complete removal of oxygen may cause injury due to anaerobic respiration. No doubt oxygen is generally injurious to long life in seed storage, but its ill effect is largely overcome by proper drying of the seeds. Drying may counteract the ill effects of oxygen, because dried seed coats are impervious to gases; also drier protoplasm may be more resistant to whatever oxygen remains in the intercellular spaces of the dried seeds. Lowering the temperature of storage also minimizes the ill effects of oxygen in storage. The three factors, moisture content, temperature, and oxygen, are interactive: plac- ing one near the optimum lowers the ill effects of the other two that are not near the optimum. Suppose that, as a matter of academic interest, somebody wanted to find out how long the several farm and garden seeds could be kept fully viable. He would have to learn the best method and the proper degree of drying each, and the best temperature of storage, whether a little below freezing or much lower, possibly even as near absolute zero as possible. He would proceed to dry each sort of seed, hermetically seal it in a vacuum or in an atmosphere free of oxygen, and to store it at the proper tempera- ture. With all this his troubles would just begin. For some of the seeds, at least, he would have to arrange with his great-great-grandchildren or later progeny to see the end of the experiment. Now let us examine a few experimental results to see how much fair storage conditions, probably far from optimum, will lengthen the life of some short-lived seeds. Delphinium seeds degenerate rapidly in open air storage. Table 10 shows the results ^' '^ of storing annual and perennial delphinium seed under various conditions. The best storage conditions used in these experi- ments were probably far from optimum. These seeds were not dried be- yond the drying in the laboratory in December, which gives a medium low water content, as is seen in Fig. 17. The seeds were corked in small vials with paraffin over the cork. This is not as good as sealing in glass tubes with a flame so far as holding the moisture content is concerned. Finally, the seeds were sealed in air rather than in a vacuum or in absence of oxygen. In spite of only moderately good conditions, annual delphinium seeds retained their full vitality in sealed storage at the 8° and 5° C (46° and 41° F) combination for 143 months, while in open air at room temperature they had degenerated noticeably in 11 months, and nearly half had lost vitality in 22 months. The perennial delphinium seeds had retained their LIFE SPAN OF SEEDS 57 Table 10. Viability of Delphinium Seeds Stored under Various Conditions Seed Storage conditions Germination percentages after months of storage 11 22 46 69 111 123 143 168 193 Annual, 72 Tc ger- minated when stored Open room temp. Sealed room temp. Open 8° C (46° F) * Sealed 8° C (46° F) * 57 75 50 70 44 80 41 67 0 50 31 66 0 15 5 80 0 0 76 0 0 71 71 48 43 Perennial, 43% germinated when stored Open room temp. Sealed room temp. Open -15°C(5°F) f Sealed-15°C(5°F)t 11 35 44 42 0 21 45 53 0 0 37 57 0 27 44 • 8 49 6 50 8 45 3 45 33 * After 7 years the temperature was changed to 5° C (41° F). t After 7 years the temperature was changed to — 5° C (23° F). original vitality after 168 months in sealed storage at — 15° C (5° F) for 7 years followed by — 5° C (23° F) for the rest of the storage period, although nearly all had lost their vitality in 11 months in open storage at room temperature, and all the seeds were dead in 22 months under this condition. Even after 193 months in sealed storage at the — 15° and — 5° C (5° and 23° F) combination of temperatures, two-thirds of the seeds were still alive. The Italian population of the United States commonly grows dandelions for greens. Dandelion seeds are very short-lived in open storage. Fig. 19 shows the effect of temperature, moisture content, and sealed storage upon retention of vitality by these seeds.^ In open storage they keep perfectly for three years at — 5° C (23° F), but degenerate rapidly at room tempera- ture and 5° C (41° F). In sealed storage ^\dth 7.9 per cent moisture they keep perfectly at -5° C (23° F) and 5° C (41° F) and fall nearly 50 per cent in vitality at room temperature for three years. The situation is similar in sealed storage with 6.2 per cent water content, except that the fall in vitality at room temperature is considerably less. Finally, with 3.9 per cent water content the seeds keep their vitality almost equally well at all three temperatures, mth only a slight fall at room temperature. Barton has found that repeated opening and resealing of sealed seeds results in more rapid degeneration than occurs if the seeds are kept sealed continuously for the whole period. She attributes this to sHght fluctua- tions in water content due to opening and exposing to the air. Let us look at another set of storage experiments (Fig. 20) where only one storage factor approached the optimum, namely, reduced and con- stant moisture content \vith no reduction in oxygen pressure and labora- tory temperature for storage.^ These seeds at the beginning bore from 8.2 to 12.5 per cent moisture, about the amount held by seeds in equi- librium with the air in mid-summer at Yonkers. The seeds that were 58 GROWTH OF PLANTS 100 YEARS OF STORAGE Figure 19. Germination of dandelion seeds on moist filter paper after storage for various periods at room temperature (R.T.), + 5^ C (41° F), and - 5° C (23° F) in open containers (upper left) or sealed in glass tubes (upper right and lower half) with moisture contents of 7.9, G.2, and 3.9 per cent. "Open" lots moisture at start was 7.9 per cent. dried with CaO for sealing had one-third of the moisture removed before sealing. It will be noted that the seeds sealed without drying degenerated most rapidly. The moisture was too high for sealed storage at such a high temperature. All the seeds kept well for six years in sealed storage after the rather slight reduction in moisture, except pepper seeds, and even they were improved by drying and sealing. More thorough drying prob- LIFE SPAN OF SEEDS 59 O'OPEN ©:AIR-DRY, SEALED t^DRI ED OVER CaO, SEALED 12 3 4 5 6 12 3 YEARS OF STORAGE Figure 20. Deturioratiou of seeds at room temperature storage. Germination tests made in ovens on moist filter pai>cr. ably would have improved their keeping qualities. At the same time samples of all these, not shown in the curves, were stored at — 5° C (23° F). At this temperature those in open storage and sealed storage without dry- ing kept almost perfectly for six years, as of course did those in sealed storage after drying. 60 GROWTH OF PLANTS Air conditioning — control of temperature and humidity of the atmos- phere — is now used extensively and on a large scale in the United States. The data given above indicate the importance of low constant moisture content of seeds and of low temperatures in conserving the vitality of seeds in storage. Already big seed firms are building large, air-conditioned seed-storage houses, which can be used for drying the seeds by putting them in loose fabric bags and cording the bags up, as well as to provide low constant moisture and temperatures after the seeds are dried. It might be desirable to use separate rooms for drying, so that higher tem- peratures wth low humidities could be used for quickly drying the seeds to the desired moisture content. Some rooms should also be run below freezing for storage of certain seeds after they have been properly dried. One large seed firm has consulted the Institute on the desirable specifica- tions for such storage houses. With the storage houses described above available, there need be no trouble in holding most farm, garden, and flower seeds in full vitality for two, three, or four years. Why Do Seeds Degenerate with Age? Many theories have been offered to explain the degeneration of seeds with age. Most of the explanations offered to date are highly theoretical and have relatively little factual substantiation. We shall consider only the more prominent of them and finish by giving the more probable one along with the facts that tend to confirm it. It has been suggested that the enzymes in seeds degenerate with age, with the result that the seeds become incapable of germination. This is not at all probable, for dry seeds are relatively low in enzymes, and the latter, both hydrolyzing and respiratory, are formed largely by the proto- plasm of the embryo in the initial and later stages of germination. The failure of enzymes to form in adequate amounts in older seeds must be sought in changes in the protoplasm itseK. Auxins persist ^^ in Zea mays seeds after 26 to 38 years of storage. It has been suggested that stored foods disappear in seeds mth long aging, and that the embryos do not get sufficient nourishment in old seeds. Most old seeds kept in dry storage contain large amounts of stored foods long after vitality has been lost. For seeds in the soil under natural con- ditions, if they absorb water readily, it is possible that exhaustion of the foods by respiration determines the life span. Hard-coated seeds in the soil will, of course, use little stored food in respiration and after many years in the ground will have an abundance of stored foods. Jones and Gersdorff ^^- ^^ find that three different types of changes occur in the proteins of the grains of wheat and corn and the seeds of soybeans in storage: (a) a decrease in solubility, (6) a partial breakdown of the pro- teins indicated by decrease in true protein content, by decrease in the amount of nitrogen precipitable A\ith trichloroacetic acid, and by increase LIFE SPAN OF SEEDS 61 in amino nitrogen, and (c) a decrease in digestibility. These changes were rapid at first and had slowed down considerably after two years of storage. They were also much more rapid at 24.5° C (76° F) than at -1° C (30° F) and in open as against sealed storage. Even in the favorable storage con- ditions the changes were easily measurable in two years. We do not know to what extent these changes occurred in the living germ or scutellum, and to what extent in the more inert endosperm and aleurone layer; but the fact that the proteins of white flour showed very much greater changes than those of the intact grains suggests that the storage proteins are in- volved in these storage changes. The changes were thought to be due to enzyme action and oxidation. The latter change may be greatly reduced in intact grains by the low oxygen permeability of the dry grain or seed coats. While stored foods are not used up to any great extent in dry stored seeds, no doubt they are being slowly denatured. Ewart -^' p-i^^ offers the following explanation: "Longevity depends not on the food materials or seed coats, but upon how long the inert proteid molecules into which the living protoplasm disintegrates when drying, retain the molecular grouping which permits of their recombination to form the active protoplasmic molecule when the seed is moistened and supplied ^nth oxygen." This explanation is not satisfactory because it is highly speculative and not capable of experimental proof. Crocker and Groves ''^ h^ve suggested that the degeneration of seeds in dry storage may be due to the gradual coagulation of proteins of the embryo. They applied Buglia's time-temperature formula for coagulation of proteins to the degeneration of wheat grains at various temperatures and at two different moisture contents, and found that the calculated longevities agreed well mth the determined longevities for high tempera- tures. The calculated longevities for lower temperatures were, however, many times as long as the life span of wheat grains in ordinary storage, but, as we shall see later, controlled storage prolongs the life span of seeds tremendously. This explanation has the fault of being very general. There are many different kinds of proteins in an embryo, and this work does not throw any light upon the particular proteins that coagulate with time. Furthermore, it throws no light on the possibility of the degenera- tion of some particular mechanism of the living cells. It is possible that in seeds in dry storage the greatly curtailed respira- tion leads to the accumulation of intermediate products of respiration that are toxic to the delicate mechanism of the cell nucleus. Stubbe ^** has suggested this as one possible cause of the degeneration of seeds in storage and of the increased mutation sho\\'n by old seeds. Schwemmle ^' offers some experimental evidence of the accumulation of inhibiting or toxic substances in seeds as they age. In certain hybrids of Oenothera herteriana the seeds germinated more slowly as they became older, and from the old seeds he could extract substances that greatly inhibited the germination of fresh seeds that showed prompt and complete germination in absence 62 GROWTH OF PLANTS of the extract. Seeds grown on the meal of the old seeds were more in- hibited than seeds grown on the extract. The inhibition on meal was so great that many of the embryos of the fresh seeds could not free them- selves from the seed coats. This work suggests that we need to examine aging seeds of many sorts of plants to learn whether accumulation of inhibiting or toxic substances increases with age and leads to degenera- tion, or whether Schwemmle's findings apply to the particular seeds he studied due in part to their hybrid natiu-e. If accumulation of metabolic products leads to the death of long-stored seeds, the good storage condi- tions are those that reduce the rate of formation and accumulation of such products. Undoubtedly low moisture content and low temperatures will fulfill these conditions. Perhaps many seeds last longer soaked up in the soil than they do in dry storage, either because such intermediate products are not formed in imbibed seeds or because they diffuse out through the soil water. The later work indicates that the fall in the vitality of seeds in dry storage is due to a gradual degeneration of the nuclei ^' of the cells of the embryo, which results in disorder in the delicate mechanism of mitotic division. This work shows that aging, heating, and x-ray treatment of dry seeds all cause a similar degeneration.. Seeds partially injured by any one of these conditions produce seedlings showing an increased number of irregularities in chromosome distribution during mitosis, chromosome muta- tion, and an increasing number of mutants in the resulting plants. Nava- shin " found that fresh Crepis lectorum seeds produced plants in which only 0.1 per cent showed chromosome irregularities in mitosis, and a cor- responding percentage of mutants among the plants. On the other hand, more than 80 per cent of the plants grown from five-year-old seeds showed cytological mutations in the roots, and the seedlings grown from such seeds showed many abnormalities. Many of the plants died before they were large enough for transplanting, and others at later stages. Many albino plants appeared. Some of those that reached maturity were par- tially or wholly sterile. Peto " has found similar changes in plants produced from old maize grains, as well as in plants produced from barley grains subjected to high temperatures. By heat treatment he produced a tetra- ploid barley plant. Avery and Blakeslee ^ find that Datura seeds mutate much more slowly when buried in the soil than when stored in the labora- tory, just as they degenerate much more slowly, as mentioned above. In a summary of the literature, Goodspeed '^ shows that irradiation of plants causes chromosome and plant mutations very similar to those produced in seeds by aging or heat treatment. The literature is accumulating rapidly on the chromosome and plant mutations caused by aging, heating, and irradiating of seeds, and the results are in general agreement. Let us summarize briefly the effects these treatments produce, realizing of course that the degree of change increases with the aging under a given set of storage conditions or with LIFE SPAN OF SEEDS 63 the intensity of the treatment: (a) on the mitotic divisions of the embryos; (6) on the resulting seedHngs. The following are some of the chromosomal and mitotic modifications brought about by these treatments: fragmenta- tion of chromosomes with fragments attaching to other chromosomes oi- remaining unattached and not entering into the constitution of daughter nuclei; change in the number of chromosomes in the daughter nuclei, sometimes resulting in polyploidy; giant nuclei; two nuclei in one cell; globules of chromatin in the cytoplasm; and ring chromosomes. The aged, heated, and irradiated seeds showed the following changes in the resulting seedlings: polyploid plants or parts of plants; new forms of plants, some of them larger and more vigorous than the parents; slower germination; slower growth after germination; death of many seedlings in early stages; greatly increased number of chlorotic seedlings and sterile plants; as well as many other morphological abnormalities. In these treatments, as the nuclear or chromosome abnormalities increase, the morphological abnor- malities also increase. Aside from the delicate mechanism of nuclear division we should not forget that some of the most complex organic compounds enter into the make-up of the cell nuclei. These may decompose in storage and in turn upset the nuclear mechanism. Also it is possible that toxic or inhibiting substances accumulated in seeds during storage may upset the nuclear mechanism. The remarkable fact is not that the delicate nuclear mecha- nism or the complex compounds of the nuclei degenerate with time, but that for many seeds under good storage conditions they stay intact for centuries. Sure it is that in nature some plants are produced from old seeds, seeds that have lain in the ground for 10, 50, or in some cases, 100 years or more. Since aging seeds produce more and more mutations as they age, we have here one of the means by which nature produces new forms of plants, or carries forward evolution. This conception of the degeneration of seeds in storage has the virtue of concrete evidence in its favor; it localizes the significant changes in the nucleus and ties the change up with one of the most delicate cell mech- anisms, mitotic division. If this explanation of seed degeneration is cor- rect, then the best storage conditions for seeds are those that best preserve complex organic compounds of the nuclei and the mitotic mechanism of the embryo cells. 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Nobbe, F., "Ueber die HartschaUgkeit von Samen," Abhandl. Naturw. Bremen, 1890 : 289-294. 45. , "Untersuchungen iiber den Quellprozess der Samen von Trifolium pratense und einiger anderen SchmetterUngsbliitler," Landw. Versuchs-Stationen, 94 : 197- 218 (1919). 46. Nobbe, F., und H. Hanlein, "tJlDer die Resistenz von Samen gegen die ausseren Faktoren der Keimung," Landw. Versuchs-Stationen, 20 : 71-96 (1877). 47. Ohga, Ichiro, "The germination of century-old and recently harvested Indian lotus fruits, with special reference to the effect of oxygen supply," Am. J. Bot., 13 : 754— 759 (1926); also in C. B.T.I.,1: 289-294 (1926). 48. Ozorio de Almeida, A., M. D. Goulart, M. lelpo, and A. Vieira Pinto, "Estudo da agao inhibidora do suco de 'Solanum lycopersicum' sobre a germinagao de sementes e crescimento de plantas," Rev. Brasil Biol., 1 : 345-354 (1941); Abstr. in Biol. Abstr. 16 : 8009 (1942). 49. Rees, Bertha, "Longevity of seeds and structure and nature of seed coat," Proc. Roy. Soc. Victoria, 23(2) : 393-414 (1911). 50. Schjelderup-Ebbe, Thorleif, "Uber die Lebensfahigkeit alter Samen," Skrifter Norske Vidensk.-Akad. Oslo, Mat.-Naturvidensk. Kl, 1935 : 1-178 (1936); Abstr. in Biol. Abstr., 11 : 10520 (1937). 51. Schwemmle, J., " Keimversuche mit alten Samen," Zeitschr. Bot., 36:225-261 (1940). 52. Shuck, A. L., "A growth-inhibiting substance in lettuce seeds," Science, 81 : 236 (1935). 53. Stout, Myron, and Bion Tolman, "Interference of ammonia, released from sugar beet seed balls, with laboratory germination tests," J. Am. Soc. Agron., 33 : 65-69 (1941). 66 GROWTH OF PLANTS 54. Stubbe, H,, "Samenalter und Genmutabilitat bei Antirrhinum majus L. (Nebst einigen Beobachtungen iiber den Zeitpunkt des mutierens wahrend der Entwick- lung.)," Biol. Zentralbl., 55 : 209-215 (1935). 55. Thornber, J. J., "Some practical suggestions concerning seed germination," Timely Hints for Farmers No. 50, in Arizona Agric. Exp. Sta. Bull., 51 : 536-541 (1905). 56. Thornton, Norwood C, "Factors influencing germination and development of dormancy in cocklebur seeds," C. B. T. I., 7 : 477-496 (1935). 57. Turner, J. H., "The viability of seeds," Kew Bull. Misc. Inform., 1933:257-269 (1933). 58. Ullman, Salomon Baruch, "On germination inhibitors. V. Essential oils, alkaloids and glucosides as inhibitors of germination and growth," Ph.D. Thesis, Hebrew Univ., Jerusalem, 30 pp., 1940. 59. Waggoner, H. D., "The viabihty of radish seeds {Raphanus sativus L.) as affected by high temperatures and water content," A?n. J. Bot., 4 : 299-313 (1917). 60. Walti, A., "Activation and inhibition of germination of lettuce seeds," Am. Chem. Soc. Div. Biol. Chem. Absts. papers, 96th meeting, Milwaukee, Wise, Sept. 1938, pp. B20-21. 61. Wiesehuegel, E. G., "Germinating Kentucky coffee tree," J. Forest., 33:533-534 (1935). CHAPTER 3 Dormancy in Seeds Significance op Dormancy in Seeds In the previous chapter we showed that seeds of many wild plants will lie in a germinator or in moist soil for years without germinating, or in cases where some germinate, the germination of a single planting of a given crop may spread out over a period of years, with now and then a few seeds germinating. In short, in seeds of wild plants, delayed or distrib- uted germination is very common both in a germinator and with nature in the soil. While this is markedly true for seeds of wild plants, it is also true to a lesser degree of seeds of many cultivated plants. In the latter, the dormancy is often transient and extends over a period of days or weeks rather than years. The seeds of some cultivated plants do have a long period of dormancy. This is true of some leguminous and other hard-coated seeds that are slow to absorb water. With the knowledge available during the last part of the past century the explanation of dormancy and delayed germination of seeds was much simpler than it is today; and it was correspondingly more vague. Hard- coated seeds, of course, did not germinate because they did not absorb water. Seeds that absorbed water and did not germinate were supposed to need some special stimulus besides the three conditions ordinarily thought necessary for germination — proper temperature, oxygen, and water supply. The stimuli were supposed to act promptly and to give immediate germination. This was thought of as a release response, and workers sought for stimuli that gave such immediate release. True, during the latter part of the last century, many workers showed that light was necessary for the germination -" of some seeds and accelerated the germi- nation of others, and early in this century some seeds were found to be prevented from or hindered in their germination by light. Since the response to light is rather quick and the light need not act for a long time, it was interpreted as a stimulus or release response. Now it seems prob- able that even light brings about biochemical or biophysical changes that lead to germination, and that its effect can be interpreted on the basis of definite chemical and physical changes rather than on the basis of the vague conception of release or stimulus response. We have already seen how long some seeds must be held in a low- temperatiu'e germinator to after-ripen the dormant embryos, and also in a high-temperature germinator with restricted oxygen pressure in order 67 68 GROWTH OF PLANTS to induce dormancy in embryos. Later we shall discuss some of the physi- cal and chemical changes occurring in embryos during after-ripening and during dormancy induction. As we shall see later, many other changes involved in overcoming dormancy also take much time and involve kno^vn biochemical changes and, like those mentioned above, can be interpreted on the basis of definite physical and chemical changes rather than by the vague term "stimulus." Later in this chapter under the topic "Categories of Dormancy" we shall point out many ways in which nature maintains dormancy in a moist soil, and several means by which seeds are throAvn out of dormancy. Contrasted mth the old explanations of seed dormancy caused by hard- coatedness on one hand and lack of a release stimulus on the other, the new data and explanations afford a much greater richness of concept and precision of conclusions. Advantages to Plants of Delayed Germination Delayed germination is advantageous to many wild plants in nature. It carries the plant over the winter in the seed stage and the young plant grows in the spring. In the Dakotas, Minnesota, Saskatchewan, and ad- joining regions wild oats are a bad weed because the grains are suffi- ciently dormant to carry over intact until spring, when they germinate. Cultivated oats and false ^vild oats are not troublesome weeds in these regions because the dormancy of these grains is so temporary that the grains after-ripen and germinate in the fall and the cold winter kills the seedlings. In the previous chapter we learned that seeds of some \vild plants lie dormant in the soil for years, or even centuries, and germinate only when the soil is stirred up or burned over. This is no doubt helpful in the persistence of the species. Advantage to Man of Dormancy in Seeds It would be a calamity for mankind if all at once seeds of cultivated plants ceased to have at least a temporary dormant period. Mangels- (JQpf 79, 80 found a number of types of maize, produced either by inbreed- ing or by crossing, in which the grains had no dormant period, but instead the embryos continued to grow in the green ear and to form seedlings. Professor William H. Eyster ^^ has kindly furnished an illustration of such an ear of corn which is shoAvn in Fig. 2L Pope and Brown ^^ forced young embryos in heads of normally very dormant varieties of barley to continue to enlarge and form seedlings in the green head by placing moist filter paper on the embryo portion of the immature grain. Apparently water deficit in the green head is a factor in inducing dormancy. Fig. 22 shows viviparous heads of barley, the photograph for which was very kindly supplied by the authors. Evidently appearance of dormancy in grains of cereals, or its failure to appear, is determined either by genetic characters, as shown by Mangelsdorf and by Eyster, or by conditions of DORMANCY IN SEEDS 69 Figure 21. An ear of maize homozygous for vivipary showing the viviparous embryos in various stages of development. {Photograph furnished by Dr. William H. Eyster, Botanical Laboratory, Bucknell University, Lemsburg, Pa.) Figure 22. Viviparous seedlings in the spikes of one spring and three winter varieties of barley. 1, Manchuria (C. I. 2330) 6 rowed, white, spring; 2, Dentil (C. I. 1260) 2 rowed, white, winter; 3, Abyssinian (C. I. 1222) 6 rowed, black, winter; 4, Meliton (C. I. 1456) 2 rowed, black, winter. {Photograph furnished by Drs. Merrill N. Pope and Edgar Broum, U. S. Dept. Agriculture, Bellsville, Md.) 70 GROWTH OF PLANTS growth, as shown by Pope and Brown. If seeds of all cultivated plants failed to have a dormant period as in this illustration, man and domestic animals would be without any food in the form of dry seeds and grains, which form a large proportion of the food of both. Such a failure of dor- mancy would also bring about a great disturbance in plant propagation by making difficult or eliminating propagation by seeds. It is a common experience that during rainy weather grains germinate in the shock. Different varieties and strains of cereals vary a good deal in the length of dormancy of the grains i^, as. ^Iso the weather conditions during ripening produce considerable variation in length of dormancy. Length of dormancy of the grains ®- is one of the factors being studied in breeding desirable new varieties of wheat. Grain dormancy will become less and less significant as the combine more and more displaces the binder in harvesting, for the grains seldom retain enough water to germinate while still standing unharvested in the field. Recently the swather has been introduced. It leaves the grain in a swath to dry and is followed after drying by the pick-up combine. Here again a period of dormancy will be important in preventing germination in the swath in case of rain. Seed Dormancy Inconveniences Man Dormancy in seeds causes man a great deal of inconvenience in at least three ways. (1) Seeds of species of plants that are persistently dormant need pretreatment to induce after-ripening, so that they will come up promptly and all at the same time when planted. As we shall see later, it takes a long tune to after-ripen some seeds. In some cases two or more very different sets of conditions must be used for after-ripening, and in others two long-time, low-temperature treatments must be applied to after-ripen the seed and later to after-ripen the epicotyl. Until one knows the conditions necessary for after-ripening any particular sort of dormant seed, he may not be able to produce any seedlings; and after he knows how, he may have a two-, three-, or four-phase job in producing seedlings. These difficulties are met in their most complicated form when one under- takes to grow certain wild flowers. (2) We have seen that some weed seeds lie in the ground for 60 years, dormant and still capable of producing plants. Fighting weeds is one of the heavy tasks of both the farmer and gardener. It is probable that if a piece of land were cultivated so thoroughly that no weed seeds matured, and weed seeds were prevented from coming in from outside, the land could be freed of all weeds far short of 60 years, because cultivation stirs up the dormant seeds and throws them out of dormancy. (3) In farm and garden operations it is customary to test seeds in advance of planting to be sure that only viable and vigorous seeds are sowed. Even winter cereals are sometimes sufficiently dormant to give trouble in testing previous to fall sowing. Harrington '^^ found that dor- mant cereal grains could be hastened in germination by various means DORMANCY IN SEEDS 71 such as using 12° C (54° F) germinators instead of 20° C (68° F) or higher, by breaking the grain coats near the embryo, by using high oxygen pres- sure, etc. Grains are more persistently dormant when they ripen during rainy weather. The Germans repeatedly report difficulty in converting "rain barley" promptly into malt because of delayed germination of the grains. Even for many seeds that have very long dormant periods, includ- ing those ^\dth dormant embryos, there are means of making prompt vitality and vigor tests, as we shall see later in the discussion of quick vitality tests. Categories of Dormancy Now let us consider the types of dormancy in seeds based on the mech- anism by which the dormancy or delayed germination is secured. As we shall see, nature has several devices for securing delayed and distributed germination of seeds. Hard Coats We have already shown the extreme importance of hard-coatedness in seeds in increasing their life span in storage as well as in the soil. Hard coats maintain a low constant moisture content in the embryos — an effec- tive storage condition — by hermetically sealing them individually. There are several families, some species of which produce seeds that will not absorb water. Leguminosae ^^ are outstanding in this respect, but the Malvaceae, Cannaceae, Geraniaceae, Chenopodiaceae, Convallariaceae, Convolvulaceae, Solanaceae and other families have species that bear hard seeds. Hard-coatedness is primarily determined genetically, but the appear- ance or degree of hardness is also modified by environmental factors. In white sweet clover "^ either hard or soft strains can be developed by selec- tion and inbreeding. The same is true of hairy vetch.^^ On the other hand, the author has observed at Yonkers that more than 98 per cent of hand-hulled, white sweet clover seeds are hard when they ripen during hot, dry weather and that 100 per cent are soft when they ripen during rainy weather. Many investigators agree that the outside layer of cells of the coats which are palisade in form prevent the entrance of water, and some claim that, of this layer of cells, only the outer half or the portion outside the "light line" is impermeable. This layer of cells in Nelumbo was illustrated and described in the previous chapter. White ^^s claims that in small leguminous seeds the cuticular layer over the palisade cells determines the impermeability, while in the larger leguminous seeds the outer portion of the palisade cells is involved as well. The question has also been raised whether the physical character or chemical composition of this thin layer gives it the remarkable resistance to water absorption. Raleigh ^® con- cludes that as the seeds of the Kentucky coffee tree harden in ripening, pectic substances change into water-resistant substances. Shaw ^"^ germi- 72 GROWTH OF PLANTS nated American lotus seeds by treating them with acetone and then plac- ing them in water. She assumed that fat-like substances were dissolved out of the stomatal cavities, which extended deeper than the palisade layer, allowing water to enter. Ether as the solvent made the coats per- meable also, but killed the embryos. Shaw also maintains that the palisade cells are impervious throughout their length. Hamly ^^ speaks of suberin caps over the palisade cells as causing the water-resistance in sweet clover seeds. McKeever " found that treatment of black locust seeds for 10 to 120 minutes Avith several wax solvents (ether, xylene, acetone, and others) was effective. All removed consider- able amounts of wax. The main effect seems to be hastening the germi- nation by 10 or 15 days rather than increasing it after 27 days. Very early Hohnel ^^ claimed that soakmg yellow lupine seeds in ether with immedi- ate transfer to water softened the coats. Perennial lupine seeds did not respond to this treatment. Verschaffelt,^26 working mainly with hard honey locust seeds, found that placing them in ethyl alcohol and trans- ferring them immediately to water led to swelling. He assumed that the alcohol filled the rifts or interstices in the hard coats and furnished a channel by which water could travel to the deeper layers of the coats. Other simple alcohols were effective, but higher alcohols, ether, and other fat solvents were not, because water is not sufficiently soluble in them to reach the deeper layers; also some of them failed to fill the rifts in the seeds. Many hard seeds of Caesalspinaceae and Mimosaceae acted like honey locust seeds, but alcohol was less effective with Papilionaceae. The first two groups have minute rifts all over the surface of the seeds, while the latter has one rift at the hilum. According to Verschaffelt's interpre- tation, the alcohol does not act as a wax or fat solvent but as a bridge for conveying the water to the deeper layers of the coats. Some regions of the hard seed surface seem to be rendered water- permeable more easily than others. Hutton and Porter ^^ showed that dry, hard seeds of Amorpha and Lespedeza, when shaken in a bottle, become water-permeable at the hilum. Hamly ^^ found that hard Meli- lotus seeds were made water-permeable by moderate heating or mechanical impact by producing a rift at the strophiole. This long and much worked problem of what physical or chemical characteristics make hard seed coats resistant to entrance of water evidently still needs thorough research atten- tion. The chemical or physical method of water-proofing may vary with different kinds of hard seeds. This is made probable by the contradictory explanations offered above; also we must remember that little attention has been given to the mechanism of hard-coatedness in several of the families of plants. In some, not even the layer of the coat involved is known. There is another reason for learning the mechanism or mechanisms of hard-coatedness in seeds. Hard-coatedness is the world's best example of highly effective water-proofing by thin layers. Man can well devote some DORMANCY IN SEEDS 73 time to learning the method or methods of this water-proofing in the hope of applying it to his needs. The seeds of any crop vary in perfection of water-proofing, as we have already seen. This insures time-distributed germination of any crop. The embryos of most kinds of hard seeds germinate readily and grow vigorously when the hard coats are broken and germination conditions furnished; but the embryos of redbud, Cercis canadensis, need several weeks' after-ripening in a low-temperature germinator or stratification in the swollen condition to prepare them for growth.^ The embryos of hard seeds of the beach pea, Lathyrus maritimus, grow much more promptly after the seeds have a period in dry storage.^^ In red clover seeds Saulescu ^"^ finds that percentage hardness increases with diminution in size and with darkness in color from yellow to purple. Grimm ^^ finds the same size-hardness relation for several clovers, but believes that lighter-colored seeds have a higher percentage of hard seeds. Lespedeza stipulacea seeds ^^ show the same relations between size and hardness. In alfalfa seeds ^^ percentage hardness seems to decrease with range in color from bright yeUow to green to brown. Hardness in Vicia saliva seeds ^^ increases with depth in color. In commercial alfalfa and clover seeds the percentage of hard seeds varies greatly with varieties, con- ditions during ripening and storage, and with conditions in machine- hulling such as closeness of cylinder and concave, and moisture content. The persistence of hardness ^^' "''• ^^^- ^^^ increases in the following order: alfalfa, red, white, and alsike clover. With spring sowing, hard alfalfa seeds swell and germinate mostly the first season with few or none carry- ing over. In the clovers some seeds carry over until the second spring and a few still longer. In storage, hard alfalfa seeds ^^ soften faster than the clover seeds, and low temperatures and high moisture favor softening during storage. Lupines ^^ and hairy vetch seeds ^^^ become hard in high-temperature, low-humidity storage, and soften in low-temperature, high-humidity stor- age. Even a few days in dry laboratory air increases the degree and per- centage of hardness in yellow and blue lupine seeds. ^- Careful attention must be given to storage condition of these seeds during the ^vinter to avoid hard-coatedness and failure to germinate when soun in the spring. Temperatures at the freezing point or lower favor softening of hard clover and alfalfa seeds whether wet or dry. 5°- ^^^ ^^ Busse ^^ found that freezing dry hard seeds of sweet clover and alfalfa to — 190° C (— 310° F) softened the coats \vithout injury to the seeds. Even repeated freezing at this temperature did not injure dry alfalfa seeds. High temperatures, 60° C (140° F) for 2 hours or 75° C (167° F) for 0.6 hour," soften hard alfalfa seeds. Both high and low temperatures are probably factors in softening hard seeds in the soil. This is especially true of low and variable tempera- tures during the winter. High hydraulic pressures "■ 24. 99 have been used to soften hard coats of 74 GROWTH OF PLANTS seeds. The pressures used varied from 20 to 60,000 pounds per square inch. Higher pressures softened the coats but injured the seeds. Lower pressures softened the coats without injury. Small seeds required higher pressure to soften the coats and also endured higher pressures without injury. In the soil such factors as weathering, bacterial and fungal action, and abrasion of agricultural instruments modify hardness. Several methods have been developed for overcoming hard-coatedness in seeds before sowing. Soaking the seeds in hot or boiling water has been long used. In the nineties Rostrup,^'"' a Swedish botanist, discovered that the outside layers of the hard coats could be eaten away with concentrated sulfuric acid followed by thorough washing to remove all acid. The lengt,h of time for either of these treatments varies greatly with different kinds of .seeds and to a degree with different crops of the same kind. This is especially true of the sulfuric acid treatment. Many different sorts of scarifying machines have been invented and used commercially. In such machines the seeds are thrown against sandpaper, needle points, etc., to scratch the hard outside surface of the seeds. Some of these have proved useless, either because the impact broke the embryos or injury laid the seeds open to infection. Porter and Brown ^^ have shown that shaking hard, black locust seeds in a bottle for 20 minutes makes them water- permeable. In small-seeded commercial leguminous seeds one will gener- ally find a much smaller percentage of hard seeds in those threshed by a mechanical huller than those hulled by hand. Even rubbing the seeds through a sieve to get rid of the hulls softens some of the seeds. In short, the threshing machine acts as a more or less effective scarifier. We have discussed only a portion of the important data on factors that induce or overcome water-impermeability of hard seeds in practice or in nature as well as in storage and in the soil; but space does not permit a fuller discussion. Light as a Factor in Dormancy Some seeds require light for germination and many others are favored by hght, while other seeds are completely or partially inhibited in their germination by light. Light-favored seeds may remain dormant when covered by soil to such a depth as to exclude the necessary light, or light- inhibited seeds may fail to germinate if they are sown with little or no cover. The first type of seeds should be sown on or near the surface of the soil or otherwise treated to overcome the light need, and the second type should be sown deep enough to prevent the inhibiting effect of light. Relatively little time can be given to this topic. Consequently, the author will quote a summary of an article which he wrote in 1936 en- titled "Effect of the visible spectrum upon the germination of seeds and fruits" 2°' p-^2°~^22 and published in the two- volume treatise "Biological effects of radiation."* This quotation will be supplemented by a brief * McGraw-Hill Book Co., Inc., New York, N. Y., 1936. Permission to quote this material is gratefully acknowledged. DORMANCY IN SEEDS 75 statement concerning a few of the more recent and more significant con- tributions to the subject. " (A) Light favors the germination of a large number of seeds and fruits. Among these are Viscum album together with many other Loranthaceae and epiphytes, all Gesneriaceae studied to date, many grasses, various species of Oenothera and Epilobium, Ranunculus sceleratus, Lythrum salicaria, and L. hyssopifolia. Viscum album and Arceuthobium oxycedri will not germinate at all without light. The former is killed in darkness within a few weeks, while the latter endures darkness for a longer period. Of 964 species of seeds studied by Kinzel, 672 or about 70 per cent were favored by light under the [very limited] conditions used in his experiments. " {B) Light interferes with the germination of many seeds and fruits. Among these are several species of Phacelia and other Hydrophyllaceae, 3 species of Nigella, several species of Allium and most other Liliaceae. Of 964 species of seeds and fruits tested, Kinzel found 258 inhibited by light under the conditions of his experiments. "(C) Some seeds and fruits germinate equally well in light and dark. This is true of the small grains, Zea mays, beans, clover, and many other legumes. Of the 964 species investigated by Kinzel, 35 were indifferent to light. " (Z)) Several conditions partly or entirely displace the effect of light in light-sensitive seeds and fruits. "(a) After-ripening in dry storage reduces or entirely eliminates the need for light in various light-favored seeds. Pea achenes [caryopses] kept in dry storage for one year germinate almost as well in darkness as in light. After-ripening partially eliminates light need in Chloris, Ranunculus, Epilobium, and Oenothera achenes or seeds. The inhibiting effect of light on Phacelia seeds falls with period of dry storage. "(b) Seed or fruit coats, or the hulls of grasses, increase the necessity for light in the germination of some light-favored seeds. The hulls render Chloris achenes light-obligate and increase the need for light in Poa. Pricking the seed coats of Oenothera increases germination in darkness. The coats also modify the action of light on light-inhibited seeds. Removal of the seed coats from Phacelia seeds overcomes the inhibiting effect of light. Pricking the coats causes 'lichthart' seeds of Nigella to germinate in part. "(c) A full atmosphere of oxygen forces the light-obligate Chloris achenes with hulls intact to full germination in darkness, and the light-inhibited Phacelia seeds to full germination in light. "(d) Knop's solution substitutes for light in a number of light-favored seeds. The nitrate of the solution is effective. The other salts of the solu- tion are not effective. Nitrites, nitric acid, ammonium salts, and urea are also favorable. Nitrates entirely displace the light need of Chloris achenes with hulls intact at temperatures above 22° C. They also increase greatly the germination at temperatures below 22° C, where light inhibits. Nitrates 76 GROWTH OF PLANTS favor the germination of the following light-favored fruits and seeds in darkness: Poa, Ranunculus, Epilobium, Lythrum, and the Gesneriaceae. The light-inhibited seeds of Phacelia and Nigella are not favored by nitrates. " (e) Weak acids substitute for light in part in the light-favored seeds of Lythrum, Scrophularia, Verbascum, and Epilobium. " (/) Either daily intermittent or high constant temperatures substitute for light in various light-favored seeds. The most favorable intermittent temperatures give better germination of Poa achenes than light with any constant temperatures. Light and nitrates increase the germination of Poa compressa achenes somewhat at the most favorable intermittent tem- peratures. Intermittent temperatures replace light with after-ripened Chloris achenes with hulls intact, but not with non-after-ripened or ' dunkel- hart' achenes. With seeds of Epilobium, Oenothera, and others intermittent temperatures substitute fully for light. "(£■) When light-favored achenes of Chloris are kept for a time in a dark germinator, they are changed in a manner that makes them inca- pable of germination later even in hght. Such seeds are said to be ' dunkel- hart.' 'Dunkelhart' achenes can be forced to germinate by breaking the coats, increasing oxygen pressures, and other treatments. When light- inhibited seeds of Nigella are kept for a time in a light germinator at a temperature above 20° C, they are changed in such a manner that makes them incapable of germination later even in darkness. Elinzel spoke of such seeds as 'lichthart.' 'Lichthart' seeds can be forced to germinate by breaking the coats, or still better by other treatments. Imbibed Phacelia seeds also become 'lichthart' when exposed to light. " {F) If imbibed Ranunculus sceleratus seeds are exposed to light, dried, and later placed in a dark germinator \vith intermittent temperatures, they still show the favorable effect of the light exposure. Chloris achenes also show this latent light effect. Since the light exposure of the seeds during ripening in the capsules varies mth the weather, the rate of dry- ing of seeds in the capsules, and the position of the capsule on the plant, Wieser concluded that the latent light effect may account in part for the great variation in the amount of light required for the germination of different collections of the same species of light-favored seeds. " (G) Several theories have been offered to explain the favoring or in- hibiting action of light upon the germination of seeds and fruits. Most of these theories postulate that the action of the light is upon the living endosperm or embryo, but some of them assert that the action is upon the non-living coats. None of these theories has adequate evidence for even a single species of seeds. It is not improbable that light has its effective action upon the endosperm and embryo of some seeds, upon the coats of others, and upon both in still others. There is need of a very thorough and detailed chemical, microchemical, and physiological study of the effect of light upon the coats and living portions of several light-favored and light-inhibited seeds and fruits. There is also need of a similar study of DORMANCY IN SEEDS 77 the changes brought about in seeds and fruits by agents and conditions which substitute for light." The recent findings of FUnt and associates 45. 46, 47. 48 qj^ ^^j^g efifect of different portions of the spectrum on the germination of lettuce seeds are of great interest. These findings deserve special consideration because of the excellence of technique on which they are based. In batches of freshly imbibed lettuce seed that required light for germination, a few seconds' exposure to light induced germination. The sensitiveness approached that of a photographic plate. The region 5200 to 7000 A (red, orange, and yellow) was stimulative, mth the critical wave length at about 6600 A; the region 4200 to 5200 A (green, blue, and violet) was inhibitive, with the critical wave lengths at about 4400 and 4800 A; and the band 7000 to 8600 A (mainly infrared) was even more inhibitive, with the critical wave length at about 7600 A. The critical regions for inhibition of germination in the visible spectrum are about the same as those for induction of photo- tropic curvature and for the inhibition of growth of plant organs. The critical inhibitive region in the infrared was not associated with assimila- , o tion or temperature effects, and 7600 A did not induce phototropic response in lettuce seedlings. Keeping seeds in a dark germinator at 5° C (41° F) for several weeks did not modify then- sensitiveness to light; but keeping them in a dark germinator at 25° C (77° F) for 24 hours did so alter their sensitiveness to light that they would not respond to standard illumina- tion. As we shall see later, a germinator at 5° C (41° F) is an excellent condition for after-ripening many dormant seeds, while a germinator at 20° to 25° C (68° to 77° F) maintains many dormant seeds in status quo. It is well established that many fern spores require light for germina- tion. Orth *^ finds two groups of fern spores in respect to their response to light: those that germinate in various bands within the region 550 to 710 m/i, and have brown exospores, and those that germinate as above and also in ultraviolet light, and have colorless exospores and much caro- tene in the cells. Within the generally favoring band there are hindering and favoring regions. In the short end of the spectrum various bands of inhibiting rays appear. The same is true in the infrared around 800, 1000, and 2400 m/i. Fern spores germinate in light that passes through green leaves, unlike light-sensitive seeds. This is due to the strongly favoring action of green-yellow overcoming the inhibiting action of the infrared. Because of the several bands of favoring or inhibiting action in the spectrum, Orth concludes that the action of light on germination of fern spores cannot be explained on the basis of quanta, as Kommerell ^^ has attempted to do for seeds, but on the basis of the specific effects of various bands of the spectrum on the spores. Flint's results on lettuce seed would seem to justify the same conclusion for seeds. Raleigh ^^ showed that thiourea forced the germination of dormant lettuce seeds (Lactuca saliva) in darkness, and in L. Serriola it increased the germina- tion in both light and darkness. 78 GROWTH OF PLANTS Muenscher ^^ shows that Ught is necessary for the germination of Lobelia inflata and that other factors will not substitute for light. These seeds gave no germination when covered with 1 cm. of soil. L. cardinalis and L. siphilitica seed also require light, while L. tenuior and five forms of L. Erinus seed germinate equally well in light and dark. Funke *^ has recently confirmed the findings of Wiesner and others on the role of light in maintaining the life and inducing the germination of Viscum album seed. The seeds are injured by two days of continuous dark. Most rapid germination is produced by removing the endosperm and subjecting the embryo to continuous illumination, artificial light at night, and sunlight during the day. The failure to germinate in Belgium during the winter, he feels, is due to low light intensity and daily duration rather than low temperatures. Sprague ^'* further confirms the fact that dry storage overcomes the need for light; Poa pratensis seed six months after harvest no longer required light or alternating temperature for germination. Jensen ^^ sug- gests that exposure of seeds to artificial light lengthens their viability in dry storage. Various workers ^^' ^^' *^ have shown that certain light rays modify enzyme content, metabolism, and growth substances in germinat- ing seeds, but in no case does this work explain how light induces or hinders germination. Oxygen Deficiency and Dormancy Growhig plants with their intracellular aeration systems connected with stomates and lenticels are much better equipped to get the needed oxygen supply from the air than are embryos of seeds which are, in the main, completely sealed within seed coats and often additionally covered with fruit coats and other structures. Using common cultivated species of plants (common bean, broad bean, cress, savory, and Hydrangea) in a special growing chamber, Schaible ^"" grew the plants and germinated the seeds in one-fourth of an atmosphere of pressure with a continuous change of the atmosphere; in one case he drew air through the chamber, thereby giving one-fourth the partial oxygen pressure of a full atmosphere of air; and in the other he used oxygen- enriched air so that the partial oxygen pressure in the chamber was equal to that in a full atmosphere of air. Plants in the reduced pressure grew much faster than plants in a full atmosphere, regardless of whether the partial oxygen was normal or one-fourth normal. The atmospheric pres- sure determined the rate. Seeds germinated a little better in the reduced pressure of oxygen-enriched air than in a full atmosphere of air, but very much worse in the reduced atmosphere without oxygen enrichment. In other words, the oxygen content of the air is far above that needed for the fastest growth of plants, but it is not so far above that needed for the germination of seeds. The latter is conditioned by the slow passage of oxygen through the seed and fruit coats. DORMANCY IN SEEDS 79 Brown ^^ estimates from his experiments with barley grains that embryos in partially imbibed grains floating on water are in equilibrium with 10 per cent of oxygen, whereas excised embryos are in equilibrium with the full percentage of oxygen of the air. The rate of uptake of oxygen and release of carbon dioxide was much higher in the excised embryos. This no doubt is partly due to the higher water content of the excised embryos as well as to higher oxygen pressure. Brown ^^ has also shown that the imbibed seed coat of Cucurhita pepo permits carbon dioxide to diffuse through it several times as fast as oxygen. StMfelt "^ shows that the amount of oxygen taken up by white mustard seeds in a germinator is increased if the partial pressure of oxygen is increased from 20 to 50 per cent. The increase is much greater for the cotyledons than for the root. The researches just cited, as well as a number of others that might be cited, show that some imbibed seeds are limited in their use of oxygen from the air because of the low permeability of the coats to oxygen. Are there cases in which seeds stay dormant because oxygen does not pass through the coats sufficiently fast to permit germination of the embryos even when they have good aeration? If this is true, two conditions must be fulfilled in such seeds: (1) the embryo must have a certain oxygen pres- sure demand in order to grow, and (2) the low permeability of the coats must limit the oxygen pressure to the embryo below the minimum neces- sary for growth. It is well established that embryos of various kinds of seeds vary greatly in the oxygen pressure or oxygen supply needed for germination. One might expect seeds of water plants that normally germinate under water to have low oxygen requirement for germination. Crocker and Davis -^ found that embryos of seeds of Alisma Plantago, with coats broken, will germinate in absence of oxygen. They heated the seeds in water in special flasks at 35° C (95° F) for 30 minutes under reduced air pressure of 0.1 mm of mercury and sealed the flasks at this temperature and vacuum. In the vacuum cultures in water the embryos grew in length 1100 to 1200 per cent in 21 days, while in the checks in water the growth in length was 1800 to 2200 per cent. In the vacuum cultures no leaf branches and no chlorophyll formed. Both developed in the controls. About 5 mm of air pressure were required for chlorophyll formation, and more than 5 cm of air pressure for leaf differentiation. While germination occurs in absence of oxygen, the growth is limited and differentiation and chlorophyll forma- tion do not occur. Takahashi ^^^ claims that the plumule in rice, another water plant, will grow in absence of oxygen, but the root will not. Taylor ^'^ determined the effect of various oxygen pressures on the germination of rice, as a water plant, and wheat, as a land plant. He says (p. 736): "In the absence of O2 the germination of rice seeds was reduced less than 1 0 per cent below that in air and was accomplished at more than half the normal rate. No germination of wheat occurred under similar conditions. Significant reduction in the extent and rate of germination of wheat 80 GROWTH OF PLANTS occurred when the O2 tension was lowered to 5 per cent, and considerably less than half of normal germination resulted in O2 concentrations below 1 per cent. At O2 tensions of 5 per cent or less, rice seedlings made approxi- mately twice as much growth as wheat seedlings (on the basis of increment in dry weight of embryo in air). Reduction in O2 tension inhibited roots of both seedlings more than shoots, and it had least effect on rice shoots." He attributes the ability of rice seeds and seedlings to grow in very low oxygen tension to a well-developed anaerobic, energy-liberating fermenta- tion system within the seed, which is lacking in wheat; in rice the energy liberation in absence of oxygen was 3^ that in air and in wheat K5. In both grains root growth was more limited by low oxygen pressure than plumule or stem growth. Morinaga^'' made a study of the germination of many seeds of land plants under water. He listed 34 species that will not germinate under tap water, 25 species that germinate better on moist filters than under water, 18 that germinate well under water, and 21 that germinate under boiled water sealed with paraffin oil. In none of these was there total absence of oxygen, for the seeds themselves contained some oxygen within the intracellular spaces and even the boiled, paraffin-sealed water permitted some diffusion from air. Among the seeds germinating in boiled water were: timothy, Bermuda grass, Canadian blue grass, lettuce, wormwood, celery, alfalfa, and Petunia. From this work it is evident that oxygen requirements for germination vary greatly even among seeds of land plants. Now let us consider seeds that will not germinate in full air pressure because the low permeability of the coats to oxygen reduces the oxygen supply to the embryo below that needed for growth. From all that has been said above, such species of seeds might be thought to be rather rare, and they probably are. It is a different story, however, when the restric- tion offered by coats is further increased by environmental factors, such as submersion in water, in water-logged or packed soil, or in soil rich in carbon dioxide. The best understood case of seed coats restricting the oxygen supply to the embryo below the minimum needed for germination is the cocklebur. Farmers had claimed that one of the two seeds in the cocklebur germinates the first season after maturity and the other the second season. Arthur ^ investigated this claim of the farmers and in the main confirmed it, although in some cases both seeds in a bur germinated during the first season and in some burs the second seed did not germinate until the third or a still later season. He found that the two seeds differed in their size, shape, and position in the bur. One was borne higher up in the bur, was smaller and convex on its outer face and concave on the inner face. He termed this the "upper seed." The other was borne lower in the bur, was con- cave on the outer face and convex on the inner face, and was called the "lower seed." The lower seed is the one that germinates in the first season DORMANCY IN SEEDS 81 after maturing. Both seeds absorb water readily, and neither the bur nor the old dried ovary wall seems to play any considerable part in the delay. Fig. 23 shows the bur, the two seeds, and the arrangement of the seeds in the bur. Without evidence except that enzyme differences were used to explain many plant responses, Arthur concluded that enzymes were more abundant or developed faster in the lower seed, and consequently it germinated more promptly. Figure 23. The cockle- bur. Upper row: upper seed. Middle row: intact bur, cros.s section of bur showing the two seeds, longitudinal sec- tion of the bur showing the two seeds. Lower row: lower seed. Crocker ^^ attempted to explain this delay in the germination of the upper seed of the cocklebur. He observed the following facts: Germina- tion failure of the upper seed, with the coat intact and either in or out of the bur, is due to the seed coat and not to the bur or ovary wall; this is true in spite of the fact that the three-layered seed coat is very thin, about 0.034 mm at the cotyledon end and 0.145 mm at the radical end of the seed. When the excised seeds or the seeds in the bur are placed in a germi- nator in air at 22° C (71° F) the lower seed only germinates. When placed in a germinator \\\ih a full atmosphere of oxygen at 22° C (71° F) both seeds germinate, but the growth in the upper seed starts in the cotyledons where the coat is thin. When placed in a germinator at 33° C (91° F) with air, both seeds germinate. When the seed coats are removed, both embryos germinate promptly, even at 18° or 20° C (64° or 68° F). Prick- ing the coat of the upper seed with a pin causes it to grow in a germinator at 20° C (68° F) in air, but the growth starts in the region of the prick. From these results Crocker ^^ concluded that both seeds would germinate in the first season after harvest if the seed bed reached a temperature as high as 33° C (91° F) at a time of adequate water and air supply; that the failure of the upper seed to germinate at lower temperatures was due to the fact that the thin seed coat reduces the supply of oxygen to the embryo below the minimum needed for germination ; and that the oxygen supply to the embryo of the lower seed was restricted by the coats, for it too germinated at a somewhat lower temperature when the coat was removed. Naturally, one inquires why the upper seed germinates during the second or later seasons. Crocker suggested that the delicate semiper- 82 GROWTH OF PLANTS meable membrane is slowly decomposed by organisms in the soil. In other types of seed dormancy to be discussed later, we shall see that microorgan- isms in the soil play an important part in eliminating coats as factors in seed dormancy; moreover, Thornton ^'^° shows that the seed coat of Xan- thium is rendered permeable by very slight injury. Whether the higher temperature increased the permeability of the coats to oxygen, or reduced the oxygen supply or pressure needed for the germination of the embryo of the upper seed and thus forced the germination, was later partly an- swered by Shull and still later by Thornton. o z 5 80 60 40 20 < o UJ a. 60 40 20 EMBRCO OF LOWER SEED AT ^I'C EMBRYO OF UPPER SEED AT 21" C EMBRYO OF LOWER SEED AT 30° C EMBRYO OF UPPER SEED AT 30' C 0.2 0.b 0.6 0.7 0.8 0.9 LO OXYGEN. PERCENTAGE BY VOLUME 1.2 Figure 24. Minimum oxygen required for germination of the naked embryos of cockle- bur during six days at 21° and 30° C (70° and 86° F). Shull,!"-'' 1"^ using Schaible's method of reduced atmospheric pressure, determined the minimum O2 pressures under which the naked embryos of the upper and lower seeds of Xanthiuvi would germinate at 21° and 31° C (70° and 88° F); for the upper embryos it is 12 mm at 21° C (70° F) and 7 mm. at 31° C (88° F) and for lowers 9.5 and 3 mm at the respective temperatures. The thin coats are extremely effective in reducing the O2 absorbed by the embryos in upper seed at 21° C (70° F); 12 mm of O2 pressure is required with naked embryos and 760 mm, or 63 times as much, for the intact seeds. Naked lowers as against intact lowers absorb 2)4 times as much O2 and naked uppers 5 times as much as intact seeds. Thornton, 120 using full atmospheric pressure with reduced or increased DORMANCY IN SEEDS 83 percentages of O2, determined the percentage germination of naked embryos and intact seeds of both seeds of the cocklebur at 21° and 30° C (70° and 86° F) at a range of O2 percentages. Upper naked embryos (Fig. 24) at 21° C (70° F) require 1.5 per cent and at 30° C (86° F) 0.9 per cent O2 for 100 per cent germination; lowers under like conditions require 0.7 and 0.5 per cent O2. Note that complete germination of the lower embryos occurs at percentages of O2 that give no germination in the upper embryos. Higher temperature in both lowers and uppers reduced the O2 percentage needed for germination. Fig. 25 shows percentage germination of both 100 eo z o z 2 (C LOWER SEED AT 2i' C UPPER SEED K ZI'C 3 6 t Z UJ 100 a a so 60 40 20 20 60 70 eo 90 100 LOWER SEED AT ao* c UPPER SEED AT 30° C 3 6 10 30 40 50 60 70 OXYGEN, PERCENTAGE BY VOLUME eo Figure 25. Minimum oxygen required for germination of the intact cocklebur seed during six days at 21° and 30° C (70° and 86° F). upper and lower intact seeds at like temperatures and range of O2 per- centage. Intact seeds require higher percentage of O2 for germination than do naked embryos. The naked embryo of the upper seeds gives 100 per cent germination in 1.5 per cent O2, the uppers ^dth coats intact require 100 per cent, or 66 times the O2 pressure. Exact quantitative comparisons between ShuU's and Thornton's data are impossible since they worked with different species and used somewhat different temperatures and dura- tion of experiments. They do, however, agree on all essential points: naked embryos of both upper and lower seeds need definite, easily measur- able minimum Go pressures for germination at various temperatures; minimum pressure needed for the upper embryo is always higher than that 84 GROWTH OF PLANTS for the lower; that the minimum oxygen pressure needed for the germina- tion of the naked embryos falls decidedly with, a rise of 9° to 10° C (16° to 18° F) in temperature; that the intact coat of the upper seeds, through its low permeability, increases the required oxygen pressure for germination more than 60-fold ; and finally, that one factor leading to the germination of the intact upper seed in air at higher temperatures is the lower minimum oxygen pressure required for the germination of the embryo. Crocker, Shull, and Thornton all agree that the failure of the upper intact seed to germinate in air at temperatures below 30° C (86° F) is due to the low permeability of the seed coat to oxygen; hence the upper seed remains dormant in the soil at lower temperatures. It is of interest but of no known significance in dormancy that on an equal dry weight basis the embryo of the lower seed is richer in catalase "^ than that of the upper seed. On the basis of his data, Shull agrees with Crocker in concluding that oxygen has its function in the germination of cocklebur seeds in producing sufficient aerobic respiration to furnish the necessary energy for growth and that it does not act merely as a. stimulus, as Becker, Lehmann, and others have assumed. The cocklebur embryos probably rank high among seeds in their need for the energy from aerobic respiration for growth. A few other dormant imbibed seeds have been found in which increased oxygen pressure will force them to grow. Atwood ^ and Johnson ^^ have found that dormant, recently harvested wild oat grains are forced to ger- minate by increased oxygen pressure, and Harrington *^ has found the same for freshly harvested cereals. Spaeth,"- for American basswood, and Stier,"^ for freshly harvested potato seeds, find that the portion of the seed coat derived from the nucellus inhibits the passage of oxygen. A number of other similar cases could be mentioned. For years we have been studying the mechanics of dormancy in seeds in the seed laboratory at this Institute and we always try increased oxygen pressure for forcing. We have found very few cases where this is effective in contrast to the many kinds of seeds that are dormant because of hard coats, because of dormancy of the embryo, or because of the coats limiting the absorption of water. The findings for the cocklebur, as interesting and definite as they are, may not explain the dormancy of any considerable number of different kinds of seeds. There is another way in which oxygen pressure is involved in dormancy of seeds. As described in a previous chapter, Davis -^ and Thornton i-" have shown that Xanthium and Ambrosia embryos can be thrown into dormancy by keeping them in germinators at higher temperatures with subminimal oxygen pressure for germination. In the case of after-ripened seeds of Ambrosia, the thin seed coats reduced the oxygen supply to the embryos sufficiently at high temperatures to induce dormancy. In Xan- thium reduced oxygen pressure was necessary, in addition to the intact coats. As mentioned in the last chapter, other unfavorable factors in the germinator also induce secondary dormancy. DORMANCY IN SEEDS 85 The oxygen pressure relation to germination is not as simple as it might seem from the statements above. Thornton found that high percentages of carbon dioxide, especially 40 to 80 per cent, lowered greatly the mini- mum oxygen pressure needed for the germination of intact upper seeds at 25° C (77° F). He did not determine whether this increased the perme- ability of the coats to oxygen or lowered the oxygen pressure needed by the embryo for germination as does a rise in temperature. Harrington ^^ found that 60 to 80 per cent carbon dioxide is effective in forcing dormant Johnson grass seeds in which oxygen supply is not a limiting factor. Later, Thornton ^-^ found that similar concentrations of carbon dioxide were effective in forcing the germination of intact lettuce seeds at 35° C (95° F), a temperature many degrees above the maximum germination tempera- tures in absence of carbon dioxide. As we have seen above, the upper seed of Xanthium is almost unique among seeds, in that the thin coat reduces the oxygen supply below the minimum needed for germination at lower temperatures. It is like^\^se peculiar in that it prevents the germination at low temperatures but not at high temperatures, while in the other two seeds studied in this respect. Ambrosia and lettuce, the thin coats prevent the germination at higher temperatures but not at lower temperatures. Dormant Seeds that Respond to a Single Period of Moist Low-Tempera- ture Stratification There is probably no other condition — unless it is breaking the coats, which is mainly impractical — that will overcome the dormancy of as many different kinds of temperate-zone seeds as placing them in germina- tion conditions at a low temperature for periods varying from a few days to many months, according to kind and condition of the seeds to be treated. In the earlier and century-old practice, the seeds and sand were laid do^\Ti in successive horizontal layers and the stratified mass exposed to low temperatures during the winter; hence the term "stratification." In recent practice the seeds are mixed ^\^th moist sand, granulated peat, or other medium and exposed to low temperature for the desired time. Many workers still use the term "low-temperature stratification" for the newer practice. For seeds of many water plants, which need little oxj'^gen, water is a good stratification medium. Low-temperature stratification imitates nature's methods of after-ripening seeds in the temperate zone; the seeds fall to the ground in the fall and are more or less covered in the cold soil during winter. Artificial stratification has the advantage of making pos- sible the holding of the several stratification factors (temperature, mois- ture, and oxygen) at the optimum, which produces the quickest possible results. In nature these factors, especially temperature, are at the opti- mum only a portion of the time. Fall, winter, and early spring sowing of many seeds that respond to low-temperature stratification is a fair substi- 86 GROWTH OF PLANTS tute for stratification and has advantages over nature's method in that the seeds are well covered with soil. For a long time it was assumed that after-ripening in stratification was brought about by freezing or by freezing and thaAving. This is almost, although not quite, entirely wrong. The changes involved in low-tempera- ture after-ripening of seeds occur in the main at temperatures above freezing, ranging from 1° to 15° C (34° to 59° F), for various sorts of seeds. Figure 26. Excised embryos of control seeds of Sorbus ancwparia showing the develop- ment of the cotyledon in contact with the moist filter paper for 21 days. These are temperatures at which essential metabolic changes occur within the living tissues of the seeds. The exceptions which may involve freezing or freezing and thawing are seeds of certain water plants, like Alisma,^^^ as well as some others in which the seed is held dormant entirely by the coats and in which freezing and thawing rupture or weaken the coats. Even such seeds are generally after-ripened by low- temperature stratifica- tion a little above freezing. It will be best to discuss the two different physiological groups of seeds that respond to low-temperature stratifica- tion under separate headings: (a) seeds with dormant embryos, and (6) seeds with non-dormant embryos held dormant solely by the coats. Seeds with Dormant Embryos. Because the embryos are dormant in this type of seed it must not be assumed that the coats are not important DORMANCY IN SEEDS 87 in their dormancy. Indeed, one cannot determine whether the embryos are dormant until they are removed and put into a germinator. The dor- mancy in the embryo does not manifest itself by complete inability to grow when the embryo is removed from the coats and placed in a germi- nator, but rather by a marked sluggishness in early growth and by a dwarf- ishness in the part of the seedling derived from the epicotyl. Once the hypocotyl starts to germinate, it forms a normal vigorous root system, just as does the hypocotyl of an after-ripened embryo. A number of investigators ^^•-''- ^^ early showed that embryos of certain rosaceous seeds are dormant. They also showed that these embryos are Figure 27. Excised embryos of seeds of Sorbus auciiparia which had been stratified for various weeks at 1° C (34° F) after three days on moist filter paper. after-ripened by low-temperature stratification and that the time required for after-ripening the embryos in stratification is shortened if the peri- carps and seed coats are removed. Sluggish growth of dormant embryos. It was left, however, to Flemion to make a thorough-going study of the physiology of dormant embryos. The sluggishness of the dormant embryo of Sorbus aucuparia ^^ is shown by Fig. 26. After the excised dormant embryos have Iain on moist filter papers in the light for 21 days, only the cotyledon in contact with the paper has grown and become green. This indicates great resistance to the movement of water through the embryo, which results in an insufficient supply of water to the other cotyledon and the hypocotyl for growth. Fig. 27 shows the great increase in vigor of growth of the embryo caused by stratifying the intact seeds at 1° C (34° F) for two months and then 88 GROWTH OF PLANTS excising the embryo and placing it on moist filter paper. The non-after- ripened embryo showed growth only in the cotyledon lying against the moist filter paper after 21 days, while the after-ripened embryo showed some enlargement of both cotyledons and a spreading apart of the coty- ledons and an elongation of several centimeters of the hypocotyl after only three days. One is struck by the enormous increase in growth vigor of this embryo induced by six weeks of low-temperature stratification. In this same figure, one seed that had four weeks' stratification partially removed the dormancy since there is some growth after three days. There is no growth in the embryo from a seed stratified only two weeks. Figure 28. The degree of after-ripening attained by seeds of Sorbus aucuparia which had been stratified for two months at various temperatures, as shown by the changes in the excised embryos after being on moist filter paper for two days. Correlation in the growth of the several organs of the dormant embryo is changed by after-ripening at low temperatures. In after-ripened embryos and embryos of non-dormant seeds the hypocotyl and radical grow first, and a root is formed with abundant root hairs. Later the cotyledons begin to grow and spread apart, and still later the epicotyl develops. In dormant embryos not only is the growth much slower but the cotyledons grow first — often only one of them if it alone is in contact ^vith water; also, the epicotyl often elongates before the hypocotyl and radical. Fig. 28 shows the effectiveness of various low temperatures in after-ripening the embryos of intact Sorhus seeds in tw^o months: 1° C (34° F) gives greatest vigor, 5° C (41° F) next, while -5° C (23° F) and weekly alternations at -5° and +5° C (23° and 41° F) are ineffective. It is evident that tem- peratures above freezing are effective, whereas freezing temperatures and I DORMANCY IN SEEDS 89 freezing and tha\ving are not. Although the optimum temperature for after-ripening of Sorhus seeds is nearer 1° C (34° F) than 5° C (41° F), that for other rosaceous seeds with dormant embryos such as some species of Rosa (Fig. 29) is about 5° C (41° F). The best optimum temperature for the stratification of Rhodotypos seeds ^* is about 5° C (41° F). Fluc- Figure 29. Rosa rubiginosa seeds: check stored dry; the others in moist sand for six months at the temperatures designated and then planted in a flat in the greenhouse. The picture shows the effective temperature for stratification, that is, 5° C (41° F). tuating temperatures ^^ithin a certain range are effective; for instance, for Rhodotypos seeds daily alternating (16 hours at low temperature and 8 hours at high temperature) or weekly alternating temperatures (half time at each temperature) of 1° and 10° C (34° and 50° F), 1° and 15° C (34° and 59° F), and 5° and 10° C (41° and 50° F) proved about as effective as, and in some cases better than, the constant temperature 5° C (41° F). Table 11 shows the optimum stratification temperatures,^- the effective range, and the length of time required for complete after-ripening of vari- ous seeds that have been studied at this Institute, with the addition of three studied earlier at the University of Chicago. The three additional ones are: Crataegus mollis, Acer saccharum, and Juniperus species. This table includes seeds with both dormant and non-dormant embryos that respond to low temperatures. This list could be considerably extended by drawing upon literature from other sources. It Mill be noticed that a number of seeds have an optimum stratification temperature of about 1° C (34° F), though most of them have 5° C (41° F) as the optimum. A number have 10° C (50° F) as the optimum, and some after-ripen equally well over a rather ^^^de range, 1° to 5° C (34° to 41° F) or 1° to 10° C (34° to 50° F). The degree of sluggishness in the early gro'\\i;h of excised dormant em- 90 GROWTH OF PLANTS Table U. Effective Moist Pretreatment for Seeds Benefiting by Period of Low Tem- perature Before Greenhouse Planting. Best Effective Days at Species temp. (°C) range C°C) best temp. . 1 1-5 30 Abies arizonica 5 5 5 5 5 5 35 Acer saccharum 1-10 30-60 Alisma Plantago-aquatica 1-^ 90 Amelanchier canadensis X *J 1-10 90-120 Aralia hispida 1-5 42 Arbutus menziesii KJ 10 1-10 100 Asimina triloba l.\J 5 5 5-10 120 Belamcanda chinensis 5-10 120 Benzoin aestivale Betula lenta * \ " lutea* B.papyrijera*\ 5 1-10 60-75 5 5 1-10 30-60 Butomus umbellatus 3-10 30-12" Garya ovata IS 1-10 90 Celastrus scandens 5 1-10 60-90 Celtis occidentalis 1-10 60-90 Cornus florida 1-10 120 " Kousa 5 5 135 Crataegus cocdnea fl 5 180 " mollis 5 5 1 5 75-90 " inollis t 5 135 " Phaenopyrum 1-10 60 Cupressus macrocarpa 5 5-10 60 Dictamnus albus 10 5-10 60 Diospyros virginiana 5 1-10 30-75 Gaultheria procumbens 1 1-5 60-90 Gentiana acaulis 5 1 1-10 60-90 " Andrew sii 1-5 30-60 " crinita i- 5 1-5 90-120 Hamamelis virginiana 5 1-5 60-90 Impatiens biflora 5 1-10 75 Iris versicolor 3 1-10 60-120 Juglans cinerea, J. nigra \J Juniperus communis, J. depressa, \ " prostrata, J. virginiana] 5 5 100 l-.'i 1-10 30-60 Libocedrus decurrens i-in 1-10 30-60 Liquidambar Styraciflua 1 XV / 1-10 1-10 70 Liriodendron Tulipifera X i.\J 5 5 1-5 150-180 Mitchella repens 1-10 90 Myrica carolinensis 10 5-10 60-90 Nyssa sylvatica 5 1-10 30-60 Physocarpus opulifolius 1 5 5 ■ 1-5 30-60 Picea canadensis, P- excelsa 1-10 30 " Omorika 1-10 42-60 " pungens 1 1-10 30-60 " sitchensis X 5 1-10 30-60 Pinus austriacus 1 5 1-5 30-60 " Banksiana 1-15 30-60 " caribaea , DORMANCY IN SEEDS 91 Table 11. — (Continued) Best Effective Days at Species temp. range best (°C) (°C) temp. Finns contorta 5 1-10 30-60 " Coulteri 5 1-10 30 " densifiora 10 1-10 30 " echinata 5 1-15 30-60 " flexilis, P. insignis 5 1-10 30-60 " koraiensis 10 1-10 30-60 " Lambertiana 5 1-10 90 " Laricin 1-5 1-10 30-60 " moniicola 1 1-10 60-90 " ponderosa 1-5 1-10 30-60 " resinosn 1-10 1-10 30 " rigida 5 1-10 30 " Strobiis 10 1-10 60 " Taeda 5 1-15 30-60 " Thunbergii 5 1-10 30 Polygonum acre 1-10 1-10 30-60 " arijolium 5 1-5 90-120 " lapathifolium 10 1-10 30 " pennsylvanicum, P. virginianum 10 1-10 60-90 Pruniis americana 5 1-5 150 " persica 5 5-10 60-90 Ptelea isophylla 1 1-5 60-120 " serrata 1 1-5 150 " trifoliata 5 5 60-90 " trifoliata var. mollis 5 5 30-90 Pyrits arbutifolia 1 1-5 90 " arbutifolia var. atropurpurea 10 1-10 60 " (French pear) 5 1-5 60-90 " Malus var. niedwetzkyana 5 1-5 60 Ribes Grossularia 5 1-10 90-120 Rosa midtiflora 5 5-8 50 Scirpus campestris var. paludosus 5 1-10 90-120 Sequoia gigantea 5 1-5 30-60 Smilacina trifolia 5 1-10 90-120 Sorbus aucuparia 1 1-5 60-120 Taxodium distichum 5 1-10 30 Thuja gigantea 5 1-10 30-60 " occidentalis 5 1-10 30 " orientalis 5 1-10 60 Typha latifolia 5 1-10 30 Vitis aestivalis 5 1-10 90 " bicolor 5 5 90-120 " (Concord grape) 5 5-10 90 " (Delaware grape) 5 5 90 Zizania aquatica 5 1-10 30 * Transferred to higher temperature ovens instead of greenhouse. t Pericarps removed. 92 GROWTH OF PLANTS bryos varies considerably with the variety and species. Apple, pear, most Crataegus species, Prunus americana, and others behave much like the embryo of Sorbus shown in Fig. 26, in which only the cotyledon in con- tact with water grows; also in some of these the epicotyl elongates with- out any growth of the radical and hypocotyl (Fig. 30) when the seeds are placed on moist filter paper. The dormant peach and Rhodotypos embryos Figure 30. Excised non-after-ripened apple embryos grown in Petri dishes 14 days at room temperature. are less sluggish than the ones mentioned above. The latter in the main show growth of both cotyledons and the hypocotyl and radical when placed on moist filter paper, but even in these the growth of the non- after-ripened embryo is very much slower than that of the after-ripened embryo. Sorhus embryos attain sufficient after-ripening after six weeks' stratifi- cation at 1° C (34° F) in intact seeds to give vigorous early gro^vth, as shown by their development when excised; but 12 weeks' stratification is needed for the intact seed to give full germination. Very recent unpub- lished work by Flemion indicates that the dwarfishness in the later growth of the embryo is not entirely overcome until the intact seeds have been stratified long enough to enable them to germinate completely. Seeds with dormant embryos will finally germinate at the optimum stratification temperatures, but they will do so more quickly if they are transferred to a somewhat higher temperature after complete after-ripening. Transferring partially after-ripened seeds to germinators at 20° C (68° F) is likely to throw them into secondary dormancy, and even fully after-ripened seeds do better if planted at relatively low temperatures such as are met in early spring planting. Indeed, Pack ^^ found that various species of after- ripened Juniperus seeds germinated faster at the best after-ripening tern- DORMANCY IN SEEDS 93 perature, 5° C (41° F), than they did at 10° C (50° F), and that 15° C (59° F) or higher temperatures were unfavorable for germination. Dwarfish plants from dormant embryos. While dormant embryos grow very slowly and show unusual correlations in the growth of their several organs, Flemion ^^' ^^ was able to get seedlings from most of them by use of proper methods; she finally obtained plants by transferring the seedlings to soil. If placed in aerated water at room temperature so that water is in contact with the whole surface of the embryo, excised Sorhus embryos slowly form a seedling with the hypocotyl-radical elongated and both Figure 31. Rhodotypos kerrioides seedlings. Leji: grown from non-after-ripened embryos. Right: grown from after-ripened embryos. cotyledons enlarged. Excised peach, apple, and hawthorn embryos can be germinated by placing them in moist peat at 25° C (77° F). This treat ment vrAX not give seedlings -^Wth dormant Sorhus embryos. The seedlings gro-UTi from dormant embrj'os at higher temperatures form dwarfish tops, although the root systems grow vigorously. Fig. 31 shows, at left, seed- lings of Rhodotypos gro\\Ti from dormant embryos and, at right, seedlings grown from low-temperature after-ripened embryos. The dwarfed seed- lings have very short internodes and thick, deep green leaves in contrast to the long internodes and thin, lighter green leaves of the seedlings from the after-ripened embryos. Fig. 32 shows, at left, a seedling grown from a dormant peach embryo and, at right, a seedling from a low-temperature after-ripened embryo. In the dwarfed peach seedlings the internodes are very short, the leaves thicker, deeper green, and shorter, and often much deformed. To date, Flemion has been able to produce dwarfish seedlings from all species and varieties of rosaceous seeds with dormant embryos that she has tested, as well as A\4th witch-hazel seeds; but her studies have not been extended to seeds of other families of plants \^'ith dormant embryos. 94 GROWTH OF PLANTS This dwarfishness in the seedUng may persist for weeks, months, or even years or until some growth condition after-ripens a bud (generally a lateral bud) so that it acquires vigor of growth. The most effective condition now Figure 32. Peach seedlings. Left: grown from non-after-ripened embryo. Right- grown from after-ripened embryo. kno^\Ti for after-ripening such a bud is a period at low temperature. If the dwarfed seedling is placed at 5° C (41° F) for six weeks and then placed at a good growing temperature, one of the buds begins vigorous growth similar to that sho^\Ti by the epicotyl of an after-ripened embryo. Fig. 33 shows a picture of a dwarfed seedling of Rhodotypos at two stages of dwarfish growth and the later vigorous growth of the terminal bud. Lam- merts '^^ has found that high temperatures will throw buds of the dwarfish peach seedling out of dormancy and that the rate of elongation of such buds is favored by a long day, especially by continuous illumination. From what has been said above, it appears that low-temperature strati- fication does two things to dormant embryos: it overcomes the sluggish- ness in their early growth and it after-ripens the epicotyl so that in its later growth it forms a vigorous rather than a dwarfish plant above ground. The epicotyl of the seed is, of course, a bud and the second phase of low- temperature after-ripening described above is a phenomenon that occurs DORMANCY IN SEEDS 95 Figure 33. A plant from a non-after-ripened embryo of Rhodotypos kerrioides photographed 122, 161, and 226 days after planting. Thi,s illustrates the normal growth of the terminal bud which took place during the interval between 161 and 226 days. 96 GROWTH OF PLANTS generally in buds of trees and shrubs of the temperate zone. The buds go into the mnter in the dormant condition and are after-ripened by the cold weather so they grow with vigor in the spring. Consider one example, the peach tree. Its leaf buds are after-ripened by the cold weather of winter and consequently take on vigorous growth in the spring. If one goes into Georgia he will note that the peach trees are smaller because of insufficient after-ripening of the buds during the short winters. If one goes far enough south, the peach no longer even persists, probably because there is not sufficient winter to after-ripen the buds even partially. The buds of trees of low-altitude tropical plants do not require low tempera- tures to after-ripen the buds and give them growth vigor. Perhaps one could select strains of peaches that had this need to a less degree and extend the culture farther south. It is evident that the epicotyls of many seeds are not dormant in the sense we have just described and consequently do not need low- temperature after-ripening. However, we shall later discuss classes of seeds having dormant epicotyls that need low-temperature after-ripening but that do not have a sluggish growth in other parts of the embryo. In classes of seeds to be described later we shall frequently meet dormant embryos that require low-temperature stratification for after-ripening. Davis and Rose " believed that the radical is the dormant organ of the Crataegus embryo, but Flemion ^^ has shown that the epicotyl is the dor- mant organ in this embryo. The radical is sluggish in the early growth of the embryo, but once started, it grows A\dth vigor. On the other hand, the eipcotyl shows a persistent dwarfishness in growth until it is after- ripened by a period of low-temperature exposure. This could be discovered only by forcing dormant embryos to form seedlings without low tempera- tures and by continuing the growth of these seedlings over long periods. Very recently Flemion and Waterbury ^'^^ have thrown some additional light on the persistence and nature of the dwarfishness in seedlings grown from dormant embryos. After-ripened peach embryos were deprived of all or part of their storage material. Death resulted when both cotyledons were removed at the time of planting; but when they were removed ten days later, small but normal seedlings were obtained. When various parts of the cotyledons were removed at the time of planting, normal plants resulted, except that the plants were smaller when only one-third of one cotyledon remained. However, none of these small plants had the tele- scoping of internodes or other dwarfing characteristics so typical of seed- lings obtained from non-after-ripened embryos. Dwarfish seedlings have apparently adequate root systems, for in dry weight determinations the ratio of root to top was always greater in the dwarfs. When the growing tip of a normal seedling was grafted on the stem of a dwarfish seedling, the result was a normal seedling, showing that the root system of the dwarf was capable of sustaining normal growth and that apparently there was no substance in the root which inhibited shoot growth. Interposing by DORMANCY IN SEEDS 97 grafting a portion of the normal stem on the dwarfs or a portion of the dwarf stem on the normal seedlings had no effect whatsoever on the sub- sequent growth of the normal seedling or in overcoming dwarfishness. When the growing tip of a dwarfish seedling was grafted on a normal seedling, the subsequent growth of this tip remained dwarfish. Thus the seat of this dwarfishness is in the growing tip and not in the root or stem. The breaking of seed dormancy in the peach by low temperature is in a sense a treatment for overcoming bud dormancy, for normal development is obtained by subjecting either the seed or the dwarfish seedling to the required period at low temperature. Chemical and physiological changes occurring in dormant embryos during low-temperature after-ripening. There have been a number of investiga- tions of the chemical and physiological changes occurring in seeds with dormant embryos during after- ripening at low temperatures. Eckerson ^^ studied several species of Crataegus seeds using microscopic, chemical, and physiological methods. The storage substances in these are in the form of proteins and fats with little soluble sugar and no other assimilable car- bohydrates. Also the foods are stored almost entirely in the cotyledons. The initial change is increased acidity. Correlated with this is an increased water-holding power; increased catalase and peroxidase activity occurred as after-ripening progressed. Soaking the seeds in dilute acids hastened the after-ripening and the changes mentioned above. Eckerson does not know whether the increased acidity is a correlative change with after- ripening or whether it holds a causal relation to after-ripening. There is some evidence for the latter. There was also a great increase in water absorption by the embryo as after-ripening progressed. Jones ^^ studied sugar-maple seeds which have proteins and fats as stor- age material but also contain more than 6 per cent cane sugar. The best after-ripening temperature is 5° C (41° F) and several weeks are required to complete the process. During after-ripening there is a great increase in catalase, a considerable increase in reducing sugar, and a slight increase in peroxidase. There is no increase in water-absorbing power and the embryo is alkaline in both mature and after-ripened seeds. Pack ^^' ^"' ^^ studied the after-ripening of seeds of several species of Juniperus. These seeds have their stored foods in the form of proteins and fats with little sugar and no starch. The optimum temperature for after-ripening is 5° C (41° F) and the time required is about 100 days. Recently Webster and Ratliffe ^^^ have shortened the stratification period of Juniperus virginiana seeds to less than two months by treating the seeds with a weak solution of lye before stratifying. Pack ^^' p-^^ lists the following changes that occur in the seeds of Juniperus during low-temperature after-ripening: "(1) rather rapid and complete imbibition, followed by a steady slow decrease in water content during after-ripening or until near germination; (2) increased H+ ion concentration, especially of the embryo; (3) an incre- ment of titratable acid; (4) a steady and enormous increase in the degree 98 GROWTH OF PLANTS of dispersion of the stored fat; (5) decrease in the amount of stored fat and protein, with an increase of sugar content and the first appearance of starch; (6) the translocation of food in the form of fat or fatty acids from endosperm to embryo; (7) a seven-fold increase in the amino acid content, and a complete disappearance of histidine from the endosperm; IS 13 >- t < UJ >- N Z u Lipase Peroxidase 5 7 9 WEEKS AT LOW TEMPERATURE FiGTJKE 34. Catalase, peroxidase, and lipase activity of embryos of Rhodotypos kerri- oides after various weeks in moist peat moss at 5° and 10° C (41° and 50° F) alternated weekly. The activities are calculated on the basis of the activity of the control expressed as one. (8) an increase of soluble proteins with a marked hydrolysis of the stored proteins; (9) slight growth of embryo; (10) very slight increase of the respiration intensity; (11) increased respiratory quotient; (12) decreased intramolecular respiration; (13) a doubling of the catalase activity; and (14) the rise in vigor of seeds as sho\vn by their resistance to fungal attack." A later study of J. scopulorum seeds by Afanasiev and Cress ^ confirms many of the results found by Pack and shows that removal of the fruit and seed coats hastens the after-ripening of the seed in low-temperature stratification. Flemion ^^' p-^^^ summarizes the chemical and physiological changes oc- curring in Rhodotypos embryos during after-ripening as follows: "Analyses of the seeds at intervals of two weeks during the after-ripening period show that the seeds increase in catalase, peroxidase, and lipase activity and also increase in water absorption power, nitrogen-soluble in 80 per cent alcohol, titrable acid, and sucrose. The ether-soluble fraction de- DORMANCY IN SEEDS 99 creases as after-ripening progresses." Fig. 34 shows the changes in catalase, Upase, and peroxidase as after-ripening proceeds. Lipase has the function in hydrolyzing the important storage substances, fats. Fig. 35 shows change in catalase, peroxidase, and lack of change in emulsin as Sorbus embryos after-ripen.^'* In this seed also there is no appreciable increase in amylase with after-ripening. This is to be expected, since there is no starch in the embryo until germination occurs. -[ — r # Catalase Peroxidase -<3 (SEmulsin control I J_ J_ _L J_ _1_ _!_ _L _L SEEDLINGS J I L 5 6 7 8 9 10 II 12 13 14 WEEKS OF STRATIFICATION 16 Figure 35. Changes in the catalase, peroxidase, and emulsin activities of Sorbus aucuparia seeds while after-ripening at 1° C (34° F). In every case reported above, catalase has sho^^^l a great increase with degree of after-ripening of the seed or embryo. This enzyme decomposes hydrogen peroxide, and also organic peroxides; it is found in all living matter, increasing in general proportion but not in strict proportion to the physiological and metabolic activity. We know little about its function in organisms; it may aid in protecting them against over-accumulation of organic peroxides. The change in catalase content has received a great deal of attention in connection with after-ripening, germination, and stor- age of seeds. In connection with after-ripening. Fig. 36 is of interest. It shows the modification in the catalase content of Sorbus embryos main- 100 GROWTH OF PLANTS tained in germinators at various temperatures. At the optimum tempera- ture for after-ripening (1° C, 34° F), except for a slight initial drop, there is a continuous rise until at the completion of the after-ripening, after control I 5 7 WEEKS OF 9 II STRATIFICATION Figure 36. The catalase activities of Sorbus aucuparia seeds stratified for periods of 1 to 15 weeks at various temperatures. 12 weeks, the catalase has increased 5-fold. At the somewhat higher and variable temperature of the icebox, which is also a poorer temperature for after-ripening, it rose 12-fold in nine weeks. The catalase also rose faster at 5° C (41° F) than it did at the optimum after-ripening temperature. At DORMANCY IN SEEDS 101 the still higher and still less favorable temperatures for after-ripening, the catalase content rose much less or actually fell after a small initial rise. While the catalase rises greatly \\dth after-ripening of dormant embryos, the amount of rise is not a strict measure of the progress of after-ripening. What is the nature of the changes brought about in seeds ^^^th dormant embryos during low-temperature stratification? First, there is an increase in enzymes, not only hydrolytic and oxidative — lipase, peroxidases, oxi- dases, catalase — but, judging from Pack's finding of a great increase in amino acid, probably proteases also; secondly, there is an accumulation of simple organic materials that can be readily used in building new tissues, sugars, amino acids, etc.; and finally, there is a transformation of insoluble, osmotically inactive substances to soluble, osmotically active ones, i.e., fats to sugars and insoluble proteins to soluble proteins, amino acids, and other nitrogenous organic compounds. The formation of osmotically active substances may account for the free movement of water in after-ripened, rosaceous embryos in contrast to the difficulty of movement in dormant ones. The hypothesis that inhibiting substances may hold embryos in dormancy should not be forgotten, especially since the long stratification in moist medium gives good conditions for the outward diffusion of such substances. Opposed to this hypothesis, however, is the fact that there is a definite optimum temperature for after-ripening of any given dormant embryo and that the effective range of temperature in many cases is very narrow — also that the optimum is very low, 1° C (34° F) in Sorhus. If after-ripening were a matter of the leaching of inhibiting substances, one should expect a Ande range of effective stratification temperatures in which high temperatures are more effective than low. As a matter of fact, high stratification temperatures make many dormant embryos still more dor- mant rather than after-ripening them. It is still harder to see how the continual dwarfishness in the epicotyl portion of the plant can be explained on the basis of inhibiting substances. The dormant slow-gro\\'ing embryos are long in contact with moist peat or actually in aerated water before they are planted in soil; moreover, after they are grown in soil there is opportunity for any soluble inhibiting substance to move from the buds back into the stem and finally to the roots. Perhaps the dormancy in embryos is brought about by organization characteristics of the proto- plasm involving insoluble substances. Seeds with Non-dormant Embryos. Many seeds that do not have dor- mant embryos, as sho\\'n by the fact that they will germinate immediately and produce vigorous seedlings if the coats are broken, respond to low-tem- perature stratification. Alisma Plantago-^ seeds germinate readily and with vigor if the coats are broken. They also respond to low-temperature stratification m water, as sho^^^l in Table 11. Barton ^^ '^''d m unpublished work has sho^vn the same to be true for Butomus umhellatus, Scirpus americanus, S. campestris var. paludosus, and Zizania aquatica, all the aquatic seeds she studied in this respect. The same is probably true of many other seeds 102 GROWTH OF PLANTS of water plants. The freshly harvested dormant cereals are forced to ger- minate by a few days of prechilling,*^^- ^"-^ or by use of low temperatures; also, breaking the coats forces these seeds to vigorous growth. Other dor- mant grass seeds have been found to respond to coat breaking and to stratification, or to prechilling in germinators. This is true of Setaria macrostachya ^^^ and other grass seeds.^^^ Most dormant grass seeds germi- nate with vigor when the coats are broken or greatly weakened by treat- ment with sulphuric acid of various concentrations, and many respond to prechilling or stratification. A careful study of dormant seeds in this family will no doubt give a big list of seeds with non-dormant embryos that respond to stratification. Barton ^ found that low-temperature stratification markedly increased the percentage germination and speed of germination of three of the south- ern pines: loblolly {Pinus Taeda), shortleaf (P. echinata), and slash (P. cari- baea). Longleaf pine (P. palustris) seeds germinated much more promptly than seeds of the other three species but were benefited somewhat by stratification. Later, Barton '' extended the studies to other pines and to several other conifers and found that seeds of many of these were benefited by low- temperature stratification. By examination of Table 11, it will be seen that Abies arizonica, Taxodium distichum, Sequoia gigantea, three species of Thuja, several species of Picea, and many species of Pinus are benefited by low-temperature stratification. Jolmstone and Clare ^^ con- firm Barton's findings for Coulter's pine and show that seeds of the Torrey, Digger, knob-cone, and pinon pine are benefited by stratification. Fig. 37 shows that one, two, or three months' stratification of Pinus rigida seeds at 5° C (41° F) gives full germination of practically all good seeds 15 days after planting, whereas the non-stratified seeds string along in their germination, giving only 30 per cent after 50 days. Fig. 38 shows the effect of stratification on the germination of loblolly pine (P. Taeda) seeds. One to four months' stratification at 5° C (41° F) gave almost complete germination of all good seeds within 20 days. The germination of untreated seeds had scarcely started after 20 days and attained 40 per cent after 100 days. In the spring sowdng of coniferous seeds in nurseries, it is important to have seeds come up promptly and completely so the seedlings \vill have attained some size before the dry, hot days of summer. A month or so of low-temperature stratification just previous to spring sowing will accom- plish this. This discovery is of great importance to nursery practice. Stratification is probably superior to fall planting, which is practiced in some nurseries. Stratified spring-so'wn seeds avoid the hazards of a winter in the soil incurring the danger of germination in mid-winter wdth later freezing and killing of the seedlings. Also the ravages of rodents are avoided. Stratification is also much simpler to apply and much more effective than light, which has been mentioned as a factor in the germina- tion of certain coniferous seeds. DORMANCY IN SEEDS PINUS RIGIDA 103 .^^ Vo GERMINATION 100 _ I MONTH CONTROL Figure 37. The effect of stratification at 5° C (41° F) for one, two, and three months on germination of the seeds of Pinvs rigida. Broken Une shows the percentage of good seeds as revealed by embryo tests. 104 GROWTH OF PLANTS We have classified coniferous seeds as seeds with non-dormant embryos that are benefited by low-temperature stratification. The main evidence that the embryos are non-dormant is the fact that conifer seeds in general will germinate and produce normal seedlings at 20° C (68° F) or above if 100^ »/o GERMINATION LOBLOLLY PINE so DAYS Figure 38. The effect of stratification at 5° C (41° F) for one, two, three, and four months on germination of loblolly pine seeds. Broken hne shows the percentage of good seeds as revealed by embryo tests. given sufficient time. One will see from the two sets of curves just men- tioned that the time is likely to be rather long — more than 50 days for some species. Flemion in unpublished work has attempted to determine whether conifer embryos show any of the dormancy characteristics such as those already described for rosaceous seeds. Fig. 39 shows, at left, the typical groAHh of an Austrian pine embryo isolated from a seed that had been held in granulated peat three days at room temperature before it was excised and put into soil. At the right is sho^vn a similar embryo taken from a seed after it had been in moist granulated peat at 5° C (41° F) I I DORMANCY IN SEEDS 105 for one month before it was planted in soil. This is the desirable stratifi- cation time and condition for these seeds. In conifer seeds much of the stored food is in the endosperm. The difference in the growth of the embryo from the low-temperature stratification and that from the non- stratified seed may be explained by the movement of nutrients and acces- sory foods from the endosperm during the month of stratification. Flemion is planning experiments that will distinguish between nutritional effects of the endosperm and possible dormancy in the embryo. Figure 39. Austrian pine seedlings. Left: seeds soaked for 3 days at 20° C (68° F) before excising the embryo for planting. Right: seeds stratified in moist peat for one month at 5° C (41° F) before excising the embryo for planting. The question naturally arises, What are the effective changes that occur in dormant seeds with non-dormant embryos when they after-ripen in low-temperature stratification? This question cannot be answered at pres- ent. We have seen that both hydrolytic and oxidizing enzymes are formed or activated in seeds A\dth dormant embryos, and that sugars, amino acids, and other soluble organic compounds are formed from more complex and less soluble compounds during low- temperature stratification. Perhaps similar changes occur in seeds Avith non-dormant embryos that give the embryos greater growing pressure. Low temperatures in plants in general lead to the formation of soluble sugars and other soluble substances. Like- wise it is possible that essential changes occur in the seed coats. We must not forget also that moist stratification gives favorable conditions for the leaching of inhibiting substances. In these seeds, however, like those with dormant embryos, there are definite optimum temperatures for stratifica- tion and the effective range of temperature is rather narrow and low. Leaching ought to progress faster at high rather than at low temperatures; also one might expect it to proceed over a wide range of temperatures. 106 GROWTH OF PLANTS Two-Year Seeds. Nurserymen often speak of two-year seeds. These are seeds that in nursery practice as well as in nature do not produce seedlings until the second spring after the seeds ripen. Work at the Institute has led to the grouping of these into three physiological categories. (1) Seeds that need a period m the soil at good growing temperatures to permit microorganisms or other factors in the soil to disintegrate the coats, followed by a period at a low temperature to after-ripen the dor- mant embryos. These will be discussed under the heading "Seeds with Resistant Coats and Dormant Embryos." (2) Seeds that need a warm-temperature period in the soil to produce a root system, followed by a low-temperature period to after-ripen the dormant epicotyl. These will be described under the heading "Dormant Epicotyls." (3) Seeds that require a period of low-temperature stratification to induce root growth, followed by a high-temperature period permitting the root to grow, followed in turn by a low-temperature period to after- ripen the epicotyl, which later requires a higher temperature for good gro^vth. These will be discussed under "Seeds That Require Two Low- temperature Exposures." Seeds with Resistant Coats and Dormant Embryos. Seeds of this class differ from those of the group just discussed in that the coats (pericarps or other structures) must be removed or partially disintegrated before the embryos are in a condition to after-ripen in low-temperature moist strati- fication. In nature the resistant coats are partially disintegrated by agents in the soil, especially microorganisms. The latter require good growing temperatures for greatest activity. Hence in nature such seeds require a few months in soil at high temperature for removing the coat resistance, followed by a few months at a low temperature for after-ripening of the embryos, after which they ^vill germinate. The coat resistance will be overcome during the first summer in the soil and embryos will after-ripen durmg the second ^^dnter so that the seeds are ready to grow the second spring. In nursery practice, if such seeds are to be grown out-of-doors without special treatment they should be sown in the sprmg. The coat resistance may be overcome by removing the coats mechanically or by corroding them with such agents as concentrated sulfuric acid. By using one of these methods of removing the coat resistance, seeds can be stratified during the first \vinter and made to grow immediately upon planting the first spring. As to seed or fruit coat, there are two classes of seeds in this group: one in which there is no suture line in the coats, and the whole surface of the coat is decomposed when the coat resistance (Symphoricarpos) is removed, and the other in which there is a dehiscent line at which the coat resistance is removed by decomposition. In the latter, the coats open into equal valves {Crataegus) or a valve comes off with dehiscence (Cotoneaster). DORMANCY IN SEEDS 107 Flemion '^ reports that one investigator was unable to obtain any ger- mination of snowberry (Symphoricarpos racemosus) seeds, while another investigator obtained only 50 per cent after two years in the soil. Flemion was successful in getting nearly complete germination of these seeds A\'ithin a year. She summarizes her results as follows: ^^' p-^"' "In order to induce germination in seeds of Symphoricarpos racemosus it is necessary that the seed coat be disintegrated. This can be accom- plished by placing the seeds for a period of three or four months in moist acid peat moss at 25° C, or by soaking the seeds in concentrated H2SO4 for 75 minutes, or by H2SO4 treatment and several weeks at 25° C. After the required changes in the seed coat have occurred it is still necessary to after-ripen the embryo, which can be brought about by a period of six months at 5° C. Of these three methods which modify the seed coats, the combination of H2SO4 treatment and a short period at 25° C is the best, and germination percentages of 60 to 90 may be obtained in this way. "For the production of seedlings on a large scale the best method is to plant the seeds in flats in spring and place out-of-doors in a cold frame which is covered with a board cover during the winter months. Germina- tion occurs the following spring. In nature, as in our laboratory experi- ments, the seeds respond to a high temperature followed by low tempera- ture. During the period at high temperature (summer months) conditions are favorable for the modification of the seed coats; the embryos are then after-ripened during the subsequent cold winter months. "That the seed coats undergo changes during dry storage at room tem- perature is sho^^'n by the fact that a suboptimal treatment of sulphuric acid followed by low temperature produced a maximum germination from seeds which had been stored about three months. Practically no seeds stored nine months or longer germinated although they were shown to be still viable when subjected to a more effective treatment. "The seeds increased several-fold in catalase and peroxidase activity during the period of after-ripening at 5° C. WTien at 25° C the activity of these enzymes does not increase, but there is instead a slight decrease." Pfeiffer ^' made a study of the building up of the seed coat during the development and maturing of the seed, as well as of decomposition of the coat in the soil. Fig. 40 shows the general structure of the seed and coats. The outer longitudinal, many-celled layer of fibers, as well as the circum- ferential inner and somewhat thinner layer of fibers are derived from the ovary wall. Both consist of long, thick- walled cells with small lumina and give the coat its marked leathery toughness. Pfeiffer summarizes her work in part as follows: ^^- p-^-^ " The components of the fiber walls and the integument epidermis include cellulose, pentosans, and lignin. Deposition of the substances is in this sequence; the greater deposit of lignin is in the integument epidermis. "These wall substances become subject to decomposition by fungi from 108 GROWTH OF PLANTS the medium when seeds are kept in moist peat moss or soil at favorable temperatures. The coats soften and readily disintegrate, thus removing a mechanical barrier in seed germination. "The coats of seeds kept free from fungi, but under similar conditions of moisture and temperature, do not undergo these changes. "In seed coats exposed to sulphuric acid for different periods of time, the fiber tissue is reduced in amount in proportion to the length of expo- sure. The longer exposures favor the development of fungi in subsequent holding in peat moss at 30° C. Too long exposure is disadvantageous for seed germination, probably because of excessive development of fungi, possibly because of change in the inner cuticle rendering it non-resistant to fungus entrance. "The fibers of the coat and the thin-walled tissue in the placenta region are permeable to water, as indicated by entrance of salts and methylene blue. The inner cuticle is apparently impermeable. Both inner and outer cuticles seem to be barriers to the progress of fungi under normal condi- tions. "The embryo is small with a short suspensor radical, short hypocotyl and cotyledons and an undeveloped stem tip. There seems to be a tend- ency toward increase in size and differentiation with keeping in moist peat moss at 5° C, which is more marked if this is subsequent to exposure to sulphuric acid and an interval at 30° C." OUTER FIBERS CRYSTAL LAYER JNNER FIBERS ENDOSPERM INTEGUMENT EPIDERMIS EMBRYO THIN WALLED TISSUE Figure 40. Diagram of median longitu- dinal section of Symphoricarpos racemosus seed (16.5 X). Later, Flemion and Parker ^'^ showed that the germination behavior of Symphoricarpos orhiculatus is similar to that of S. racemosus, except that the former responds to a higher stratification temperature. Flemion "' found that addition of nitrates or other nitrogen compounds to the peat during the warm temperature period in the peat hastened the decomposi- tion of the tough seed coats. It is a well-established fact that when micro- organisms decompose cellulosic materials in the soil, they consume much DORMANCY IN SEEDS 109 available nitrogen and are often limited in the decomposition by lack of available nitrogen. We have already learned that nitrates and other nitro- gen compoimds substitute for light in light-favored seeds. Gassner ^- found that nitrates substitute for light in forcing the germination of Chloris ciliata seeds, although the enveloping tissues are not permeable to nitrates. This raises the question as to what extent nitrates favor the germination of light-favored seeds in darkness by furthering the decom- position of the coats by organisms. Table 12. Percentage Germination of Symphoricarpos orhiciilafvs Seeds When Mixed in Moist Peat Moss and Kept at Various Temperatures.* Temp. (°C) 1 5 10 15 20 Percentage germination 0.5 year 0 1 5 0 0 1 year 2 years 3 years 4 years 0 0 1 2 2 3 3 5 43 62 66 70 0 0 1 2 0 0 0 0 5 years 3 10 74 5 0 * Duplicate lots of 200 seeds of 1934 crop; experiment started March 6, 1935. We have seen that the coats of Symphoricarpos seeds are decomposed by organisms most rapidly in the soil at relatively high temperatures, and that the embryos after-ripen best at about 5° C (41° F). Can an inter- mediate constant temperature be selected that will permit both processes to occur in succession, followed by germination? Table 12 shows the ger- mination of seeds of S. orbiculatus at constant temperatures, 1°, 5°, 10°, 15°, and 20° C (34°, 41°, 50°, 59°, and 68° F). Only 10° C (50° F) gave any considerable germination, amounting to 43 per cent after one year and finally rising to 74 per cent after five years. At 5° C (41° F) there was only 10 per cent germination after five years and at 15° C (59° F) only 5 per cent after five years. Temperatures much below 10° C (50° F) per- mit only very slow dismtegration of the coats, and temperatures much above 10° C (50° F) do not lead to after-ripening of the embryos. Flemion "^^ has found that seeds of various species of Crataegus differ markedly in their requirements for germination. C. cordata [Phaenopynim] and C. coccinea belong to the class previously discussed. They need two and one-half to five months' low-temperature stratification to after-ripen. If planted outside in the fall, they after-ripen during the ^vinter and pro- duce seedlings the following spring. They are not two-year seeds. The seeds of C. fiava, C. punctata, C. Crus-galli, and C. rotundifolia are two-year seeds. Fall planting will not produce seedlings the first spring, but seed- lings are produced the second spring after fall planting. This gives a summer in the soil for overcoming coat resistance. Spring planting of these produces seedlings the next spring. This gives a summer in the soil no GROWTH OF PLANTS for coat changes and a winter for embryo after-ripening. Fig. 41 illustrates this behavior. Seeds of C. rotmidifolia were planted in flats m the tall ol 1932 and placed in three sorts of cold frame at Yonkers. Row A shows photographs of these flats in June of 1933 after one winter m soil. None MULCH BOARDCOVER OPEN GREENHOUSE 21° C ■f «11:3^'- 54 % Figure 41 Per cent germination of various Crataegus seeds when planted m the fall of 1932 and placed in various cold frames. A and B, C. rotundifolia photographed June, 1933 and July, 1934 respectively. C, C. flava photographed July, 1934. has germinated. Row B shows photographs of the same flats in July of 1934. Row C shows the seedling production for C. flava the second spring after fall planting in flats in cold frames. No seedlings of either species were produced when the flats were kept continuously in the greenhouse. Fig. 42 shows that seeds of three of these more resistant species^ of Crataegus A\all not germinate after five or even nine months in soil at 5° C (41° F) followed by six weeks at good growing temperature. They all give abundant seedling production if the flats are kept four months at 21° C (70° F), then at 5° C (41° F) for five months followed by six weeks at a higher temperature. In these the four-months' period of high temperature can be shortened to two or three weeks by decomposing the coats partially with concentrated sulfuric acid before the high-temperature period. By such treatment the total high- and low-temperature after-ripening periods DORMANCY IN SEEDS 111 can be reduced to less than six months. In this way the more resistant Crataegus seeds can be made to germinate the first spring after maturity. In the case of less resistant seeds Flemion says: •*^' ^■*" "Although seeds of C. amoldiana, C. carrierei, C. mollis, C. sanguinea, and C. tomentosa germinate after a period at low temperature, more seedlings are obtained when the seeds have been treated in a moist medium for several weeks at 21° or 25° C prior to the low-temperature treatment." Figure 42. Seedling production of various Crataegus species. Lots of 500 seeds each were planted on Xov. 26, 1932, in flats and kept for various periods at 5° C (41"" F) with and without a previous four months at 21° C (70° F). Photographed six weeks after being transferred to a warm greenhouse. Cotoneaster seeds "' behave much as do Crataegus; C. Dielsiana and C. Zabelii show 100 per cent germination within four months when kept in a germinator at 10° C (50° F), while C. acutifolia, C. apiculata, C. hori- zontalis, C. lucida, and C. divaricata show very little germination in this condition even after ten months. These five all respond, as do the more resistant species of Crataegus seeds, to a period in soil at high temperatures for overcoming coat resistance, followed by a period at low temperature to after-ripen the embryos. Like\\'ise the high temperature can be par- tially or entirely eliminated by proper treatment with concentrated sul- furic acid. Table 13 shows the data on the seeds described above and a number of other seeds ^- °*- ^® belonging to this class that have been studied at the Institute. It gives the best temperatures for the high-temperature period, as well as the low-temperature period that follows; the range of effective temperatures for each treatment; the number of days required for each 112 GROWTH OF PLANTS period; and the length of time for concentrated sulfuric acid treatment if it is used to overcome the coat resistance. Table 13. Effective Pretreatment for Seeds Requiring Periods at Both High and Low Temperatures before Planting in the Greenhouse. Species Best temp (°C) Effective range of temp. (°C) Days at best temp. Effective H2SO4 time High Low High Low High Low Aralia racernosa * 25 5 14-25 1-10 30-60 90-120 10 min. Arctostaphylos Uva-ursi t 25 10 20-25 5-10 60-90 120-240 2-4hr.t Cornus canadensis * 25 1 25 1-5 30-60 120-150 10-30 min. Cotoneaster divaricata 14-25 5 14-25 1-5 90-120 90-120 2.5 hr. " horizontalis * 14-25 5 14-25 1-5 90-120 90-120 1.5 hr. Crataegus Crus-galli 25 5 25 5 120 180 2hr. flava 25 5 25 5 120 180 2hr. " Oxyacantha 25 5 25 5 90 180 2hr. " punctata 25 5 25 5 120 180 2hr. " rotundifolia 25 5 25 5 120 180 2.5 hr. Halesia Carolina * 20 5 14-27 1-5 30-90 60-90 — Rhodotypos kerrioides * 25 5 25-30 1-10 30 90 — Symphoricarpos orbiculatus 25 10 25 10 90-120 150 30-40 min. " racemosus 25 5 25-30 5 90-120 180 75 min. Taxus cuspidaia 20 5 20-25 1-5 90 120 — Tilia americana * 20 5 14-20 1-5 120 90-150 20 min. * Give some germination with pretreatment at low temperature only but much better with high plus low. t Neither high temperature nor H2SO4 alone sufficient to overcome coat effect. Best results when these two treatments were used together. t Effective length of treatment depends on whether entire nutlet stones, stone pieces, or single seeds are used. One should realize that there is no hard-and-fast line between seeds of this class and those of the previous class. Seeds with only moderately resistant coats may respond fairly well to simple low-temperature stratifi- cation; yet they give a higher percentage of seedlings if first exposed for a period to a high temperature in the soil, followed by low-temperature stratification. This is true of Rhodotypos kerrioides seeds. Pfeiffer has sho^vn beyond doubt that organisms in the soil decompose the non-dehiscent coats of Symphoricarpos seeds. We have assumed that organisms decompose the coats with dehiscent lines at these lines. This, however, is questioned by some workers. Miiller ^^ claims that seed coats with dehiscent lines are weakened at these lines only by water absorption and not by soil organisms, acids, and other agents unless these lines (planes) are composed in part or wholly of cellulosic materials. In this group of seeds the fact that the coat resistance is overcome only at higher tempera- tures that permit action of microorganisms is evidence against Miiller's conclusion. DORMANCY IN SEEDS 113 Dormant Epicotyls. At the Institute we have found a number of seeds in which the epicotyl has to be after-ripened by a period of low-temperature exposure after the radical and hypocotyl have grown. Table 14 shows the seeds studied to date that belong to this group. In all of these the germi- nation and formation of the root system — a process that takes place at Table 14. Effective Treatment for Producing Plants from Seeds with Dormant Epicotyls. Requirement for Pretreatment for root production shoot production Species Temp. (° C) Time (mos.) Temp. (° C) Time (mos.) 15-30 * or Asarum canadense 10-30* 3 5 3 Liliutn auratum 20 3-6 1-10 2-3 " canadense II 3-6 1-10 2-3 " japonicum II 3-6 5-10 3-4 " rubeUum 11 3-6 1-10 3 " superhum it 3-6 5-10 2-3 " szovitsianum II 3-6 10 3-4 Paeonia (herbaceous) 15-30 * 2-3 5-10 3 (tree) 20 or 2-4 5-10 2-3 Viburnum acerifolium 20-30* 6-17 5 2-3 " dentatum 11 6-17 5-10 i-2 " dilatatuhi II 7-9 5-10 3-4 " opidus II 2-3 3-15 1-2 " prunifolium << ' 7-9 3-15 1-2 * Daily alternation. higher temperatures — is rather slow, requiring from two to three months in the herbaceous peony and Viburnum Opulus, to 6 to 17 months in V. acerifolium and V. dentatum. One might expect low-temperature strati- fication of the seeds to hasten the growth of the root system, but such has not proved to be the case. It is evident that there ought to be some way of getting more prompt root production in this type of seed, but it has not been discovered to date. In some of these seeds the percentage of germination is also low. All these seeds will produce some seedlings the second spring if the seeds are properly planted early in the spring. The tree and herbaceous peonies and V. Opulus will give a good stand of seed- lings the second spring if planted in late spring or early summer. Some of the seeds of forms like V. acerifolium, V. dentatum, V. dilatatum, and V. prunifolium are likely to carry over to the third spring or later for complete seedling production. With the conditions met in nature, which are generally far from the optimum, seedling production in all these may extend over several years. Fig. 43 shows the behavior of tree peony seedlings ^ with hypocotyls 1 to 3 cm planted in pots and placed at 1°, 5°, 10°, 15° C (34°, 41°, 50°, 114 GROWTH OF PLANTS I DORMANCY IN SEEDS 115 59° F) , and greenhouse temperatures for 2, 2}/^, and 3 months, and then transferred back to greenhouse temperatures and photographed after three weeks and seven weeks. Examination of this figure shows that 5° C (41° F) Figure 44. Tree jx-ony seedlings, one year and seven weeks after transfer to green- house at 13° C (55° F) following a pretreatment of two and one-half months. Kept in board-covered cold frame over winter. is the best temperature used for after-ripening the epicotyls. This tem- perature gave a good epicotyl growth after 2 months of low temperature, followed by greenhouse temperature. There was somewhat less growth from the 10° C (50° F) exposure and still less at 1° C (34° F) exposure for 2 months. With 23^ and 3 months' exposure 1° C (34° F) and 10° C (50° F) were only slightly less effective than 5° C (41° F). The exposure at 15° C (59° F) gave little after-ripening of the epicotyl and the control at 20° C (68° F) gave none. Fig. 44 shows some of the seedlings in Fig. 43B after an additional year's growth. Fig. 45 shows the seedling production by Figure 45. Seedling production of tree peony in May 1933 from seeds planted in flats which were placed in a board-covered cold frame. Seeds planted: A, Dec. 1931; B, Feb. 1932; C, March 1932; D, May 1932; and E, July 1932. 116 GROWTH OF PLANTS tree peony seeds when the seeds are planted in flats at different times of the year and kept in cold frames, with board covers. Those planted during the spring and summer of 1932 gave no seedlings until the spring of 1933. During the summer of 1932 the roots grew and during the winter of 1932- 1933 the epicotyls after-ripened and seedhngs came up in the spring of 1933. Figure 46. The effect of low temperature on shoot development of Viburnum species. A, V. acerifolium. B, V. dilatatum. C, V. -prunifoUum. The May planting gave the best results. This gave the roots adequate time to grow mthout gromng so long that foods of the seeds were exhausted before the epicotyl developed ready to manufacture foods. The earlier plantings may have led to exhaustion of the foods of the seeds before the epicotyls functioned, but the early decay of some of the seeds at the low temperatures was also a factor. The July planting did not give sufficient time for root development before the cold weather set in. The May plant- DORMANCY IN SEEDS 117 ing gave 25 per cent seedling production, which is a good yield for com- mercial production but below that attained with good seeds by more fully controlled inside culture. Fig. 46 shows the effect of various low tempera- tures for three months on epicotyl after-ripening or shoot development of three species of Viburnum.^'" For V. acerifoUum and V. dilatatum 5° and 10° C (41° and 50° F) are both effective temperatures for epicotyl after- ripening. Viburnum prunifolium has a less limited range of temperature, 3° to 15° C (37° to 59° F) being effective. In this as in V. acerifoUum there is a small percentage of epicotyl development without cold exposure. Also it will be noted that the cold exposure period required by V. prunifolium is only I5 months contrasted with 3 months for the other two. Figure 47. A, Viburnum acerifoUum. The effect of various planting times during summer on seedling production the following spring. Left to right: Planted April 1st, June 1st, September loth, 1936. B, The effect of storage on seedling production of V. acerifoUum planted May 1, 1936. Left to right: Room temperature cleaned, in pulp; 5° C (41° F) cleaned, in pulp. Seedling production of the most stubborn Viburnums can be accom- plished out-of-doors by early spring planting. Of course, the seedlings do not come until the second spring. Fig. 47A shows the effect of planting V. acerifoUum seeds at different times during the summer upon seedling growth the next year. April 1 was better than June 1, and September 1 gave no seedlings. The earliest planting gave the slow-gro^^^ng roots 6 to 8 months to grow and get established before ^^^nter set in. Evidently 4 to 6 months, as shown by the June 1 planting, was not sufficient for many of the slower gro'U'ing seeds to form roots. To avoid the hazard of two \\'inters in the soil, these seeds should be spring planted. Fig. 47B shows that it is better to store the seeds of this species during the Annter at 5° C (41° F) than at room temperature, whether they are dried in the pulp or 118 GROWTH OF PLANTS cleaned. Giersbach found that V. nudum and V. scabrellum did not need epicotyl after-ripening and offered no problem in germination. These are more southerly forms. It must not be forgotten that the temperature relations described in this and previous sections, as well as those to be described later, probably apply to colder temperate zone plants and prob- ably not to torrid zone plants unless they are high-altitude forms. The species of Lilium needing low-temperature after-ripening of the epicotyl after roots are formed were studied by Barton." In unpublished work, Flemion'has found that Chionanthus virginiana and Symplocos paniadata seeds have dormant epicotyls that require low- temperature after-ripening after the roots start to grow. In the first of these seeds the roots grow much more promptly than in most seeds of this class. In fact, many of the roots start soon after the seeds fall to the ground and before winter sets in. In nature, the epicotyl will after-ripen the first \Adnter and the seedling will come up the first spring. These are not two-year seeds. In seeds that need a high -temperature period in a germinator to dispose of coat resistance and a low temperature to after-ripen the embryo, we noted that a constant intermediate temperature could be used that per- mitted both changes to occur. The time required for both processes and complete germination, however, was greatly lengthened by using this com- promise, intermediate constant temperature instead of the optimum tem- peratures for each individual process. No doubt in the type of seeds being discussed in this section such an intermediate constant temperature could be selected that would permit development of the roots followed by epicotyl growth. In all these seeds temperatures as high as 10° C (50° F) permit epicotyl after-ripening, and in two Viburnums even 15° C (59° F) is effective. No doubt constant temperatures of 10° C (50° F) or higher would permit both processes to go on. This, however, is of no importance in practical horticulture, for it would greatly lengthen seedling production time and it has no significance in nature, because long-maintained constant temperatures do not occur in the temperate zone. One might question the wisdom of having seeds that produced a root one year and had to wait until the next year for the epicotyl to develop foliage for feeding the root. These seeds ripen in the fall and go through the hazards o( one winter before even a root forms. If the roots start early in the spring they draw on the stored foods all summer and no doubt exhaust them before ^\'inter. The tardiness of the root formation in many of these seeds probably delays root gro^vth until late summer or fall, which lowers the draft on stored foods the first year. It is probal^le that a rela- tively small percentage of the seeds of this group ever produce seedlings in nature. This may also be true of most of the seeds of wild plants that have such a complex system of after-ripening and germination. The situa- tion is quite different in horticultural practice. Once one knows the tricks of a given seed he can put it under the optimum conditions for each phase DORMANCY IN SEEDS 119 of after-ripening and germination and come out with a high percentage of seedUng yield in minimum time, although in some cases the time is not so short at that. Seeds Requiring Two Low-temperature Exposures. Barton '^ has made an extensive study of the germination of Trillium grandiflorum seeds. The following method of treating the seeds gave the highest percentage of seedlings in the shortest time: the seeds were planted in pots in moist soil and kept for three months at 5° C (41° F) for after-ripening; then they had three months in a greenhouse, which produced a root system; then five months at 5° C (41° F) to after-ripen the dormant epicotyls; and finally a period in the greenhouse to grow the epicotyl and develop a com- plete seedling with top and root. This means tw^o low-temperature periods to after-ripen the seed and the epicotyl respectively, each followed by a high-temperature period for gromng the root and finally the epicotyl. This method requires 12 to 14 months to produce a high percentage of seedlings. In nature the seeds would after-ripen the first ndnter, the root system would grow the next summer, the epicotyl would after-ripen the second winter, and the plant appear above ground the second summer. This is a two-year seed in the sense the nurserymen use this term. In nature, the four different periods would not be at the optimum conditions a large percentage of the time either as to temperature or as to duration of exposure. Consequently there must be a high wastage of seeds in nature. Table 15. Trillium grandiflorum, 1940 Crop. Root and Shoot Production after Various Temperature Treatments from Duplicate Lots of 100 Seeds Each. Planted in Soil in Pots. Treatment Percentage seedling production First low temperature period Months in greenhouse Second low temperature period Percentage roots Percentage epicotyl °C Months °C Months growth None None None None 17 6 None 10 None 3 7 35 6 32 1 0 0 0 5 5 3 3 3 3 .5 5 3 5 83 90 84 86 54 80 38 80 10 10 3 3 3 3 10 10 3 5 86 69 78 81 63 63 46 72 Table 15 shows the seedling production from various periods of tempera- ture exposure. Contmuous greenhouse temperatures for 17 months gave 7 and 6 per cent of root gro^^i,h and 1 and 0 per cent of epicotyl gro\\i,h. Six months of greenhouse exposure followed by three months at 10° C 120 GROWTH OF PLANTS (50° F) gave 35 and 32 per cent of root growth and no epicotyl growth, because there was not a low-temperature period to after-ripen the epicotyls after the roots had grown. Judging from the later figures in this table, the six months' initial period in the greenhouse mterfered with the after- ripening for root growth. Compare 35 and 32 per cent with the percent- ages below in the same columns (Table 15). Thi-ee months at 5° C (41° F), followed by three months in the greenhouse and then three months at 5° C (41° F), gave 83 and 84 per cent of root production and 54 and 38 per cent of shoot production. When the second cold period was lengthened to five months, there was about the same root production but much higher shoot production — 80 and 80 per cent against 54 and 38 per cent. As Table 15 shows, 10° C (50° F) is almost as effective as 5° C (41° F) for after-ripening for both root and shoot growth. Table 16 lists several other seeds that belong to this class — Caulo- phyllum thalidroides, Smilacina racemosa, and Trillium erectum strictly so because there is very little root production ^\'ithout a prechilling period. Polygonatum commutatum, Sanguinaria canadensis, and Convallaria majalis belong in part to this class of seeds and in part to the previous class, for there is considerable root production ^\ithout a prechilling period; but such a period increases considerably both the percentage and speed of root production. For instance, Convallaria seeds give 46 per cent root production after several months in the soil without prechilling, whereas seeds that have been stratified for three months at 5° C (41° F) give 92 per cent root production rather promptly. If one examines the length of time each of the low-temperature periods (and also the high-temperature periods) requires according to the optimum conditions Barton has found for getting the epicotyl ready to grow into a plant, one will realize not only the complexity of the process but its time-consuming nature. This adds up to 8.5 months for Caulophrjllum seeds, 7 to 18 months for Con- vallaria, 11 months for Polygonatum, 12 months for Sanguinaria, 14 to 19 months for Smilacina, 9 to 14 months for Trillium erectum, and 9 to 12 months for T. grandiflorum. So far as persistence of the species is con- cerned, one wonders whether nature has not overdone dormancy in these seeds, certainly as to complexity and perhaps as to time required. Yet if nature wanted to become even more complex and difficult she could do so right in line with the things we have noticed in the several classes of dor- mancy mentioned above. Seeds that required initially a high-temperature period in the soil to overcome coat resistance, followed in succession by a low-temperature period to after-ripen for root growth, a high-temperature period for root growth, a low-temperature period for epicotyl after-ripening, and a high-temperature period for growth of the plant would add one more step to the after-ripening process. If such a temperate zone seed existed and matured in the late fall, it would not germinate until the third spring after maturing: it would be a three-S^ear seed. Has nature gone to this extreme with any seed? If so, the folly has not been discovered. DORMANCY IN SEEDS 121 a o3 a) a H I o c ID w o b£ .£ *c §-.2 O -.J o *^ o a W C S O Q 4) 0) CO (x o c s «5 Si o3 H O O Pi o 3 o o d F fC 1 t CO ^ CO 1 S CO lO »— I I CO o o o lO 1— t f— 1 lO 1 lO iC CO ^ iC iC >-o lO lO lO 30? o li°- 2: = S 03 (N CO to CO c^ I CO o a 3 O 0! iC CO ^ CO o 05 fe-^5 s (N !0 CO I CO =^ bD . j^ c a w2H o o lO 1 ^H lO 1 CO lO iO 1— 1 o £^ pa v°^ lO lO lO iC iC lO lO 02 * M ^ ^ H_ -e ^ •2 o e •*.* ;^ s -e "5^ 1 s * :S .e e ^ •?* << e s o •^ g to 5a, e o o e O O a. ^ * ^ I s c ■^ qj a 4J ri -u o f ; 3 1 01 a a o 03 o -tj (1 1) o «*« 01 rf} b ■T-! 3 -tJ QJ i=S >H O s ^ S ^ *j o o3 Q) u T) Ji CI ti-i Tl a d t!i o 3 -^ -tJ u o3 3 a 2 S a, 0) -i-5 -<-> ^ 2 ^ T) ^ 1 3 CJ cr fl 122 GROWTH OF PLANTS There is one other pecuharity that Barton and Schroeder ^* found in certain seeds mentioned in Table 16: the epicotyl could not be after-ripened by a low-temperature exposure until it had lengthened sufficiently to break the epicotyl sheath. This stands m contrast to the situation with the peony and other seeds described in the previous section of this chapter; Figure 48. Stages in the development of the seedlings of Convallaria majalis. Left to right: Stage 1, the protrusion of the radicle and hypocotyl; Stage 2, the first evidence of shoot development; Stage 3, further growth of the shoot to break through the coty- ledonary sheath. in these cases the epicotyl could be after-ripened as soon as the root started, without the necessity of previous elongation. This has been worked out in detail for Convallaria majalis and Smilacina racemosa seeds. Fig. 48 shows three stages in the early development of the seedlings of Convallaria.^'^ It must reach the stage sho^vn in "3" of this figure before a cold period for after-ripening the epicotyl is effective. The epicotyl goes into dor- mancy at this stage of growth and not at earlier stages. This is very similar to the situation with Botrychium lanccolatum and with many of our early spring flowers that grow up every spring from bulbs or tubers. The bud that forms the spring shoot is formed the year before and elongates considerably before it goes into dormancy, no doubt to be after-ripened by the cold of winter ready for early spring growth. We have seen for Syynphoricarpos orbiculatus seeds that an intermediate constant temperature, 10° C (50° F), can be chosen that will permit all the after-ripening processes to proceed, including overcoming coat resist- ance, after-ripening of the dormant embryo, and finally growth of the seedling. This temperature is not optimum for any of the processes, and consequently lengthens the time for seedling production, in contrast to using the optimum temperatures for each individual process. No doubt in all the cases described above, where temperature is an important factor 1 DORMANCY IN SEEDS .123 in the various phases of after-ripening and growth of seeds, such a con- stant intermediate temperature could be used to consummate the complete process; but nature in the temperate zone does not deal in constant tem- peratures and the practical grower cannot easily maintain such a constant temperature with other necessary growth conditions, nor can he wait so long for the plants. The facts of seed dormancy stated m this and in previous sections show the inadequacy of ending germination studies with the mere growth of the root. Studies thus terminated would entirely miss epicotyl dormancy and the dw^arfishness in seedlings due to the growth of non-after-ripened epi- cotyls. We always follow the growth of the seedling for many months or even years to catch any pecuharities in the later development of the seedlings that might result from seed dormancy. A Period or Dry Storage After-Ripens Many Seeds A period of dry storage may be quite as important a factor in after- ripening dormant seeds as low-temperature stratification, especially if one considers the number of species that respond to each treatment. Many light-sensitive seeds -^ lose then- light-sensitiveness partially or completely if kept for several months in dry storage. Viscum album and Arceuthobium oxycedri seeds are notable exceptions, for the first is killed by continuous darkness, and neither germinates under any conditions without light. There are probably other exceptions 'among light-sensitive seeds. Most seeds of culti\'ated grains and other grasses after-ripen m dry storage. In the cereals the period of after-ripening varies ^\'ith species, varieties, and races from a few days to several months. According to Pietruszczynski,^^ it is longest in oats, shorter in barley and wheat, and shortest in rye. The period is shorter in whiter than in spring cereals, in early than in late ripening varieties, and in dry than in wet seasons. In wild grasses, several months of dry storage are generally required for complete after-ripening. Most weed seeds studied in this respect after-ripen m dry storage. Even seeds ^^^th dormant embryos ~^' ^^ can be at least partially after-ripened by dry storage, although this treatment is not nearly as effective as low- temperature stratification. Kroeger ^^ has recently studied the progressive after-ripening of Impa- tiens halsamina seeds with increasing periods of dry storage. An examina- tion of lot 1 in Fig. 49 shows that, as the dry storage period mcreased from 0 weeks to 43 weeks, the speed and total percentage of germination m- creased. The fresh seeds gave 32 per cent germmation after 20 weeks in the germinator, while the seeds that had been dry-stored for 43 weeks gave 100 per cent germination in one week. Even four weeks of dry storage raised both the speed and final germination, which reached 70 per cent after 20 weeks. The speed and final percentage of germination rose as the dry storage period was lengthened progressively to 9, 16, and 25 wrecks. 124 GROWTH OF PLANTS Evidently, after-ripening was complete after 43 weeks of dry storage. Lot 10 in Fig. 49 shows a similar relation between the period of dry storage and degree of after-ripening. In this case, the later collection of seeds WEEKS IN GERMINATOR AT 25'C. Figure 49. Effect of weeks of dry storage at room temperature and date of harvest on after-ripening of seeds of Impatiens halsamina. Lot 1 was collected August 14-19 and Lot 10, October 14-16. shows higher germination of the fresh seeds, perhaps due to partial after- ripening on the plants; also it appears that after-ripening was not complete after 34 weeks of dry storage. Kroeger also found that for fresh seeds or DORMANCY IN SEEDS 125 for partially after-ripened seeds the percentage and speed of germination was increased by two weeks' moist stratification at 5° C (41° F). Various grass seeds respond similarly to both dry storage and low-temperature stratification, or prechilling. Obviously, we would like to know the essential changes that occur in dormant seeds in dry storage that enable them to germinate. We asked the same question about low-temperature stratification and found the answers only in small part satisfactory. Answers to this question are also only partially satisfactory. There are, however, a number of hypotheses and facts that throw some light on the question. On the basis of culture of excised embryos from dormant and after-ripened barley grains on nutri- ent gelatin and on the basis of transplanting embryos from dormant and after-ripened grains on endosperm from dormant and after-ripened grains in all possible combinations, Windisch ^-^ concluded that the embryos from dormant grains themselves show dormancy, and that during dry storage of the grains the epithehum of the scutellum is essentially modified. There is much evidence that dormancy in many kinds of seeds of the grass family is determined by intactness of the coats, and that breaking the coats causes prompt, vigorous germination giving no evidence of embryo dormancy (Crocker and Harrington ^^ for Johnson grass; Harrington®' for wheats, oats, and barley; and Atwood ^ for wild oats). Many investi- gators have found that dormant cereals mil germinate promptly if placed in a germinator at lower temperatures, such as 10° to 12° C (50° to 54° F) or 14° to 15° C (57° to 59° F). Flemion, in preliminary unpublished work at the Institute, has found that wheat and oats gro\\Ti from dormant grains, by opening the coats, show the same vigor in the later growth of fhe seedlings as those grown from after-ripened grains. As we have seen from Flemion's work cited above, dwarfishness or lack of dwarfishness in the seedling is the best criterion for embryo dormancy. There is the possibility that freshly harvested seeds contain inhibiting substances that volatilize or decompose during dry storage. Shuck '*'^* '*'* finds that fresh lettuce seeds form inhibiting substances when put into a germinator at a high temperature, 25° C (77° F), in darkness. Repeated growth of batches of seeds on the same filter paper leads to the accumula- tion of inhibiting substances on the paper that inhibit later sowings. Water and soil are more favorable germination media for these seeds than filter paper. He believes this is the case because they have greater power to absorb the inhibitors. Even soaking in cold water seems to remove the inhibitors. He also finds that light and low-temperature germinators over- come the inhibitors. We have already discussed other cases where inhibit- ing substances prevent germination. This hypothesis deserves serious examination in all seeds that after-ripen in dry storage. Cereal grains are hastened in after-ripening by heating at 35° to 40° C (95° to 104° F) ■* for 2 to 4 days; the heating is effective without drying, although dry heat adds to the effectiveness. Of course, heat will drive off 126 GROWTH OF PLANTS volatile inhibitors if they are present. Gadd ^^ thinks that at first seed coverings are alive and thus deprive the embryos of oxygen, and that when the coats die oxygen gets to the embryo. We have found that freshly har- vested lettuce seeds that will not germinate at 30° C (86° F) in air also will not germinate at this temperature under one or two atmospheres of pure oxygen. They will, however, germinate promptly at this temperature if the coats are broken. It might seem that a 10-fold increase in oxygen pressure would be sufficient to allow some to reach the embryos. Death of living cells in the coats might also favor the outward diffusion of in- hibitors. Table 17. Percentage of Water Held by Seeds of Impatiens balsamina after Different Periods of Soaking; Freshly Harvested Seeds; Seeds Dry-Stored 8 Weeks. Dry Weight Basis. Temperature of Soaking 20° C. Weighings Made on DupUcate Lots for Each Soaking Period. Storage period, weeks Percentage water in seeds after hours soaking 0 3 6 12 24 30 48 0 12.1 20.9 34.6 46.1 50.4 50.8 50.9* 8 6.2 23.0 38.9 51.9 56.7 56.8* * Maximum water absorption. Barton, in unpublished work at the Institute, has found that certain seeds that after-ripen in dry storage increase greatly in the initial rate of water absorption and somewhat in the final total amount held by them when fully imbibed as the dry storage period increases. Table 17 shows this relation for Impatiens seeds freshly harvested and dry-stored in the laboratory for eight weeks. It will be noted that freshly harvested seeds bore 12.1 per cent water while the dry-stored seeds bore 6.2 per cent. In spite of this initial difference, after three hours' soaking, the dry-stored seeds contained 23.0 per cent water against 20.9 per cent for the freshly harvested; in three hours of soaking the dry-stored seeds had absorbed 16.8 per cent of their dry weight while the fresh ones absorbed 8.8 per cent. She got similar results with Rumex, Amaranthus, and lettuce seeds. The initial rate of water absorption and the total amount absorbed increase with the period of dry storage as after-ripening progresses, and is above that which occurs from drying without after-ripening. While the statements above throw some light on the changes involved in after-ripening of seeds in dry storage, the great number and range of species and varieties of seeds in tliis category demand that many of these seeds, including several representatives from every family of plants in- volved, be examined in the light of all the hypotheses and facts mentioned above, and in the light of new hypotheses that will be suggested by such investigations. DORMANCY IN SEEDS 127 In examining all the foregoing discussion on dormancy of seeds, it will be seen that nature secures delayed germination in seeds by a great variety of methods and not by a single method. It is not improbable that dry storage likewise leads to the after-ripening of seeds by several different essential changes in the many kinds of seeds involved. Temperature a Factor in Overcoming Dormancy We have already spoken of the importance of daily alternating tempera- tures in substituting for light in light-sensitive seeds. There are many other records in the literature of seeds that germinate much better at alternating temperatures than at optimum constant temperatures. This is so important that alternating temperatures are used as a regular pro- cedure in the commercial testing of many seeds. Seeds in nature in the soil experience such a daily alternation of temperatures — warm in the da\i:ime and cool at night. There are many seeds that germinate at tem- peratures at or near the freezing point, and some of them germinate only at such low temperatures. There are other seeds that require high tem- peratures for germination. Schroeder and Barton ^"^ found that seeds of some high-altitude alpines (Calochortus macrocarpus, Camassia LeichtUnii, Lewisia rediviva) germinate only at low temperatures. The first germinate best at 5° C (41° F) and will not germinate fully at temperatures much above this. Annual del- phinium seeds do not germinate well at temperatures above 15° C (59° F). These low-temperature seeds can be successful!}^ growTi at temperatures above the low maximum for germination by pregerminating ^^ at favor- able low temperatures before planting at higher temperatures. We have already mentioned the fact that many seeds that need low- temperature stratification will germinate when they are after-ripened right at the optimum stratification temperature. As will be seen in Table 11, this temperature is 1° C (34° F) for several seeds studied. We have also em- phasized the fact that seeds after-ripened at low temperatures may go back into secondary dormancy if put in a germinator at too high a tem- perature. \Miile one generally thinks of physiological processes in plants increasing in intensity as the temperature rises above the freezing point, the after-ripening of seeds goes on fastest near the freezing point and falls off as the temperature rises; and in some seeds the germination proceeds fastest just a little above the freezing point. Just as some kinds of seeds are attuned to very low temperatures for after- ripening and germination, others require relatively high temperatures for germination. At the Institute we have tested a number of crops of Amaranthus retro flexus seeds for their temperature requirements for ger- mination immediately after harvest and after various periods of dry stor- age. Immediately after harvest these seeds require a temperature of 35° to 40° C (95° to 104° F) for germination, and they germinate promptly at this temperature. As they remain longer and longer in dry storage they 128 GROWTH OF PLANTS will germinate at lower and lower temperatures, until after several months of dry storage they germinate, although slowly, at 10° C (50° F). We have already mentioned the fact that the intact upper seed of the cocklebur requires about 33° C (91° F) for prompt and complete germination, and that an excised embryo has a minimum germination temperature of 18° C (64° F) . It is probable that high-temperature requirements for germina- tion determine the late appearance of crab grass, Panicum sanguinale,^^^ Portulaca oleracea, and other weeds ^^ rather late in the gro\ving season in this latitude. From what has just been said it is evident that many seeds remain dormant in a germinator because the temperature is too low or too high or lacks variation. Quick Vitality Tests for Dormant Seeds Seeds of farm and garden plants are tested for viability before they are put on the market. This is relatively easy to do by germination, for some of these seeds will germinate fully within four or five days under the proper conditions; and even the slower ones, like the blue grasses, will germinate within 28 days under the good conditions provided in the seed-testing laboratories. The testing of farm and garden seeds for viability and purity has returned many-fold the expense of maintaining thoroughly equipped state, government, and private laboratories. No such adequate methods and equipment have been available for comparable tests of forest and horticultural seeds that show great delays. For many of them, ordinary germination tests are not available, because it takes so long to after-ripen and germinate the sample that there is not enough time left to after-ripen the main part of the seeds for early spring planting. In two ways it is more important to be sure of high viability in these seeds than it is for farm and garden seeds; considerable effort and expense must be put on stratification, and failure of a crop due to poor seeds cannot be recouped to any degree the same year by replanting. On the other hand, the total crop value from farm and garden seeds is many times that from dormant forest and horticultural seeds. Probably due to its work on dormant seeds, the Institute in the early thirties began to receive samples of dormant seeds from nurseries for test- ing. If the samples came in as late as January it was impossible to stratify the seeds and later germinate them so that the viability tests could be furnished to the grower in time for him to stratify the seeds and have them ready for early spring planting. Flemion had been excising embryos from dormant seeds and growing them on moist filter papers to determine whether the embryos were the seat of dormancy and how the dormancy of the embryo expressed itself in the later gro\vth of the seedling, as de- scribed in a previous section. This led to her method of quick vitality tests for dormant seeds, which consists of growing the excised or partially excised embryo at room temperature on moist filter paper. DORMANCY IN SEEDS 129 For a long time botanists have been trying to get reliable methods for determining quickly the viability of seeds without bothering to germinate them. Flemion ^^ gives a review of this literature to which one may turn for citations. As early as 1876 Dimitriewicz found that sections of good grain seeds turned a deep rose color in sulfuric acid in five minutes, whereas sections of poor seeds required 15 minutes. Lesage used dilute potassium hydrate for determining viability in garden cress seeds. Heat of respiration, electrical response ("blaze current"), and electrical conduc- tivity have been suggested as means of quick vitality tests. Catalase con- tent has been used. This offers difficulties because many dead seeds contain catalase, and dead Amaranthus seeds ^^ are as rich in catalase as viable seeds. Davis -^ found that soaking seeds in warm (32° C, 89° F) water overnight lowered the catalase in dead seeds and increased it in live seeds, and on this basis developed a viability method. This method, how- ever, has been questioned as to general usefulness. Some dyes penetrate dead embryos readily, but Hving embryos much less readily. Indigo car- mine 1:2000 is the best dye for this purpose. Several chemicals enter the seeds and are reduced by the respiratory activity of seeds producing color reduction products. Soaking seeds in solutions of para- or ortho-dinitro- benzene for 20 hours, followed by treatment with ammonia for one hour, gives an orange coloration in live seeds. Tellurites, tellurates, selenites, and selenates enter seeds and are reduced to the elements tellurium and selenium, giving purple color with the former and yellow with the latter. Tetrazolium salts " have been very recently recommended as substitutes for selenium salts because they are less toxic. They are reduced to red formazanes. In three papers '^' ^''' '*- Flemion describes in detail the methods of excis- ing embryos and of running the viability tests on 38 different species of plants representing 10 different families. In later unpublished w^ork she has extended this method to 15 other species of dormant seeds represent- mg 3 additional families. In some of the seeds, excising of the embryo without injury is a fairly difficult process and requires considerable tech- nical skill. This is especially true of Symphoricarpos, which has a tough, leathery coat and a rudimentary embryo embedded in the endosperm. A quick vitality test on this seed not only requires much work but 6 weeks to get results. This seed represents an extreme case and is of no practical significance. In most seeds of practical interest the excising of the embryo is relatively simple; and the period for running the viability tests ranges from 3 to 14 days, about the time required for germination tests of farm and garden seeds. In all cases, Flemion finds that viability tests deter- mined with excised embryos check closely with germination obtained by after-ripening the dormant seeds and later germinating them. Even the relative vigor of the several embryos is clearly sho^vn, as it is with the speed of germination of the after-ripened seeds. 130 GROWTH OF PLANTS Let us examine the application of the Flemion method to several different donnant seeds shown in Figure 50. In A are embryos of Rkodotypos kerrioides. Beginning at the left is an embryo just removed from a swollen seed of 1937 crop; the next 5 in succession are sample embryos from the seed crops of 1932, 1934, 1935, 1936, and 1937, all removed and kept in the germinator five days in 1937. Note that the embryo of the 1932 crop is disintegrating and note the increased growth from the 1934 crop to the 1937. In excised "dormant" Rkodo- typos embryos both the cotyledons and the roots grow rather promptly, though not nearly as promptly as the low-temperature after-ripened embryos, as we have already seen; and the rate of growth increases with vigor of the embryos and decreases with the age of the seed. The behavior of embryos from the 1932 and 1934 embryos kept on moist filter paper 5 addi- tional days, or 10 days in all, are shown in B. The 1932 embryo is dead and decaying, while the 1934 embryo has such low vigor that it has not enlarged, although it has developed more clilorophyll. It is evident that this test shows not only the embryos that are alive but also the relative vigor of the living embryos of the several crops. Apple embryos are shovvm in C: the one at the left, a freshly excised embryo, followed by 2 dead embryos and 2 hve ones after 6 days on moist filter paper. The apple embryo is more sluggish in its early growth than the Rkodotypos embryo. There is merely an enlargement and the greening of the cotyledon in contact with moist filter paper in some embryos; in others both the cotyledons and the roots grow in the 6-day test. In D Crataegus Cnis-gaUi embryos are shown in a 10-day test run started February 1, 1938. Begiiming at the left, first, second, and third are embryos from 1928, 1930, and 1933 crops respectively, all non-viable. The fourth embryo is of the 1935 crop which has not yet had time to enlarge. The fifth embryo is of the 1936 crop in which the intact carpel had been stratified one year at 5° C (41° F) ; but the embryo had not after-ripened under this condition, due to the resistant coat, as shown by the growth of only the cotyledon in contact with the moist filter paper. Crataegus embryos are very sluggish in their early growth, and manifest their viability in these tests by the enlargement and greening of the cotyledon in contact with the moist filter paper and their lack of viabiUty by decay. The test period for these should run for several weeks. Sorbus aucuparia embryos are shown in E: beginning at the left is a freshly excised embryo of Sorbus aucuparia; embryo of the 1930 crop, decaying; and embryo from the 1937 crop with cotyledons enlarged and green. The second and third have been on moist filter paper for 6 days beginning November 18, 1937. The dormant Sorbus embryos are more sluggish in their growth than dormant Rkodotypos embryos and less sluggish than apple and Crataegus embryos. Witch-hazel embryos appear in F after 5 days on moist filter papers (November 18, 1937), 1934, 1935, and 1936 crops. The one-year-old embryos gave good growth, the two-year-old embryos little growth, and the three-year-old none. In G is shown the reaction of the 1934 embryos (dead) and the 1936 embryos, high viability, to para-dinitrobenzene-ammonia treatment; in H is the reaction of the same to 1 per cent potassium tellurite treatment; and J shows the development of these embryos after 5 days on moist filter paper. The color diEfer- ence from the reduction of the two chemical compounds is not very conclusive as to degree of vitality. Prunus americana embryos are shown in K, all from viable seeds after several weeks on moist filter paper. These embryos are extremely sluggish in their early growth and show viability mainly by slow growth and greening of the cotyledon in contact with the moist filter paper and by the occasional growth of a root. The dead embryos soon disintegrate. The fringe-tree embryos appear in L. In order, beginning at the left, are: dead embryo and live embryos after 4, 6, and 20 days. In M are Douglas fir embryos: beginning at left, freshly excised, viable, and non-viable embryos after 4 days. Pinus rigida embryos are shown in N. From left to right, freshly excised, dead embryo, embryo with low vitality, and one with high vitality after 8 days on moist filter paper. In selecting the embryos for this plate (Fig. 50), the embryo that most nearly represented the average growth or behavior in duplicate cultures of at least 50 embryos was used. Figure 50. Development of excised embryos on moist filter paper in Petri dishes at room temperature: K, natural size; F,, M, and N 4 X ; all others 2 X. {See explanation on opposite page.) DORMANCY IN SEEDS 131 What are the relative merits of viabihty tests based on color reactions and those using excised embryos? The color reaction requires only 24 to 48 hours, whereas the excised method requires 4 to 14 days. In both cases the embryos must be at least partially removed, but for the color tests less care needs to be taken against injuring the embryo. Finally, Flemion claims that the main advantage of the excised embryo method is its relia- bility. Often worthless embryos show considerable color with the color tests. In the excised embryo method the embryo shows its viability by a growth reaction of some sort. It is a direct viability test. Let us select two of many cases where the excised embryo method was applied to practice. A seedsman reported that he had a chance to pur- chase 2000 bushels of Douglas fir cones that had been found in a squirrel cache. The seeds looked good, but a test for viability was desired; also the offer had to be accepted in a few days, whereas an ordinary germina- tion test required about 60 days. Within 5 days by the use of this quick viability test Flemion was able to advise that 95 per cent of the seeds were dead and the other 5 per cent showed very low vitality. The largest dis- tiller of witch hazel has always used wild growth as the source of wood for distillation. This source became scarce and had to be gathered at greater and greater distances from the still. A few years ago the distiller decided to start a grove near the still as a wood source. A quick vitality test showed that the peck of seeds they had for this purpose was entirely dead. The collection of seeds the next autumn proved viable and was after-ripened with three months of stratification at 5° C (41° F). Several hundred thousand seedlings are now ready to transplant into' the proposed grove. The excised embryo test for viability of dormant horticultural and for- est seeds is used extensively at Boyce Thompson Institute, and is gradu- ally being adopted by seed-testing laboratories. Chemicals as Forcing Agents for Dormant Seeds As we shall see in a later chapter, Denny and co-workers have been very successful in forcing dormant buds with chemicals. Among the chemicals tested to date, few have proved successful in forcing dormant seeds. Nitrogen compounds for light-favored seeds and concentrated sulfuric acid for hard seeds and seeds with resistant coats are notable exceptions. The latter, however, is a corrosive action. A few kinds of dormant seeds are forced by one or more of the following substances: carbon dioxide, mercury salts, hydrogen peroxide, anesthetics, etc., but none of these is generally effective as a forcing agent. We have tried bud- forcing chemicals, hormones, and other compounds, on both dormant seeds and embryos, without promising results either in forcing the dormant seeds to germinate or dormant embryos to grow with vigor. It is possible, however, that later researches \vill find chemicals that will parallel tem- perature manipulations in eliminating seed and embryo dormancy. 132 GROWTH OF PLANTS Summary We have seen the advantages to plants of delayed and time-distributed germination of seeds in furthering the persistence of species, and the advantages to man of at least a temporary dormant period in seeds in furnishing him seeds for food and for propagation of plants. We have seen how delayed germination presents difficulties in propagation and in fight- ing weeds on the farm and in the garden. We have seen that delayed ger- mination of seeds in nature is secured by a variety of means: by hard coats that allow no water to enter until decay or other factors corrode the coats; by requiring light or darkness for germination so that the first seeds are prevented from germinating if they are covered Avith soil and the latter if they are not; by coats, very thin in the upper seed of the cocklebur, that reduce the oxygen supply below that needed for germmation; by dormancy in embryos that gives them sluggishness in early growth and manifests itself by dwarfishness in the later growth of the seedling, unless previously given a low-temperature period in the soil which after-ripens the embryos and perhaps aids in coat destruction; by dormancy of the epicotyl that is after-ripened by a period of low-temperature exposure only after the root has started; by demanding a high-temperature period in the soil for decomposition of coats by soil agents, especially organisms, followed by a low-temperature period for after-ripening the embryos; by demanding a low-temperature period to after-ripen the seed so that the root can grow, followed by a high-temperature period for root growth, and in some seeds for epicotyl elongation and rupture of cotyledonary sheath, followed again by a low-temperature period for epicotyl after-ripenmg, and a high-temperature period for growth of the epicotyl. At every phase of this study we have seen the great significance of seed and fruit coats, non-livmg or in the main non-living, in securing this delay. We have seen that two big factors in after-ripening temperate zone seeds and preparing them for germination are periods of low-temperature moist exposure and periods of dry storage; and hardly less important is the coat-dismtegrating action of soil agents, especially organisms which act best at gro^^•ing temperatures. We have discussed various methods for quick vitality tests of seeds that require long periods for germination, the best of which seems to be isolating or partially isolating the embryos and placing them on moist filter paper. Fmally, the mam problems in delayed germination and dormancy of seeds are yet to be solved. The work to date has thrown much light on the way dormant seeds of various types behave m nature; it has put into the hands of commercial and amateur growers methods of growing many kinds of seeds that could not be grown before; and it has done much to define the big physiological and biochemi- cal problems still to be solved. No doubt further research A\ill shorten the time needed to get complete germination of the seeds that are the most difficult to handle. DORMANCY IN SEEDS 133 Literature Cited 1. Afanasiev, Michel, "Germination of redbud seed," Am. Nurseryman, 69 (11) : 3 (June 1, 1939). 2. Afanasiev, M., and M. Cress, "Changes within the seeds of Juniperus scopulorum during the processes of after-ripening and germination," J. Forest., 40 : 798-801 (1942). 3. Arthur, J. C, "Delayed germination of cocklebur and other paired seeds," Proc. Soc. Prom. Agric. Sci., 16 : 70-79 (1895). 4. Atterberg, Albert, "Om sadesvarornas eftermognad," K. Landtbruks-Akad. Stockholm. Handl. o. Tidskr., 38 : 227-250 (1899). 5. Atwood, W. M., "A physiological study of the germination of Avena fatua," Bot. Gaz., 57 : 386-414 (1914). 6. Barton, Lela V., "Hastening the germination of southern pine seeds," J. Forest., 26 : 774-785 (1928); also in B. T. I. Prof. Pap., 1 : 58-69 (1928). 7. , "Hastening the germination of some coniferous seeds," Am. J. Bot., 17 : 88- 115 (1930); also in C. B. T. I., 2 : 315-342 (1930). 8. , "SeedUng production of tree peony," C. B. T. I., 5 : 451-460 (1933). 9. , "Dormancy in Tilia seeds," C. B. T. I., 6 : 69-89 (1934). 10. , "Germination of delphinium seeds," C.B.T.I.,7: 405-409 (1935). 11. , "Germination and seedhng production in Ldlium sp.," C. B. T. I., 8 : 297-309 (1936). 12. , "Experiments at Boyce Thompson Institute on germination and dormancy in seeds," Sci. Hort., 7 : 186-193 (1939). 13. , "Some seeds showing special dormancy," C. B. T. I., 13 : 259-271 (1944). 14. Barton, Lela V., and Eltora M. Schroeder, "Dormancy in seeds of Convallaria majalis L. and Stnilacina racemosa (L.) Desf.," C. B. T. I., 12 : 277-300 (1942). 15. Brown, R., "An experimental study of the permeabiHty to gases of the seed-coat membranes of CucurUta Pepo," Ann. Bot., 4 : 379-395 (1940). 16. , "Studies in germination and seedhng growth. I. The water content, gaseous exchange, and dry weight of attached and isolated embryos of barley," Ann. Bot., 7:93-113 (1943). 17. Busse, W. F., "Effect of low temperatures on germination of impermeable seeds," Bot. Gaz., 89 : 169-179 (1930). 18. Chang, S. C, "Length of dormancy in cereal crops and its relationship to after- harvest sprouting," J. Ain. Soc. Agron., 35 : 482-490 (1943). 19. Crocker, W., "Role of seed coats in delayed germination," Bot. Gaz., 42 : 265-291 (1906). 20. , "Effect of the visible spectrum upon the germination of seeds and fruits," in Duggar, B. M., editor, "Biological effects of radiation," 2 : 791-827. McGraw- Hill Book Co., New York, 1936. 21. , and W. E. Davis, "Delayed germination in seed of Alisma plantago," Bot. Gaz., 58:285-321 (1914). 22. , and G. T. Harrington, "Catalase and oxidase content of seeds in relation to their dormancy, age, vitality, and respiration," /. Agric. Res., 15 : 137-174 (1918). 23. Davies, P. A., "High pressure and seed germination," Am. J. Bot., 15 : 149-156 (1928). 24. , "The effect of high pressure on the percentages of soft and hard seeds of Medicago sativa and Melilotus alba," Am. J. Bot., 15 : 433-436 (1928). 25. Davis, W. E., "The use of catalase as a means of determining the viability of seeds," Proc. Assoc. Off. Seed Anal. N. Am., 18 : 33-39 (1926); oko in B. T. I. Prof. Pap., 1 : 6-12 (1926). 134 GROWTH OF PLANTS 26. Davis, W. E., "Primary dormancy, after-ripening, and development of secondary dormancy in embryos oi Ambrosia trifida," Am. J. Bot., 17 : 58-76 (1930); also in C. B. T. I., 2 : 285-303 (1930). 27. Davis, W. E., and R. Catlin Rose, "The effect of external conditions upon the after- ripening of the seeds of Crataegus mollis," Bot. Gaz., 54 : 49-62 (1912). 28. Deming, G. W., and D. W. Robertson, "Dormancy in small-grain seeds," Colorado Agric. Exp. Sta. Tech. Bull. No. 5, 12 pp., 1933. 29. Dinnis, E. R., and S. Jordan, "The germination of freshly harvested and of stored seeds of sea pea," J. South East. Agric. Coll. (Wye, Kent), 44 : 140-142 (1939). 30. Eckerson, S., "A physiological and chemical study of after-ripening," Bot. Gaz., 55 : 286-299 (1913). 31. Esdorn, I., "Der Einfluss der Lagerung auf die Keimfiihigkeit der gelben Lupine," Fortschr. Landw., 3 : 346-353 (1928). 32. , and H. Stiitz, "Die Bewertung harter Leguminosensamen," Landw. Versuchs- Stationen, 114 : 137-147 (1932). 33. Eyster, W. H., "Vivipary in maize," Genetics, 16 : 574-590 (1931). 34. Flemion, F., "After-ripening, germination, and vitality of seeds of Sorbus aucuparia L.," C. B. T. L, 3 : 413-439 (1931). 35. , "Physiological and chemical studies of after-ripening of Rhodotypos kerrioides seeds," C. B. T. I., 5 : 143-159 (1933). 36. , "Dwarf seedUngs from non-after-ripened embryos of Rhodotypos kerrioides," C. B. T. /., 5 : 161-165 (1933). 37. , "Physiological and chemical changes preceding and during the after-ripening of Symphoricarpos racemosus seeds," C. B. T. I., 6 : 91-102 (1934). 38. , "Dwarf seedlings from non-after-ripened embryos of peach, apple, and hawthorn," C. B. T. I., 6 : 205-209 (1934). 39. , "A rapid method for determining the germinative power of peach seeds," C. B. T. I., 8 : 289-293 (1936). 40. , "A rapid method for determining the viability of dormant seeds," C. B. T. /., 9 : 339-351 (1938). 41. , "Breaking the dormancy of seeds of Crataegus species," C. B. T. I., 9 : 409- 423 (1938). 42. , "Further studies on rapid determination of the germinative capacity of seeds," C. B. T. /., 11 : 455-464 (1941). 43. , "Effect of the addition of nitrogen upon germination of seeds of Symphori- carpos racemosus," C. B. T. L, 12 : 485-489 (1942). 44. , and E. Parker, "Germination studies of seeds of Symphoricarpos orbiculatus," C. B. T. I., 12 : 301-307 (1942). 44a. , and E. Waterbury, "Further studies with dwarf seedlings of non-after- ripened peach seeds," C. B. T. /., 13 : 415-422 (1945). 45. Flint, L. H., "Sensitivity of dormant lettuce seed to light and temperature," J. Washington Acad. Sci., 25 : 95-96 (1935). 46. , and E. D. McAhster, "Wave lengths of radiation in the visible spectrum inhibiting the germination of light-sensitive lettuce seed," Smithsonian Inst. Publ. No. 3334, 11 pp., 1935. (Smithsonian Misc. Coll. v. 94. No. 5.) 47. , and C. F. Moreland, "Response of lettuce seedlings to 7600 A radiation," Am. J. Bot., 25 : 12s (1938). 48. , , "Response of lettuce seedlings to 7600 A radiation," Am. J. Bot., 26 : 231-233 (1939). 49. Funke, H., "Beitriige zur Kenntnis von Keimung und Bau der Mistel," Bot. Centralbl. Beih. A, 69: 235-274 (1939). 50. Gadd, I., "Ueber die Natur der Hartschaligkeit der kleinsamigen Leguminosen und den Einfluss der Lagerung auf dieselbe," Internat. Seed Testing Assoc. Proc, 10 : 146-174 (1938). DORMANCY IN SEEDS 135 51. Gadd, I., "On methods for the ehmination of seed dormancy in seed control work," Internal. Seed Testing Assoc. Proc, 11 : 108-118 (1939). 52. Gassner, G., "Beitrage zur Frage der Lichtkeimung," Zeitschr. Bot., 7:609-661 (1915). 53. Giersbach, J., "After-ripening and germination of Cotoneaster seeds," C. B. T. I., 6:323-338 (1934). 54. , "Germination and seedling production of Ardoslaphylos uva-ursi," C. B. T. /., 9 : 71-78 (1937). 55. , "Germination and seedling production of species of Viburnum," C. B. T. I., 9 : 79-90 (1937). 56. , and L. V. Barton, "Germination of seeds of the silver bell, Halesia Carolina," C. B. T. I., 4 : 27-37 (1932). 57. Grimm, K , "Uber die Keimung des Klees und aussere Einfliisse auf diese," Bot. Arch., 21: 344-445 (1928). 58. Hamly, D. H., "Softening of the seeds of Melilotus alba " Bot. Gaz., 93 : 345-375 (1932). 59. Harrington, G. T., "Agricultural value of impermeable seeds," /. Agric. Res., 6 : 761-796 (1916). 60. , "Further studies of the germination of Johnson grass seeds," Proc. Assoc. Off. Seed Anal. N. Am., 9 10(1916-1917) : 71-76 (1917). 61. , "Forcing the germination of freshly harvested wheat and other cereals," /. Agric. Res., 23 : 79-100 (1923). 62. Harrington, J. B., and P. F. Knowles, "The breeding significance of after-harvest sprouting in wheat," Sd. Agric, 20 : 402-413 (1940). 63. Hohnel, F. von, "Ueber die Ursache der Quellungsunfahigkeit von Legumenosen- samen und der Einfiuss der chemisch-physikahschen BeschafiFenheit der Pallisa- denschicht auf die Keimfiihigkeit derselben. WissenschaftUchpraktische Untersuch," Gebiete Pflanzenbaues, 1 : 80-88 (1875). 64. Hiibner, R., "Untersuchungen liber die Hartschahgkeit der Zottelwicke und ihre Behebung auf ziichterischem Wege," Landw. Jahrb., 85 : 751-789 (1938). 65. Hutton, Mary Erne-Jean, and R. H. Porter, "Seed impermeability and viability of native and introduced species of Leguminosae," Iowa State Coll. J. Sci., 12 : 5-24 (1937). 66. Jensen, C., "Is it possible that seeds through treatment with light may keep their germinating power through a longer span of years than normal?" 16 pp., J. D. Quist & Co., Copenhagen, 1941: Abstr. in Exv. Sta. Rec, 86:343 (1941). 67. Johnson, L. P. V., "General preliminary studies on the physiology of delayed germination in Avena fatua," Canadian J. Res. (Sec. C), 13 : 283-300 (1935). 68. Johnstone, G. R., and T. S. Clare, "Hastening the germination of western pine seeds," /. Forest, 29 : 895-906 (1931). 69. Jones, H. A., "Physiological study of maple seeds,"Bot. Gaz., 69 : 127-152 (1920). 70. Jones, J. P., "A physiological study of dormancy in vetch seed." New York [Cornell] Agric. Exp. Sta. Mem. No. 120, 50 pp., 1928. 71. Kommerell, E., "Quantitative Versuche iiber den Einfluss des Lichtes verschiedener Wellenlangen auf die Keimung von Samen," Jahrb. Wiss. Bot., 66 : 461-512 (1927). 72. Kroeger, G. S., "Dormancy in seeds of Imvatiens balsamina L.," C. B. T. I., 12:203-212 (1941). 73. Lakon, G., "Topographischor Nachweis der Keimfahigkeit der Gotreidefriichte durch TetrazoHumsalze," Ber. Deutsch. Bot. Ges., 60 : 299-305 (1942). 74. Lammerts, W. E., "Effects of photoperiod and temp)erature on growth of embryo- cultured peach .seedlings," Am. J. Bot., 30 : 707-711 (1943). 75. Lute, A. M., "Alfalfa seed made permeable by heat," Science, 65 : 166 (1927). 136 GROWTH OF PLANTS 76. Mcllvaine, H. R. C, and H. W. Popp, "Further studies on growth substances in relation to the mechanism of the action of radiation on plants," J. Agric. Res., 60 : 207-215 (1940). 77. McKeever, D. G., "A new black locust seed treatment," J. Forest., 35 : 500-501 (1937). 78. MacLachlan, P. L., "Fat metabolism in plants with special reference to sterols," J. Biol. Chem., 113 : 197-204 (1936). 79. Mangelsdorf, P. C, "The inheritance of defective seeds in maize," /. Heredity, 14: 119-125 (1923). 80. , "The genetics and morphology of some endosperm characters in maize," Connecticut [New Haven] Agric. E.xp. Sta. Bull. No. 279 : 509-614, 1926. 81. Martin, J. N., "Structure of seed coat and environmental factors pertaining to germination of weed seeds," Iowa Agric. Exp. Sta. Report on agricultural re- search for the year ending June 30, 1942, Pt. 1, p. 144. 82. Middleton, G. K., "Size of Korean lespedeza seed in relation to germination and hard seed," /. A7n. Soc. Agron., 25 : 173-177 (1933). 83. Midgley, A. R., "Effect of alternate freezing and thawing on the impermeabihty of alfalfa and dodder seeds," J. Am. Soc. Agron., 18 : 1087-1098 (1926). 84. Morinaga, T., "Germination of seeds under water," Am. J. Bot., 13: 126-140 (1926); aUo in C. B. T. I., 1 : 67-81 (1926). 85. Miiller, G., "Beitrage zur Keimungsphysiologie. Untersuchungen liber die Spreng- ung der Samen- und Fruchthlillen bei der Keimung,"/a/ir6. Wiss. Bot., 54 : 529- 644 (1914). 86. Muenscher, W. C., "Seed germination in Lobelia, with special reference to the influence of hght on Lobelia inflata," J. Agric. Res., 52 : 627-631 (1936). 87. Murakami, R., "The influence of monochromatic lights on the action of enzymes," J. Agric. Chem. Soc. Japan, 16: 15 (1940); Abstr. in Biol.Abstr., 16:469 (1942). 88. Orth, R., "Zur Keimungsphysiologie der Farnsporen in verschiedenen Spectral- bezirken," Jahrb. Wiss. Bot., 84 : 358-426 (1937). 89. Pack, D. A., "After-ripening and germination of Juniperus seeds," Bot. Gaz., 71 : 32-60 (1921). 90. , "Chemistry of after-ripening, germination, and seedhng development of juniper seeds," Bot. Gaz., 72 : 139-150 (1921). 91. , "Dispersion of hpoids," Bot. Gaz., 79 : 334-338 (1925). 92. Pfeiffer, N. E., "Morphology of the seed of Symphoricarpos racemostis and the relation of fungal invasion of the coat to germination capacity," C. B. T. L, 6 : 103-122 (1934). 93. Pietruszczynski, Z., "After-ripening of cereals," Polish Agric. & Forest Ann., 15: 206-235 (1926). Abstr. in Biol. Abstr., 6 : 21627 (1932). 94. Pope, M. N., and E. Brown, "Induced vivipary in three varieties of barley possess- ing extreme dormancy," /. Am. Soc. Agron., 35 : 161-163 (1943). 95. Porter, R. H., and E. O. Brown, "Investigation of impermeability, longevity, dormancy, viabihty and germination of seeds," Iowa Agric. Exp. Sta. Report on agricultural research for the year ending June 30, 1935, p. 87. 96. Raleigh, G. J., "Chemical conditions in maturation, dormancy, and germination of seeds of Gymnocladus dioica," Bot. Gaz., 89 : 273-294 (1930). 97. , "The germination of dormant lettuce seed," Science, 98 : 538 (1943). 98. Rancken, M., "Der Einfluss von Feuchtigkeit und Temperatur auf die harten Kleesamen wahrend der Aufbewahrungszeit," Internal. Seed Testing Assoc. Proc., 9 : 263-271 (1937). 99. Rivera, R., H. W. Popp, and R. B. Dow, "The effect of high hydrostatic pressures upon seed germination," Am. Jour. Bot., 24 : 508-513 (1937). 100. Rostrup, O., "Report of the Danish seed control for 1896-1897," 37 pp., Copen- hagen, 1898; Abstr. in Exp. Sta. Rec, 10 : 53-54 (1899). DORMANCY IN SEEDS 137 101. Saulescu, N., "Untersuchungen liber die Hartschaligkeit beim Siebenburger Rotklee," Pflanzenbau, 11 : 180-186 (1934). 102. Schaible, F., " Physiologische Experimente uber das Wachsthum und die Keimung einiger Pflanzen unter vermindertem Luftdruck," Beitr. Wiss. Bot., 4 : 93-148 (1900); Ahstr. in Bot. Centralbl., 82 : 52-54 (1900). 103. Schaumann, K., "Uber die Keimungsbedingungen von Alisma plantago und anderen Wasserpflanzen," Jahrb. Wiss. Bot., 65 : 851-934 (1926). 104. Schroeder, E. M., and L. V. Barton, "Germination and growth of some rock garden plants," C. B. T. I., 10 : 235-255 (1939). 105. Schwendiman, A., and H. L. Shands, "Delayed germination or seed dormancy in Vicland oats," J. Am. Soc. Agron., 35 : 681-€88 (1943); also in News Letter Assoc. Off. Seed Anal, 17(4) : 14 (Oct., 1943). 106. Shaw, M. F., "A mitrochemical study of the fruit coat of Nelumbo lutea," Am. J. Bot., 16 : 259-276 (1929). 107. Shuck, A. L., "The formation of growth inhibiting substance in germinating lettuce seeds," Internat. Seed Testing Assoc. Proc, 7 : 9-14 (1935). 108. , "Light as a factor influencing the dormancy of lettuce seeds," Plant Physiol, 10 : 193-196 (1935). 109. Shull, C. A., "The oxygen minimum and the germination of Xanthium seeds," BoL Gaz., 52 : 453-477 (1911). 110. , "The role of oxygen in germination," Bot. Gaz., 57 : 64-69 (1914). 111. , and W. B. Davis, "Delayed germination and catalase activity in Xanthium," BoL Gaz., 75 : 268-281 (1923). 112. Spaeth, J. N., "Dormancy in seeds of basswood, Tilia americana L.," Am. J. Bot., 19 : 835 (1932). 113. Sprague, V. G., "Germination of freshly harvested seeds of several Poa species and of Dactylis glomerata," J. Am. Soc. Agron., 32 : 715-721 (1940). 114. Staifelt, M. G., "Die Permeabilitat des Sauerstoffs in vervvundeten und intakten KeimUngen von Sinapis alba," Biol Zentralbl, 46 : 11-23 (1926). 115. Stevenson, T. M., "Sweet clover studies on habit of growth, .seed pigmentation and permeabiUty of the seed coat," Sci. Agric., 17 : 627-654 (1937). 116. Stier, H. L., "The effect of certain seed treatments on the germination of recently harvested potato seeds," Proc. Am. Soc. Hort. Sci., 35(1937) : 601-605 (1938). 117. Stiitz, H., Uber den Einfluss verschiedenartiger Lagerung auf die Hartschahgkeit von Kleesamen," 42 pp.. Diss., Hamburg, 1933. 118. Takahashi, T., "Is germination possible in absence of air?" Bull Coll Agric. Tokyo, 6 : 439-442 (1905). 119. Taylor, D. L., "Influence of oxygen tension on respiration, fermentation, and growth in wheat and rice," Am. J. Bot., 29 : 721-738 (1942). 120. Thornton, N. C., "Factors influencing germination and development of dormancy in cocklebur seeds," C. B. T. I., 7 : 477-496 (1935). 121. ," Carbon dioxide storage. IX. Germination of lettuce seeds at high tempera- tures in both Hght and darkness," C. B. T. I., 8 : 25-40 (1936). 122. Toole, E. H., and V. K. Toole, "Progress of germination of seed of Digitaria as influenced by germination temperature and other factors," J. Agric. Res., 63 : 65-90 (1941). 123. Toole, V. K., "Notes on the viabihty of the impermeable seed of Vicia viUosa, hairy vetch," Proc. Assoc. Off. Seed Anal N. Am., 31 : 109-111 (1939). 124. , "Germination of seed of vine-mesquite, Panicum obtusum, and plains bristle- grass, Setaria macrostachya," J. Am. Soc. Agron., 32 : 503-512 (1940). 125. , "Factors affecting the germination of various dropseed grasses (Sporobolns spp.)," /. Agric. Res., 62 : 691-715 (1941). 126. Verschaffelt, E., "Le traitement chimique des graines a imbibition tardive," Rec. Trav. BoL Ncerland., 9 : 401-435 (1912). 138 GROWTH OF PLANTS 127. Webster, C. B., and G. T. Ratliffe, "A method of forcing quick germination of Juniperus virginiana L. seed," J. Forest., 40 : 268 (1942). 128. White, J., "The occurrence of an impermeable cuticle on the exterior of certain seeds," Proc. Roy. Soc. Victoria, 21 (pt. 1) : 203-210 (1908). 129. Windisch, W., "Warum keimt die getrocknete bezw. abgelagerte Gerste besser als die frisch geerntete?" Wochenschr. Brau., 22(7) : 89-92 (1905). 130. Witte, H., "Some international investigations regarding hard leguminous seeds and their value," Internnt. Seed Testing Assoc. Proc, 6 : 279-312 (1934). 131. , "New international investigations regarding the germination of hard legu- minous seeds," Internal. Seed Testing Assoc. Proc, 10 : 93-122 (1938). CHAPTER 4 Physiological Effects of Ethylene and Other Unsaturated Carbon-containing Gases Early Experiments Beginning at the University of Chicago and continuing later at Boyce Thompson Institute in a much more detailed way, a group of investigators have made extensive studies of the effects of gases upon plants. In the later phases, the researches were extended to the effect of some of the gases upon animals. In regard to the physiological effect of the gases upon plants we may speak of two groups: (1) gases low in lethal action and high in anesthetic and growth-modifying effects, among which are certain un- saturated C-containing gases such as ethylene, acetylene, propylene, and carbon monoxide; (2) gases mainly lethal that have only minor physio- logical action other^^ise, including hydrocyanic acid, mercury vapor, sulfur dioxide, ammonia, chlorine, and hydrogen sulfide. The physiologi- cally active gases will be discussed in this chapter and the lethal gases in the follo\\ing chapter. This work was initiated by a question from a Polish greenhouse operator, a good grower but painfully short on English, "What is the effect of illumi- nating gas on carnations?" Answering his question raised others; as a result, much of the w^ork reported in this and the two chapters that follow was an outgrowth of his question. Since the hormone work at the Institute originated in the anesthetic and formative effects of ethylene upon plants, all the work reported in the hormone chapter is a continuation of the answer to the Polish greenhouse operator's query. The author has been assigned two botany problems in his time. One was the reason for the dif- ference in behavior of the two seeds in the cocklebur, assigned by Professor Chas. F. Hottes of the University of Illinois (1902), and the other was the illuminating gas problem (1908) here discussed. The previous two chapters show what has come of the first assignment, and this and the next two chapters set forth the results of the second. Each, of course, has involved the cooperation of a number of investigators, not only in the laboratories with wliich the author has been associated, but in laboratories throughout the world. The literature on the remarkable physiological effects of the unsaturated C-gases has become almost voluminous smce 1908, though most of it has appeared since 1930; only a portion can be cited in this popular discussion. Which assigned the better problem, the learned uni- versity professor or the practical greenhouse operator, quite innocent of higher learning? 139 140 GROWTH OF PLANTS The greenhouse operator had lost the carnation crop in several of his greenhouses each fall for two years after a cold spell had frozen crust on the ground. Near the greenhouses affected was a honey-combed illuminat- ing gas pipe. When there was no crust on the ground the gas escaped upward into the air. With a frozen crust on the ground the gas moved horizontally and escaped into the greenhouse. With the ventilators closed during the cold spells the gas was held in the houses. Crocker and Kjiight "^ tested the effect of the Chicago illuminating gas (a water gas) on carnations. The buds and flowers proved most sensitive. One part of illuminating gas to 40,000 of air stopped the growth of young and medium-aged buds, and 1 : 80,000 caused freshly opened flowers to close, "go to sleep," within 12 hours, and they wou^d not open again even in absence of the gas. As manufactured illuminating gas is a mixture of many gases, the question naturally arose, Which of the constituents are effec- tive? Neljubow ^^ had previously investigated the reason for the peculiar growth of certain plants in "laboratory air" and found it due to a trace of illuminating gas. He found that ethylene produced the "horizontal nuta- tion" of the garden pea epicotyl in concentrations as low as 1 ppm of air. The ethylene in the air changed the geotropic equilibrium position of the garden pea epicotyl gradually from the vertical to a more and more declined position as the concentration of ethylene increased, until 1 ppm of ethylene brought the tip of the seedling into a horizontal position or made it diageo- tropic. Fig. 51 shows the results of repeating Neljubow's experiments 43 years later upon another variety of garden pea. The controls show the great amount of elongation in the epicotyls in three days, and also that they are negatively geotropic, i.e., grow vertically. One ppm of ethylene reduced the rate of elongation markedly, caused the portion of the pea that grew w^hile in the gas to take a declined position; 1.33 ppm reduced the elongation much more and caused the part gro\\ang while in the gas to become diageotropic and the gromng part to increase in diameter; and 4 ppm reduced elongation still more, with a consequent shorter swollen and diageotropic region. It is interesting that these results check so closely with Neljubow's, although a different variety of pea was used under a different set of conditions. He got horizontal nutation in 1 ppm of ethylene; Crocker and Knight got it mth 1.33 ppm of ethylene. They found further that 1 ppm of ethylene prevented the growth of young carnation buds and 0.5 ppm of ethylene caused open flowers to close. From this work Knight and Crocker concluded that ethylene was the effective constituent of illuminating gas in bringing about the injury to the carnation buds and flowers. The crude methods of analysis available at that time indicated that the gas bore about 3 per cent ethylene, whereas it should have borne 4 per cent according to the flower and bud response. The discrepancy can be explained by errors in analysis and variation in the sensitivity of the buds and flowers, as well as variations in the ethylene concentration of the gas. Later work has also shoAvn that, while certain PHYSIOLOGICALLY ACTIVE GASES 141 plant organs respond to very low concentrations of ethylene, the concentra- tion may vary many per cent without a noticeable difference in response. The authors also spoke of the toxicity of ethylene. Later work has showTi that ethylene is not a highly lethal chemical, that is, tissue-killing, but Figure 51. Response of the etiolated epicotyl of garden pea to ethylene. Seedlings 3 to 4 cm high exposed to ethylene for 3 days room temperature. A {left to right) : control, 1 ppm ethylene, 1.33 ppm ethylene, 4 ppm ethylene. B: (left) control; (right) 1.33 ppm ethylene. rather an anesthetic and a growth modifier. The failure of the buds to grow was due partly to anesthetic action, and the closing of the flowers was an irreversible gro^\i:.h response called hyponasty. As ethylene is hard to detect chemically in the low concentrations that produce plant responses, Knight and Crocker ^^ attempted to find plant responses that would serve as reliable and delicate tests for traces of ethyl- ene. They tried various sorts of peas as test plants and found certain varie- ties of sweet peas, Gladys Umvin, for example, even more sensitive than garden peas. One-tenth ppm of ethylene inhibited the elongation of the 142 GROWTH OF PLANTS epicotyl of this pea measurably; 0.2 ppm caused slight declination, together with much greater inhibition of growth; and 0.4 ppm caused still greater reduction in elongation, a greater declination from the vertical, and a swell- ing of the declined portion. This constituted the "triple response" of Knight and Crocker. They proposed the use of either the declination or triple response of the sweet pea seedling as a means of detecting the presence of ethylene. Later, several workers 4, s, it, 35 y^q^yq sho\vn that ethylene causes a modification in the relative rate of growth on the upper and lower sides of the petioles of leaves of many kinds of plants so that the leaves curve downward, giving the epinastic response. Fig. 52 shows this response in the tomato plant. Figure 52. Tomato plants after exposure for 28 hours in various houses of a com- mercial range suspected of gas injury. No. 1, a house in which Acacias showed almost complete bud and leaf fall; No. 8, a house in which newly forced roses were showing leaf fall; No. 7, a house in which roses were showing similar injury to No. 8; No. 6, a house in which roses showed no injury. Besides ethylene, acetylene, and propylene, carbon monoxide (CO) and perhaps butylene will induce the declination or the triple response in the sweet pea seedling and the epinasty of leaves, but these three gases must be used in much higher concentrations than ethylene to be effective. Table 18 shows the minimum effective concentrations of the several unsaturated C-gases for inducing declination of the sweet pea seedling and epinasty in the tomato leaf.^ The gases of the olefin series (ethylene, CH2=CH2, propylene, CHaCH^CHz, and butylene, CHaCH^CH-CHs*) fall off rapidly with increase in length of the chain. Acetylene, CH^CH, with a * Formula given is one of three isomers. PHYSIOLOGICALLY ACTIVE GASES 143 Table 18. Comparative Effectiveness of Gases in Producing Declination in Sweet Pea Seedlings and Epinasty in Tomato Petioles. Minimum parts per million needed to produce Gas Declination in sweet pea seedlings, according to Knight and Crocker * Epinasty in tomato petiole t Ethylene Acetylene Propylene Carbon monoxide Butylene 0.2 250 1000 5000 0.1 50 50 500 50,000 * 3 daj's' exposure used. t 2 days' exposure used. triple bond and chemically more active than ethylene, is much less effective in inducing either response; more than 1200 times the concentration is needed to induce the pea response and 500 times the concentration to induce the tomato response. Other unsaturated short chain compounds ^ such as allyl alcohol, CH2=CH— CH.OH, acrolem, CH2=CH— CHO, and isoprene, CH2=CH — C(CH3)=CH2, did not induce these responses. Allyl alcohol killed the tomato in 125 ppm of air and acrolein in 30 ppm of air. Denny ^^ has found three out of 77 other volatile chemicals tested that produce leaf epinasty: ethyl bromide, ethyl iodide, and propyl chloride. Later we shall see that most so-called plant hormones induce this reaction. The latter in general have low volatility and neither the three halides Denny mentions nor the hormones are likely to be present at all, or at least in a way that will interfere with the tests we describe below for ethyl- ene and other effective unsaturated C-gases. Responses Induced by Gases Containing Unsaturated Carbon The four effective gases mentioned above induce the following responses in plants: (1) epinasty of leaves; (2) proliferation of tissues; (3) abscission of leaves, flowers, and fruits; (4) anesthesia and inhibition of gro'«i:h; (5) coloring and other ripening changes in fruits; (6) other metabolic changes in living plant tissues; (7) root and root-hair initiation; and (8) other phj^siological effects. Epinasty of leaves. The effective gases cause epinasty in leaves by in- ducing a rapid elongation in the upper side of the leaf or petiole. This is sho\^■n in Fig. 53.^ In this case only the older basal leaves of the tomato plants in which the base of the petioles had ceased to elongate were used. The gas starts gro^vth again in the tissue on the upper side of the base of the mature petiole. In young leaves in which the petiole is still growing even at the base, the gas causes gro^\i,h on the upper side of the petiole in excess of that on the lower side, and thus turns the leaf downward, the curvature extending over a great part of the length of the petiole. After 144 GROWTH OF PLANTS leaves have responded in gas and the plants are put in gas-free ah, the young leaves recover their former position completely, and the older leaves partially, by growth on the lower side, or hyponastic growth. Leaves of plants rotated on a horizontal clinostat show much less epinastic response to ethylene than do leaves of plants in a stationary, upright position; and leaves of inverted plants show little or no epinastic response to ethylene. With the plant upright in air, the position of the leaf is determined mainly by its geotropic equilibrium position, but in part also by autonomic epi- FiGURE 53. Tomato plants showing change in length of upper faces of petioles when sealed in Wardian cases for 24 hours: left, in air; right, in 1 part of Yonkers gas to 10,000 of air. nasty; ^ in some leaves light plays a part. With a little ethylene in the air, either the geotropic equilibrium position is changed or the geotropic effect is so weakened that autonomic epinasty plays the main role in leaf position. In some leaves no doubt geotropism is still a factor in determin- ing the ethylene position of the leaf, while in others autonomic epinasty is the main determinant. We shall see later that petioles of some leaves in the presence of ethylene continue to grow on the upper side until they actually form coils. In such cases it would seem that autonomic epinasty and not geotropism is the determining factor. Out of 202 species and varieties of plants tested,^ 89 gave leaf epinasty in the presence of ethylene and 113 did not. Fig. 54 shows the type of response in four different plants. In Fuchsia the petioles decline somewhat, but the blade shows the greatest epinastic curvature. In buckwheat and sunflower the gro^^'th on the upper side of the petiole is so extreme that it PHYSIOLOGICALLY ACTIVE GASES 145 Figure 54. Ethylene-induced epinasty of leaves. All plants sealed in Wardian cases for the periods and in the concentrations of gases mentioned below and photographed immediately after removal from the cases: 1. Fuchsia, check; 2. Fuchsia in 500 ppm of ethylene for 24 hours; 3. Buckwheat, 1 part to 10,000 of Yonkers gas, equivalent to 3 ppm of ethylene, for 72 hours; 4. Sunflower, 1 part to 10,000 of Yonkers gas, 48 hours; 5. Paper White narcissus, 1 part to 10,000 of Yonkers gas, 96 hours. 146 GROWTH OF PLANTS produces coils in some of the petioles. In narcissus only the young leaves respond, and the inequality of growth on the two faces of the leaves is sufficient to produce coils at the tips of the leaves. The epinastic response of tomato leaves has already been shown in Figs. 52 and 53, and it will be shown for other plants in later figures. Figure 55. A, Marigold plants. Left: control. Right: in 1 part of ethylene to 1 billion parts of air for 20 hours. B, Potato plants. Left: control. Right: in 1 part of ethylene to 300 million parts of air for 24 hours. Since leaf epinasty is a growth response, it is most easily induced in thrifty plants growing under optimum conditions; but there is a great dif- ference in the minimum concentration of ethylene or other effective gases needed in the air to induce leaf epinasty in various kinds of plants. The tomato is moderately sensitive. The leaves of thrifty plants in good growth condition will respond to 1 part of ethylene to 10 to 20 million of air. In old leaves under long exposure epinasty will occur in 1 to 25 million or less of ethylene (Fig. 59). As is shown in Fig. 55, leaves of the young potato plant respond to 1 part of ethylene to 300 million of air, and the African marigold leaves to the extreme dilution of 1 part of ethylene to 1 billion of PHYSIOLOGICALLY ACTIVE GASES 147 air. A medium-sized thimble holds about 2.5 cubic centimeters. One such thimbleful of ethylene placed in a room 80 feet long, 32 feet wide, and 32 feet high gives 1 part of ethylene to 1 billion parts of air, or just enough, after it is equally diffused throughout the room, to induce epinastic response in the leaves of the African marigold. An- is a rather rare medium compared ^^^th the density of water or plant tissue, also ethylene has low solubility m water and probably in plant tissues, especially at 25° C (77° F), a good grounng temperature. The percentage by weight of ethylene in the plant tissue in the atmosphere described above must be almost fancifully small. The epinastic response of the marigold to ethylene must rank high in the extreme sensitiveness of an organism to a chemical. Vitamins and hormones are noted for producing physiological effects in extreme dilutions and, as we shall see later, there are good reasons for considering ethylene a plant hormone. Destructive or dry distillation or mcomplete combustion of carbon com- pounds produces ethylene, carbon monoxide, and other carbon gases. As a result, there are several sources of ethylene and carbon monoxide in the air: artificial illuminating gas; automobile exhaust and exhaust from other internal combustion engines; furnaces when the oxygen supply is made- quate; improperly trimmed or adjusted oil or gas stoves or torches; "^ pipe, cigarette, or cigar smoke; and burning brush or rubbish piles. Finally, burning a sheet of paper ^"^ in the air produces some ethylene and carbon monoxide because the heat is not great enough to burn all distillation gases. There are three kno\\-n natural sources of ethylene: it is given off by coal in the mine or in storage ; one natural gas ^ is kno^vn to contain a trace of ethylene; and respiring living plant tissues produce ethylene. In traces of the gas mixtures mentioned above, it is the ethylene that induces the re- sponses and not the other three. This is because ethylene is so much more effective than the others; also propylene and acetylene when present are generally in lower concentrations than ethylene in these mixtures. In some, CO is more concentrated than ethylene; hi water gas there are about 24 per cent CO and about 3 per cent of ethylene. But in the lowest concen- tration of this gas in air that will mduce epinasty, because of the ethylene present, CO exists in about 3^25 sufficient concentration to induce the response. Epinasty does, however, serve as an indirect test for CO for it generally accompanies ethylene in the gases mentioned above. Epinastic response of leaves has proved a very useful and delicate test for traces of unsaturated C-gases both in practice and in research.i^. i5 This test has been accepted m courts as evidence of illummatmg gas m greenhouses.'* Fifteen or twenty years ago there were frequent and some very extensive injuries to plants in commercial greenhouses by artificial illuminating gas that escaped from leaking pipes, seeped along under the frozen crust, and came up into greenhouses. Numerous researches on the effect of illummating gas and its constituents, mainly ethylene, upon green- house plants have reduced this loss tremendously. The epinastic response 148 GROWTH OF PLANTS has given a very sensitive and exact means of detecting the presence or absence of the gas in the greenhouse. This and various other detrimental changes caused in plants by traces of ethylene have been impressed upon the minds of both the greenhouse o^vners and the gas companies. As a result, the greenhouse owners now recognize gas injuries in their incipiency and, what is more important, the gas companies take great care in testing for leaks and repairing them in the neighborhood of greenhouses. There is one precaution that has not received enough attention, namely, the trapping of water drains from greenhouses. We have observed cases where gas leaks several blocks from a greenhouse caused injury. The gas escaped into the storm sewer, traveled through it, and into the greenhouse through the untrapped drainage tile. Water drainage lines from green- houses should have cemented joints so as to be air-tight. They should also have a trap outside the greenhouse ^\dth an upright vent outside the trap. While this would prevent injury lq some cases, ia many others it would not. Gas mil seep many rods thi^ough the ground under a frozen crust and escape up into the greenhouse through the unfrozen ground. The real way to prevent injury is to see that there are no gas leaks even in the general neighborhood of a greenhouse. The epinastic response has been used in submarines ^ to determine whether any exhaust gases are escaping into the hull. This test even has its place in home and social adjustment. The local gas company had trouble in convincing two lady school teachers that there was not a gas leak in their apartment. The ladies were sho^vn that the tomato could detect about Mooo the least concentration the human nose could detect. The tomato plant indicated no gas in the apartment. The teachers agreed that they were smelling something else. The author gave a judge's wife a beautiful Crassula arhorescens plant. After it had been in the house two weeks many of the leaves had fallen. The tomato plants indicated leaks in the 20-year-old gas stove, but a much bigger leak in the gas meter under the front room. The judge's wife got a new stove and the gas company repaired the meter. The judge heard from his neighbors for his penuriousness in failing to buy a new stove until the evidence was overwhelmingly against him. Oort\njn Botjes ^^ used the epinasty of tomato leaves to show that ripening apples produce ethylene. Other inves- tigators 16, 25. 26, 55 have used epmasty in tomato, potato, and marigold leaves and horizontal nutation in the pea seedling to demonstrate ethylene emanations from respiring tissues of several kinds of plants. Many other uses of this response in research will be mentioned below. Proliferation of tissues. Ethylene induces the proliferation of plant tis- sue, especially of cork cambium, as well as enlargement of cells. Harvey and Rose ^^ showed that when illuminating gas flowed slowly through soil in which plants grew it caused the development of massive soft white tissue at the base of the stem and on the larger roots of Hibiscus, also on roots and lower part of the stem of Catalpa seedlings. This tissue resulted both PHYSIOLOGICALLY ACTIVE GASES 149 from a multiplication of cells (hyperplasia) and from an increase in size of some of the cells (hypertrophy). Fig. 56 shows the effect of ethylene in inducing proliferation of tissue in the lenticels of young Hibiscus stems. In Figure 56. Twigs of Hibiscus cut from tree in lat« January and placed in moist chambers for 10 days. Left: control in air. Right: treated in ethylene 10 ppm. Note that ethylene induced undifferentiated outgrowth from the lenticels and stopped bud growth. the picture the outgrowths look like roots. Instead they are undiiTeren- tiated tissue. It will be noted also that ethylene hindered the grou-th of buds. The enlargement in the tip of the sweet pea seedling (Fig. 51) in- duced by ethylene probably results mainly from cell enlargement. When the illuminating gas flowed through the soil more rapidly, this response did not occur, but the roots and base of the stem were killed. As 150 GROWTH OF PLANTS we shall see later, illuminating gas contains cyanides which are soluble; in heavier flow of gas these accumulate in the soil in sufficient concentration to kill the plants, so the ethylene response can not occur. Wallace ^^ found that intumescences in the apple stem in response to stimulation by ethylene gas arise through three fundamental changes in the tissues affected, namely, solution of cell walls already showing marked secondary thickening, en- largement of cells, and proliferation of cells. Ethylene induced intumes- cence in apple tudgs in the extreme dilution of 1 to 100 million of air. This approaches the sensitiveness of the potato and African marigold leaves which require over 1 to 300 million and 1 to 1 billion of ethylene in the air, respectively, to induce epinasty. We shall see later that many of the plant hormone types of chemicals induce proliferation of tissues. Abscission of leaves, flowers, and fruits. One of the more general effects of ethylene and other effective gases is to cause leaf, petal, flower, and fruit fall by inducing growth in cells of the abscission layer. This is in part because the flat cefls of the abscission layer enlarge and become spherical (hypertrophy) , although proliferation of cefls may occur in some cases. Xo doubt abscission is also furthered by the tendency of ethylene to induce the solution of msoluble pectins of the middle lamella of the cell walls. We shall discuss this effect of ethylene later in this chapter. Fig. 57 shows leaf fall in Crassula arhorescens and petal fall in Salvia induced by ethylene in cigarette smoke in the first, and by ethylene in illuminating gas in the second. Ethylene has been used in Oregon to defoliate roses at time of digging. For this purpose a moderately tight chamber having high humidity and a temperature of 70° to 75° F (21° to 24° C), not exceeding 85° F (30° C), is used. Tank ethylene may be used at the optimum rate of 1 cu. ft. to 100,000 cu. ft. of space. Apples stored in the chamber give the optimum ethylene concentration when there is a bushel to every 400 to 500 cu. ft. of space. Kerosene stoves and rose hips proved a less desirable source of ethylene because the ethylene concentration was not sufficiently high. The defoliation required four days under optimum conditions. Ethylene has been used to loosen the shucks of English walnuts ^^ and pecans,-- -^ ^\^th improvement in the color of the product in the former. In both cases the nuts could be harvested earlier, as soon as the meats were ripe, mthout waiting for the shucks to loosen on the tree. This prevented later injury in the dry, hot air of California and Arizona. The optimum conditions in the shucking chambers were 1 to 1000 of ethylene and a temperature some- what above 70° F (21° C) with a period of two to four days. Emanations from pecan shucks induced epinasty in potato plants; the pecan tissue pro- duces ethylene, but evidently not enough to induce quick shuckmg. Ethylene probably induces earlier gro^vth in the abscission layer between the shuck and shell. The cytological changes, however, have not been studied. Anesthesia and inhibition of growth. Doubt" found that the proper concentration of ethylene produced complete rigor in Coleus, from which the PHYSIOLOGICALLY ACTIVE GASES 151 plant recovered A\'ithout injury when the gas was removed. In proper con- centrations of ethylene, Mimosa pudica was unable to respond to the con- tact or heat stimulus; it recovered after removal from the gas, but showed some later injury. Neljubow ^^ and Knight and Crocker ^^ showed still Figure 57. A, Crassula arboresceris showing leaf fall. Left: control. Right: sealed in a 10-liter bell jar with 3 puffs of cigarette smoke for 5 days. B, Salvia showing petal fall. Left: control. Right: treated 24 hours with 1:10,000 illuminating gas. earlier that the rate of elongation of etiolated pea seedlings was reduced by very low concentrations of ethylene in the air, and that as the con- centration of ethylene increased, the rate of elongation fell until in relatively low concentrations the elongation ceased, that is, growth rigor was pro- duced. Anesthetized pea seedlings showed no killing of tissue and resumed 152 GROWTH OF PLANTS a rapid rate of elongation when removed from the gas. There is an increase in diameter in the growing region near the tip of the seedling, even when it is in a concentration of ethylene that stops all elongation. This forms a knob near the tip, as is shown in Fig. 51, with the high concentration of ethylene. A number of later studies 6- s. 69 have been made on the anesthetic action of ethylene, acetylene, propylene, and carbon monoxide upon plants and lower animals. In plants the order of effectiveness of the several gases, that is, the minimum concentration inducing rigor, is the same as for epi- nasty and declination of the pea seedling. It took very high concentrations of these gases to induce rigor in insects and centipedes and the order of effectiveness of the several gases showed no relation to that found for plants. Butylene, which is almost without effect on plants, is the most effective in anesthetizing insects and centipedes, requiring from 5 to 40 per cent for the various species. It also partially paralyzes the organisms. Ethylene, which is the strongest anesthetic for plants, along with acetylene, was the least effective on insects and centipedes, requiring a complete atmosphere for anesthesia; 30 to 75 per cent of propylene and 80 to 90 per cent of carbon monoxide were required to anesthetize these organisms. It is evident from the facts stated above — and will be borne out by those to be stated later — that besides anesthesia these gases have many other effects on plants, namely, epinasty, proliferation of tissue, abscission, etc. Probably most of these secondary effects can be avoided by using gases in concentrations that produce complete rigor. Epinasty of leaves, however, is induced in concentrations of ethylene that result in complete rigor in other parts of the plant, as shown by time-lapse motion pictures ^ of tomato plants in air and in air containing 2 ppm of ethylene. The photographing was continued 24 hours with the treated plant in the gas, followed by 24 hours with the treated plant in ethylene-free air, in order to get both the response and the recovery. The exposures were made 96 times per hour to give a speed of movement on the screen 600 times that occurring in the plants themselves. The top of the check plant was in continuous movement due to unequal rate of growth on the several flanks of the stem; also the leaves on the control plant showed movement during the course of the experiment. Especially conspicuous was the sleep movement during the first night of the experiment. This movement was less conspicuous during the second night due to bad gro^vth conditions furnished by the experiment. The movements of the gassed plant must be considered under two heads: the response movements and the recovery movements. Soon after the plant was placed in the anesthetic, the tip of the plant ceased to move and showed no movement during the rest of the exposure. It was in rigor. Moreover, the sleep movement of the leaves did not occur. Instead, noticeable epi- nasty of the petioles began within two to three hours after exposure. The first leaf to show this was the third from the top, followed successively by PHYSIOLOGICALLY ACTIVE GASES 153 the leaves below on the gro\\nng part of the stem and then by the two tip leaves. Several hours later the older leaves on the non-elongating part of the stem showed epinastic movement. The leaves on the growing part of the stem had completed their epinastic movements Avithin 8 or 10 hours and the leaves on the more mature part of the stem withm 15 or 20 hours. Younger leaves curved throughout the length of the petioles, while in the older leaves the curvature was limited to the base of the petioles. After the leaves had come into equilibrium by epinastic response, all movement in them ceased for the duration of the exposure. The whole plant was in rigor. Two or three hours after the gassed plant was put into ethylene- free air, the recovery from the epinasty began and continued in about the same order and, for the younger leaves, with nearly the same speed as the response. The leaves on the growing part of the stem showed complete recovery to the original position, while the older leaves showed only partial recovery. Also within two or three hours after removal of the plant from the anesthetic, vigorous movement started in the tip and continued throughout the period. The tip of the plant recovered completely from the rigor. The time-lapse pictures of the sunflower showed similar behavior. Com- parable time-lapse pictures were made of the sensitive plant, using 0.5 per cent of CO as the anesthetic. Complete anesthesia was induced in this plant, and complete recovery occurred after removal from the gas, as shown by loss of power to respond to contact stimuli while in the gas and recovery of this power after removal from the gas. When this plant is anesthe- tized, however, there is a slow, non-correlated movement of the individual leaves and leaflets that gives the plant a disorganized appearance (Fig. 58). The movements of the leaves and leaflets of this plant are brought about by changes in osmotic pressure in cells of the pulvini at the base of leaves and leaflets, and not by growth. The slow, non-correlated movements in the leaves and leaflets in the anesthetized plant would have been entirely- overlooked if it had not been for the time-lapse pictures. When plants are exposed to ethylene in concentrations below the rigor- producing dosage, the rate of growth is retarded, with or without other secondary effects, depending upon the kind of plants treated and the con- centration of the gas used. A study was made of the effect of low concen- trations of ethylene (1 part to 10 million and 1 part to 25 million of air) on the growth rate of seeds and seedlings of wheat, buckwheat, tomato, clover, and corn growing in soil in pots. These experiments were carried out in two continuous and regulated air-flow Wardian cases in a special greenhouse. This apparatus will be illustrated and described in the next chapter. The rate of air flow through each chamber was 200 cubic feet per minute. By means of a calibrated capillary flowmeter a measured amount of ethylene was added to the air passing through one flow-chamber to give the concentration of ethylene desired. Six pots each of seeds and seedlings of each kind of plant were used in the control and treated chamber. 154 GROWTH OF PLANTS FiGTJRE 58. The anesthetic effect of carbon monoxide on. plants. A, Mimosa pudica control. B, The same plant as "A" after exposure to one per cent carbon monoxide for 18 hours. Note the change in the normal equilibrium position of the leaves and the disturbance of normal correlation. Fig. 59 and Table 19 show the effect of these low concentrations of ethylene upon the growth rate of seeds and seedlings. For the illustration a typical pot was selected in each case and photographed, and for the table all seedlings of each kind and lot were measured at the beginning and end of the experiments and the average elongation during the experiment cal- culated. Examination of the figure shows that 1 part of ethylene to 10 million of air causes a marked reduction in growth rate and 1 part to 25 million an evident reduction. There is no killing of tissue; the growth inhibition is an incipient anesthetic effect. The table shows that 1 part of ethylene to 10 million of air reduced the rate of elongation by the following percentages: clover, 50; tomatoes and buckwheat, 40; and wheat, 25. Table 19. Per Cent Inhibition of Growth in Length by Ethylene. 1 part of ethylene to 10 million of air. Continuous fumiga- tion for 4 weeks Clover Tomatoes 50 40 Buckwheat Wheat 40 25 PHYSIOLOGICALLY ACTIVE GASES 155 Measured by volume or wet or dry weight increase, the reduction m percentage growth would be much greater. It will be noted that the lower concentration mduced epinasty in the older leaves of the tomato plant. Even so, the question arises whether epinasty of tomato leaves is sensitive enough to detect the lowest concentration of artificial illuminating gas in greenhouses that will retard gro\nh. If not, epinasty m the leaves of potato or marigold vnW do so. Greenhouse operators will do well to keep young plants of all of these growmg in their greenhouses continuously if there is any danger of injury from leaking gas pipes. From observing the work on the effect of ethylene on plants done m the botany department at the University of Chicago, Dr. Luckhardt of the animal physiology department of the same institution became interested in the effects of ethylene upon animals. He and associates *^' ^'^ found it to be a rather remarkable anesthetic for higher animals and man, but it must be used in high concentration, 80 per cent or more, i^-ith oxygen. The authors cited describe its use m 800 operations at the Presbyterian Hospital at Chicago. They mention several advantages of ethylene as an anesthetic in surgery under the headings: (1) ease of induction and rapidity of re- covery; (2) relaxation \dthout cyanosis; (3) absence of sweating; (4) ab- sence of respiratory u-ritation; and (5) narrow anesthetic margin. They list as disadvantages: (1) odor; (2) oozmg from wound; and (3) danger of explosion. They state that it is no more explosive, however, than ether. In 1938 Luckhardt was awarded the Alpha Omega medal as discoverer, with J. Bailey Carter, of ethylene as an anesthetic with qualities superior to nitrous oxide. Chipman,^ an anesthetist of Washington, D. C, ranks ethylene-oxygen first among anesthetics kno\\Ti and in use up to 1931. Dr. H. M. Living- stone, Chief Anesthetist at the University of Chicago Clmics, says that ethylene-oxygen is enjoying its greatest popularity in the midwest and some in California. She describes"^ the use of ethylene-oxygen in 6590 cases, mostly \\'ithout the accompaniment of other anesthetics. She also states that to date she has used ethylene-oxygen in about 50,000 cases ^^^thout death or explosion. Luckhardt states that John S. Lundy ^^ of the Mayo Clinic used ethylene-oxygen in an even greater number of cases udthout mishap, but that lately he is using mainly an intravenous anesthetic. Dr. Poe ^2 considers ethylene-oxygen the best anesthetic kno\vn to date and finds no danger of explosion if it is properly applied. Drs. Guthrie of Sayre, Pa., and Woodhouse of Cedar Rapids, Iowa, confirm this view and describe 35,500 operations conducted with ethylene-oxygen either as the sole anesthetic or m conjunction with ether. They say:^^' pi^^" "It is our opinion that ethylene is an excellent anesthetic agent for general surgical use and that an unjust prejudice exists in the mmds of the surgical pro- fession ^^^th respect to its widely heralded disadvantages." The anesthetic used by anesthetists seems to depend to a great degree upon the anesthetic in use where they were trained. On the whole, however, ethylene-oxygen 156 GROWTH OF PLANTS e CO O S o OS o l-H PHYSIOLOGICALLY ACTIVE GASES 157 ^ CI r" ^ ni te (U Ji o3 S3 a; « "^fQ "5 • — ■ t- " "2 02 1> -3 in , S 02 a C c O o Bj 03 3 -^ CO -o -3 -^ C O i^ S • fl g _g a; ^ __ ~ d, 02 •-- _** 03 c 03 o 03 o3 5P>0 „ ^ "" 03 fi .22 03 ^ 03 ^ hC 0) 03 02 c o o3 o C o C 03 -O fi c ^ o 03 o bO b£ O c bC C o 03 -S =1^ ^ -t^ O 535 O *^ ^ - S . ''0 o -(J 03 a o 03 ■is o 03 03 o3 -c: - ^ -. !- O O '^ 03 *^ t*-. <-l -e ^ -S LT M 03 C StI M fi g ^ -fi ^ 03 r< 03 03 > o3 03 C5 ^^ ^ 03 5 "S,-^ 03 03 03 ^ 03 C ~ -Ki g O «S (N O 03 'o 03 03 & O 02 158 GROWTH OF PLANTS has had rather extensive use and is in great favor with the anesthetists who do use it. Coloring and ripening of fruits. Denny 9. i". " was the first to note the effectiveness of ethylene in coloring citrus fruits. When commercially mature as determined by their size, color, or chemical composition, citrus fruits are often still green in color. For years the citrus growers had been burning kerosene stoves in their storehouses to hasten the proper coloring of the fruits. At first they thought the stoves did this by raising the tem- perature, but it was discovered that the stoves gave off some gas that increased the rate of chlorophyll decomposition. Denny found that low concentrations of ethylene in storage houses would hasten the coloring. Any concentration from 1 : 5000 to 1 : 5 million of the air had some effect, with 1 : 5000 to 1 : 1 million representing the most effective range of concen- tration. Very high concentrations, such as 80 per cent, slowed the process. The process was fastest at about 82° F (28° C) . Besides accelerating the coloring of lemons, ethylene increased the rate of respiration 100 to 250 per cent, depending upon the concentration of the gas and other conditions. Acetylene and carbon monoxide were effective, but must be used in much higher concentrations; and butadiene showed some effect. In practical application of this discovery the rooms were gassed two to four times a day and aired out at least once a day. Ethylene came to be very generally used instead of the kerosene stoves. The significance of this discovery was well put by the late Dr. Henry G. Knight, former Chief of the U. S. Bureau of Agricultural Chemistry and Engineering, on the occa- sion of his receipt of a medal from the American Institute of Chemists for his achievements in agricultural chemistry. Science ^^ reports a portion of his speech as follows: "As a single, dramatic example of the returns obtain- able on small outlay in research, Dr. Knight cited the case of the ethylene gas treatment of oranges, to bring a bright color to the skins of some types of fruit that persist in staying green after the oranges themselves are ripe. 'The treatment bleaches out the predominant green color and leaves the orange a beautiful natural yellow. The chemical investigations leading to the development of this treatment, which is now in rather general use, cost the taxpayers of the country about $4,000 and is estimated to be worth about $4,000,000 a year to the producers of citrus fruits in Florida alone and about the same amount to producers in California. And yet some people say that research doesn't pay. . . .'" Ethylene induces the decomposition of chlorophyll in many different plants and plant organs. This is not a direct effect of ethylene upon the chlorophyll but an indirect effect through the protoplasm. In high con- centrations, such as 80 per cent, ethylene inhibits the decomposition, as Denny showed for the lemon, because it produces partial rigor in the protoplasm and lowers the speed of many of its activities. Fig. 60 shows that illuminating gas (the ethylene portion) induces the decomposition of chlorophyll in rose leaves.''* PHYSIOLOGICALLY ACTIVE GASES 159 R. B. Harvey ^^ recommends the use of ethylene, 1000 ppm or more dilute, for blanching celery. Acetylene was less effective. Hibbard ^^ con- firmed Harvey's conclusions that ethylene hastens the blanching of celery, but found that it also reduces the growth. It hastens the yellowing of the green part of Mcintosh apple skin but had no effect on the development of the red pigment. Other investigators have found that ethylene hastens several other ripening processes in fruits. These will now be described. Figure 60. At left: control. At right: leaves of Rosa (hybrid tea) variety Madame Butterfly showing the yellowing of the veins of leaflets 3 days after removal from a 48-hour treatment in 1 : 5000 illuminating gas. Other metabolic changes in living plant tissues. E. M. Harvey ^® was one of the early workers to make a detailed study of the effect of ethylene upon the metabolism of plant tissues. He also reviewed the earlier literature on the effects of anesthetics upon plant metabolism. He grew inch-long epicotyls of the sweet pea for 72 hours in air containing 1 ppm of ethylene and compared the chemical composition of these with epicotyls gro^\Ti in air. Ethylene caused the simple soluble substances to increase at the expense of higher soluble and insoluble forms. Sugars, amino acids, amides, polypeptides, lipoids, etc., soluble in hot alcohol-ether, increased 8 to 9 per cent while the insoluble substances — proteins, starch, cellulose, ligno- celluloses, etc. — showed a corresponding decrease. Reducing sugars in- creased about 11 per cent and non-reducing sugars decreased about 3 per cent. Amino acids plus amides increased and the polypeptides de- creased. The fats were lower in the treated epicotyls and the cellulose and proteins were about 3 per cent lower. The acidity was not changed. Respiration was lowered, the author believed, because the ethylene was 160 GROWTH OF PLANTS used in too high a concentration. Denny ^^ points out that Harvey got increased respiration in one of his determinations, namely, the one with shortest exposure, probably before the anesthetic effect became evident. A number of investigators ^^- ^" have determined the effect of ethylene upon the metabolism of celery and a number of fruits, citrus and pomaceous, generally using 1000 ppm of ethylene in the ah. Ethylene caused an in- crease in reducing and simple sugars, at the expense of polysaccharides if the latter were present. If not, the simple sugars were decreased because ethylene increased respiration. In all the fruits ethylene hastened coloring and ripening. Ethylene decreased the tannin and catalase *~ and increased the peroxidase content. Ethylene, 1 ppm in air,^^ increased the respiration of potatoes in storage from the first and increased the soluble sugar content in later storage. There was one exception to tliis; in tubers rich in sugars caused by a period of storage at low temperature, ethylene did not increase respiration. If such tubers were de-sugared by a storage period at high temperature, ethylene again stimulated respiration. Hansen ^^- ^^ has confirmed the findings mentioned above for the effects of ethylene upon fruits and added another .very important metabolic change. Ethylene in 1000 ppm in air hastens the hydrolysis of insoluble protopectin of cell walls of the fruits into soluble pectin. This accounts for the more rapid softening of fruits when ripened with ethylene. Hansen also found that ethylene hastened the formation of pectin from protopectin in citrus rinds and English walnut shucks. This no doubt is a factor in shuck- ing walnuts with ethylene. Hansen also found that little ethylene was pro- duced by green fruits and that ethylene production increased as the fruit ripened. Ethylene did not induce ripenmg and softening of fruits after they had been m cold storage at 31° F ( -0.5° C) for three weeks. Lynch ^^ suggests that ethylene acts as a respiratory coenzyme in the ripening fruits, and Nord ^^ suggests that ethylene increases the enzymatic formation of plant hormones. Englis and Dykins -° find that ethylene does not modify the rate at which salicin, a glucoside, is hydrolyzed by the enzyme emulsm. They conclude that ethylene, in modifying the metabo- lism and hastening the ripening of fruits, does not act directly upon enzymes but indirectly upon the living protoplasm of the fruit. Root and root-hair initiation. The discovery that the unsaturated C-gases induce rooting was accidental. For some years Zimmerman and Hitchcock had been studying the rooting of cuttings, and during this time had been seeking unthout success chemicals that would induce rooting. On the side they were working with the author on the effect of illuminating gas and its constituents on greenhouse plants, a problem that would seem to have no relation to their main researches. In general the exposures of the plants to the gases were for a duration of three to four days. On one occasion a tomato plant was exposed to 1 per cent CO in a bell jar, and because of a holiday making a long week-end was kept in the gas several days more than usual. The gassed plant showed many roots along PHYSIOLOGICALLY ACTIVE GASES 161 the stem and the control plant none. The results are Ulustrated in (A) con- trol and (B) treated plant in Fig. 61. So far as the author knows, this was the first time an effective root-inducing chemical was reported. Carbon Figure 61. Tomato plants treated with carbon monoxide gas. A, Control m Wardian case for 11 days. B, Plant exposed to one per cent carbon monoxide gas for 5 days and then held in Wardian case for 6 more days at the end of which time it was photographed Note the slender root growth made after the plant was removed from gas. C, Control plant. D, Plant with a flask of carbon monoxide sealed over a leaf. Note the epmastic response of the leaves, indicating that the gas is taken in through the leaf and transmitted to all parts of the plant. 162 GROWTH OF PLANTS monoxide ^^ showed root-inducing effects on many plants. The paper reporting these results was awarded the A. Cressy Morrison Prize in Experi- mental Biology in 1932 by the New York Academy of Sciences. Later /^ Figure 62. Nicotiana tabacum (Turkish variety) exposed to one per cent carbon mon- oxide gas. A, Control plant kept in Wardian case. B, Plant exposed to gas 15 days, then allowed to stand in air two days, after which time it was photographed. C, An enlarge- ment of the rooting region of B. D, Tobacco cuttings from control plants in Wardian case for 10 days. Photographed 5 days after having been placed in rooting medium. E, Cuttings from plants treated with one per cent carbon monoxide for 10 days and then placed in rooting medium for 5 days, after which time they were photographed. PHYSIOLOGICALLY ACTIVE GASES 163 ethylene, propylene, and acetylene, like CO, were found to induce rooting. These gases were effective in lower concentrations than CO, as has been found for other plant responses, but the minimum root-inducing concen- tration was not determined for any of them. In some plants roots were induced along considerable stretches of the stem, as shown m Fig. 61 for Figure 63. African marigold to show the effect of acetylene on orientation of roots to gravity and on formation of root hairs. Left: normar growth of roots six days after a three-day exposure to the gas. Right: the same roots after a 48-hour exposure to 0.25 per cent acetylene. Root hairs were induced and the roots changed their direction of growth. 164 GROWTH OF PLANTS tomato and in Fig. 63 for the African marigold. In other plants the roots grew only in the region of the stem that was elongating at the time it was subjected to the gas, as in tobacco (Fig. 62) and in a number of other plants. In still other plants the roots developed only at the nodes. The gases also induced development of roots on leaves and roots. The greatest growth of roots was obtained in mtact plants if they were subjected to the gas for the induction period and then placed in gas-free moist air for further growth. While the gases induce rooting they also inhibit later elongation of the roots. These gases induce root-hair formation also, as is shown in Fig. 63- While in one respect Zimmerman and Hitchcock had found the thing they had long been seeking, namely, chemicals that induce rooting, these chemicals could not be used in propagation. Since they are gases they move readily throughout the plant, ^^^ " probably traveling through the extensive intercellular system of plants. Because of this they induce roots at places where roots are not desired, all along the stem in some cases and at espe- cially susceptible zones in others. Consequently Zimmerman and Hitchcock sought non-volatile or slightly volatile chemicals that would induce root- ing only at the point of application. This led to the study of other plant hormones discussed in a later chapter. Other physiological effects. Several investigators have found that the unsaturated C-gases induce the earlier formation of flowers in pmeapple *''• ^^ and flower and leaf buds in mango.^^ In the Hawaiian Islands acetylene has been used to induce earlier flowering of pineapples and to spread the harvest over a greater period of the year. Use of these gases on tobacco ^' ^s during fermentation improved the color and smoking quality and increased the rate of fermentation of the leaf. Ethylene, 100 ppm,^^ is said to improve the germination and baking performance of freshly harvested and high- moisture wheat in storage. Respiring Plant Tissue Produces Ethylene Elmer ^^•'^^ in 1932 observed that apples gave off a volatile substance that inhibited the growth of potato sprouts. A year later Oortwjn Botjes ^^ reported that emanations from apples caused epinasty m tomato plants and horizontal nutation of the etiolated pea seedling. She found that ethylene absorbents would remove the effective gas from apple emana- tions. Gane -^ found that aerobically gro^ving yeast (but not anaerobically growing yeast) produced a substance that prevented the growth of a pea seedUng. The substance was absorbed by bromme water, an ethylene absorber. He showed ^^ that ripe apples in ah- produce a substance that modifies the growth of several kinds of plants. In an atmosphere of nitro- gen the apple did not produce emanations that modified the gro\vth of the pea seedling, but seemed to hasten ripening of bananas. Apples killed by freezing no longer produced effective gases. The effective gases from apple were absorbed in bromine water ^^ and ethylene dibromide was identified PHYSIOLOGICALLY ACTIVE GAJSES 165 in the absorbent. He found ^^ that green Bon Chretien pears stored at 0° C (32° F) produced vapors that ripened bananas. Denny and Miller ^^ and Denny ^^^ ^^' ^* found that most of the many kinds of living plant tissues tested emanated a chemical that induced leaf epinasty in the potato. Among the tissues giving the response were fruits (mature and green), seeds, flowers, leaves, leafy stems, young shoots, roots, tubers, anthers, pistils, and petals of a number of different kinds of plants. A few living tissues gave negative results: potato tubers (whole or cut into pieces) ; seedlings of wheat, corn, and oats; mycelium of Rhizopus nigricans, two mushrooms, and baker's yeast. By absorbing emanations from a greater number of seedlings of oats, wheat, corn, and several other seedlings in a mercuric nitrate-nitric acid reagent and later releasing the absorbed gases with HCl, a positive response was obtained. It may be that all living plant tissues produce ethylene, but some in very low amounts. Ripe apples produce relatively large amounts. The flesh of the squash and dandelion flowers are less active. Seedlings of Cruciferae tested produced both toxic (probably mustard oils) and leaf epinasty-induciag emanations. Denny found that tomato stems lq the horizontal position, out of equilibrium with gravity, produced more of the effective emanations than smiilar stems in the vertical position. Quantitative determinations have been made of the amount of ethylene produced by fruits and contained in fruits, as well as of the effect of stage of ripening and other conditions upon the amount of ethylene produced. Niederl, Brenner, and Kelley ^^ converted the ethylene from ripening bananas to acetylene and determmed the latter as silver acetylide. They estimate that 100 pounds of bananas produce 0.1 to 0.2 cc of ethylene dur- ing the ripening period. Nelson ^^ finds that Mcintosh apples after nine months of storage contain 0.12 mg of ethylene per kilogram of apples. He also found ^^ that for six varieties of apples studied, the ones with longer storage lives showed less capacity to produce ethylene. There is a general correspondence between the ethylene content and respiratory activity of apples. Ethylene has a hydrolytic action on ripening fruits, but the effect is probably not directly upon the enzymes. Ethylene may be consumed as well as produced by the ripening bananas. Hansen ^^' P-55&-557 summarizes his quantitative measurements of ethylene production by pears in part as follows: "In fruit in air at 20° C the rate of ethylene production increases during the climacteric rise in respiration, reaches a peak at the respiratory climax, then declines during the post- climacteric period. During the climacteric rise, ethylene production in- creases seven- to eighty-fold, while rate of respiration approximately doubles. Each variety was found to have a characteristic maximum rate of production. The maximum rate for Bartlett, a variety which maintains its capacity to ripen for only a short period of time when kept at a storage temperature of 0° C, is 3.25-i.48 ml per kg-day. The maximum rate for Anjou, a variety which maintains its capacity to ripen for a long period of 166 GROWTH OF PLANTS time when kept at cold storage temperatures, is 0.57-0.78 ml per kg-day. The maximum rate of respiration for Bartlett is approximately double that for Anjou. Under anaerobic conditions, the production of ethylene is either greatly retarded or entirely inhibited. In the fruits used for these experiments, there was found but little difference in the production of CO 2 under aerobic and anaerobic conditions. The maximum rate of ethylene production occurs at 20° C. At higher temperatures production decreases and is totally inhibited at 40° C. Respiratory activity as measured by either CO2 production or O2 consumption is greatly accelerated between 20° and 40° C." The question naturally arises whether living plant tissues produce the other effective gases: acetylene, propylene, and carbon monoxide. As has been stated above, wherever the minimum effective concentrations of the several gases for producing a plant response have been determined, ethyl- ene has proved effective in very much lower concentrations than the other gases. Hansen and Christensen ^'* state that solubility tests indicate that ethylene is the active gas evolved by the fruits, with similar unsaturated hydrocarbon gases, such as acetylene, propylene, and butylene, not present in sufficient amounts to be detected by the bromination procedure. Lang- don ^^ found that the atmosphere in the float and air channel of giant kelp, Nereocystis leutkeana, is 1.1 to 12.2 per cent CO, varying widely from plant to plant and little with time of day. Langdon and Gailey ^^ believe the CO is a product of aerobic respiration, since it occurs only when oxygen is present and equally abundant in light and darkness. Rigg and Henry ^* confirm the previous work on the origin of the CO in the kelp. As we have seen, the concentration of CO in the kelp float is sufficient to cause many formative changes in various plants. We have no evidence, however, that CO is produced by most plants and even if it is, probably not in sufficient concentration to have formative effects. The same is true of acetylene, propylene, and butylene. Since plant products, especially fruits, produce ethylene which has marked physiological effects upon other living plant organs, some dis- crimination must be used in storing various plant products in proximity to each other. Cut carnations ^^ in cold storage rooms with apples become sleepy. Ripe apples and high ethylene-producing apples ^^ hasten the respiration and ripening of slow-maturing apples stored with them. A number of storage practices may be modified by these findings on the production of ethylene by plants and the effect of ethylene on plants. In the case of apples being used to defoliate roses, ^* the association is beneficial. Summary The researches described above and carried out mainly in the United States have shown that ethylene, acetylene, propylene, and carbon mon- oxide have many far-reaching and interesting effects upon plant develop- ment and plant metabolism. These researches were early stimulated by the PHYSIOLOGICALLY ACTIVE GASES 167 work of Crocker and Ivnight (1908) in answering the practical question on the effect of artificial illuminating gas upon carnations. Denny's dis- covery (1923) of the effectiveness of ethylene in coloring lemons was the second great stimulus to work in this field. Qualitatively the four gases act similarly on plants; so far as tested, each will produce the responses that any other produces provided they are all used in the proper concentrations. Quantitatively they are very different; the minimum effective concentration of ethylene is very much lower than that of acetylene and propylene, and the minimum effective concentration of carbon monoxide is much higher than that of the last two. In general, the effectiveness can be expressed as ethylene > acetylene and propylene > carbon monoxide. Ethylene modifies the geotropic equilibrium position of plant organs, causing such changes as declination or horizontal nutation of pea seedlings, leaf epinasty, and change in the orientation of roots. Leaf epinasty of the tomato seedling and potato shoot has been used rather extensively in prac- tice in detecting the presence of ethylene in the air and through it the presence of traces of a number of gas mixtures, such as illuminating gas and exhaust gases from internal combustion engines. Leaf epinasty has been especially useful in scientific research for detecting the emanation of ethylene from respiring plant tissues. The declination or horizontal nuta- tion of the pea is the least sensitive of the tests. With a very sensitive variety of sweet pea, using declination as the indicator, one might detect 0.2 ppm of ethylene in the air. With leaf epinasty of the tomato one can detect easily 0.1 ppm of ethylene in the air. If one observed only the mature leaves of the tomato and gave a long exposure, 0.04 ppm of ethylene could be detected. The leaf epinasty of the potato furnishes a considerably more delicate test than that of the tomato. Finally, leaf epinasty of the African marigold is the most sensitive test kno^^^l for ethylene. It will detect 0.001 ppm of ethylene in air. On the basis of the small amount of the chemical needed to induce the response, this is one of the most delicate responses of an organism to a chemical kno^^^l to date. Ethylene causes cells that are dormant and would otherA^se not grow, to enlarge and divide, forming soft, rather massive tissues. This is especially true for lenticular and cortical tissue. Traces of ethylene ^\'ill even cause cells with thickened walls to dissolve the walls and to enlarge and multiply. The proliferation of tissue is induced by concentrations of ethylene as low as 0.01 ppm in the air. Ethylene induces the abscission of leaves, flowers, petals, and fruits by inducing flat cells of the abscission layer to enlarge and become spherical, thus furthering organ drop. Hydrolysis of the insoluble protopectins of the middle lamella of cell walls is also a factor in abscission. Ethylene is used for defoliating plants when desirable in horticultural practice. This is probably the response that makes ethylene useful in shucking English walnuts and pecans. 168 GROWTH OF PLANTS Ethylene is an excellent anesthetic. Ethylene will anesthetize certain plant organs in concentrations as low as 1 ppm in air. It is interesting, how- ever, that while it anesthetizes one part of the plant, it may start growth in another part. In concentrations far below the anesthetic dosage it still slows the gro\vth, and the inhibition of growth falls very slowly as the con- centration falls. Dilutions of 0.1 ppm of ethylene reduce the rate of elonga- tion of plant organs 25 to 50 per cent, and 0.04 ppm gives easily measurable reduction in rate of elongation. It is likely that much lower concentrations will inhibit elongation in some of the more sensitive plants. Butylene, which is almost inactive with plants, will anesthetize centipedes and various insects in concentrations 5 to 40 per cent of the air and partially paralyzes the organisms in higher concentrations; 30 to 75 per cent of propylene is required; and 80 to 90 per cent of carbon monoxide; ethylene and acetylene must be used as a full atmosphere. The most effective of these for insects and centipedes is least effective for plants and the most effective anesthetic for plants is the least effective for insects and centipedes. Ethylene is an excellent anesthetic for mammals, lacking as it does some of the undesirable qualities of ether and nitrous oxide. It must be used in high concentration, 80 per cent or a higher concentration, with oxygen. It has been used extensively in surgery. Many anesthetists are very enthusi- astic about its use and others are quite as antagonistic. Ethylene induces the decomposition of chlorophyll in the living plant and has been used extensively to hasten the proper coloring of ripe citrus fruits, especially lemons, and to bleach celery. It also hastens many other ripening changes in fruits and has been used commercially for this purpose with tomatoes, bananas, and other fruits. Ethylene induces or hastens many other metabolic changes in living plants. These changes are mainly hydrolytic, such as transformation of insoluble protopectins to soluble pectins, higher carbohydrates to soluble sugars, proteins to polypeptides and amino acids. Ethylene and the physiologically similar gases induce root and root-hair formation. Finally, many if not all respiring plant tissues produce ethylene. The amount of ethylene produced varies greatly with the kind of plant and with the organ of the plant, as well as with the age of the organ. Apples and pears produce much ethylene at the climacteric stage of the fruit; bananas produce less; and the potato tuber little, if any. Since ethylene is produced by plants, often in sufficient concentrations to modify develop- ment, it may be considered a phytohormone. Literature Cited 1. Asmaev, P. G., "The effect of ethylene on the gas exchange and respiration ferments during starvation period of tobacco leaves and other vegetative objects [sic]," Proc. Agric. Inst. Krasnodar, 4 : 100 (1937). 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F., "Effects of ethylene on the plant growth hormone," Science, 83 : 284 (1936). 61. Oortwijn Botjes, Je, "Aethyleen als vermoedehjke oorzaak van de groeiremmede werking van rijpe appels," Tijdschr. Plantenziek., 39 : 207-211 (1933). 62. Poe, J. G., "Ethylene anesthesia," /. Am. Med. Assoc, 105 : 66-67 (1935). 63. "Research in agricultural science," Science, 93(2421) : 8s-9s (1941). 64. Rigg, G. B., and B. S. Henry, "On the origin of the gases in the float of bladder kelp," Am. J. Bot., 22 : 362-365 (1935). 65. Rodriguez, A. G., "Influence of smoke and ethylene on the fruiting of the pine- apple (Ananas sativus Shult)," /. Dept. Agric. Porto Rico, 16 : 5-18 (1932). 66. Smock, R. M., "The influence of one lot of apple fruits on another," Proc. Am. Soc. Hort. Sci., 40 : 187-192 (1942). 67. Sorber, D. G., "The use of ethylene gas for loosening walnut hulls," Diamond Walnut News, pp. 3-4 (June, 1934). 68. Wallace, R. H., "Histogenesis of intumescences in the apple induced by ethylene gas," Am. J. Bot., 15 : 509-524 (1928). 69. Zimmerman, P. W., "Anesthetic properties of carbon monoxide and other gases in relation to plants, insects, and centipedes," C. B. T. I., 7 : 147-155 (1935). 70. , W. Crocker, and A. E. Hitchcock, "Initiation and stimulation of roots from exposure of plants to carbon monoxide gas," C. B. T. I., 5 : 1-17 (1933). 71. , and A. E. Hitchcock, "Initiation and stimulation of adventitious roots caused by unsaturated hydrocarbon gases," C. B. T. I., 5 : 351-369 (1933). 72. , , and W. Crocker, "The movement of gases into and through plants," C. B. T. I., 3 : 313-320 (1931). 73. , , , "The effect of ethylene and illuminating gas on roses," C. B. T. I., 3 : 459-481 (1931). CHAPTER 5 Effect of Certain Lethal Gases upon Plants and Animals The study of the effect of physiologically active gases upon plants led a group of investigators at the University of Chicago and later a group of research workers at Boyce Thompson Institute to investigate the effect of other gases upon plants and animals. Injury to Trees and Shrubs from Leaks of Artificial Illuminating Gas When we discuss illuminating gas injury to trees and shrubs along streets or in parks, there are three factors that must be considered that are not involved in gas injuries in greenhouses: (1) soluble constituents of the gas will accumulate in the soil about the roots in the first case, whereas they will be absorbed by the soil and not reach the air of the greenhouse in the second; (2) the roots as well as, or even more than, the tops of the plants must be considered in tree and shrub injury; and finally, (3) there is a free movement of air outside, that will take the gases away from the tops of the plants instead of holding them about the plants, as is the case in the greenhouse. Harvey and Rose ^^ found that when root systems of plants were sealed in the soil and subjected to moderate concentrations of Chicago artificial illuminating gas various abnormal growths occurred. These responses were the same as those occurring when ethylene was added to the soil in amounts equal to that in the gas. The following groA\i:h changes occurred in one or another of the several kinds of plants treated: coiling or swollen growth near the tip of roots, proliferation of cortical cells on the upper part of roots and lower parts of stems, and fall of leaves. In the last case the ethylene was absorbed by the roots and passed up through the stem to induce leaf fall. In high concentrations of illuminating gas the roots were killed and the whole plant died. Harvey and Rose also flowed illuminating gas through soil bearing plants, very slowly in some cases, and very rapidly in others. In the former they observed ethylene-induced growth responses and in the latter death soon occurred, which they attributed in part to the lack of oxygen, though admitting that toxic substances in the gas may have played a part. They observed that with slow flow of gas, all the starch disappears from the cortex of the roots, and believe that this, together with the proliferation of tissue on roots and lower parts of the stems, may serve 172 LETHAL GASES 173 as a point for diagnosing gas injury, if not on the trees killed, at least in adjoining shrubs and trees that received a lower dosage of the gas. Hitchcock, Crocker, and Zimmerman '^ later reviewed the literature and carried out many experiments on injury to plants by illuminating gas seeping through the soil. Exposure of Roots in Soil to Flowing Gases The tomato plant proved to be very sensitive to illuminating gas flowing through the soil in which it grew, and was much used in this investigation. Fig. 64B shows that 2 cubic feet of Yonkers illuminating gas flowing through a pot bearing a small tomato plant leads to the final death of all the roots and the lower part of the stem, so that the plant later collapsed. Even 1 cubic foot of gas killed the root system. Table 20 shows the effect of flo^ving several different gases through the soil of pots bearing six differ- ent kinds of plants. The most toxic of these gases is unscrubbed Yonkers illuminating gas. Even 1 cubic foot of this killed all the roots, and 4 cubic feet killed all the underground parts of the tomato plant, which collapsed. The injury decreased in the following order for the other plants: willow, maple, cherry, silver bell, and privet. Scrubbing the gas through water before passing it through the pots reduced the toxicity measurably for all Table 20. Effect on Potted Plants of Passing Illuminating Gas and Certain of Its Unsaturated Hydrocarbon Constituents Through the Soil for a Period of 30 Minutes Vol. of gas in cu. ft. Relative degree of injury to roots caused by different gases * Plant Illuminating gas Ethyl- ene Propyl- ene Butyl- ene Not filtered Filtered through Acetyl- ene Water NaOH Tomato 1 4 + + + + + + t + + + + + + t + + + 0 ++ 0 + + 0 ++ + + + Willow 1 4 + + + + + + + + + + + 0 + + + + + + + + Maple 1 4 + + + + + + + + + + 0 + + + + + + + + + +++ + + + + + Cherry- 1 4 + + + + + — + + 0 +++ + + ++ +++ + 4- + + + Silver beU 1 4 + + + + — + 0 + + + + + + ++ + + + + Privet 1 4 + + + + + + + 0 + + + + ++ + + * 0 signifies no noticeable injury, + slight discoloration, + -f noticeable discoloration and death of part or all of many roots, -f- + + aU roots badly discolored and most slender roots killed. t All underground parts killed. 174 GROWTH OF PLANTS the plants except the tomato and willow, and scrubbing it through NaOH solution reduced the toxicity markedly for all. The investigators showed that there was a close agreement between the amount of cyanides in the volume of gas that was needed to cause a given Figure 64. Comparison of injuries on tomato due to residues of illuminating gas left in the soil and those caused by flowing gas and residues. A, Seedlings placed in soil previously gassed. B, Roots of seedhngs in soil subjected to flowing gas. Left to right in (A) and (B): control, one cubic foot, two cubic feet. LETHAL GASES 175 injury and the amount of cyanide that had to be added as a water solution of Ca(CN)2 or KCN in order to produce the same degree of injury. In short, the highly toxic or tissue-killing constituent of the gas was HCN. Scrubbing the gas with water removed some of the HCN, and scrubbing it with NaOH changed the HCN to NaCN and took out most of it. The amount of cyanides in solution ia water necessary to cause the collapse of a small potted tomato plant was very small, 4 to 7 mg. It is interesting to find that it took 20 to 24 times as much illuminating gas that had been thoroughly scrubbed through NaOH solution to give the same amount of killing as was produced by a given amount of unscrubbed gas. Artificial illuminating gas is always scrubbed to remove HCN, and the amount in the gas probably varies considerably from time to time with the thorough- ness of the scrubbing. It is evident that if the scrubbing were thorough enough to remove the last trace of HCN, the killing of trees and shrubs by many small leaks would be avoided. With such thoroughly scrubbed gas it is probable that the killing would result from compounds such as phenol, toluene, xylene, etc. Flowing 1 cubic foot and 4 cubic feet of each of the unsaturated gaseous hydrocarbons (ethylene, propylene, butylene, and acetylene) in general produced less injury than the unscrubbed illuminating gas, and the order of sensitiveness of the several plants changes; the tomato was little injured by any of these, even ^vith 4 cubic feet, while the maple was considerably injured, especially by 4 cubic feet of propylene, butylene, and acetylene, the cherry by 4 cubic feet of ethylene, butylene, and acetylene, and the silver bell by acetylene. The tomato, which was the most sensitive to unscrubbed illuminating gas, was among the least sensitive to the un- saturated C-gases, and some plants which were more resistant to un- scrubbed illuminating gas were most injured by the C-gases. The results sho^\^l in Table 20 throw out of perspective the significance of the C-gases as toxic factors in illuminating gas because they make up such a small percentage of the gas. The Yonkers gas s. pise contained 3 per cent of ethylene, a fraction of a per cent of propylene, and still less butylene and acetylene. If the Yonkers gas were used as the source of ethylene, one w^ould have to flow 33 cubic feet of it through the pot to get 1 cubic foot of ethylene and many times this volume to get 1 cubic foot of each of the other three unsaturated C-gases. So far as killing roots and stems is con- cerned, the unsaturated C-gases can be disregarded as causes of injury from illuminating gas. In order to get gro^vth response from ethylene and perhaps propylene and acetylene, the experiments in Table 20 were run in the wrong way, since all the gas passed through the soil in 30 minutes. Had 1 cubic foot of ethylene or even 1 cubic foot of illuminating gas containing 3 per cent of ethylene been flowed very slowly through the pot during several days or a week, no doubt ethylene responses (leaf epinasty, yellowing of foliage, prolifera- tion of tissues on root and stem) would have appeared. Ethylene would 176 GROWTH OF PLANTS have dissolved in the soil and been absorbed by the plant in sufficient amounts to induce these changes with such slow flow. These statements are in accord with the findings of the experiments of Harvey and Rose, where they flowed illuminating gas very slowly through the soil. With a very slow flow of gas, it is also probable that HCN would be disposed of by microorganisms in the soil, so it would not accumulate in tissue-killing con- centrations. It is evident from what has just been said that two different Figure 65. Effect on the tomato of residues left in soil by ethylene and by illumi- nating gas. Left: control. Center: 10 cubic feet of ethylene. Right: collapse of roots and lower part of stem in soil caused by the residues from three cubic feet of illuminating gas. Photographed six days after seedlings were planted in the soil. types of injury may appear in trees and shrubs along leaking gas lines: if the leak is slight, ethylene responses will appear; if the leak is large, the root systems and plants will be killed and none of the growth responses will occur. These investigators ^^ found that the carbon monoxide of the Yonkers gas, although it constituted 13 per cent of the gas, was not a factor in the injury of plants in the soil outside. Toxicity of Gas Residues in the Soil A comparison of Fig. 64A and B shows that the tomato plant is injured to about the same degree whether it is in the soil while the illuminating gas flows through the soil or is set in the soil immediately after gassing. LETHAL GASES 177 Fig. 65 shows that flowing 3 cubic feet of Yonkers gas through the soil leaves a residue that kills the root system and the lower part of the tomato plant set in the soil, but that flowing 10 cubic feet of ethylene through the soil does not leave a killing residue. It is of interest in replanting trees and shrubs in place of those killed by illuminating gas to know how long the toxic residues remain in the soil and what are the best means of getting rid of them. Fig. 66 shows that gassed soil sealed in a can at 24° C (75° F) loses practically all its toxicity within Figure 66. Effect of low temperature storage on the toxicity of soil through which illuminating gas had been previously passed and which was then sealed for one week. Left to right: control soil -15° C (5° F), gassed soil -15°C (5° F), gassed soil 3° C (37° F), gassed soil 24° C (75° F). The tomato plants in gassed soil previously stored at -15° C (5° F) and at 3° C (37° F) were killed, but the soil kept in the laboratory at 24° C (75° F) caused only a slight retardation of growth. a week, whereas similar soil samples sealed at —3° and —15° C (26° and 5° F) do not lose their toxicity. At proper temperatures and other con- ditions for growth the microorganisms of the soil use the hydrocyanic acid — which probably is mainly cyanides because of neutralization by bases of the soil — as a nitrogen source. Leaching the soil with water removes a part of the residual toxic substance. Fig. 67 shows the gro"^i:h of a tomato plant in gassed soil that has been leached ^A-ith water equal to ten times the volume of the soil, in contrast to the gro^^i:h of a similar plant in gassed soil ^\'ithout leaching, and in soil not gassed. It is evident that leaching removed a portion of the poisonous residue, but there are still substances that reduce the growth rate far below that of the check. Leaching is not a very effective way of removing the toxic residues, and it might be even less effective outside where the soil is likely to be packed and poorly drained. In practice, it is probably best to remove the gassed soil and replace it with good soil. If this is not feasible, the soil could be loosened up to the proper depth and limed if it is acid, after which two to three weeks should elapse before planting. In case of acid soils, the lime will neutralize any residual HCN and increase the activity of organisms that consume the cyanides. 178 GROWTH OF PLANTS ^^^RI^^Sp ■ ^s BB^ 1 pjk BP^lfi Wm ^■u^9(j - ji^H imij ^^^n 'i ' ''^^^^1 il Figure 67. Effect on growth of tomato of leaching highly toxic gassed soil with water. Left to right: control; soil from a 65-liter lot through which 7210 cubic feet of illuminating gas had been flowed; a similar sample of gassed soil after leaching with an amount of water equal to ten times its volume. Photographed three weeks after planting. Addition to the Soil of Other Compounds Present in Illuminating Gas Drip oils, i.e., oils condensed from illuminating gas at low temperatures, caused the young tomato plant to collapse when 1 gram was mixed mth 450 grams of soil. The injury caused by these oils could not be distinguished from injury caused by adding solutions of cyanides or passing unscrubbed illuminating gas through the soil. The weight of drip oil that had to be added to produce a given injury was more than 100 times the weight of hydrocyanic acid or cyanides necessary to produce the same degree of injury. It is not uncommon to find the soil near leaks in gas lines dis- colored by drip oils. They persist in the soil for a long time and of course ^\^ll injure roots growing in the soil bearing them. Drip oils were more toxic than equal weights of toluene or xylene which, along with these, exist in illuminating gas in small amounts. Phenol in high concentrations killed the tomato, as did illuminating gas. It also caused a deep pink or reddish coloration of roots, stems, and leaves of the tomato and of the roots of privet and sunflower. Heavy dosages of illuminating gas produced similar color changes. The color change induced by phenol is one of the symptoms that should be looked for in diagnosing illuminating gas injury to trees and shrubs. Diagnosis of Tree and Shrub Injury by Artificial Illuminating Gas As we have seen, it is easy to diagnose a case of illuminating gas injury in a greenhouse. At the time the injury is occurring, reliable test plants can LETHAL GASES 179 be used. Several days after the leak has been stopped one can judge rather accurately from the behavior of the various kinds of plants in the green- house whether ethylene has been present. Some greenhouse men show great accuracy in observing and describing unusual responses in the plants they are gro^^^ng. With accurate descriptions of the responses, one can judge fairly accurately whether ethylene was involved long after the injury has occurred. It is more difficult to determine gas injury to trees or shrubs outside, especially when the plant is killed quickly. No physiological responses of ethylene appear. One can locate the leak and observe the degree of injury to plants at various distances from the leak. On the out- skirts of the area of injury he may find ethylene responses in plants that are not killed. One should also observe various plants within the zone of injury for the red color in the roots due to phenol. Samples of soil should be taken from different depths and from various distances from the leak and test plants exposed to the soil samples under bell jars to detect the presence of ethylene. The samples should be taken with as little stirrmg as possible and kept tightly sealed until ready to be placed under the bell jars for the test. The author has often obtamed ethylene responses from such tests, which of course show that gas has been present. Hitchcock, Crocker, and Zimmerman did not find chemical tests of soils for the presence of various constituents of gas of very much value in diagnosing gas leaks. Among these were treating soil extracts with bromine water to detect ethylene, etc., reactions for phenol and cyanides in soil extracts. Drip oils do often discolor the soil in the immediate region of the leak. Leaks are more likely to develop in the winter mth freezing and thawing of the ground. If there is a frozen soil crust after a big leak develops, the gas is likely to move for considerable distances under the crust in sufficient amounts to cause injury over a sizable area. If the roots are killed in the early spring the foliage may open out, but the leaves remain very small and die later. If the roots are killed in midsummer the full-sized leaves die and dry up. Injury from Natural Gas With one knouTi exception natural gases do not contain ethylene or other similar growth-modifying gases and, in the one exception claimed to date, the percentage of ethylene is extremely low.^ Natural gases ^- ^^ also contain no HCN, phenol, toluene, xylene, etc. Natural gases have methane (CH4) as the main constituent. In some, methane is the sole combustible constituent. Others contain in addition the higher homologs of methane — ethane, propane, and butane as gases, and vapors of pen- tane, hexane, heptane, and octane. Natural gases from some fields contain hydrogen sulphide and organic sulphur compounds. In some of these the hydrogen sulphide runs as high as 15 per cent. Hydrogen sulphide, if present, is supposed to be completely scrubbed out before the gas is de- 180 GROWTH OF PLANTS livered to consumer pipes. As we shall see later, hydrogen sulphide, although highly toxic to mammals, has a rather low order of toxicity to plants. Some N2 and CO2 are present in most natural gases. Nitrogen may run as high as 40 per cent and CO 2 over 1 per cent. Some natural gases contain helium. All these constituents except the hydrogen sulphide have relatively low toxicity to plants. If the hydrogen sulphide, when present, is completely scrubbed out, the natural gases are almost inert so far as plants are concerned. The most toxic constituents are the higher homologs of methane, especially pentane, hexane, heptane, and octane, and they are present in very low concentrations. Solheim and Ames ^^ found that certain natural gases of the northwestern United States showed very low toxicity to plants. Tomato, potato, sun- flower, castor bean, and geranium plants were uninjured by 50 per cent of natural gas from the Billy Creek field in Sheridan, Wyoming, or gas from the mains at Laramie after four days of exposure. Cut carnations did not exhibit any symptoms of injury when they were exposed to 2 per cent of the gas for four days. Fuchsia petals showed slight bro^^^ling and wilting when the plants were exposed to 4 to 50 per cent of the gas for one to four days. SchoUenberger ^* found that a natural gas composed mainly of methane and ethane rendered soil highly toxic to wheat and oats. Analysis of this soil showed a marked increase in soluble manganese and ammonium nitro- gen, and lesser but distinct increases in sodium, potassium, and calcium. Since these changes frequently occur as a result of water-logging, puddling, or other conditions which favor reducing actions, SchoUenberger con- cluded that the toxic efTect of the natural gas in soil was due primarily to reduced oxygen pressure. Injury to Plants in a Greenhouse by Mercury Vapor Zimmerman and Crocker ''^' ^^ were asked to investigate injuries to roses in a greenhouse at Attleboro, Mass. The injuries were quite different from those caused by illuminating gas; also the injuries could not be attributed to insects or fungal and bacterial pests, for the plants were almost free from such pests and the injuries were not such as would be caused by them. The only hint at a possible source of injury was the fact that the soil in some of the benches had been treated with a solution of mercuric chloride, HgCl2, to kill earthworms. The soil had also been fertilized with tankage. The injury, however, appeared in all benches in the house, regardless of whether they had been treated mth HgCU solution and tankage. Later, rather extensive experimentation led the investigators to the conclusions that the injury was caused by the HgCl2 solution added to the soil of some of the benches, and that the organic matter of the soil reduced the chloride to metallic mercury, which had sufficient vapor pressure to move through the air and injure roses in untreated benches. Ratsek ^^ later reported similar results from treating soils in a rose house with HgCU, but he explained the LETHAL GASES 181 injury to the roses in untreated benches in another way. He beheved that the HgCl2 passed as a vapor through the air and injured the roses at a distance. There is considerable evidence in favor of the Zimmerman-Crocker inter- pretation. Organic matter reduces HgCls to mercurous chloride,20- p-833-834 and vegetable and animal substances -"■ p-^"^ reduce HgCl to metallic mercury. The vapor pressure of metallic mercury is considerably higher than the vapor pressure of HgCl2 throughout a considerable range of tem- perature, and especially at temperatures suitable for the growth of plants. Fig. 68 shows the vapor pressure of mercury, HgCl2, and HgCl at various 2 2 q: ^ (A i/) UI CC a. tr o Q. 10 20 30 40 50 60 70 80 TEMPERATURE 90 100 110 120 130 140 150 FiGtJKE 68. Curves showing the relationships of the vapor pressures of metallic mer- cury, mercuric chloride, and mercurous chloride at different temperatures. All data taken from International Critical Tables. temperatures. For instance, the vapor pressure of mercury at 40° C (104° F), a temperature often attained in a greenhouse, is 66 per cent higher than the vapor pressure of HgClo at 60° C (140° F), practically a pasteurizing temperature. The vapor pressure of mercury at 60° C (140° F) is five and one-half times that of HgClo. About one-fourth the HgCl2 molecule is chlorine. At saturation at 60° C (140° F), the air will contain more than seven times as much mercury in the form of mercury vapor as in the form of HgCl 2 vapor. WTien a current of air is drawn through a column of HgCU crystals and then over a gold foil, no mercury is deposited on the foil; but if air is drawn through a rich soil treated with HgCU solution, mercury is deposited on the foil, showing that the soil reduces the HgCl2 to the metal. If air was drawn through a bell jar containing a glass 182 GROWTH OF PLANTS plate atomized with HgCl2 solution, no mercury was deposited on gold foil by such air. If in addition the bell jar contained a potted plant, mercury was deposited on the gold foil. The plant evidently gave off some material that reduced the chloride, or HgCU vapor reached the plant and was reduced to the metal. Addition of HgCl2 solution to tankage injvired untreated plants enclosed with it much more than did HgCU solution added to sand or powdered charcoal. Mercurous chloride dust, which has much lower vapor pressure than HgCU, is not toxic to a growing plant with which it is enclosed. If HgCl2 is added to soil and the soil enclosed with a plant, the plant is in- jured. Zimmerman and Crocker found that various inorganic mercury compounds as well as several organic mercurial fungicides, Dubay, Nu- Green, Semesan, and Uspulun, when added to fertile soils, gave off emana- tions that injured plants in untreated soils in the same enclosure. Finally, Daines ^ says that in soils where mercurials are effective as fungicides, mercury compounds are reduced by the soil to metallic mercury, which migrates in the soil as mercury vapors, and that any factor that prevents the conversion of the mercury salt to metallic mercury destroys the fungicidal effects of the mercurial. Walker, et al.^^ give a similar explanation for the effectiveness of HgCU solution in controlling club root of cabbage when applied at the time of transplanting. This is the type of evidence we have that fertile soils reduce mercury compounds to metallic mercury, and that it is vapors of the metallic mercury that travel through the air and injure the plants in un- treated soil in the same enclosure. Regardless of the correctness of this interpretation of the mechanics by which additions of mercury compounds to the soil in greenhouses injure plants in untreated soils in the same house, there is no doubt that mercury compounds, organic and inorganic, must be used with caution in greenhouse soils, because of the release of mercurial vapors in the air. In the use of mercurials in outside planting the danger of injury is through the roots. The air will be kept relatively free from dangerous vapors by diffusion and wind currents. That mercury vapor in the air is injurious to both plants and animals has long been known. The only new claim here introduced is that adding mercury compounds, even those with very low vapor pressures and solu- bilities, to a greenhouse soil releases mercurial vapors into the air in suffi- cient concentrations to injure plants throughout the greenhouse, including those in untreated soils. Zimmerman and Crocker ^2- p-^'^^ state: "In 1797 there was made known in a letter addressed to Van Mons by Lauwerenburgh that four Dutch chemists, Deiman, Paats, Van-Troostwyck, and Lauwerenburgh,^'' had discovered the deleterious effects of metallic mercury vapors on plants. The results of 15 experiments mentioned in the letter showed that where beans, mints, or spiraea we're enclosed in bell jars with metallic mercury the leaves became spotted after 24 hours, and if left exposed to the vapors Figure 69. Briarcliff rose buds. A, left to right: 1, normal control bud; 2, bud from plant in muslin cage where the soil bearing 14 other plants had been watered with 0.05 per cent mercuric chloride; 3 and 4, buds from plants in muslin cage in which the soil bearing the plants had been watered with 0.05 per cent mercuric chloride. B, left to right: 1, normal bud from control glass case; 2, 3, and 4, buds from plants in glass case where the soil bearing three other plants had been watered with 0.05 per cent mercuric chloride. LETHAL GASES 183 for several days the plants died. In 1867 Boussingault ' repeated some of the experiments, obtaining results comparable to those of the Dutch scien- tists." Since that time several German workers have discussed the poisonous effects of metallic mercury vapors, but no publications have been found which show that the air becomes contaminated where mercuric compounds are applied to the soil." The buds of the Briarcliff rose proved especially sensitive to mercury vapor in the air. Very young buds and a region of the stems just below them were killed. In older buds the petals were killed and turned brown, the whole corolla abscissed, and stamens and pistils became black. In old buds the petals which had begun to open lost most of the pink pigment and turned bro^^^l at the edges. Fig. 69 shows the nature of these injuries. It will be noted from Fig. 69B that buds showed the same type and degree of injury whether they grew in pots to which HgCl2 solution was added to the soil or in pots in the same enclosure in untreated soil. The injury in all cases was due to vapor in the air. While the buds were the most sensitive part of the plants, the leaves were also injured with more severe treatments. There was considerable variation in the sensitiveness of different varieties of roses. Of the seven varieties tested (Columbia, Templar, Killarney, Fernet, Madame Butterfly, Mrs. Calvin Coolidge, and Briarcliff), Briar- cliff was the most sensitive and Templar the most resistant. Plants of 65 different genera were injured by vapors from metallic mer- cury or from soils treated with HgCl2 solution. Broad bean, butterfly weed, oxalis, and sunflower were especially sensitive. Aloe, croton, and sarcococca were very resistant. The sensitiveness probably depended upon the ma- turity and degree of dormancy of the plants at the time of treatment as well as upon the species. In actively growing peach seedlings a foot or more high, the old leaves were most sensitive and the young leaves most resistant. The degree of injury caused by vapors from mercury or soil treated with mercury compounds depended upon several factors. Temperature was very important, as would be expected from the fact that the vapor pressure of mercury rises rapidly with the temperature. Fig. 70 shows that all the leaves are killed on peach seedlings at 75° F (24° C) by vapors from both mercury and soil treated with HgCl2 solution. The injury is less severe at 60° F (16° C), slight at 50° F (10° C), and nil at 40° F (4°C). With mercury the injury increased not with the total volume of mercury in the enclosure but with the surface exposed. Covering the mercury with a layer of water 1 cm thick prevented injury. The mercury was determined in the leaves of various plants after ex- posure to mercury vapor. No relation existed between the amount of mercury absorbed and the degree of injury produced in the various kinds of plants. Under a given exposure the leaves of the Briarcliff rose absorbed about one-half as much mercury as leaves of Killarney and Coolidge, and yet the leaves of the former were more injured than those of the latter. 184 GROWTH OF PLANTS LETHAL GASES 185 The Jerusalem cherry absorbed much mercury from mercury vapor in the atmosphere but was httle injured by it. Evidently there is a great differ- ence in the resistance of protoplasm of different plants to injury by mercury. Gray and Fuller ^^ found that dry seeds of pea, corn, bean, radish, sun- flower, and cucumber stored in fairly tight chambers with an open beaker of mercury for six months showed no injury. There was a slight delay if the seeds were germinated in the presence of mercury vapor, but the percentage of germination was not affected. Seedlings of these plants were injured by addition of mercury to the substratum or mercury vapor in the air. The injury was somewhat greater under the first condition. In both cases the seedlings were stunted, showed yellowing of leaves, early leaf fall, and failure of leaf development. Kincaid ^^ found mercury vapor in the air toxic to germinating tobacco seeds, especially at higher temperatures and with exposure of large surfaces of the metal. Harrington ^' states that presence of mercury forces dormant Johnson grass seeds to germinate promptly at temperatures that gave no germination in absence of mercury. Giese ^^ has recently emphasized the danger of laboratory workers being poisoned by mercury fumes. A cubic meter of air saturated with mercury at 25° C (77° F) contains 19.5 mg of mercury. A stream of air passing over 10 cm^ of mercury at 25° C (77° F) at the rate of 1 liter per minute becomes about 15 per cent saturated and contains 3 mg of mercury per cubic meter. He cites researches to show that prolonged exposure of some individuals to as little as 0.01 mg of mercury per cubic meter and of many individuals to 0.25 mg results in chronic mercury poisoning. Mercury is eliminated from the body slowly and consequently accumulates with continued ex- posure. Giese mentions that Faraday, Pascal and other physicists and chemists suffered from chronic mercury poisoning -svdthout realizing it. Among the symptoms of mercury poisoning are irritability, headaches, and recession of the gums. Effect of Sulphur Dioxide on Plants Fifty years ago it was not unusual to see large areas around smelters com- pletely denuded of plants by the large amount of sulphur dioxide released into the air. Extensive studies have been made in both Europe and America on injury to vegetation and even to animals by smelter fumes. These studies have resulted in three great accomplishments. First, very accurate methods have been developed for determining the effect of SO2 on plants. This phase has been especially marked by the designing of accurate instru- ments for automatic recording of the SO2 content of the atmosphere and for fumigating plants A\'ith regulated and automatically recorded SO2 content. Secondly, a great deal of information is now available on the injury to plants by SO 2, the relative sensitiveness of different plants and plant parts to SO2, and the degree of injury necessary to reduce crop yield. Finally, smelters have made use of this scientific knowledge in reducing the injury by SO2 about smelters. They have also removed solid matter from smelter 186 GROWTH OF PLANTS fumes by the use of Cottrell precipitators. This prevents other toxic substances from reaching the atmosphere about the smelters and gives valuable by-products. Research by Hill, Thomas, and associates. The advance in methods and instrumentation was started by Wells of the Selby Smelter Commission and later further developed and perfected by scientists of the Department of Agricultural Research, American Smelting and Refining Company of Salt Lake City, Utah, early by O'Gara and later by Hill and Thomas and associates. The outstanding advance in apparatus was the Thomas autometer for continuously analyzing and recording low concentrations of SO2 in the atmosphere. This has later been adapted for measuring and recording low concentrations of many other gases and vapors in the atmos- phere. The automatic recording fumigating apparatus used for SO 2 and other gases at the Institute is illustrated in Fig. 71,2'^ and a full description of the apparatus and method of its operation was made by Setterstrom.^^ Thomas and associates ^^ have recently improved this apparatus for long- time large-scale study of the effect of SO 2 fumigation on the nutrition and physiology of plants. The new apparatus gives control of the soil conditions as well as the atmosphere about the plants. Besides doing so much to develop suitable apparatus, the group at the American Smelting and Refining Company has contributed much accurate knowledge on the physiological effect of SO2 in the air on plants. Space will permit the mention of only two of the later contributions. They studied the effect of SO2 fumigations on the rate of photosynthesis and respira- tion 3" as measured by the CO2 absorbed or released by alfalfa. Heavy fumigations, 0.7 to 1.26 ppm, of durations too short to kill any tissue of the leaf reduced the rate of photosynthesis during the exposure; but imme- diately after exposure the photosynthesis rose to normal or greater than normal, so the net effect of such fumigations was practically zero. Light fumigations for long periods, 0.24 ppm for 3 days, 0.19 ppm for 11 days, 0.14 ppm for 39 days, either showed no effect or suggested stimulation. Even when heavy fumigations were long enough to cause extensive killing of leaf tissue, much of the photosynthetic power was restored by the development of new leaves within 10 or 15 days. The workers conclude that fumigations that do not produce visible injury (spotting of leaves) do not produce "invisible injury" (reduction of photosynthesis and yield). Thomas and co-workers, "^ in a study of sulphur nutrition of alfalfa in the improved culture chambers, found that sulphur-deficient plots were im- proved in yield by light SO2 fumigations. We shall later discuss the sig- nificance to soil fertility of release of SO 2 into the atmosphere by mdustries. American Smelting and Refining Company has faced realistically the problem of possible injury to vegetation by the fumes from its smelters, and has built and manned a fine laboratory to work out in detail the effect of SO2 upon plants. They have contributed materially to improved instru- mentation in plant and industrial science and to fundamental knowledge LETHAL GASES 187 Figure 71. Part of the apparatus used for studying the effects of gases on plants and animals. A, Greenhouse which houses most of the apparatus. B, Close-up of scrubber and metering devices with cabinet and autometer in the background. 188 GROWTH OF PLANTS of plants. The findings have been made generally available to industry, to the public, and to science. Much credit is due the director, Dr. George R. Hill, for the public-spirited and thoroughgoing scientific way in which the researches have been managed, and to Dr. Moyer D. Thomas for his unique ability in designing apparatus and planning and carrying through experiments for solving difficult problems in plant physiology. Research by National Research Council of Canada. The best case history we have of fumes from a larger smelter is that of the Trail Smelter in the upper Columbia Valley, British Columbia, about ten miles north of the international boundary. The researches were conducted by eight specialists of the National Research Council of Canada, and are published m a 447- page volume. 21 The book reports the observations and experiments of this group and reviews the earlier literature on the subject. Part I (206 pages, including several inserted two- or three-page plates) covers the field studies. The introduction gives the history of Trail Smelter, the amount of SO2 emitted by it yearly from 1900 to 1935, and also in 1937, SO2 abatement methods adopted, and history of the international tribunal dealing \\ith claims of United States farmers for SO2 injury. Other subjects covered by the first part are: SO 2 content of the atmosphere m industrial regions, including the Trail region; symptoms of SO 2 injuries on plants; and the effect of the SO 2 content of the air on the sulphur content of trees and shrubs, on the acidity, base-exchange capacity, and sulphur content of soils, and on the gro^^'th in diameter of trees. Part II describes numerous fumigation experiments on forest and crop plants, including a study of the effect of environmental factors on susceptibility of barley and alfalfa to SO 2 injury. It discusses the effect of SO 2 fumigation on stomatal behavior; on crop yield when the fumigation is in tissue-killing and sub-killing dosages; and on photosynthesis, respiration, and chemical composition of plants. The Thomas recorders were used extensively in these investigations and modifications of the Hill and Thomas fumigatmg apparatus were used throughout the fumigating experiments. This work has added many accurate data and sound generalizations to our knowledge of the effect of SO2 on vegetation. Space will permit the mention of only two of the generalizations. The "invisible injury" findings of Hill and Thomas were confirmed, that is, there was no reduction in crop yield unless the fumigations were sufficient to kill leaf tissue. Ethylene is an ideal gas for producing "invisible injury " because it is a growth inhibitor and does not kill tissue, as pointed out in the last chapter. It will, however, reduce growth rate only so long as it is in the air surrounding the plant. The SO2 in the leaves was oxidized to sulphates, apparently completely in light fumigations and partially in heavier fumigations. It is the unoxidized SO2 or sulphites that kill leaf tissue. Leaves of evergreens growing m the SO2 belt some distance from Trail showed three to four times the normal content of sulphur without any effect on the color of the leaves and without any retardation in growth. It is likely that the oxidation of SO2 to sulphates accounts for the lack of "invisible injury." LETHAL GASES 189 Experiments at Boyce Thompson Institute. Beginning in 1930, Zimmer- man and Crocker ^*'' *^ made a study of the effect of SO2 on more than 30 species of plants representing more than a dozen families and including farm, garden, ornamental, and wild species. The experiments were carried on under a considerable range of conditions as to SO 2 concentration, dura- tion of fumigation, light, moisture, and other factors. These experiments were made before automatic self-recording fumigating apparatus was available. The concentration of the SO2 was adjusted by flow meters and the concentration of SO2 checked in the fumigating chamber by periodic analyses of air samples. Figure 72. Buckwheat leaves from plants treated for six hours with various con- centrations of sulphur dioxide gas. Left to right: (1) control, (2) 1.0 ppm, (3) 0.7 ppm, (4) 0.5 ppm. The experiments led to the following conclusions. There is a great range in the sensitiveness of different species of plants: buckwheat proved the most sensitive; it showed killing of leaf tissue in 0.46 ppm A\dth seven hours' fumigation. Some species of orchids were very resistant, withstanding 60 ppm for several hours without injury. The cereals and various weeds were sensitive and carnations, gardenias, and rhododendrons were rather resistant. The leaves were injured, but not the stems and buds. In dicoty- ledons middle-age leaves were more sensitive than young or old leaves; interveinal parenchyma was more injured than the veins, and small veins more than large ones. Fig. 72 shows the leaf injury on buckwheat by 1.0, 0.7, and 0.5 ppm with six hours' exposure. The plates (some colored) in the Trail Smelter book show the type of injury caused in leaves by SO 2, and the Report of the Selby Smelter Commission contains colored plates of leaf injury by SO2. The Trail report speaks of marginal as well as veinal killing of leaf tissue in dicotyledons. Both duration of exposure and concentration of the SO2 were important in determining the extent of injury. Sulphur dioxide is very soluble in plant tissues and accumulates with time. In subtoxic concentrations time 190 . GROWTH OF PLANTS is not a factor. As the National Research Council of Canada group found in such concentrations, the SO2 is oxidized to less harmful sulphates before enough accumulates to kill the tissue. With responses from ethylene the situation is quite different. Here time of exposure is of prime importance. Ethylene has low solubility in plants and consequently does not accumulate with time. Moreover, it must be present until the response occurs and the response ceases soon after the ethylene disappears from the surrounding air. Wilted plants were more resistant than similar plants that were turgid. The difference was considered to be due, at least in part, to the condition of the stomata. The investigators believe that low air humidity and low water content of the soil make plants more resistant by modifying the water content of the plant. Plants fumigated at night showed more resistance than similar plants treated during the day. Plants also gained in resistance if they were placed in the dark two hours before they were fumigated in a dark case. Shading while plants were being exposed to the gas was not effective. If this work was important at the time it was done in furthering the physiological point of view in SO2 injury, it has since been greatly out- distanced even in this respect by the extensive and very accurate researches by Hill, Thomas, and associates, and by the National Research Council of Canada. Later work by Setterstrom and Zimmerman -^ confirmed the earlier work of Zimmerman and Crocker and added the following facts: plants are more resistant at 40° C (104° F) than at higher temperatures, and also when previously grown in good rather than in poor light and when exposed to full light rather than in deep shade (65 per cent or greater reduc- tion of light intensity). The sulphate nutrient supply and previous treat- ment \vith SO 2 did not affect the susceptibility if in the latter case sufficient time was allowed for recovery from previous treatment. Sulphur Dioxide Content of Air at Boyce Thompson Institute. The SO2 content of the air at Boyce Thompson Institute ^s. 28 ^ras re- corded continuously with minor interruptions for two years, November 1, 1936 to November 1, 1938, by use of the Thomas recorders. The average readings including zero readings were 0.033 ppm for the first year and 0.035 ppm for the second. The maxima were 0.75 ppm for the first year and 0.53 ppm for the second. Both the averages and the maxima are almost identical with those at Northport, Washington 21 ■ p-^s during the growing season, where claims were made for injury to crops and forests. The high SO2 content of the air at Yonkers was in the winter when plants outside were not gro'wdng. Setterstrom and Zimmerman ^s. p-i78 conclude: "Correlation of SO2 con- centrations with the Avind direction indicates that the sulphur dioxide comes largely from New York City (15.4 miles SSW to Times Square which marks the approximate center of the metropolitan area). A study of the relationships between concentrations of sulphur dioxide of the atmosphere LETHAL GASES 191 and of the air of a greenhouse shows that greenhouse concentrations are approximately 90 per cent of atmospheric when ventilators are partly open, 60 per cent when ventilators are closed. The fact that the many plants gro^vn throughout the year in the Institute greenhouses are con- sidered comparable to plants grown in areas where there is no sulphur dioxide, is an indication that exposure to sulphur dioxide in the prevailmg concentrations and durations has no unfavorable effect on plant life." Sulphur Dioxide of Atmosphere as a Sulphur Source for Plant Nutrition Setterstrom, Zimmerman, and Crocker ^'^ found that sulphur deficiencies in the soil for growth of alfalfa could be supplied in part by fumigating the air with non-marking concentrations of SO2. The Cruciferae, which are very rich in organic sulphur, gave negative results. As we have already seen, Thomas and associates ^^ found later that sulphur deficiency for aKalfa could be supplied in part by SO 2 fumigation. Available sulphur is deficient ^-^ in some soils for maximum yield of protein-rich legumes like alfalfa. Indeed, it is so deficient in some soils in Washington, Oregon, and Idaho that sulphur additions will increase alfalfa yields as much as five-fold. As measured by crop needs in general, sulphur supply of soils is about equal to phosphorus supply, and as measured by the needs of high-sulphur crops like certain legumes and crucifers, the sulphur supply in the soil is below the phosphorus supply. Moreover, sulphur in the form of sulphates is leached from the soil in large amounts, w^hereas phosphorus is held rather tenaciously. In the United States enormous amounts of sulphur, largely in the form of SO2, are released into the atmosphere by smelters and 'by industrial and home consumption of coal and other fuels. This finally reaches the soil by being washed down by ram or absorbed directly by the soil or vegetation. This averages many pounds per acre per year for the w^hole country, and of course is high near industrial and population centers and very low in remote rural sections. This replenishment of sulphur in the soil may be an important factor in soil fertility around big population centers. Together with the later use of acid phosphate, which is about half calcium sulphate, this may account for the fact that gypsum ^ was very effective on clover in colonial days but has far less effect in eastern United States today. Also it is possible that fumes from the Trail Smelter may be improving and not injuring the yields of alfalfa in the upper Columbia Valley in the United States. This is made probable by the fact that soils in neighboring valleys both east and west are deficient in sulphur for maximum alfalfa production. Effect of Sulpbtur Dioxide on Animals Men and animals, like plants, ^^ are subjected to sulphur dioxide fumes. In the atmosphere of smoky cities sulphur dioxide sometimes reaches con- centrations of 10 ppm. Workers engaged in the manufacture of refrigerants 192 GROWTH OF PLANTS are exposed to 30 to 100 ppm at times. The disaster of the Meuse Valley in 1930 during six days' fog when 63 people died has been attributed to sulphur dioxide, which was estimated to reach concentrations as high as 38 ppm. It has since been questioned whether these deaths were due to SO2 and whether it reached anywhere near such a concentration. There are many other statements in the literature concerning the effect of SO2 in the air on health, most of them not well backed by facts. We seem long on assumptions and short on measurements of the effect of SO 2 in various concentrations and for various durations upon animals. The earlier meas- urements of the effects of SO2 on animals were on the dosage basis, that is, 180 360 540 720 TIME TILL 50 PER CENT MORTALITY (HRS.) 900 Figure 73. Time till 50 per cent mortality versus concentration toxicity curves for mouse and guinea pig in various SO2 concentrations. the animal was enclosed in a case and a single dose added, generally a large one, and the effect observed. Sulphur dioxide is absorbed readily by the walls of the case and more readily by the animal. Some later experiments were performed by the continuous air flow method but the concentration was not regulated and recorded accurately. Weedon, Hartzell, and Setterstrom " used the accurate continuous flow fumigation chambers at the Institute to study the effect of SO 2 on guinea pigs, mice, grasshoppers, and cockroaches. The concentrations used were 10, 35, 65, 100, 150, 300, and 1000 ppm and the duration of exposures ran up to more than 1000 hours in some cases. No significant mortality or signs of distress occurred in healthy animals in 33 ppm, even with 400 or LETHAL GASES 193 500 hours of exposure. One realizes the great resistance of animals as com- pared with the most sensitive plants when it is stated that leaf tissue in buckwheat is killed by seven hours' exposure to 0.46 ppm, though animals are not injured by 500 hours' exposure to 33 ppm. At high concentrations a longer total exposure to gas was necessary to cause death when the SO2 treatments were given in intermittent doses. Fig. 73 shows the length of exposure to various concentrations of SO2 necessary to kill 50 per cent of mice and guinea pigs. Above 200 ppm the guinea pigs are more resistant than the mice, and below that concentration the mice are much more resistant than the guinea pigs. Fifty per cent of the mice were still alive after nearly 900 hours in 150 ppm. The suscepti- bility of both grasshoppers and cockroaches approximated that of the mice. Symptoms in vertebrates at higher concentrations of SO 2 were lethargy, nasal catarrh, lachrymation, coughing, conjunctivitis, difficult breathing, distension of abdomen, weakness, and paralysis of hindquarters. In the highest concentration insects cleaned their mouth parts and antennae, showed lack of coordination of muscular movements, and paralysis of posterior legs. The authors describe the internal pathologic changes in mammals resulting from high dosages: ^^' p-^'"^ "Pathologic changes in vertebrates include general visceral congestion of slight to moderate degree, slight to moderate edema of the lungs ^^^th hemorrhages at higher concen- trations, acute dilation of the right heart at higher concentrations, gross distention of the stomach with multiple ulcers and hemorrhages at the higher concentrations, distention of the gall bladder except at lowest concentrations." Effect of Hydrogen Sulphide on Plants In connection with spray injury from Ume sulphur, McCallan and associates " ran continuous-flow fumigation experiments with hydrogen sulphide on 29 different species of plants. The fumigations were carried on in shaded glass cases outside during the summer. The duration of the fumigation was five hours in each case and was carried on at midday. The mean temperature was 74° to 81° F (23° to 27° C) and the relative humidity from 82 to 100 per cent in the several experiments. The symptoms of injury were scorching of young shoots and leaves and basal and marginal scorching of next older leaves with the older and mature leaves unaffected. This differs strikingly from the injury by SO2 which kills the parenchyma of the middle-aged leaves in lower fumigations while the young and mature leaves are more resistant and the stems uninjured. The authors describe further results of their experiments: '^' p-^^^^" "The different species varied \\idely in their response: carnation, purslane, Boston fern, apple, cherry, peach, strawberry, and coleus showed no appreciable injury at concentrations below 400 ppm; pepper, rose, nastur- tium, castor bean, gladiolus, sunflower, buckwheat, and cornflower, slight to moderate injury at concentrations from 40 to 400 ppm; and soybean, 194 GROWTH OF PLANTS Turkish tobacco, aster, kidney bean, cucumber, tobacco (A^. glaiwa), salvia, poppy, tomato, clover, radish, calliopsis, and cosmos, slight injury below 40 ppm and severe injury and death above 400 ppm. Temperature is as important as concentration, injury increasing rapidly with increases in temperature. In some cases wilted plants appear less sensitive to hydrogen sulphide injury than normal turgid plants. Plants tested for lime sulphur injury (aster, buckwheat, sunflower, and tomato) showed symptoms iden- tical Avith those produced by hydrogen sulphide." Here again injury by H2S differs from injury by SO2 in two respects: the minimum concentration of HoS for injury to plants is many times that for injury by SO2, and wilting of plants furnishes less protection against H2S than it does against SO2. We have already seen that the degree of stomatal opening is an important factor in SO2 injury. Hydrogen sulphide may enter through the cuticle rather than through the stomates, or the young tissues may be much more susceptible to H2S injury than older tissues. Effect of Chlorine Gas and Chlorinated Water on Plants AND Animals The staff of the Institute has been called in on several cases of injury to plants by chlorine gas escaping from tanks used to treat water for swimming pools and on injury to plants by fumes from laundries. The question whether there is enough residual chlorine in the tap water of a city to injure greenhouse plants watered with it, and water plants and fish hving in it, is a perennial question. Our observations and unpublished experiments indicate that chlorine gas m the air affects plants much as does SO2. The middle-aged leaves are most sensitive and the spotting of the leaves is similar to that caused by SO 2; also, as reported later in this chapter, CI2 is much more toxic to leaves in clear than in cloudy weather as is SO2, indicating further similarity of behavior between these two gases. It will also be observed that CI2 in the air spots leaves in even lower dosages than SO 2. It is also more toxic to animal pathogens and about equally toxic to plant pathogens. Our un- published experiments likewise proved leaves more sensitive to CI2 than to SO2. Zimmerman and Berg ^^ ran extensive experiments on the effect of chlorinated water on land and water plants and on goldfish. Land plants proved rather resistant to chlorinated water, but water plants were sensi- tive. The authors summarize their results on plants as follows: p"^"''^ "None of the species of plants grown in loam soil and watered, syrmged, or watered and syringed with chlorinated water having 50 ppm or less of chlorine in water, were injured or retarded when grown in pots in cold frames, on open benches in the greenhouse, or under bell jars in the green- house. Chlorine concentrations of 100 and 150 ppm injured or retarded some plants but had no effect on others. Concentrations of 200 and 300 ppm always produced some degree of injury to the tops, but had no LETHAL GASES 195 appreciable effect on roots. Concentrations of 500 and 1000 ppm retarded the emergence of seedlings, burned and usually killed the tops and roots of plants eventually. A combination of watering and syringing generally pro- duced greater and more rapid injury than watering the soil alone. Syringing the tops usually was somewhat less injurious than watering the soil with the same solutions. Injury from all treatments was m most cases more serious and more rapid in the greenhouse than m open cold frames, and worse under bell jars m the greenhouse than on the open greenhouse bench. "Tops of plants grown in a mixture of equal parts of loam and sand were retarded in top growth by solutions of 50 ppm and 100 ppm but not by 5 ppm. Roots were never injured in this soil. Plants grown in sand were retarded in root and top development by solutions of 5, 50, and 100 ppm chlorine, the degree being somewhat in relation to the concentration of chlorine in the solution. Chlorinated water applied to sand and to loam soil for three weeks had no significant effect on acidity of the media. ''Roots from tomato cuttings in chlorinated water were retarded in size directly as the chlorine concentration mcreased from 10 ppm, but they were not affected by 5 ppm. Cut flowers were not affected by free chlorine ui water up to 10 ppm. Fifty parts per million mjured gerbera and snap- dragons but not gladiolus or roses. Cahomba and Elodea were discolored by chlorinated water containing 3 ppm in one week when the water was refreshed daily. A concentration of 5 ppm produced injury m two days and death in four days." From what has been said above, it is evident that CI2 in the air is much more toxic to land plants than when in water solution; it takes less than 1 ppm by volume of CI2 in the rare medium, air, to spot more sensitive plants, whereas it requires several ppm by weight in the dense medium, water, to injure these plants. The CI2 enters the tissue of land plants through the stomates much more readily as a gas than it does through the general cuticular structure in solution. The greater injury from keeping plants sprayed with chlorinated water under bell jars or in greenhouses as compared with open air was due to gas escaping from solution and being held about the plants by the enclosures so it could enter through the stomates. Since water plants, such as Cahomba and Elodea, are poorly cutinized, CI2 in solution enters the plant readily. Concerning the effect of chlorinated water on goldfish the authors say: ^^^ p"^ "Under the conditions stated in the text goldfish were killed by chlorine solutions of 1.5, 2.0, and 3.0 ppm where the water was changed daily. Where water was constantly renewed, concentrations of 1.0 and 1.5 ppm were toxic. Fantail variety appeared to be less resistant than the Common variety of goldfish, while the Common variety was less resistant than the Shubunkin variety. Aquatic plants appeared to counteract ui some degree the toxic effects of chlorinated water on fish." The authors point out that Yonkers city water at the tap frequently contains 0.5 ppm of residual CI2, occasionally 1.0 ppm, and rarely 1.5 ppm. 196 GROWTH OF PLANTS Hence it is probable that the free chlorine in city water supplies sometimes reaches a concentration that \vill injure or kill fish and even water plants, but it is questionable whether it ever contains enough free chlorine to be dangerous for watering or syringing land plants. Comparative Effect of Five Toxic Gases on Plants and Animals Several members of the Institute staff 2. is. la. s^, ss ugg(^ o^. continu- ous-flow fumigation apparatus to study the relative toxicity of the following five gases upon various animals, plants, and plant organs: ammonia (NH3), chlorine (CI 2), hydrogen cyanide (HCN), hydrogen sulphide (H2S), and sulphur dioxide (SO2). Each gas was studied at concentrations of 1, 4, 16, 63, 250, and 1000 ppm of the air and the periods of exposure were 1,4, 15, 60, 240, and 960 minutes. The full set of experiments produced a great number of detailed facts, many of which are of interest. Space demands, however, that we must present only the general findings with a few of the more interesting details in connection with some of the organisms. Fig. 74 shows the number of minutes of exposure required for the highest concentrations of the gases used, 1000 ppm, to kill one-half of the various organisms (LD50) insofar as 960 minutes was sufficient to accomplish this end. One must realize in this figure that, due to the limited page size, the time is recorded as a geometric series on the ordinate rather than as an arithmetical series. If the figure were made on the arithmetical basis and each minute given the same space as the first minute, the figure would be about 45 inches high and the points in the upper part would be very much farther apart, increasingly so as the top of the figure is approached. With this in mind, let us see what the figure shows. Beginning with the eight actively growing plant pathogens, it is evident that they vary greatly from each other in their resistance to the several gases. They are arranged from left to right in their increasing order of resistance. These pathogens are in general most readily killed by SO2. The one exception is Rhizoctonia tuliparum, which proved a little more sensitive to CI2 than to SO2. Chlorine is the next most toxic. Then follow NH3, H2S, and HCN in succession, with no significant difference between the last two. Each of the last two failed to kill 50 per cent of four of the eight plant pathogens even in 960 minutes. In the case of the two animal pathogens tested, CI2 proved by far the most toxic, with SO2 second. The other three gases showed low toxicity. Sclerotia of fungi and seeds proved resistant. These reproductive organs are well protected by non-living coats. Soaked seeds, of course, were more sensitive than dry ones. Green leaves were, on the whole, the most sensitive to the five gases of any organisms tested, with CI2 the most toxic, SO2 next, followed by NH3 and HCN, and with H2S least toxic by a large margin. As has already been mentioned, CI2 and SO2 both caused interveinal spotting of the blades of the leaves. Ammonia in much higher dosages brought about spotting of the leaves, but the spots were often black instead LETHAL GASES 197 \ 1 — ■ r 1 *> ^■^ - <©o © ® • 1 (0 -I • <© O © « • 4; V> < 5 ■ <© O © ® • 3-^ o^ Z < . g 8 © - * to in 8 • © • - 7 - ©« §• ■" e (2 < Q. Z LJ U _ « • ©8> 1: - « © • o © - -CI 2 I - « • © o © E <8 -b -> - q§ - <© 0© o _ <5 - to Q UJ UJ 10 . 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C rl 03 o3 ■5b c 03 3^ taoo CO 03 •c 0) o 02 a) > 03 a; H-5 -^ (N « t-^ Ol 01 9J m a CJ -3 ■a -n C <: a 03 S3 '^ 03 a 02 3 i^ ^ ^aim CO a S3 •a c 3 f:^ S3 c/2 03 02 <; a 3 o u bC T3 O ti -O cl S3 03 -a CO a 0) u C 43 to 33 •a bC O l-H (M CO LETHAL GASES 199 of yellow or reddish brown due to the presence ^ of tannins in the leaves. Darkness also protected leaves from injury by NH3 but to a lesser degree than it did against SO2. The petioles of the younger leaves ^* were first killed by HCN. In the higher dosages CI2 and SO2 showed marked acidula- tion of the leaves, CI2 being more effective than SO2, whereas H2S caused only slight acidulation. Ammonia made the leaves more alkaline. The pH of the soil was lowered by CI2 and SO2 and raised by ammonia. Stems were much less sensitive on the whole than leaves, wdth httle if any significant difference between the several gases. The toxicity of the five gases to houseflies and mammals shows a great difference in effectiveness \\dth no overlapping of results. They rank as follows: HCN > H2S > CI2 > SO2 > NH3. Table 21 shows (A) order of toxicity of gases to classes of organisms, and (B) order of sensitivity of classes of organisms to the five different gases based on the highest concentration used, 1000 ppm. In general, CI2 and SO2 are most toxic to plants and HCN and H2S to animals. Table 22. Time Till 50 Per Cent Mortality of Gas-Treated Animals (In Minutes) Gas Animal Concentration, ppm 1000 250 63 16 NHj Flies Mice Rats > 960 > 960 > 960 Ch Flies Mice Rats 45 28 53 240 440 440 840 > 960 > 960 >960 HCN Flies Mice Rats 3.3 1.2 1.4 <8 5.1 8.7 8.2 66 40 48 > 960 > 960 H2S Flies Mice Rats 7 18 14 > 960 410 > 960 804 > 960 > 960 > 960 SO2 Flies Mice Rats 120 132 910 720 786 > 960 > 960 Up to now we have discussed the effect of only one concentration, 1000 ppm, mth only the time of exposure varying. If the total dosage (time X concentration) were a constant for producing a given physiological effect, this would be adequate at least for one kind of organism. Calculating from the figures in Table 22 will show that the product law does not hold. There is another reason why various concentrations as well as various times 200 GROWTH OF PLANTS must be considered when one organism is compared with another; flies, for instance, resist 1000 ppm of HCN more than twice as long as mice or rats, but mth 63 ppm mice and rats endure HCN five to seven times as long as flies. At 16 ppm neither rats nor mice showed any deaths or even signs of injury during 960 minutes, while 50 per cent of the flies were killed in 48 minutes. For SO2 we have already mentioned that guinea pigs resisted higher concentrations better than mice, but that mice were much more resistant to low concentrations. It is certain that flies could be killed in the presence of mammals by use of HCN in perfectly regulated low concentra- tions for long periods. It is regrettable that several other insects were not included in these studies to see how generally very low regulated concentra- tions of HCN proved fatal to insects. Those that are interested mil want to read the original articles, especially for details on symptoms and internal pathological changes caused in ani- mals and on types of injuries produced in green plants by the several gases. Summary As we saw in the previous chapter, ethylene is the constituent of artificial illuminating gas that injures plants in greenhouses when this gas seeps through the soil and into the houses. Hydrocyanic acid is the most deadly constituent of artificial illuminating gas to plants growing outdoors near leaking gas pipes. ISIost natural gases have very low toxicity to plants because they contain no ethylene or other unsaturated hydrocarbons, and no HCN or other highly toxic gases. Some natural gases contain H2S which might injure plants if the gas were not thoroughly scrubbed. Mercuric chloride, calomel, or organic mercury fungicides must be used with caution on soils in greenhouses or other enclosed spaces because the soils reduce these compounds to metallic mercury which has sufficient vapor pressure, especially at higher growing temperatures, to injure plants throughout the enclosed space. Use of mercury in respirometers or to seal apparatus may vitiate experiments in plant physiology either by injuring or stimulating the plants. The health of laboratory workers is endangered by exposure of large surfaces of mercury to the air, as in the case when pellets of mercury are allowed to lie on the floor or laboratory tables. Injuries from SO 2 from smelters have led to the development of accurate apparatus for recording the SO2 concentration in the air and for fumigating plants and animals in continuous-flow chambers with the SO2 regulated accurately and continuously recorded. The Department of Agricultural Research of the American Smelting and Refining Company did much to develop this apparatus and contributed greatly to knowledge of the physi- ological effect of SO2 on plants. The National Research Council of Canada in connection with Trail Smelter injury added much to our understanding of the effect of SO 2 on vegetation. Boyce Thompson Institute added to the knowledge of the physiological effect of SO2 on plants and animals. The most delicate plants are injured by 0.46 ppm of SO2 with seven hours' LETHAL GASES 201 exposure. Animals endure 33 ppm for 500 hours without injury. Sulphur dioxide kills the leaf parenchyma of the medium-aged leaves, thus cutting doA\Ti assimilation, but there is no reduction of assimilation or growth if no tissue is killed, that is, there is no "invisible injury." Darkness and partial wilting increase the resistance of plants to SO 2, partly at least by closing the stomates. Many continuous records have been made of SO2 content of the air about industrial and population centers. At Boyce Thompson Institute the average and highest aimual concentration of SO 2 in the air, largely blowing in from New York City, is about the same as that in the upper Columbia Valley at Northport, Washington, during the growing season, where injury from the Trail Smelter is claimed. The highest concentration of SO 2 in the air about cities is during the A\inter when most coal is being burned. Finally, the SO2 given off by industries and cities aids soil fertility by replenishing sulphur deficiency of the soU. Hydrogen sulphide differs from SO 2 in several ways as to its effect upon plants; it requires a much higher concentration to injure plants, 40 to 400 ppm ; it kills the young leaves and stems rather than spotting middle- aged leaves, and its toxicity is not so greatly reduced by darkening and by •wilting the plants. Chlorine acts much like SO2 on plants and spots them in even lower con- centrations. Chlorinated water has relatively low toxicity for land plants when used either for syringing or watering them. Chlorinated water bearing 1 .0 to 1 .5 ppm of chlorine will kill fish, and water plants are a little less sensitive. City water supplies may at times contain enough chlorine to injure fish and water plants. By use of the continuous air-flow method, a study was made of relative sensitiveness of plant and animal pathogens, sclerotia, seeds, green plants, and houseflies, rats, and mice to the five gases, CI2, HCN, H2S, NH3, and SO2. Chlorine and SO2 showed high toxicity to pathogens and other gases low toxicity. Sclerotia and seeds were little injured by any of the gases. Green leaves were very sensitive to these gases, and the gases show^ed the followmg order of toxicity : Cl2>S02>NH3>HCN >H2S. Green stems were more resistant than leaves, with no significant difference in the degree ' of toxicity of the five gases. Animals were readily killed by HCN and H2S and the order of toxicity for animals was HCN>H2S>Cl2>S02>NH3. Literature Cited 1. Alway, F. J., "A nutrient element slighted in agricultural research," J. Am. Soc. Agron., 32 : 913-921 (1940). 2. Barton, L. V., "Toxicity of ammonia, chlorine, hydrogen cyanide, hydrogen sulphide, and sulphur dioxide gases. IV. Seeds," C. B. T. I., 11 : 357-363 (1940). 3. Boussingault, "Sur Taction deletere que la vapeur cmanant du mercure exerce sur les plantes," Compt. Rend. Acad. Sci. [Paris], 64 : 924-929 (1867). 4. Bredeman, G., and H. Radeloff, "Ueber Schadigung von Pflanzen durch Ammoniak- gase und ihren Nachweis," Zeitschr. Pflanzenkrankh. u. Pflanzenschntz, 42 : 457- 465 (1932). 202 GROWTH OF PLANTS 5. Burrell, G. A., and G. G. Oberfell, "Composition of the natural gas used in twenty- five cities; with a discussion of the properties of natural gas," U. S. Bur. Mines Tech. Pap. No. 109, 22 pp., 1915. 6. Crocker, W., "The history of agricultural gypsum," Gypsum Indus. Assoc, 36 pp., Chicago, 111., 1922. 7. , "The necessity of sulfur carries in artificial fertilizers," J. Am. Soc. Agron., 15: 129-141 (1923). 8. , P. W. Zimmerman, and A. E. Hitchcock, " Ethylene-induced epinasty of leaves and the relation of gravity to it," C. B. T. I., 4 : 177-218 (1932). 9. Daines, R. H., "Some principles underlying the fungicidal action of mercury in soils," Phytopath., 26 : 90 (1936). 10. Deiman, Paats, Van-Troostwyck, and Lauwerenburgh, "Experiences sur Taction du mercure sur la vie vegetale," Ann. Chim. Phys., 22 : 122-126 (1797). 11. Giese, A. C, "Mercury poisoning," Science, 91 : 476-477 (1940). 12. Gray, N. E., and H. J. Fuller, "Effects of mercury vapor upon seed germination," Am. J. Bot., 29 : 456-459 (1942). 13. Harrington, G. T., "Further studies of the germination of Johnson grass seeds," Proc. Assoc. Off. Seed Anal. N. Am., 1916-1917 : 71-76 (1917). 14. Harvey, E. M., and R. C. Rose, "The effects of illuminating gas on root systems," Bot. Gaz., 60 : 27-44 (1915). 15. Hitchcock, A. E., W. Crocker, and P. W. Zimmerman, "Toxic action in soil of illuminating gas containing hydrocyanic acid," C. B. T. I., 6 : 1-30 (1934). 16. Kincaid, R. R., "Toxicity of mercury vapor to germinating tobacco seeds," Plant Physiol, 11 : 654-656 (1936). 17. McCallan, S. E. A., A. Hartzell, and F. Wilcoxon, "Hydrogen sulphide injury to plants," C. B. T. I., 8 : 189-197 (1936). 18. , and C. Setterstrom, "Toxicity of ammonia, chlorine, hydrogen cyanide, hydrogen sulphide, and sulphur dioxide gases. I. General methods and correla- tions," C. B. T. I., 11 : 325-330 (1940). 19. , and F. R. Weedon, "Toxicity of ammonia, chlorine, hydrogen cyanide, hydrogen sulphide, and sulphur dioxide gases. II. Fungi and bacteria," C. B. T. /., 11 : 331-342 (1940). 20. Mellor, J. W., "A comprehensive treatise on inorganic and theoretical chemistry," Vol. 4, 1074 pp. Longmans, Green & Co., London, 1923. 21. National Research Council of Canada, Associate Committee on Trail Smelter Smoke, "Effect of sulfur dioxide on vegetation," 447 pp., Ottawa, Canada, 1939. 22. "Natural gas." In Encycl. Brit. 14th ed., 16 : 163-164 (1929). 23. Ratsek, J. C, "Injury to roses from mercuric chloride used in soil for pests," Flor. Rev., 72(1858) : 11-12 (July 6, 1933). 24. Schollenberger, C. J., "Effect of leaking natural gas upon the soil," Soil Sci., 29 : 261-266 (1930). 25. Setterstrom, C, "Sulphur dioxide content of air at Boyce Thompson Institute. II," C. B. T. I., 10 : 183-187 (1939). 26. , "Effects of sulfur dioxide on plants and animals," Ind. Eng. Chem., 32 : 473- 479 (1940). 27. , and P. W. Zimmerman, "Apparatus for studying effects of low concentrations of gases on plants and animals," C. B. T. I., 9 : 161-169 (1938). 28. , , "Sulphur dioxide content of air at Boyce Thompson Institute," C. B. T. I., 9 : 171-178 (1938). 29. , , "Factors influencing susceptibihty of plants to sulphur dioxide injury. I," C. B. T. I., 10 : 155-181 (1939). 30. , , and W. Crocker, "Effect of low concentrations of sulphur dioxide on yield of alfalfa and Cruciferae," C. B, T. I., 9 : 179-198 (1938). LETHAL GASES 203 31. Solheim,"W. G., and R. W. Ames, "The effects of some natural gases upon plants," J. Colorado-W yarning Acad. Sci., 3 : 38 (1941); Abstr. in Biol. Abstr., 15 : 19835 (1941). 32. Thomas, M. D., R. H. Hendricks, T. R. ColUer, and G. R. Hill, "The utilization of sulphate and sulphur dioxide for the sulphur nutrition of alfalfa," Plant Physiol., 18 : 345-371 (1943). 33. , , J. O. Ivie, and G. R. Hill, "An installation of large sand-culture beds surmounted by individual air-conditioned greenhouses," Plant Physiol., 18 : 334- 344 (1943). 34. , and G. R. Hill, "Relation of sulphur dioxide in the atmosphere to photosyn- thesis and respiration of alfalfa," Plant Physiol, 12 : 309-383 (1937). 35. Thornton, N. C., and C. Setterstrom, "Toxicity of ammonia, chlorine, hydrogen cyanide, hydrogen sulphide, and sulphur dioxide gases. III. Green plants," C. B. T. I., 11: 343-356 (1940). 36. Walker, J. C., M. A. Stahmann, and D. A. Pryor, "Efficacy of fungicidal trans- planting liquids for control of clubroot of cabbage," Phytopath., 34 : 185-195 (1944). 37. Weedon, F. R., A. Hartzell, and C. Setterstrom, "Effects on animals of prolonged exposure to sulphur dioxide," C. B. T. I., 10 : 281-324 (1939). 38. , , , "Toxicity of ammonia, chlorine, hydrogen cyanide, hydrogen sulphide, and sulphur dioxide gases. V. Animals," C. B. T. I., 11 : 365-385 (1940). 39. Zimmerman, P. W., and R. O. Berg, "Effects of chlorinated water on land plants, aquatic plants, and goldfish," C. B. T. I., 6 : 39-49 (1934). 40. , and W. Crocker, "Sulfur dioxide injury to plants," Proc. Am. Soc. Hort. Sci., 27 (1930) : 51-52 (1931). 41. , , "The injurious effect of mercury vapor from bichloride of mercury in soil of rose houses," Flor. Exch., 81(21) : 13 (May 27, 1933); alsoiuB. T. I. Prof. Pap. 1 : 222-225 (1933). 42. , , "Plant injury caused by vapors of mercury and compounds of mercury," C. B. T. /., 6 : 167-187 (1934). 43. , , "Toxicity of air containing sulphur dioxide gas," C. B. T. I., 6 : 455- 470 (1934). CHAPTER 6 Plant Hormones by P. W. Zimmerman The mysterious forces which regulate the growth and movement of plants have always been a subject of major interest to botanists. For many years efforts were centered around essential mineral elements in fertilizers and soil, with the thought that properly balanced nutrient solutions might lead to an understanding of growth regulation. While plants grew at varying rates according to the kinds and amounts of minerals supplied, there was no evidence that tropisms, correlation of organs, flowering, and maturation are regulated by fertilizers. The effect of the growing stem tip on growth of other organs, the bending of stems toward light, the capacity of plants to right themselves when placed m a horizontal position, the production of adventitious roots, and the polarity of shoots and roots were phenomena which were not controlled by mineral nutrients. There was, perhaps, some- thing made by the growing plants, natural substances, which regulate growth. The results of recent investigations take away some of this mystery and indicate that, as in the animal kingdom, growth, movement, and matura- tion of plants are regulated by chemical substances (hormones) produced by the organism itself. In fact such substances, extracted from plants and animals and re-introduced into normal tissue, cause hormone-like responses. Physiologically active chemicals were prepared synthetically in the labora- tory to take the place of natural hormones. As ^^^th animals, a single active compound has several different effects on plants. For example, a single treatment of a growing plant with a-naphthaleneacetic acid may cause cell elongation, resulting in curvature of stems and epinasty of leaves, proliferations involving cell division and induction of adventitious roots, mhibition of buds, and regulation of rate of growth. These are all hormone- like responses. The term "hormone" was borrowed from the animal field, where it referred to a regulating substance produced in a particular ductless gland but having its effects on organs or tissues some distance away. In plants the terminal bud produces a substance which regulates growth of axillary buds. Botanists have a number of terms used more or less synony- mously with the word " hormone " — growth substance, growth regulator, phytohormone, and auxin. The term "hormone" should be reserved for natural substances, but it has a popular appeal and has been used loosely. Many controversial views were held before the modern growth substance 204 PLANT HORMONES 205 concepts were established. In order to present a brief historical picture, a few of the outstanding contributions from the time of Darwin are cited. Darwin ^ in 1881 showed that the coleoptiles of Phalaris and Avena curved toward the light and that when only the tip was unilaterally illuminated the influence traveled downward. When the tip was shaded, the remaining stump was unable to make a phototropic response. This definitely showed that the tip of the coleoptile was a place of great importance in connection A\'ith phototropic curvatures in plants, though Darwin did not recognize the influence as being of a chemical nature. The beginning of the chemical substance idea with proof to support it goes back to 1907 when Boy sen- Jensen i- 2. 3 started his classic experiments to show that the stimulus (chemical substance) could cross a discontinuity in the coleoptile of Avena. He found that when excised coleoptile tips were replaced on the stump mth a layer of gelatin, phototropic curvatures resulted after unilateral illumination of the tip, as with normal coleoptiles. That is, a substance which was formed in the tip drained into the gelatin and then diffused through this non-living material into the stump, where it accelerated growth on the dark side, causing bending toward the lighted side. Boysen-Jensen also showed that he could intercept the substance by inserting a small piece of mica into the coleoptile. If the mica was inserted on the illuminated side, phototropic curvature occurred normally; if in- serted on the dark side, very little or no bending occurred. Similar experi- ments were performed ^^-ith geotropically stimulated coleoptiles. If mica was inserted in the horizontally placed organs on the upper side of the tip, negative geotropism resulted, as in normal coleoptiles; if the mica was in- serted on the lower side, little or no bending occurred. The interpretation which Boysen-Jensen put upon the results of these experiments was that the stimulus originating in the tip was of a chemical rather than a physical nature, and that it acted in regulating gro^^'th. He thought there was an increased transmission of the groArth-promoting substance on the dark side — a view which is still tenable. Paal ^2' ^^ from 1914 to 1918 confirmed Boy sen- Jensen's results and fur- ther showed that if an excised coleoptile tip was replaced on one side of the stump, gro\\i:h was accelerated on that side, resulting in curvature. For this response no special stimulation of the tip was necessary, thus showing that the tip was continually making the growth hormone in the dark also. Paal also demonstrated that the stimulus passed through an interposed gelatinous membrane 0.1 mm in thickness between the tip and the stump of the coleoptile. He concluded from his experiments that the transmis- sion of the phototropic stimulus was brought about by means of a diffusible substance. ^^' p-^^^ Stark " in 1921 made the next big advance by investigating the transmis- sion of phototropic, traumatotropic, and haptotropic stimuli. He expressed the sap from coleoptiles and mixed it A\ath agar. Out of the agar plate, blocks were cut and placed unilaterally on decapitated coleoptiles. A sub- 206 GROWTH OF PLANTS stance drained out of the block into one side of the coleoptile, retarding growth and causing curvature. Seubert ^^ in 1925 extended the experiments of Stark by infiltrating agar with substances of both plant and animal sources — diastase, malt extract, saliva, etc. — demonstrating the existence of both accelerating (causing curvatures) and inhibiting substances. The field was further advanced from 1920 to 1934 by Purdy,^^ Soding,^^ Cholodny,^' •'' ^ Went,^^' ^o- ■*! Dolk,^'' Zimmerman, Crocker, and Hitch- cock,^^* ^^ Boysen- Jensen,^ Laibach et al.,^^ Thimann and Went,^* and Kogl et al.^'^ Hitchcock ^^ in 1935, experimenting with indole acids, demonstrated that /3-indoleacetic acid and j8-indolepropionic acid induced curvatures, pro- liferations, and adventitious roots when applied to intact plants. Hitchcock also found phenylacrylic (cinnamic) and /3- (phenyl) -propionic acids to be physiologically active when applied to intact plants. Zimmerman and Wilcoxon ^* in 1935 brought to light seven new hormone- like substances, giving support to the assumption that there are many physiologically active compounds, both natural and synthetic. Table 23 gives a list of hormone-like substances kno\vn in 1935. Two acids in this list, j3-indolebutyric and a-naphthaleneacetic, were pointed out as the most effective root-inducing substances. Many practical applications have been made with these and they still rank among the most important. In 1939 Zimmerman, Hitchcock, and Wilcoxon ^^' ^^ listed a total of 54 different growth substances which showed activity when applied to plants in the vapor form. The greatest possibility for locating large num- bers of active acids came when Zimmerman and Hitchcock ^^ in 1942 showed that substituted phenoxy and benzoic acids were active and that this activity varied wth kind, number, and location of the substituent groups or atoms. ^*' ^^- ^^' ^^ This is illustrated in Table 24. Table 25 shows a long list of active substituted phenoxy acids. Of the halogen substituents, chlorine and bromine caused approximately the same degree of activation. Iodine substituents in phenoxy acids did not activate to the same degree as bromine and chlorine. However, iodine substituted in the ring of benzoic acids made very active molecules. ^^' *^' ^^ Chlorine substituted in the ortho position to make 2-chloro-3,5-diiodo- benzoic acid furnished a molecule that acted very much like 2,3,5-triiodo- benzoic acid. Methods and applications. Physiological activity in substances or ex- tracts was first detected by use of the Avena coleoptile as a test object in a dark room. The method is somewhat complicated and requires a consider- able amount of equipment. Since it has been described elsewhere ^^ it need not be repeated. A simple method perfected in the Boyce Thompson Institute laboratories requires only a growing plant in light or dark. The young tomato plant, which has long been a standard test object for detecting the presence of PLANT HORMONES 207 Table 23. A List of Physiologically-Active Acids Known and Available in 1935 CHoCOOH NH X\ CHaCHjCOOH NH CH2CH2CH2COOH 3-Indoleacetic acid /3-(3-Indole)-propionic acid 7-(3-Indole)-n-butyric acid /XCH^COOH /XCHjCHjCOOH /\CH:CHCOOH CH2COOH Phenylacetic acid /3-(Phenyl)-propionic acid Phenylacrylic acid (cinnamic acid) a-Naphthaleneacetic acid H2C — CH2 H2COOH CH, Acenaphthene-(5)-acetic acid — CH2COOH Fluoreneacetic acid — CH2COOH Anthraceneacetic acid 208 GROWTH OF PLANTS Table 24. Dependence of Activity for Cell Elongation Upon the Position of Substituents, Groups, or Atoms CH2COOH 1NO2 CH2COOH Inactive JNO2 Active CH2COOH NO2 Inactive CH2COOH A NH2 CH2COOH i Inactive ,NH2 Inactive CH2COOH A NH2 Active CH2COOH CH2COOH i Active CI Active CH2COOH O CI Active CH2COOH O CH2COOH A NO2 NO2 Inactive CH2COOH ^CH3 CH3 Active ethylene gas, is a satisfactory species. The coleoptile can be used only to detect the cell-elongating power of a substance. With one treatment the tomato plant detects the capacity of a chemical to induce cell elongation, cell division, adventitious roots, and formative effects. The time required varies \vith the response. Detection of cell elongation requires only from 20 minutes to two hours, cell division 48 to 72 hours, initiation of roots five to ten days, and formative effects four to ten days. The four types of response are illustrated in Figs. 75 and 76. If the chemical does not induce at least one of these responses, it is listed as inactive. PLANT HORMONES 209 Table 25. Comparative Activity of Phenoxy and Substituted Phenoxy Derivatives of the Lower Fatty Acids. Tested as Lanolin Preparations Cell elongation Modification (epinasty). of tomato leaves. Substances Threshold concn. Threshold concn. nig/g mg/g Phenoxyacetic acid 20 Inactive a-(Phenoxy)-propionic acid 5 5 a-(Phenoxy)-n-butyric acid 5 5 2-Chlorophenoxyacetic acid 1 0.25 a-(2-Chlorophenoxy)-propionic acid 1 Inactive a-(2-Chlorophenoxy)-n-butyric acid 1 Inactive 3-Chlorophenoxyacetic acid 0.5 Inactive a-(3-Chlorophenoxy)-propionic acid 0.5 Active a-(3-Chlorophenoxy)-n-butyric acid 0.5 Inactive 4-Chlorophenoxyacetic acid 0.25 0.06 a- (4-Chlorophenoxy)-propionic acid 0.5 Inactive a-(4-Chlorophenoxy)-n-butyric acid 1 Inactive 2,4-Dichlorophenoxyacetic acid 0.015 0.003 a- (2,4-Dichlorophenoxy)-propionic acid 0.5 Inactive a-(2,4-Dichlorophenoxy)-n-butyric acid 0.5 Inactive 2,4,5-Trichlorophenoxyacetic acid 0.06 Inactive a-(2,4,5-Trichlorophenoxy)-propionic acid 0.03 Inactive a-(2,4,5-Trichlorophenoxy)-«-butyric acid 0.1 Inactive The simplest method knowTi for testing new chemicals is by means of the lanoHn preparation made by mixing 10 to 20 mg of the substance ^\^th one gram of lanolin. When these are thoroughly mixed, a small amount is applied with a glass rod to the upper side of a young tomato leaf and to one side of the adjacent stem. The angle between the stem and the leaf before treatment is usually near 45 degrees. If the chemical is active, causing cell elongation, the leaf moves downward and the stem curves away from the treated side, thus increasing the degree of the angle (Fig. 75). The same plant is kept for 10 to 12 days to determine the effect on cell division, root-inducing activity, and formative effects. If activity is indi- cated by the first test, the chemicals are then studied in comparison with a standard, such as a-naphthaleneacetic acid, which is active when used at 0.001 per cent in lanolin. Comparisons can also be made by applying the chemical to the soil of the potted plant. For example, 1 to 10 mg of an active chemical in 50 cc of water applied to the soil will cause the entire plant to show an epinastic response. This then is usually followed by the three other responses described above. Induction of adventitious roots. To date practically all the recorded growth substances which induce cell elongation (causing epinasty or stem curvatures) are active also for inducing adventitious roots. This capacity may be associated with the power to induce cell division. It is a fact that growth substances which induce mature cells to make further growth also 210 GROWTH OF PLANTS Figure 75. Tomato plants showing induced cell elongation and cell division. A, left to right: control plant; three plants treated on the upper side of a leaf petiole with lanolin preparations containing three different concentrations of a-naphthaleneacetic acid (0.0005 per cent, 0.00025 per cent, and 0.0001 per cent, respectively) which causes cell elongation, the degree of response increasing with increasing concentration. Photograph taken after 24 hours. B, left: control plant; right: lanolin preparation containing 1.5 per cent a-naphthaleneacetic acid applied around the upper end of the stem causing increased cell division and swelling of leaves and entire stem. Photograph taken after 48 hours. stimulate cell division. The actual initiation of root primordia involves some form of growth regulation which is not well understood. Root pri- mordia usually are associated with proliferations involving stimulated cell division. Several types of induced rooting are shown in Fig. 77.^'' The many groAvth substances do not all have the same degree of root- inducing power, and there are many quahtative differences in the induced responses. From a concentration standpoint alone the requirements vary from 0.1 mg/1 or less for a- (2,4-dichlorophenoxy) -propionic acid to 40 mg/1 for /3-indolebutyric acid. Again the species vary in their capacity to respond. For example, Ligustrum (privet) responds to a-naphthaleneacetic acid but not to i3-indolebutyric acid; but Evonymus (strawberry bush) is just the opposite, being sensitive to /S-indolebutyric acid and not to a-naphtha- leneacetic acid. The usefulness of a root-inducing preparation can be ex- tended and made to cover a wider range of species by including two or more PLANT HORMONES 211 Figure 76. Plants showing two different responses to "plant hormones." A, Kal- anchoe plants. Left: control plant; right: treated around the stem near the tip with 1.0 per cent /3-indolebutyric acid to induce adventitious roots. B, Tomato shoots. Left: control plant; right: growth showing modification after the tip had been sprayed with a solution containing a methyl substituted phenoxy acid. Many variations of this can be induced with different substances having a formative influence. growth substances. Considerable attention has been given to compara- tive activity ^^' ^^ and effects obtained with mixtures of root-inducing substances.-" Propagation of plants. Substances detected by means described in the preceding paragraphs may or may not have value for practical propagation. For example, iS-indoleacetic acid is a very effective substance for inducing epinasty when applied to tomato plants. It is not, however, as effective for inducing roots as /3-indolebutyric acid, a-naphthaleneacetic acid, or many of the substituted phenoxy acids. The best root-inducmg substances are determined only by testing various substances and concentrations. By this method it has been found that species of plants vary in their sensitivity to the different substances. Generally speaking, /3-indolebutyric acid and a-naphthaleneacetic acid together cover practically all the species require- 212 GROWTH OF PLANTS FiGUKE 77. Adventitious roots induced with plant hormones. A, Gladiolus corms showing (left) normal condition; {right) adventitious roots induced with /3-indolebutyric acid. B, Tomato plants with tops removed. Left: control having the cut surf ace treated with pure lanohn; right: lanohn preparation containing 1 per cent /3-indolebutyric acid appUed to cut surface of stump. C, Helianthus tuberosus tubers showing control on left; and right: tubers growing adventitious roots 20 days after having been immersed 48 hours in a solution of a-naphthaleneacetic acid (50 mg/hter of water). D, An internal piece of potato tuber induced to root by treatment with /3-indoleacetic acid solution [see publi- cation by Guthrie ^^]. E, Tomato plant treated with the first preparation of a-naph- thaleneacetic acid used as a hormone. F, Camellia cuttings showing natural rooting from callus on non-treated control (left); and right: roots induced along stem of cuttmg dipped into a mixture of /3-indolebutyric acid -f- a-naphthaleneacetic acid, equal parts, making a total concentration of 20 mg per cc of water. PLANT HORMONES 213 ments. In some cases substituted phenoxy acids show considerably more activity for inducing roots than these two, but they have not been thor- oughly tested for practical purposes. Three methods of treating stem cuttings are to immerse the basal end of the cutting in the given concentration of the chemical in water solutions; to dip the basal end of the cutting into a powder preparation containing a Figure 78. Propagation with aid of root-inducing chemicals. A, Evonymous cuttings showing increased rooting with increasing concentrations of /3-indolebutyric acid. Left to right: non-treat-ed control, next three treated at base with solution of /3-indolebutyric acid — 0.5 mg/hter, 1.0 mg/liter, and 2 mg/lit«r, respectively. The basal end of cuttings were immersed in the solution for 24 hours, then planted in rooting medium. Photograph taken after 20 days. B, Ilex cuttings showing the effects of the growth substance alcohohc dip method. Left to right: control dipped in 50 per cent alcohol; basal end dipped in 5 mg per cc of 50 per cent alcohol; 10 mg; and 20 mg, respectively. Cuttings were in rooting medium 33 days before being photographed. given amount of the chemical; or to dip the basal end of the cutting into an alcoholic solution of the substance or a concentrated water solution of the substance. In a 24-hour immersion treatment, jS-indolebutyric acid is used at a concentration of 0.5 to 80 mg of the acid per liter of water, the optimum varying ^A-ith the species (Fig. 78A). or-Naphthaleneacetic acid is effective over a range of 0.5 to 60 mg of the acid per liter of water. Halogen- substituted phenoxy compounds must be used in low concentrations. For example, a-(2,4-dichlorophenoxy)-propionic acid is effective over a range of concentrations from less than 0.1 to 10 mg/1, the exact requirement varying with the species. A second method, which has been found particularly effective, is kno^n as the solution dip method. One to 10 mg of iS-indolebutyric acid in 50 per cent alcohol makes an effective range of concentrations which can be used 214 GROWTH OF PLANTS on a large number of species. The basal end of the cutting is dipped directly into the alcohoUc solution and planted immediately in the rooting medium. • Similarly, a soluble salt of a-naphthaleneacetic acid may be used in water instead of alcohol (Fig. 78B). This is a simple method since it does not require preliminary soaking before planting. Figure 79. The powder dip method for propagating cuttings with growth substances illustrated with Camellia species. A, Moist Camellia stems dipped into powder prepara- tions containing 10 mg of /3-indolebutyric acid per g of talcum powder. B, Left: control treated with pure talcum powder; right: rooted cuttings which had been treated with talcum powder preparation containing 10 mg of /3-indolebutyric acid per g of talc. The third and the most extensively used method at the present time is the powder dip method (Fig. 79). This involves a mixture of 1 to 10 mg of /3-mdolebutyric acid, a-naphthaleneacetic acid, or other substances per PLANT HORMONES 215 gram of talcum powder in which the basal end of the cutting is dipped and then planted in the rooting medium. Enough of the mixture clings to the basal end of the stem to induce roots the same as the other treatments. All three of the methods described have their supporters among the ama- teurs and commercial growers. It is generally recommended that interested growers familiarize themselves with all the methods and then select the one which is most satisfactory. Also, for the average grower it is better to use a recommended commercial preparation than to attempt to measure and prepare the substances in small quantities. Details for propagating plants are given in several Boyce Thompson Institute papers.^^- ^^"^^ i3-Indolebutyric acid, which accelerates callus formation, has been found effective also for accelerating union between the scion and the stock where the grafting method is used. The basal end of the scion is dipped into a water solution containing 50 to 80 mg of /3-indolebutyric acid per liter of water and then grafted as usual. Lily scales which are used for growing bulbils may be treated by any of the methods described for cuttings and planted in the usual way. jS-Indole- butyric acid accelerates rooting of the scale, thus keeping it in good condi- tion until the bulbils are formed. Though various claims have been made, it has not been proved that any of the known growth substances to date initiate shoot buds. Saintpaulia leaves used for propagation purposes can be induced to form many roots by treatment with well-known growth substances. In no case, however, has acceleration of buds been proved. Generally, bud initiation is inhibited by the treatment. Preharvest apple drop. The practical use of growth substances has been greatly extended by the work of Gardner et al}^ in connection with abscis- sion layers and premature falling of fruit. Of all the substances listed in Tables 23 and 24, it appears that a-naphthaleneacetic acid is the most effective substance for preventing preharvest apple drop. The water solu- tion containing 10 to 50 mg of a-naphthaleneacetic acid per liter of water is sprayed on the entire tree at the time the apples begin to fall prematurely. The chemical prevents the separation of the abscission layer, thus causing the apples to stay on the tree. It has been reported that trees normally losing 50 to 75 per cent of their apples before harvest time have been reduced to 5 to 10 per cent drop. Many of the active substances listed in this chapter are effective for preventing abscission of leaves, but so far none has proved as effective for preharvest apple drop as a-naphthalene- acetic acid. Not all varieties of apples respond alike. For example, the Mcintosh variety is very resistant, the results being negative in practically all reports. Williams variety is perhaps among the most sensitive, the treat- ment preventing practically all the apples from dropping. The effective- ness for other varieties fluctuates between these two extremes. Variations are also reported for a given variety in different locations over the country. 216 GROWTH OF PLANTS Inhibition of growth. The use of chemicals for inhibiting growth is often as important as for accelerating growth. The same chemicals which stimu- late root gro^vth (cause cell elongation, cell division, etc.) may be used also to inhibit growth. Hitchcock ^^ in 1935 tested seven substances for com- parative effectiveness in inducing epinasty. He listed them in decreasing order of activity as follows: a-naphthaleneacetic, /3-indoleacetic, /3-indole- butyric, /3-indolepropionic, phenylacetic, (3- (phenyl) -propionic, and phenyl- acryhc (cinnamic) acids. The same order holds for inhibition. When Hitchcock applied the substances to the cut surface of decapitated stems, axillary buds down along the stem were inhibited. The buds nearest the cut surface (that is, nearest the chemical) were inhibited most. Lanolin preparations of two unsaturated hydrocarbon gases, ethylene and propyl- ene, applied to the cut surface also inhibited the uppermost buds. Sprouts and seedlings treated with a-naphthaleneacetic acid were first accelerated in growth and then inhibited (Fig. 80). The stems later in- creased in diameter but not in length, and the buds failed to develop. ^^' ^^- ^* Stem cuttings of some species, treated wdth a-naphthaleneacetic acid to induce basal roots, showed inhibition of buds up along the stem after roots were well established. Roots differed from stems in response to growth substances by being inhibited immediately without a preliminary acceleration period.'*^ Aerial roots of Cissus, which normally elongate 5 to 10 inches within a 24-hour period, practically stopped when a lanolin preparation containing 0.1 to 1.0 per cent a-naphthaleneacetic acid was applied at the tip. Guthrie ^^ showed that methyl a-naphthaleneacetate applied as a vapor was effective for inhibiting buds of potato tubers. He placed layers of filter paper impregnated with the ester among the stored tubers, and effec- tively prevented growth. About 500 mg of methyl a-naphthaleneacetate per kg of potatoes were sufficient. Denny, Guthrie, and Thornton ^ im- proved the method and worked out other methods for preventing potato tubers from sprouting by use of methyl a-naphthaleneacetate. Dusting potatoes with a talcum powder preparation of the ester was found to be effective. Though not fully tested, the new aerosol-growth substance method has been effective when used in the laboratory. Another use for bud inhibition is to delay flowering of fruit trees until danger of frost is past. This is done by spraying in the previous summer and autumn when the buds are forming. A solution containing 100 mg of ct-naphthaleneacetic acid per liter of water applied in August with a sprayer delayed flowering for two to three weeks. Some delay was accomplished also by spraying branches when the buds started showing color in the spring, but much higher concentrations were required. It was predicted that tropical fruits and others could be staggered throughout the year by this method. Flowering shrubs which have one flush of flowers in the spring might also be staggered in time of flowering. PLANT HORMONES 217 FiGUKE 80. Inhibition of growth followed by increase in diameter of stem. A, Sweet pea seedhngs showing controls (right), and seedlings treated at the tip with lanohn prep- aration containing 1 per cent a-naphthaleneacetic acid. B, Windsor bean seedhngs showing upright controls, not treated, and treated seedlings which are inhibited and swollen. C, Potato sprouts showing upright controls {right) and curved and swollen sprouts resulting from treatment with lanolin preparation containing about 1 per cent /3-indoleacetic acid. D, Potato sprouts showing inhibition of growth and swelling after treatment with lanolin preparation containing about 1 per cent a-naphthaleneacetic acid. Controls shown iu C. 218 GROWTH OF PLANTS Fruit set and seedless fruit. A long list of growth substances capable of causing fruit set A\dthout pollination of the flower is given m Table 26. In addition to inducing seedless fruit (Fig. 81), these chemicals may, under certam conditions, increase the size of fruit resulting from pollinated Table 26. Growth Substances Active for Parthenocarpy, Fruit Set of Tomatoes, and Activity or Inactivity for Modification of Leaves. Applied as Spray to Flower Clusters Phenoxy acids Q:-(Phenoxy)-propionic acid a-(Phenoxy)-n-butyric acid 2-Chlorophenoxyacetic acid Of- (2-Chlorophenoxy)-propionic acid a-(2-Chlorophenoxy)-n-butyric acid a-(2-Methylphenoxy)-propionic acid a-(3-Chlorophenoxy)-propioiiic acid a- (3-Chlorophenoxy )-n-butyric acid 4-Chloroj)henoxyacetic acid a-(4-Chlorophenoxy)-propionic acid a-(4-Chlorophenoxy)-w-butyric acid 2,4-Dichlorophenoxyacetic acid a-(2,4-Dichlorophenoxy)-propionic acid a-(2,4-Dichlorophenoxy)-n-butyric acid 2,4-Dimethylphenoxyacetic acid a-(2,4-Dimethylphenoxy)-propionic acid 2,5-Dichlorophenoxyacetic acid a-(2,5-Dimethylphenoxy)-propionic acid a-(2,5-Dimethylphenoxy)-n-butyric acid 3,4-Dimethylphenoxyacetic acid a-(3,4-Dimethylphenoxy)-propionic acid 2,4,5-Trichlorophenoxyacetic acid a-(2,4,5-Trichlorophenoxy)-propionic acid a-(2,4,5-Trichlorophenoxy)-n-butyric acid 2,4,5-Triniethylphenoxyacetic acid 2,4,6-Trichlorophenoxyacetic acid /^-(2,4,6-Trichlorophenoxy)-/3'-chlorodiethyl ether /3-Naphthoxyacetic acid /3-Naphthoxypropionic acid /3-Indolebutyric acid 2,5-Dichlorobenzoic acid Effective range of concentrations for fruit set in mg/1 of water 100-200 100-200 200-300 25-50 50-200 50-100 50-200 Active 50-100 50-200 50-200 5-10 50-100 50-100 300-450 300-450 25-100 100-300 Active Active 300-500 25-100 10-50 25-100 25-100 Active Active 50-100 50-100 500-1,000 100-300 Activity or inactivity for modification of leaves Active Active Active Inactive Inactive Inactive Inactive Inactive Active Inactive Inactive Active Inactive Inactive Active Active Inactive Inactive Inactive Active Active Inactive Inactive Inactive Active Active Active Active Active Inactive Active flowers.^' " In the latter case the resultmg fruit may be partially seedless, but once the eggs are fertilized the substances do not inhibit their growth. Under certain conditions only the wall of the fruit may be stimulated by the chemical; as a result, it develops more rapidly than the gelatmous pulp. When this happens there may be a space between the pulp and the wall. Although the flavor of the fruit may not be impaired, this condition makes the fruit unsatisfactory for commercial purposes. Several methods for applying growth substances to tomato flowers have been developed. Perhaps the simplest is the application of spray to the PLANT HORMONES 219 flower cluster Avith an atomizer. When two or more flowers are open, the entire cluster may be treated to set the fruit on both buds and open flowers. The recommended concentration for the chemical in solution is shown in Table 26. Spreaders may be used in the solution but they are not necessary. FiGUKE 81. Induced fruit set and seedlessness. A, Left to right: normal tomato fruit resulting from pollinated flowers; parthenocarpy and seedlessness resulting from treat- ment of flower cluster with 2,4-dichlorophenoxyacetic acid (5 mg/liter) when three flowers were open; parthenocarpy and seedlessness resulting from treatment of flower clusters with 2,5-dichlorobenzoic acid (100 mg/hter). B, Left to right: cross section of normal control with seeds; cross section of seedless tomato induced with /3-naphthoxy- acetic acid (100 mg/liter). Also lanolin emulsion has been tested and found effective, but is no more satisfactory than water solution. In fact there is some indication that the emulsion brings about some undesirable effects, such as scarring the surface of the tomato or increasing blossom end rot. Carbowax (polyethylene- glycol 0.5 to 1 per cent) is a good spreader and is fully as effective as lanolm emulsion. It has been recommended for fruit sprays. In spraying the flower cluster it is well to apply the solution to the peduncle as well as to the open side and back of the flowers. For a high percentage of fruit set it 220 GROWTH OF PLANTS has been found that spraying only open flowers causes approximately 100 per cent set. Another method of applying growth substances is to remove the style with small scissors and apply a lanolin preparation directly to the cut surface. For this purpose 0.1 to 0.3 per cent of ^-indolebutyric acid in lanolin has been recommended. Less of other chemicals is required, as fol- lows: i3-naphthoxyacetic and propionic acids 0.05 per cent, Q!-(2-chloro- phenoxy)-propionic acid 0.01 per cent. 2,4-Dichlorophenoxyacetic acid is not recommended for this method since it causes modification of leaves even when used in very low concentrations. The vapor method of treating flowers throughout the entire house is effective, but mhibition of growth of the entire plant after the treatment may result. If used, the ethyl or methyl esters of /3-naphthoxyacetic acid or Q:-(2-chlorophenoxy)-propionic acid are recommended. Twenty-five to 50 mg per 1000 cubic feet of space vaporized by means of a warm electric plate are recommended. The heat should be applied so that the vapor rises slowly in the course of an hour. The air should be agitated by means of an electric fan. The aerosol method of applying growth substances has recently been emphasized. It is a kind of modification of a fine spray. One of the best methods of applying aerosol is to place the gro^\^h substance with sesame oil in the cylinder with Freon (dichlorodifluoromethane) . The internal pressure resulting is approximately 150 pounds. The mixture, dispensed through a small aperture, makes a mist or fog which affects the entire plant. When used in a greenhou^^e of 2000 cu. ft. capacity with tomato plants having open flowers, 50 to 450 mg of ethyl a-(2-chlorophenoxy)- propionate were found sufficient to set fruit on all the plants. The aerosol method is subject to the same objection as the vapor method. 2,5-Dichlorobenzoic acid applied to the soil of potted tomato plants mduced seedless fruit. One to 5 mg in 50 cc of water applied to the soil of a 4-inch pot were sufficient to induce fruit set of open flowers and also flowers growing on the plant for one month thereafter. This method has more theoretical than practical value at the present time. The formative influence of growth substances. A new line of plant hormone research was started when it became known that some substances had a formative influence on growth, modifying organs in size, shape, pattern, and texture. ^o- 43. 44. 45, 46, 57. 58. 59 Formative effects were evident on new growth which occurred within days or weeks after the plant was treated. This was in contrast to induced curvatures due to cell elongation which took place within a few minutes after treatment. Fig. 82 illustrates a type of formative influence which 4-chlorophenoxyacetic acid has on the growth of tobacco plants. Three groups of chemicals, (3-naphthoxy, substituted chlorophenoxy, and substituted benzoic acids, were outstanding for their formative mfluence, PLANT HORMONES 221 FiGTJRE 82. A, Tobacco plants to show formative effects induced with 4-chloro- phenoxyacetic acid 200 mg/liter applied at the tip when the plant was approximately 10 inches in height. Left: control. B, Enlargement of plant in A. C, Series of leaves taken from base to tip of treated plant. 222 GROWTH OF PLANTS in addition to many other hormone-like effects. The following graphic formulas illustrate the structure of one acid in each group: 0— CH2— COOH -O— CH2COOH COOH I I ^-Naphthoxy acetic acid 4-Chlorophenoxyacetic add 2 ,4,5-Tniodobenzoic acid Table 27. Comparative Physiologica Activity of Xylenoxy Compounds Xylenoxy acids and esters Cell elongation and epinasty of tomato leaves. Threshold concn. mg/g of lanolin Modification of leaves. Threshold concn. mg/g of lanolin 2,5-Dimethylphenoxyacetic acid Active Inactive 5 at 20 mg/g a-(2,5-Dimethylphenoxy)-propionic acid Active Inactive 0.5 at 20 mg/g a-(2,5-Dimethylphenoxy)-n-butyric acid Active Inactive 5 at 50 mg/g a-(3,5-Dimethylphenoxy)-malonic acid Inactive Active 3,0-Dimethylphenoxyacetic acid Inactive Active at 20 mg/g 10 Q;-(3,5-Dimethylphenoxy)-propionic acid Inactive Active 0.25 a-(3,5-Dimethylphenoxy)-n-butyric acid Inactive Active 50 Inactive /3-(3,5-Dimethylphenoxy)-7i-butyric acid Inactive at 20 mg/g at 20 mg/g 2,4-Dimethylphenoxyacetic acid Active Active 0.5 0.25 a-(2,4-Dimethylphenoxy)-propionic acid Active Active 0.5 0.5 a-(2,4-Dimethylphenoxy)-n-butyric acid Active Active 20 20 3,4:-Dimethylphenoxyacetic acid Active 1 Active Active 1 a-(3,4-Dimethylphenoxy)-propionic acid 1 Active 1 0.25 a-(3,4-Dimethylphenoxy)-n-butyric acid Active Active 5 5 The degree of activity varied with the groups or elements substituted, with the number and location of substitutions, and in higher homologs with the place of attachment of the chain to the ring of the molecule. The comparative influence of groups and elements and the location of the substi- tution are illustrated in Table 24. The activity referred to in this case is the power to induce cell elongation. When a formative influence is involved, still other differences are evident. The illustration shows that the NO2 PLANT HORMONES 223 group brings about activity for cell elongation when substituted in the meta position of the benzene ring, and the NH2 group in the para position ; chlorine activates the molecule in the ortho, meta, or para positions. To date it has not been possible to predict physiological activity by study of the molecular configuration alone — biological assay is necessary. The study of configuration in relation to activity, however, has helped to locate a large number of active compounds. Table 25 shows a long list of phenoxy acids in relation to activity and inactivity for cell elongation and formative influences. Table 27 ^'' shows a similar list of xylenoxy acids, and Table 28 derivatives of benzoic acid. Table 28. Active and Inactive Derivatives of Benzoic Acid Substances Cell elongation Formative effects Benzoic acid Inactive Inactive 2-Iodobenzoic acid Inactive Inactive 3-Iodobenzoic acid Inactive Inactive 4-Iodobenzoic acid Inactive Inactive 2,4-Diiodobenzoic acid Inactive Inactive 3,5-Diiodobenzoic acid Inactive Active 2-Bromo-3-nitrobenzoic acid Active Active 2-Chloro-5-nitrobenzoic acid Inactive Active 2-Amino-5-chlorobenzoic acid Inactive Inactive 2,3,5-Triiodobenzoic acid Inactive Active 2-Chloro-3,5-diiodobenzoic acid Inactive Active 2-Amino-3,5-diiodobenzoic acid Inactive Inactive The formative influence of derivatives of benzoic acid ^^ is illustrated in Fig. 83. Some of these compounds modify the flowering habit and change the correlation of organs. In addition to the nature of the molecule, the constitution of the receptor tissue in the plant is important. First, the genetic constitution of the tissue plays an important part, and secondly, the location in the organ and the age of the tissue are determining factors. Though in the same family group, tomato and potato tissue do not respond alike to a given substance, due perhaps to the difference in their genetic constitution. Apple and lilac stem cuttings can be induced through chemical treatment to produce adventitious roots in the spring of the year, but not in autumn or winter. Though the tissue is receptive at an early age, the capacity to respond to the chemicals is soon lost. With still other species young tissue does not respond to chemical treatment, whereas older tissue is susceptible. Many other illustrations could be given to indicate that there are complex internal and external influences playing upon the living protoplasm and that the sum total of these regulates the gro^\'th and development of the plant. 224 GROWTH OF PLANTS Figure 83. Tomato shoots and plants to show the effect of 2,3,5-triiodobenzoic acid on configuration, flowering habit, and correlation of organs. A, Left: control; right: upper portion of main shoot of plant 51 days after 1.25 mg of the substance had been added to the soil. Note that the terminal shoot ended in a flower cluster with an abnormal pedicel. Arrow points to node-Hke structure. B, Left: normal plant; right: plant show- ing response to the substance 21 days after 5 mg had been added to the soil. The plant was approximately 6 inches in height when treated. Note curvature of stem at nodes and modification of new leaves which were formed after treatment. PLANT HORMONES 225 There are ample facts to prove the existence of formative influences exerted by hormone-like substances, but we are far from understanding the mechanism through which the chemicals act. It seems logical to assume that the nucleus is the stable element, and that the "normal" form of the plant results from this stable element actmg in combination ^\-ith the "com- plex influences" from the cytoplasm. If one considers the cytoplasmic influence the less stable, then it may be assumed that the modifications in form may be brought about by the action of a chemical environment upon the cytoplasm. Each chemical constitutes a different environment and therefore permits different potentialities of the protoplasm to develop. "■*• "^ Other Subjects of Interest Involving Plant Hormones Only brief discussions are included. If details are needed, the citations wall be helpful. Absorption and movement of growth substances. The controversial issues on direction of movement of hormones were somewhat moderated when it was demonstrated that roots can absorb gro^^h substances from the soil.i^ The fact that the active substances were absorbed and translocated throughout the plant was indicated by the response of the aerial parts. The rate at which the substances were absorbed and moved in the plant varied with the rate of transpiration, increasing with increasing transpha- tion. Applied at the tip of the stem by means of sprays or lanolin prepara- tions, the substances moved downward, causing a systemic response. When applied to the middle of the stem the substances caused a response above and below the treated region. In short, it was sho^\-n that growth substances can move in all directions through the plant. Light and dark effects. Young gromng tomato plants maintain their power to respond to growth substances while in light and for a period of hours or days (depending on size and age) when transferred to a dark room. They first lose their capacity to right themselves while in darkness, when placed in a horizontal position. When this condition first occurs, the plants may use synthetic substances to bring about tropic responses. Later, however, young plants lose their power to respond to either natural hormones or synthetic substances. Plants attached to storage organs (potatoes, Jemsalem artichoke, etc.) or having storage tissue m the pith (tobacco) mamtained their power to respond to growi:h substances for more than 40 days after being placed m a dark room.^ Tropic curvatures. Natural tropic curvatures, such as bending of the stem toward light or away from the earth, are assumed to be due to unequal distribution of the natural hormones.^-- ^^ Stems treated unilaterally \\ath preparations of groT\i;h substances underwent pronounced negative bend- ing when in darkness or when light came from all directions. If, however, the plants were allowed to make natural tropic responses before the sjm- thetic growth substances were applied, the final responses were greatly modified. When the substance was applied opposite the side that natural 226 GROWTH OF PLANTS hormones were assumed to accumulate, the degree of curvature was greatly reduced; when they were applied on the other side, the curvature was greatly increased. Anesthesia. In Chapter 4 (p. 150) there is a discussion of ethylene-induced anesthesia in plants. It need only be mentioned here that a condition can be induced with growth substances which in many respects resembles ethylene-induced anesthesia. The most effective growth substances for anesthesia are a-naphthaleneacetic, /3-naphthoxyacetic, /3-naphthoxy- propionic, 2,4-dichlorophenoxyacetic, 2,4,5-trichlorophenoxyacetic, and o:-(2,4,5-trichlorophenoxy)-propionic acids. Activation of cinnamic acid with ultraviolet light. Trans-cinnamic acid is not an active growth substance. If, however, it is exposed to ultraviolet light, an active substance results. The light changes the trans to the cis form, which is active. During the investigation of this subject it was found that the vapors of the substance were active and induced responses similar to ethylene. ^^ The vapor method has since become of considerable impor- tance. Natural influences. Evidence continues to accumulate to show that the growing plant produces natural hormones and that these vary with en- vironment and orientation of the plant organs. Potted plants which were placed in a horizontal position gave a pronounced geotropic response. When the pots were again set upright, the stems recovered from the first response but the leaves developed epinasty.^^ The final effect was as if synthetic hormones had been applied. Unusual physiological responses were also induced on intact plants by capping wdth black cloth.^^ Capping the tomato plant for 3 to 14 days is assumed to bring about an over-production of natural hormones which causes epinasty of leaves, swelling of stems, proliferations (including in- tumescences), inhibition of growth, uiitiation of roots, and a disturbance of correlation of organs. Comparative effectiveness of acids, esters, and salts. The part of the molecule which brings about a physiological response is still unknown. It may be the entire molecule or one of its component parts. It has been de- termined, however, that the application of acids, esters, and salts brings about similar results. For solutions it is convenient to use acid and salts, while for vapor treatment it is best to use the esters, due to their high vapor pressure. ^^ Literature Cited 1. Boysen-Jensen, P., "tlber die Leitung des phototropischen Reizes in Avenakeim- pflanzen," Ber. Deutsch. Bot. Ges., 28 : 118-120 (1910). 2. , "La transmission de I'irritation phototropique dans VAvena," Kgl. Danske Videnskab. Selskabs. Forhandl, 1911(1) : 3-24. 3. , "tJber die Leitung des phototropischen Reizes in der Avenakoleoptile," Ber. Deutsch. Bot. Ges., 31 : 559-566 (1913). PLANT HORMONES 227 4. Boy sen- Jensen, P., "Die Bedeutung des Wuchsstoffes fiir das Wachstum und die geotropische Krummung der Wurzeln von Viciafaba," Planta, 20 : 688-698 (1933). 5. Cholodny, N., "Uber die hormonale Wirkung der Organspitze bei der geotropischen Kriiminung," Ber. Deutsch. Bot. Ges., 42 : 356-362 (1924). 6. , "Beitriige zur Analyse der geotropischen Reaktion," Jahrb. Wiss. 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(Ser. 14), No. 89 : 9-11 (1939). 27. , "Additional information on the use of root-inducing substances as an aid in propagating dahhas," Bull. Am. Dahlia Soc. (Ser. 14), No. 92 : 10, 19 (1940). 28. , "Effect of indolebutyric acid on the root response of evergreens," Am. Nursery- man, 71(8) : 9-12 (1940); also in B. T. I. Prof. Pap., 1 : 273-280 (1940). 29. , "Rooting rose cuttings with chemicals," Am. Nurseryman, 72(10) : 7-9 (Nov. 15, 1940); also in B. T. I. Prof. Pap., 1 : 291-296 (1940). 228 GROWTH OF PLANTS 30. Kogl, F., A. J. Haagen-Smit, and H. Erxleben, "Uber ein neues Auxin ("Hetero- auxin") aus Harn. II. Mitteilung uber pflanzliche Wachstumsstoffe," Hoppe- Seyler's Zeitschr. Physiol. Chem., 228 : 90-103 (1934). 31. Laibach, F., A. Muller, and W. Schafer, "Uber wurzelbildende Stoffe," Naturwiss., 22 : 588-589 (1934). 32. Padl, A., "tJber phototropische Reizleitungen," Ber. Deutsch. Bot. Ges., 32:499- 502 (1914). 33. , "liber phototropische Reizleitung," Jahrb. Wiss. Bot., 58 : 406-458 (1918). 34. Purdy, H. A., "Studies on the path of transmission of phototropic and geotropic stimuli in the coleoptile of Avena," Kgl. Danske Videnskab. Selskabs. Biol. Med., 3(8), 29 pp. (1921). 35. Seubert, E., "Uber Wachstumsregulatoren in der Koleoptile von Avena," Zeitschr. Bot., 17 : 49-88 (1925). 36. Soding, H., "Zur Kenntnis der Wuchshormone in der Haferkoleoptile," Jahrb. Wiss. Bot., 64 : 587-603 (1925). 37. Stark, P., "Studien iiber traumatotrope und haptotrope Reizleitungsvorgange mit besonderer Beriicksichtigung der Reiztibertragung auf fremde Arten und Gat- tungen," Jahrb. Wiss. Bot., 60 : 67-134 (1921). 38. Thimann, K. V., and F. W. Went, "On the chemical nature of the rootforming hormone," Proc. Akad. Wetensch. Amsterdam (Sec. Sci.), 37 : 456-459 (1934). 39. Went, F. W., "Wuchsstoff und Wachstum," Rec. Trav. Bot. Neerland., 25 : 1-116 (1928). 40. , "Die Erklarung des phototropischen Krtimmungsverlaufs," Rec. Trav. Bot. Neerland., 25 A : 483-489 (1928). 41. , "A test method for rhizocaUne, the rootforming substance," Proc. Akad. Wetensch. Amsterdain (Sec. Sci.), 37 : 445-455 (1934). 42. , "Auxin, the plant growth hormone," Bot. Rev., 1 : 162-182 (1935). 43. Zimmerman, P. W., "Growth regulators of plants and formative effects induced with /3-naphthoxy compounds," Proc. Nat. Acad. Sci., 27 : 381-388 (1941). 44. , "Formative influences of growth substances on plants," Cold Spring Harbor Symposia Quan. Biol, 10 : 152-157 (1942). 45. , "Present status of 'plant hormones,'" Ind. Eng. Chem., 35:596-601 (1943); also in B. T. I. Prof. Pap., 1 : 307-320 (1943). 46. , "The formative influences and comparative eflfectiveness of various plant hormone-like compounds," Torreya, 43 : 98-115 (1943). 47. , W. Crocker, and A. E. Hitchcock, "Initiation and stimulation of roots from exposure of plants to carbon monoxide gas," C. B. T. I., 5 : 1-17 (1933). 48. , and A. E. Hitchcock, "Initiation and stimulation of adventitious roots caused by unsaturated hydrocarbon gases," C. B. T. I., 5 : 351-369 (1933). 49. , , "The response of roots to 'root-forming' substances," C. B. T. I., 7 : 439- 445 (1935). 50. , , "Effect of light and dark on responses of plants to growth substances," C. B. T. I., 8 : 217-231 (1936). 51. , , "Comparative effectiveness of acids, esters, and salts as growth sub- stances and methods of evaluating them," C. B. T. I.,8 : 337-350 (1937). 52. , , "Tropic responses of leafy plants induced by application of growth sub- stances," C. B. T. I., 9 : 299-328 (1938). 53. , , "The combined effect of light and gravity on the response of plants to growth substances," C. B.T.I.,9: 455-461 (1938). 54. , , "Response of gladiolus corms to growth substances," C. B. T. I., 10 : 5- 14 (1938). 55. , , "Activation of cinnamic acid by ultra-violet light and the physiological activity of its emanations," C. B. T. I., 10 : 197-200 (1939). PLANT HORMONES 229 56. Zimmerman, P. W., and A. E. Hitchcock, "Experiments with vapors and solutions of growth substances," C. B. T. I., 10 : 481-508 (1939). 57. , , "Formative effects induced with /3-naphtho.xyacetic acid," C. B. T. I., 12:1-14 (1941). 58. , , "Substituted phenoxy and benzoic acid growth substances and the rela- tion of structure to physiological activity," C. B. T. I., 12 : 321-343 (1942). 59. , , "Flowering habit and correlation of organs modified by triiodobenzoic acid," C. B. T. I., 12 : 491-496 (1942). 60. , , "The aerosol method of treating plants with growth substances," C. B. T. I., 13 : 313-322 (1944). 61. , , "Substances effective for increasing fruit set and inducing seedless to- matoes," Proc. Am. Soc. Hort. Sci., 45 : 353-361 (1944); also in B. T. I. Prof. Pap., 2 : 13-21 (1944). 62. , A. E. Hitchcock, and E. K. Harvill, "Xylenoxy growth substances," C. B. T. /., 13 : 273-280 (1944). 63. , , and F. Wilcoxon, "Responses of plants to growth substances applied as solutions and as vapors," C. B. T. I., 10 : 363-376 (1939). 64. , and F. Wilcoxon, "Several chemical growth substances which cause initiation of roots and other responses in plants," C. B. T. I., 7 : 209-229 (1935). CHAPTER 7 Dormancy in Buds A rest period is general for buds of tubers, bulbs, and woody plants. This is especially true of temperate-zone plants. The duration of dormancy in buds of potato tubers varies from 9 to 12 weeks, depending on the variety, when stored at an- temperatures. It is not shortened by storage at low tem- peratures but is shortened ^^ considerably by storage at 35° C (95° F), as well as by storage with high moisture.^' Low-temperature storage is very significant in shortening the rest periods of buds of many bulbs, such as the gladiolus. It is also important in inducing flowering in bulbs and in certain biennial ^ and perennial plants. After havmg plants of Crassula rubicunda about the greenhouse for years without flower production, Arthur induced profuse flowering by exposing the plants to low tempera- ture for a few months. Flower induction by low-temperature exposures has been termed ^ thermoperiodism. When seeds are made to produce plants that flower earlier by exposing them to low temperatures the process is referred to as yarovization or vernalization. The physiological changes involved in thermoperiodism and yarovization are probably similar. Buds of temperate-zone trees and shrubs go into dormancy in late summer and are thrown out of this condition by the cool weather of faU, winter, and sprmg. As in low-temperature after-ripening of seeds, temperatures be- tween 0° and 10° C (32° and 50° F) are effective ^ for buds of various kinds of trees and shrubs, but higher temperatures and temperatures below freezing are not. Dormancy in Potato Buds Attempts have been made to force dormant buds by the use of anesthe- tics 68. 69, 70 without great success. As we have already seen, ethylene and certain plant hormones which are good anesthetics inhibit bud growth, at least as long as they are in contact with the buds. McCallum ^^ found ethyl bromide especially effective in overcoming dormancy of potato buds. He exposed the tubers to 1 to 2 cc of the chemical for 24 hours in a 5-liter air-tight jar. Appleman ^ found that buds of potato tubers grew more rapidly if the tubers were kept moist. He concluded that moisture pre- vented the development of the corky periderm which reduced the oxygen supply to the buds. Thornton,^^ {^ ^ paper that was awarded the A. Cressy Morrison Prize as an outstanding paper in biology in 1938 by the New York Academy of Sciences, confirmed Appleman 's result that potato 230 DORMANCY IN BUDS 231 buds lose their dormancy quicker in the moist than in the dry condition, but gave quite the opposite interpretation. He found by anatomical studies that moisture favored the rapid development of periderm which, in turn, cut down the oxygen supply to the buds and favored growth ; and that freshly harvested tubers germinated rather promptly under 5 to 10 per cent oxygen pressure rather than at normal pressure. Peeling the tubers about the eyes shortened the rest period because it led to the rapid development of effective periderm. Two to 10 per cent of oxygen elimi- nated apical dominance, so that several buds grew from each eye instead of one bud from one of the apical or, seed end, eyes. Increasing the oxygen above 20 per cent prolonged the dormant period. This is one of few cases on record where reducing oxygen pressure below the normal favors growth of flowering plants. Certain seeds are favored in germination by reduced oxygen pressure. Perhaps storage at 35° C (95° F) ^^ hastens after-ripening of potato tubers by increasing the development of corky periderm. About the time the Institute opened we had an inquiry from Bermuda for some means of thro^^^ng freshly dug potatoes into immediate gro^\'th. Bermuda had been using seed grown in Long Island, which had been har- vested early enough in the summer to after-ripen before plantmg time in Bermuda in early October. This seed was unsatisfactory; it became in- fected ^\^th virus diseases during the season due to the abundance of insect vectors on Long Island. It was estimated that the virus diseases carried in the tubers reduced the yield about 50 per cent. Northern-growTi seed could be obtained which was practically free from leaf roll and mosaic. However, it was harvested in September and was not ready 'to grow for some weeks after planting time in Bermuda. The Institute decided to organize a project to study dormancy in buds much as it was studying dormancy in seeds. Dr. F. E. Denny, who had recently developed the ethylene method of coloring citrus fruits, was asked to head the project. Later a grant from the Herman Frasch Foundation for Research in Agricultural Chemistry enabled the Institute to add two well- trained biochemists, Drs. John D. Guthrie and L. P. IVIiller, as associates on the project. These three scientists, with assistants, carried on the project under the Frasch grant for eleven years, and it has been continued with a slight change in the staff for nine additional years. As a result of these studies some very effective chemicals have been found for throwing buds out of dormancy and for inducing or maintaining dormancy. Much has been learned about the metabolic changes brought about by the dormancy- modifying chemicals in buds and the plants to which the buds are attached. In this chapter we can cover only a few of the points of more general inter- est. The details are published in more than seventy-five different articles. Chemicals That Force Dormant Buds Ethylene chlorhydrin and thiocyanates. In his original study of the effect of 224 different chemicals for forcing dormant potato buds, Denny ^- ^ 232 GROWTH OF PLANTS found two chemicals, ethylene chlorhydrin (CICH2CH2OH) and thio- cyanates (K, Na, or NH4) that are very effective in throwing potato buds into prompt growth. The first proved especially desirable because there is a wide margin between forcing dosage and toxic dosage; moreover, as it r Figure 84. Effect of dip treatment of potatoes with 40 per cent ethylene chlorhydrin after dipping tubers left in a closed container 22 hrs. Left to right: check, dipped in H2O; dipping solution, 15 cc per liter; dipping solution 30 cc per liter. is soluble in water and has a high vapor pressure, it can be used either for dip or vapor treatment. The thiocyanates have a narrow margin of dosage and must be used by a soak treatment. Fig. 84 shows the effect of ethylene chlorhydrin and Fig. 85 the effect of sodium thiocyanate upon the growth of dormant potato tubers. These treatments completely eliminate the need of the rest period, that is, the dormant tubers treated with the chemicals grow with as high vigor and apparently give as high crop yields as time- after-ripened tubers; unlike the hormone-like chemicals discussed in pre- vious chapters, they do not produce any formative modifications in the resulting plants, such as partial or complete anesthesia, extra root forma- tion, modification of leaves and stems, and parthenocarpy. They merely activate the normal growth of dormant buds. Figure 85. Effect of sodium thiocyanate solution upon the germination of freshly harvested potato tubers. Cut tubers soaked one hour. Left to right: check (H2O); 34 per cent; 1 per cent. By treatment of dormant tubers just after harvest, new sizable tubers can be produced before dormant untreated tubers have germinated (Fig. 86), so that several crops of potatoes can be produced in a year. This has been very useful to potato breeders, who can hurry tuber multiplication by elimi- nating the rest period and to plant pathologists who are studying potato DORMANCY IN BUDS 233 diseases, especially those carried in the tubers. It is now common practice to force northern-grown seed potatoes in the greenhouse during the winter to determine whether the seed is virus-free. This gives a preview of the next year's crop. This control of tuber dormancy is also useful m practice, not merely because it enables southern growers of the fall crop to produce Figure 86. Second crop of potato tubers in same year in Institute gardens from tubers treated with ethylene chlorhydrin. (Left) Check not treated. lot from northern-grown disease-free tubers, but also to produce a second crop from the small tubers of a first crop. Many other chemicals were found more or less effective in forcing dor- mant potato tubers, but ethylene chlorhydrin and the thiocyanates showed advantages over the others for one or more of the following reasons: high effectiveness in forcing; wide margin between forcing and killing dosages; or cost of the chemical for treatment. Among the other chemicals that Denny early found to be more or less effective were di- and trichloroethylene, 234 GROWTH OF PLANTS carbon disulphide, ethylene dichloride, xylene, and ethyl bromide. The last was very effective, as McCallum found, but it is rather expensive and must be used as a vapor, since it is not soluble in water. Later, Miller ^^- ^^ found a number of sulphur compounds more or less effective in breaking the dormancy of potato tubers: ammonium dithiocarbamate, thiosemi- carbazide, hydrogen sulphide, ethyl mercaptan, ethyl disulphide, and Figure 87. Individual eyes of Bliss Triumph potato tubers cut out with a large- sized cork borer and later soaked one hour in 3 per cent thiourea solution. Note that chemical forced the growth of several buds from most of the eyes. several others. Guthrie ^^ synthesized what he believed was thiocyano- hydrin (CH2SCN-CH20H), a compound resembling both of the very effec- tive compounds mentioned above, and found it effective in breaking tuber dormancy. Ethyl carbylamine and glutathione ^^' *^ were also effective. Thornton ''^ found 10 to 60 per cent of carbon dioxide in combination '^\dth 21 or higher percentages of oxygen effective in forcing dormant buds of potatoes. Since carbon dioxide was effective with normal or higher than normal oxygen, the action of carbon dioxide was not due to anaerobiosis. None of these compounds shows advantages that would lead them to dis- place ethylene chlorhydrin and the thiocyanates as bud forcers. Dennj^ ^s has also found a combination treatment with ethylene chlorhydrin followed by thiocyanate of advantage in greenhouse tests for virus in potato tubers. This gives approximately 100 per cent germination, which is necessary in these tests. Thiourea as a bud forcer. Thiourea has proved to be a fair bud-forcing chemical, but it differs from the other effective chemicals in that it breaks up the growth correlation between the several buds of an eye and between the eyes in a seed piece. In normally after-ripened tubers and in dormant DORMANCY IN BUDS 235 tubers treated with ethylene chlorhydrin or thiocyanates only one bud generally grows from one apical eye of the seed piece. Thiourea may force the growth of all the bud primordia in an eye. This may be as high as eight. Fig. 87 shows the effectiveness of thiourea ^ in inducing the growth of several buds from a single potato eye. Fig. 88 indicates the effect of thiourea in breaking up the correlation between the several eyes of the Figure 88. Seed pieces of non-dormant Early Ohio potato. Top row: soaked in 2 per cent thiourea one hour before planting. Bottom row: soaked in water one hour before planting. Note that thiourea forced the growth of buds from eyes throughout the length of the seed piece and that water induced growth of only one bud from an eye at the seed end. potato, so that eyes all along the seed piece from the seed end to the stem end produce multiple sprouts. We have noted above that reduced oxygen pressures '^^ had similar effects on eye and bud correlations in potato tubers. In western Nebraska and in Colorado the potato tubers grow too large for economical use as seed. Treating the seed pieces with the proper dosage of thiourea gives more than one stem from the seed piece wath a correspond- ingly greater number of smaller tubers per hill, but with the same yield per acre. In this way treatment of tubers with thiourea has proved of service to seed-potato growers. Thiourea as an antioxidant. Denny observed that the cut surfaces of seed pieces of potatoes treated with thiourea remained white for a long time after they were planted, whereas cut surfaces of seed pieces treated with other bud-forcing chemicals, or not treated, turned bro^vn very readily. A further study showed that thiourea interfered with the oxida- tion system in the tuber that produced browTiing. Denny i^- ^o. 24 concluded that it was the peroxide in the system that was inactivated or destroyed 236 GROWTH OF PLANTS rather than the peroxidase. Recent work ^^ indicates that thiourea also inactivates the oxidase system in fruits that destroys vitamin C. Fig. 89 shows the effectiveness of thiourea in preventing the browning of shced apples and apple juice. If the thiourea is placed in the juice immedi- ately upon pressing, no browning occurs; also the early browning of the juice is completely or almost completely reversed if the thiourea is added within an hour after pressing, but the later oxidation in the juice is irreversible. The amount of thiourea required to prevent broAvning is very small. Sliced fruit is dipped momentarily in a 0.1 per cent solution and then drained, after which each pound of fruit retains about 0.027 gram of thiourea. If used to treat the juices, the thiourea content is less than 0.05 gram per pound. The same treatment is quite as effective in preventing sliced apri- cots, bananas, nectarines, peaches, pears, and plums from browning. In peaches a 0.05 per cent solution is effective. It has been suggested that thiourea dip be substituted for sulphur dioxide treatment for drying, canning, and quick-freezing of fruits. Thiourea is also excellent for treating sliced fruits for salads and desserts. Thiourea ^'' has lower acute toxicity dosage for mammals than table salt, and life- time feeding of mammals *^ with dosages many times that which can be consumed in treated fruit has no effect on either the weight or life span of the animals. For several years * it has been know^n that plants of the cabbage family cause thyroid enlargement, i.e., are goitrogenic. It has also been found ^^ that allyl thiourea, or some similar compound released from mustard oils by glucoside-splitting enzymes, causes this goitrogenic action and that various organic sulphur compounds (thiourea, thiouracil, etc.) in high doses have goitrogenic action. Astwood ^ used thiourea and thiouracil as therapeutic agents against hyperthyroidism. The initial doses of 1 to 2 grams of thiourea a day were used until the basal metabolism was reduced to the proper level and then 0.5 of a gram a day to maintain it. The effect of the thiourea ceases soon after its use is discontinued. When 1 to 2 grams per day were fed to normal individuals for 13 to 17 days, no change in metabolism was noted. Very recently ^^ it has been observed that rats fed a diet containing 0.5 per cent thiourea for 12 or more days are able to endure an atmospheric pressure of 200 mm of Hg, equiva- lent to an altitude of 32,000 feet, for two hours, whereas the majority of those not fed thiourea were killed by a like exposure to low pressure. Leblond ^ found that rats given 1 per cent of thiourea in their drinking w^ater for three months endured an atmospheric pressure of 100 mm of Hg much better than did the controls. These are extremely high doses. It has been suggested that aviators might be able to endure rarefied air better if given thiourea in their diet. The therapeutic and goitrogenic doses are enormous compared with the intake obtainable from eating treated fruits. Even if all the largest annual apple and peach crops the United States has ever produced were treated with thiourea to prevent browning and were all eaten by the people of the United States, each person would get on the DORMANCY IN BUDS 237 Figure 89. A, Slices of Mcintosh apple allowed to dry in air. B, Slices of Mcintosh apple soaked one minute in 0.1 per cent thiourea solution and allowed to dry in air. C, Brown apple juice, check lot. D, Brown apple juice decolorized by adding 1 mg of thiourea per 10 cc of juice. E, Thiourea added to juice at once after pressing; F, after being exposed to oxidation in air for 15 minutes before add- ing thiourea; G, for one hour; H, for two hours; K, for four hours. Residual liquid after decolorizing action of thiourea shows increasing amounts of an unreducible pigment as duration of the preliminary oxidation was increased. 238 GROWTH OF PLANTS average only 2 grams of thiourea in a year. Only a minor fraction of these crops would ever be treated with any antioxidants. The chances are poor that any individual would get as much as a gram of thiourea in a year by Figure 90. Inducing dormancy in potato tubers with the potassium salt of a-naph- thaleneacetic acid and then breaking it with ethylene chlorhydrin. A, Pieces treated with the potassium salt of a-naphthaleneacetic acid, 100 mg per liter for 4 days. B, Con- trol pieces treated with water. C, Pieces hke those shown in A treated with ethylene chlorhydrin, 24 hr. dip, 25 cc of 40 per cent per Uter. D, Pieces hke those in A, treated with water. eatmg fruit treated with it. A year's dose from treated fruit would be less than a daily dose for goitrogenic or therapeutic effects and is far below the threshold for either goitrogenic or therapeutic effects. In spite of these facts the United States Food and Drug Administration prohibits the use of thiourea as an antioxidant on fruit. DORMANCY IN BUDS 239 A Chemical That Inhibits Potato Buds In previous chapters we have mentioned that ethylene and other hor- mone-like chemicals inhibit bud growth. Guthi-ie *^' *^ found a-naphtha- leneacetic acid and its salts and esters very effective in inhibiting the growth of buds of the potato. Fig. 90 shows the effectiveness of the potassium salt of this acid in inhibiting potato buds. It also shows that ethylene chlor- hydrin throws potato buds out of chemically induced dormancy, though perhaps not as effectively ^^ as it does out of natural dormancy. With the FiGUKE 91. Irish Cobbler potato tubers stored in paper bags for five and one-half months after treatment with different dosages of the methyl ester of a-naphthaleneacetic acid applied in talc powder. Temperature varied from 10° to 16° C (50° to 60° F). Dosages (above): check without talc, check with talc only; (below, left to right): 25 ppm of weight of tuber, 50 ppm, 100 ppm of methyl ester. use of these two chemicals, potato buds can be thro^vn into and out of dor- mancy almost at w^ill. The methyl ester of a-naphthaleneacetic acid ^^ is desirable for use for potato storage because it has sufficient vapor pressure to insure its entrance into the buds; the most efficient way to apply it to the tubers is in a talc or clay dust on the surface of the tubers at 55° F (13° C) or above so that the chemical vdW have sufficient vapor pressure. Fig. 91 shows the effect of different dosages of this chemical in maintaia- ing dormancy in potato buds. It is evident that 25 parts of the chemical to a million parts of the tubers by weight is sufficient to maintain complete dormancy. It is possible that even a low^er dosage applied by this method will be effective, especially if the treatment is made at room temperature. It has been showTi that only 5 ppm of the chemical is absorbed by the tubers when the dosage is 100 ppm, and of the chemical absorbed four-fifths is held in the skin. Unpublished experiments at the Institute have sho^\^l that a-naphthaleneacetic acid and its salts and esters have low toxicity for 240 GROWTH OF PLANTS mammals. It has been showTi that tubers treated with this chemical can be stored for a year or more in bins at 10° C (50° F) or a somewhat higher temperature without sprouting or shrivelling. To prevent sprouting and shrivelling by cold storage for a year the temperature could be very little above 6° C (43° F). Such low storage temperatures are not only expensive and often unavailable but they lead to transformation of much of the starch of the tuber to soluble sugars. As we shall see later, tubers that contain considerable reducing sugars give dark bro^^^l potato chips. The world's annual potato crop is approximately seven and one-half billion bushels a year. Any improvement in storage that will give a net average saving of one cent a bushel is worth seventy-five million dollars a year. Dormancy of Gladiolus Corms and Cormels Forcing Dormant Corms by Chemical Treatment and Temperature Storage Corms of some varieties (Souvenir, Maiden's Blush, and Alice Tiplady) of gladiolus 9- 1^, n could be successfully forced one week after harvest ^vith ethylene chlorhydrin vapors. The dosages were 3 to 4 cc of 40 per cent solution per hter of the enclosure containing the corms and the exposure was two to four days. Ethylene and ethyl ether were not effective. Corms of the Halley variety did not respond to the treatment until one month after harvest; and the corms of Remembrance did not respond to chemical treatment at any time after harvest, but after-ripened ^vith sufficient period of storage mthout treatment. To msure good flowering of fall- forced corms natural light may have to be supplemented with artificial light. In many varieties the chemical treatment was successful only after a cold storage period of three to six weeks at 5° C (41° F). High-temperature storage (30° to 35° C, 86° to 95° F) was not effective if applied immediately after harvest, but had good forcing action for some varieties after the corms had been kept at room temperature for 52 days before they were transferred to high temperatures. Because of the great variation in be- havior of corms of different varieties, both as to depth of dormancy and factors that overcome dormancy, it is evident that each variety must be studied separately to determine the best forcing methods. In some varieties chemical treatment is effective soon after harvest; in others it is effective only after a period at proper storage temperatures, and in still others proper storage alone is the best method of forcing. The attempts at chemical forcing of corms to date should not be considered final. It is possible that other chemicals can be used in combination with ethylene chlorhydrin or still other chemicals found that will give perfect forcing soon after harvest. Ethylene chlorhydrin treatment ^~- *'^ increased peroxidase, catalase, pH, sulphydryl, soluble organic nitrogen, and sucrose and decreased the reducing sugar of the corms. Most of these changes occurred regardless of the dormancy of the corms, and there is no evidence that any of them hold a causal relation to the breaking of dormancy. DORMANCY IN BUDS 241 Maintaining Gladiolus Corms in the Dormant Condition Gladiolus corms can be kept sound and in the dormant condition ^^ for 18 months or more, depending upon the variety, by storing the freshly har- vested corms in moist soil at room temperature or preferably at 27° C (80° F) . Corms stored in this condition have a very low rate of respiration ^^- ^^ and consequently use up stored foods very slowly, which permits of long survi- val. When these corms are taken from the soil and placed in respirometers at the storage temperature, the respiration begins to rise within a few hours and reaches a maximum after 20 to 30 hours, after which it gradually falls back to the original low rate. The rise in respiration is 5-fold, 10-fold, 30-fold, or even larger. Such corms may be placed in soil again and continue m the dormant condition for weeks longer, the duration depending upon the state of dormancy and the variety. Corms stored in soil for longer periods, as mentioned above, are in very delicate equilibrium so far as dormancy is concerned. They grow readily when treated ^^'ith ethylene chlorhydrin or after exposure to low temperatures (0° to 5° C, 32° to 41° F), for a few hours.-^ We have already seen that a brief period of chilling various dor- mant seeds in a germinator throws them into active growth. Dormancy in Gladiolus Cormels Gladiolus cormels are generally more dormant than the corms, and the depth of dormancy increases with decrease in size of the cormels. Treat- ment of cormels of five varieties (Alice Tiplady, America, Halley, Remem- brance, and Souvenir) with ethylene chlorhydrin forces them out of dor- mancy."- -* The greatest forcing action was not immediately after harvest FiGiTRE 92. Gladiolus cormels, variety Souvenir, stored at room temperature until January. Left: untreated control. Fight: 100 grams of cormels sealed in a Mason jar with 1.5 cc of 40 per cent ethylene chlorhydrin for four days before planting. 242 GROWTH OF PLANTS but one to three months later. Fig. 92 shows the forcing action of ethyl- ene chlorhydrin on dormant cormels. By April cormels of many varieties after-ripen in storage and do not need chemical treatment to induce growth. Storage of cormels of these varieties at 3° to 10° C (37° to 50° F) overcame the dormancy more readily than higher storage temperatures. Cormels of seven varieties (Giant Nymph, Mr. W. H. Phipps, Dr. E. F, Bennett, Mrs. F. C. Peters, Minuet, Willbrinck, and Golden Measure) are especially dormant.^*' When these cormels are stored at the best low temperature until May most of the cormels of each variety fail to produce plants during the summer when planted in soil without treatment. Proper ethylene chlorhydrin treatment in the spring increased the number growing by many-fold in three varieties and by 100 to 200 per cent in two varieties. Two varieties could be forced by ethylene chlorhydrin only after this long period of cold storage. It is important to get a high percentage of germination of cormels of gladiolus in order to have them develop as quickly as possible into flowering- sized corms. Fig. 93 shows the great increase in size and number of corms Figure 93. Yields obtained in the autumn from 150 gladiolus bulblets, variety Minuet, stored over winter at 15° C (59° F) and planted in the spring. Left: bulb- lets soaked 3 days in H2O and planted. Right: bulblets soaked 3 days in H2O, then exposed to vapor of ethylene chlorhydrin, using 1 cc of 40 per cent ethylene chlorhydrin per 100 g of bulblets, weight before soaking. produced during the summer when a given number of cormels of a dormant variety are treated with ethylene chlorhydrin in the spring. This is the chief way of multiplying flowering stock of the variety. But in general the grower does not care to have them germinate until time for spring plant- ing outside. The situation is different with dormant corms for they are often forced for winter flowers. Cormels of varieties that are only moder- ately dormant should be stored at a low temperature, about 5° C (41° F), until spring and planted without treatment. Cormels of very dormant varieties should be similarly stored and treated with ethylene chlorhydrin before planting in the spring. In case of new very choice varieties it is possible that by combination of temperature and chemical treatment one could get two growing periods during the year, one in the greenhouse and DORMANCY IN BUDS 243 another outdoors, and hasten greatly the production of flowering corms. Denny is using other chemicals in combination with ethylene chlorhydrin, seeking even more effective forcing action. Denny 23 gives the following simple description for forcing dormant cormels: ''The amount of chemical to use depends upon the amount of bulblets to be treated, e.g., seven drops of the chemical per ounce, one and one-fourth teaspoonfuls per pound, or one pint per 100 lbs of bulblets. The amounts do not need to be exact, but reasonable care should be taken. For small lots, glass fruit jars Avith wide mouth are used as containers. The bulblets are weighed and put in the jars; several varieties may be included in one jar if the bulblets of the different varieties are tied in cheesecloth bags properly labeled. On top of the bulblets lay a small piece of paper toweling and on top of this place a piece of cheesecloth (in a loose pile) containing the right amount of the ethylene chlorhydrin. A little practice will show how large the piece of cheesecloth is required to take up the hquid without serious dripping. The piece of paper will absorb any excess drops. Then seal up the jar and let it stand at room temperature, approximately 70° to 75° F, for four days. The bulblets are then removed from the jar and are ready for planting. If the weather is unfavorable the treated bulb- lets may be placed in paper bags and planted when the weather is favorable. A delay of a week before planting will do no harm. "With small quantities treated ui glass jars, the distribution of the vapor to all parts of the jar during the four-day period of treatment seems to be good. If a large quantity, say 100 lbs, is to be treated in large containers such as ash-cans, it is recommended that a wire screen core be placed in the center of the ash-can with the bulblets poured into space between the can and the screen. The cheesecloth containing the ethylene chlorhydrin can then be suspended from the top of the screen into the central core space, and such a procedure will assist in getting penetration of the vapors to all parts of the can. "After the bulblets have been soaked, the excess chemical should be rinsed off with two or three changes of water. This is to avoid over-treating and to prevent the bare hands from contact with the chemical in planting, a precaution which is probably unnecessary but which may be worth taking if large amounts of bulblets are to be planted by hand." Forcing Dormant Buds of Deciduous Trees and Shrubs The buds of deciduous trees and shrubs can be forced in the fall by treat- ment with ethylene chlorhydrin vapors, thus eliminating the necessity of a low-temperature period for after-ripening the buds. Several other chemicals (propylene chlorhydrin, ethylene dichloride, vinyl chloride, carbon tetra- chloride, etc.) proved more or less effective. These, like ethylene chlorhy- drin, could be applied as vapor which is desirable for treating trees and shrubs because of the diflficulty involved in soak treatment. On the whole, 244 GROWTH OF PLANTS I I DORMANCY IN BUDS 245 however, ethylene chlorhydrin proved most desirable; it is cheap, effective, and has a relatively wide dosage margin between forcing action and tox- icity. Fig. 94 shows ^ the effectiveness of ethylene chlorhydrin vapors in forcing bloom in Azalea nudiflora. The plants are simply sealed in con- tainers "with the required amount of ethylene chlorhydrin for 24 hours and the chemical allowed to volatilize and enter the buds. From the figure it is evident that 6.7 cc of 40 per cent ethylene chlorhydrin per 100 liters of FiGTJHE 95. Left: method of treating an individual dormant lilac bud with ethylene chlorhydrin; a drop or two of the chemical is placed in the test tube and the tube sealed over the bud with modelhng clay. Right: later growth of a bud treated in this way. space is adequate for forcing blooming and that 0.75 cc has some forcing action. This treatment is effective in forcing both flower and foliage buds of deciduous plants. Besides azalea, lilac, flowering almond, Bechtel's crabapple, and Deutzia respond well to the treatment. Of those treated only the snowball, Viburnum tomentosum, failed to respond. It is likely that all deciduous forms can be forced by the proper concentration and time of treatment or by the combination of a cold period followed by chemical treatment. A more thorough study is needed of these and many other forms in order to make the method highly useful to practical growers. The chemical is toxic to leaves; consequently its use may be limited to deciduous forms not in foliage. The seat of dormancy ^^ seems to be in the individual buds rather than in the plant as a whole. Fig. 95 shows the method of treating an individual dormant bud of lilac and the later gro'vvi^h of the treated bud. (Note that the effect of the treatment is strictly local; the opposite bud on the same stem one-fourth of an inch away remains dormant.) The roots and stems of the plant are not dormant but are able to furnish the bud the necessary water and other nutrients at any time that the bud is out of dormancy and ready to grow. As we have already mentioned under Dormancy in seeds, buds that are only partially after-ripened due to insufficient period 246 GROWTH OF PLANTS of low-temperature exposure grow with low vigor or are dwarfish. This may happen if the forcing chemical is used in too low a dosage, but if the chemical is used in sufficient dosage the vigor of growth is the same as that induced by adequate low-temperature exposure. Various physiological and chemical changes ^^ were induced in dormant lilac buds by optimum forcing dosages of ethylene chlorhydrin. There was a marked increase in catalase, and an increase in water content and soluble nitrogen compounds, but no significant change in amylase. There was marked increase in respiration ranging from 20 to 100 per cent from early to later stages. These changes were greater in the buds than in the twigs. It is possible that even more effective bud-forcing methods may be found by mixing other effective chemicals with ethylene chlorhydrin. This is especially hopeful if the mixtures show more than additive or synergistic effect. Denny is investigating this possibility. In the case of trees and shrubs it is desirable to find effective chemicals that can be added to the soil and reach the buds through the roots and stems. It is possible that if such chemicals can be found, buds of evergreens can be forced without foliar injury. This is a good deal to hope for, since the leaves have rapid transpiration and may accumulate more of the chemical than the dormant buds. Metabolic Changes Induced by Chemicals That Force Dormant Potato Buds Extensive studies were made of the effects of bud-forcing chemicals upon the metabolism of potato tubers. These studies were directed at answering two questions. In the soak treatment the seed pieces absorbed large quan- tities of ethylene chlorhydrin, and upon exposure of the seed pieces to the air the chemical disappeared from them faster than could be accounted for by evaporation alone. Explaining the disappearance of ethylene chlorhy- drin led to the discovery that not only this but several other foreign and more or less toxic chemicals, when absorbed by plants, are tied up with glucose to form glucosides or with other sugars to form other glycosides which are, on the whole, less toxic to plants than the chemicals themselves. The second object of the metabolic studies was to see whether some meta- bolic change or changes brought about by the several forcing chemicals would explain why the chemicals changed the buds from the dormant to the active conditions. While no definite positive answer was gained for this question, since various forcing chemicals showed opposite effects on such basic proc- esses as respiration, the studies as a whole added much to our knowledge of plant metabolism and the modification of metabolism by chemicals. Plants Transform Certain Toxic Foreign Chemicals into Less Toxic Glyco- sides Miller ^^ early showed that the disappearance of ethylene chlorhydrin in treated potato tubers was due in part to the chemical being transformed DORMANCY IN BUDS 247 to a iS-glucoside by the living tubers. Extracted juice did not form the gki- coside. He found later ^° that corms of gladiolus when treated with ethyl- ene chlorhydrin likewise transformed the chemical to a glucoside and identi- fied the glucoside as /3-(2-chloroethyl)-d-glucoside. This ^^ was proved identical with the glucoside formed in the potato tuber. Wheat plants ^^ furnished with ethylene chlorhydrin in nutrient solution synthesized the same glucoside. When gladiolus corms ^" were exposed to the vapor of o-chlorophenol, much of the chemical was absorbed and later transformed by the corm into j3-o-chlorophenyl-gentiobioside. In this case the chemical was tied up with a disaccharide, gentiobiose, instead of glucose. It seems probable that the chemical induces the formation of the disaccharide, gentiobiose, as well as the glycoside, since there is little if any gentiobiose in untreated corms. Tomato roots ^^ respond similarly to o-chlorophenol. When gladiolus corms ^^ were fumigated alternately Avith ethylene chlorhydrin and o-chloro- phenol, both /3-glucoside and )3-gentiobioside were formed simultaneously in the corm. The foreign chemical, or aglycon, added determines the sugar with which it ties up. In this case the ethylene chlorhydrin was tied up as a glucoside and the o-chlorophenol as a gentiobioside. When growing tomato plants ^'^ were supplied with trichloroethyl alcohol, chloral hydrate or chloral cyanohydrin, j(3-trichloroethyl-gentiobioside accumulated in both the tops and roots of the plants. The first chemical is built into the glycoside without modification ; chloral hydrate is reduced before it is installed in the glycoside, and chloral cyanohydrin is first hydro- lyzed and then reduced before becoming a part of the glycoside. Tomato plants normally contain little or no gentiobiose, so the synthesis of this sugar may be induced as well by the chemicals. Not all of Miller's work on inducing the synthesis of foreign glycosides in plants by feeding them unusual chemicals can be discussed here, but one other piece of research should be described. When tobacco plants are sup- plied with chloral hydrate in the nutrient solution, both a jS-glucoside and a /3-gentiobioside are formed. The roots store up only the latter and the leaves accumulate both glycosides. An analysis of one set of leaves showed that the two glycosides constituted 13 per cent of the dry weight of the leaves. This shows the marked degree to which the organic chemical com- position of plants can be modified by supplying plants with a foreign chemi- cal. Probably further research in this direction will show that even greater accumulation of foreign compounds in plants is possible. In this work in every case the foreign chemical synthesized, so far as known, is a glycoside. Can plants be induced to synthesize other foreign chemicals, such as alka- loids, when supplied with organic chemicals not ordinarily found in them? This work suggests the possibility of further researches in this field that might be of great scientific interest and practical value. It has long been assumed that formation of glucosides or glycosides is a means plants have for detoxication of poisonous products of their own 248 GROWTH OF PLANTS metabolism. The fact that foreign toxic chemicals, when supplied to living plants, are also tied up as less toxic glucosides or glycosides tends to con- firm this explanation. Because there are many biologists who are allergic to teleology we have to be careful just how we word this conception. The fact is established, however, that some toxic products of metabohsm and some foreign toxic chemicals, when supplied to plants, are tied up as parts of less toxic glycosides. How the plants acquired this synthetic power is another question. Did those that lacked this power commit suicide with their oa\ti poisons or by absorbing foreign poisons and those that had the power persist in spite of these poisons, or did the power to tie up self or foreign poisons into less toxic glycosides come about in some other way? Other Metabolic Changes Caused by Bud-Forcing Chemicals Fig. 96 shows several metabolic changes ^'^ brought about by treating intact dormant potato tubers with one of the very effective bud forcers, 600 600 400 300 u UJ I o u. o H Z UJ o ^ 200 100 CATALASE 48 72 96 Hours after beginning of treatment FiGUBE 96. Some effects of ethylene chlorhydrin vapor on the metabolism of potato tubers. ethylene chlorhydrin. It is evident that respiration increase as measured by carbon dioxide output is one of the earliest changes induced by ethylene chlorhydrin treatment ; it is also the one showing the greatest magnitude of change, over 400 per cent of increase 65 hours after the beginning of treat- ment. After the maximum is reached the rate falls rather rapidly and con- DORMANCY IN BUDS 249 tinuously, so that after 144 hours it is about 125 per cent above the control and after a longer period it equals the control. The decrease in citric acid and H-ion concentration ^^ starts at the same time as the increase in respiration, and they reach their mmimum at about the same time that respiration reaches its maximum. Citric acid is no doubt one of the substrata for respiration and its oxidation reduces the H-ion concentration. The H-ion concentration is still further reduced by the consumption of both the sulphate and nitrate radicals,^^ probably in the synthesis of the tripeptide, glutathione. The content of these radicals begins to fall when the rise in glutathione begins and continues with the rise in glutathione, as would be expected if they were used up in the synthesis of the latter. The two enzymes, catalase and peroxidase, begin to rise some hours after the start of the respiration rise, and continue to do so for many hours after respiration has reached its maximum. Catalase reaches its maximum before the 144th hour, while peroxidase is still rising at this hour. The increase in content of these two enzymes does not hold a causative relation to respira- tion rise. The rise in sucrose content starts still later and continues at a nearly uniform rate to the end of the determinations. It is quite apparent that the respiration rate is independent of concentration of sucrose present as a substrate for oxidation, and that no positive causal relation exists between respiration intensity and sucrose content. Of course with rise in sucrose content there is a fall in starch content, but the percentage fall in the latter is rather small because starch constitutes a large percentage of the weight of the tuber. The rise in glutathione content was the latest of the changes recorded in the curve. This rise started at the 48th hour and continued a little beyond the 96th hour, after which it remained constant at nearly 200 per cent above the check. Metabolic Changes Induced by Ethylene Chlorhydrin Compared With Effects of Other Chemicals Including Other Bud Forcers Respiration. It might be thought that the great increase induced by these chemicals in the fundamental process of respiration explains their bud- forcing action, but such seems not to be the case, as further facts show. Treatment of potato tubers ^^- ^^ with many other chemicals (ethylene bromide, hydrogen sulphide, acetaldehyde, hydrocyanic and hydrochloric acids, ethyl mercaptan, alkyl, alkylene, and alkylidene halides, etc.) shows practically the same respiration curves as does ethylene chlorhydrin. Hydrocyanic acid has only moderate bud-forcing action, and hydrochloric acid less. Methyl, ethyl, and isopropyl alcohols,^'' which have moderate bud-forcing action, decrease respiration of the tubers. When intact potato tubers are treated with vapors of both ethyl alcohol and ethylene chlorhy- 250 GROWTH OF PLANTS drin the vapors counteracted each other so far as the respiration is con- cerned. Cutting the tubers ^^ into seed pieces increases the respiration rate enormously but does not force the dormant buds. Perhaps the best evidence that this early chemically-induced flush in respiration is not causally related to bud forcing is the fact that it precedes bud growth and has receded to normal considerably before bud gro^vth begins. No doubt after bud growth begins there is a second great rise in respiration. It has long been knouTi that in darkness various succulents, Bryophyllum, cacti, etc., do not oxidize the sugars completely to carbon dioxide and water but partly to the organic acid stage; hence citric, malic, and oxalic acids accumulate in such plants during darkness. Under illumination such plants complete the oxidation of these acids. Guthrie '^ has shown that treating Bryophyllum leaves in darkness with ethylene chlorhydrin vapors induces them to oxidize citric and probably malic acid, in this way render- ing the tissues less acid. This is similar to the effect of ethylene chlorhydrin on potato tubers. Enzymes. Since starch is the main food storage in the potato tubers, investigators seem justified in asking whether bud-forcing chemicals are effective by increasing the activity of the amylase already present in the tuber ("direct effect"), or by increasing the amount of amylase produced by treated tubers ("indirect effect"). Possible correlations between either of these effects on amylase and the bud-forcing action of chemicals were sought. In the main, the good bud-forcing chemicals ^' did not increase the activity of either plant or animal amylases in vitro. Potassium thiocya- nate ^^ did increase the activity of animal amylase in low pH, had no effect in intermediate pH, and inhibited that action in high pH. Also hydrocyanic acid," slightly effective as a bud forcer, increased the amylase activity of undialyzed potato juice. Neither of these throws any light on bud-forcing action. Ethylene chlorhydrin treatment " of tubers led to a later great increase in the amylase activity of the tubers. Treatment of tubers with sodium thiocyanate, another good bud forcer, led to some increase in the amylase activity of the tubers if the tubers were not too dormant; but often dormant tubers showed no increase in amylase activity when treated with this chemical. Denny concludes that the bud-forcing action of chemicals cannot be explained either on the direct or indirect effect on amylase activity. Dormant tubers were treated mth ethylene chlorhydrin, sodium thiocya- nate, and thiourea,^^ and the later effect of these treatments was determined on the catalase and peroxidase activity and reducing power of the juice of the tubers. The last was determined by the power of the juice to reduce methylene blue, indophenol, iodine, in phosphotungstic reagents. The increase in catalase and peroxidase activity began about 24 hours after the treatment, a little earlier with ethylene chlorhydrin than with the other two treatments; also the former chemical gave much greater increases than DORMANCY IN BUDS 251 the last two. None of the chemicals increased the enzjTne activity of the extracted juice of untreated tubers, so their effect was in inducing the tubers to form more enzymes or more active enzymes. While the thiocyanate increased the formation of catalase, it inhibited its activity in the extracted juice until the chemical was partially dialyzed out. The enzyme changes were greater in the tissue nearer the eyes than in that more distant from the eyes, but the treatment of tubers with eyes removed gave some increase in the enzymes. While there was a general correlation between increased enzyme activity and the sprouting response, this correlation was not very close. Sodium thiocyanate and thiourea were much less effective in increas- ing enzyme acti\aty than would be expected on the basis of the favorable sprouting response. Ethylene chlorhydrin treatment gave much greater increase in the enzyme activity in whole tubers than in cut tubers, although it forced sprouting much better in the latter. Sugars. Potato tubers treated ^A-ith ethylene chlorhydrin, thiocyanates, or thiourea ^^ showed marked increases in sucrose but no significant changes in reducing sugar. IVIany of the other chemicals ^^' ^^ that brought about a great increase in respiration of potato tubers also caused marked increases in sucrose, but the latter followed the respiration increase by many hours. In many cases the sucrose of the treated tuber was lower than that of the check when the respiration was at maximum. It is evident that the rise in sucrose does not account for the breaking of the dormancy, for ethyl alcohol, which is a fair breaker of dormancy, causes a fall in respiration and httle change in sucrose; and acetone, which does not break the dormancy, causes a considerable rise in sucrose. Permeability. Freshly harvested tubers or seed pieces of tubers ^^ were treated with the most effective sprout-inducing dosages of ethylene chlor- hydrin, potassium thiocyanate, and thiourea, and the electrical conduc- tivity of the tissue and the leaching of electrolytes from the tissue were later measured. Ethylene chlorhydrin treatment produced small but significant increases in the conductivity of the tissue and in the leaching of electrolytes from the tissues when placed in water. Potassium thiocyanate treatments produced changes somewhat smaller, but similar to those pro- duced by ethylene chlorhydrin, while no significant change was produced by thiourea threatment. Here again the change in permeability induced by the three bud-forcing chemicals is not in proportion to their sprouting effects and does not furnish an adequate explanation for bud forcing. Synthesis of glutathione and related changes. Ethylene chlorhydrin treatment *'" of potato tubers increases the glutathione content of the tubers as much as six-fold. There are two possible sources of the increased glutathione: induced hydrolysis of proteins in the tuber, and induced synthesis of the physiologically significant tripeptide. Guthrie ^^' '^^ thinks the second is the method of origin, for sulphuric and nitric acids decrease in the tubers parallel with glutathione increase. The two acids furnish the sulphur and nitrogen respectively necessary for the synthesis. Since more 252 GROWTH OF PLANTS than enough sulphuric acid disappears to account for the glutathione in- crease, some other sulphur compound is also synthesized. The bud-forcing chemicals " that did not contain sulphur, ethylene chlorhydrin, ethyl alcohol, etc., increased glutathione, which contains bivalent sulphur, much more than do the compounds containing bivalent sulphur, thiocyanates, thiourea, etc. Hydrocyanic acid seems to be an exception. It has about the same forcing action as ethyl alcohol, but un- like the latter causes only a slight increase in glutathione. On the whole, compounds containing bivalent sulphur seem to be important in bud forcing, whether the forcing compounds contain bivalent sulphur or induce the formation of glutathione which contains it. Although it is hard to get into the protoplasm, Guthrie ^^ found glutathione effective in forcing dor- mant buds of potatoes, pears, and peaches, and thinks it may act as one of the effective intermediate chemicals in the forcing action of ethylene chlorhydrin. Certain yeast extracts which are rich in glutathione are good bud forcers, but other chemicals in these extracts are more effective than glutathione. The pH of the tubers treated with ethylene chlorhydrin and other non- sulphur but effective forcing compounds " began to rise soon after the 24-hour treatment started. This is not caused by the direct effect of the chemical, for ethylene chlorhydrin is slightly acid due to the presence of a small amount of HCl formed by hydrolysis. The fall in acidity is due to the induced consumption of citric acid by respiration and sulphuric and nitric acids in glutathione synthesis. The maximum change in pH Avas reached about 72 hours after the beginning of treatment. The pH change began at the surface of the tuber and worked inward. The maximum change was near the surface and the least near the center. Pieces of tubers free from eyes showed a rise in pH when treated with the chemical. In non-dormant tubers the treatment induced less change in pH. The pH rise, glutathione increase, and the increase in reducing power of the juice ^^ are correlated; the pH rise was partly due to the use of sulphuric and nitric acids in gluta- thione synthesis, and the increased reducing power for the juice, especially for iodine in acid solution, was due in part to the increased sulphydryl in the cysteine of the glutathione molecule. Potato tubers held for a long time in storage gradually fall in ascorbic acid (vitamin C) content. The glutathione does not show a parallel fall. Old tubers low in ascorbic acid showed a rise in this chemical after ethylene chlorhydrin treatment."*" The rise was especially fast and the high content was maintained for a long time if the tubers were cut in pieces and exposed to air after treatment. The rise in the cut pieces did not occur if oxygen was excluded from the surface. Fresh tubers with high ascorbic acid con- tent showed no increase in this vitamin after ethylene chlorhydrin treat- ment, but the treatment maintains the ascorbic acid at the high level. There is a marked increase of ascorbic acid in cut surfaces of old untreated tubers but it is maintained for only a few days. There is no correlation between rise in ascorbic acid and sprouting. DORMANCY IN BUDS 253 No one metabolic change induced by effective bud-forcing chemicals throws much light on the mechanics by which these chemicals initiate the gro\vth of dormant buds. The situation is little, if any, better if one con- siders all the metabolic changes. Perhaps this is to be expected since the changes studied were largely in the storage organs rather than in the grow- ing parts of the buds themselves. Another difficulty is the fact that so many metabolic changes induced by these chemicals have been found already, and there are probably many others still to be discovered. The great number of changes make it impossible to select any one that holds a causal relation to bud forcing; indeed it is a question whether any one hiduced metabolic change holds such a relation. This failure of the study to connect some one metabolic change with initiation of bud growth does not subtract from the value of the study. The facts learned and principles established add much valuable knowledge on plant metabolism and on the effect of chemicals upon plant metabolism. The whole study, and espe- cially the work on the synthesis of glycosides, shows to what a degree the chemical composition of plant organs can be modified by the introduction of a foreign chemical into plant organs. Summary A rest period is common for buds of tubers, bulbs, and trees of the tem- perate zone. Buds of potato tubers remain dormant for a period of 9 to 12 weeks in ordinary storage. The period is shortened by storage at a high temperature, 35° C (95° F), and by moist storage that favors development of cork periderm. Buds of corms and cormels of gladiolus, of many bulbs, and of trees and shrubs are thrown out of dormancy by periods of low- temperature storage, 1° to 15° C (34° to 59° F). Cormels of some varieties of gladiolus do not completely after-ripen even after six or seven months of cold storage. A low-temperature period is necessary for the initiation of flower buds in many plants or plant organs. In the researches reported in this chapter several chemicals were dis- covered that throw buds out of dormancy. Ethylene chlorhydrin proves especially desirable in practice; as it is soluble in water and has a high vapor pressure, it can be used either for dip treatment or for vapor treatment. It has a rather wide margin of dosage between forcing and toxic action. Sodium, potassium, and ammonium thiocyanates in water solution are also effective as bud forcers for potato tubers. These must be used for soak treatment because of lack of vapor pressure. Thiourea in water solution proved fairly effective as a bud forcer. It shows peculiar effects in that it breaks up the growth correlations between the several buds in an eye or the several eyes in a seed piece or whole tuber. As a result, thiourea treatment of potato tubers causes several buds to grow in each of several eyes instead of one bud from an apical eye, as occurs in storage-after-ripened tubers, or tubers treated with other bud-forcing chemicals. 254 GROWTH OF PLANTS Thiourea prevents the browning of cut surfaces of fruits and other plant organs by inactivating the peroxide of the tissue. One dip of the cut organs in a 0.05 to 0.1 per cent solution is sufficient to prevent the browning of fruits for drying and freezing, and for salads and sliced desserts. In high dosages thiourea has goitrogenic action and reduces hyperthyroidism, but the dosages that can be consumed in treated fruit are very far below these therapeutic doses. The lethal dose of thiourea for mammals is even higher than that of table salt. Methyl ester of a-naphthaleneacetic acid proved very effective in inhibit- ing the gro\vth of potato buds. Tubers treated with 25 mg of this ester appUed in talc powder to a kilogram of tubers inhibited the growth of the buds so the tubers could be stored at 10° C (50° F) or even higher for more than a year without sprouting or shrivelling. In the fully inhibiting dosage the tubers absorb about five-millionths of their weight of the chemical and four-fifths of the chemical absorbed is held by the skin. The chemical has a low order of toxicity for mammals. This discovery should prove of great value in farm and commercial storage of potatoes. After two to three weeks of cold storage, dormant gladiolus corms can be forced by ethylene chlorhydrin vapor treatment. The cormels are more dormant, but many varieties are fully after-ripened by storage at 5° C (41°F) during the winter. The cormels of other varieties are more dormant and, in addition to low-temperature storage during the wdnter, require ethylene chlorhydrin treatment for a high percentage of germination. The produc- tion of corms of desirable dormant new varieties can be greatly accelerated by ethylene chlorhydrin forcing of cormels. Corms of gladiolus can be held dormant and in good condition for eight- een months or more if immediately after harvest they are placed and kept in moist soil at room temperature or preferably 27° C (80° F). Such corms are thrown into active gro^vth by a few hours' exposure to 5° C (41° F). Buds of dormant deciduous trees and shrubs can be thrown into vigorous growth in the fall by treatment with vapors of ethylene chlorhydrin, alkyl halides, and other volatile chemicals. If only one bud is exposed to the effective chemical it alone grows, showing that the dormancy dwells in the individual buds. Even more effective bud forcers are being sought by using other chemicals in combination with ethylene chlorhydrin in the hope of synergistic action of the chemicals. Ethylene chlorhydrin disappeared from potato tubers faster than could be accounted for by evaporation alone. It is tied with glucose in the potato tuber and gladiolus corm, forming a less toxic glucoside. Several different foreign more or less toxic chemicals, when supplied to plants, are tied up with various sugars forming less toxic glycosides. This seems to be a means that plants have of rendering innocuous poisonous products of their own metabolism and certain foreign poisonous chemicals when absorbed. When tobacco plants were furnished chloral hydrate in the nutrient solution they DORMANCY IN BUDS 255 synthesized glycosides in sufficient amounts to constitute 13 per cent of the dry weight of the leaves. Foreign chemicals can induce plants to change their chemical composition to a marked degree. Treating of potato tubers with ethylene chlorhydrin and many other bud forcers, as well as some chemicals that do not force buds, cause an early many-fold increase in respiration. Methyl, ethyl, and isopropyl alcohols decreased respiration but showed some bud-forcing action. Citric acid was used as an important substratum for respiration increase so that a fall in acidity accompanied the great rise in respiration. Treatment of Bryophyl- lum leaves mth ethylene chlorhydrin causes them to oxidize the citric and probably malic acids in darkness, rendering the leaves less acid. Ordinarily light causes the oxidation of these acids. Many of the chemicals cause a much later and marked increase in the sucrose content of the tubers but little change in reducing sugars. Some chemicals that are not effective as bud forcers cause an increase in sucrose. Ethylene chlorhydrin in the main did not affect the activity of amylase, catalase, peroxidase, or reducing power of extracted juices of potato tubers. It did induce the formation of more of these enzymes in treated tubers. The thiocyanates and thiourea did not induce an increase in these enzymes that was commensurate with then* bud-forcing action. Ethylene chlorhydrin treatment of intact potato tubers increases the permeability of pieces sliced from treated tubers to electrolytes. Thiocya- nates caused a slighter increase in permeability and thiourea induced no increase. The induced increase in permeability was not proportional to the bud-forcing action of the several chemicals. Treatment of dormant potato tubers with ethylene chlorhydrin causes a later increase in glutathione amounting to as much as six-fold. Bud-forcing chemicals that contain bivalent sulphur cause much less increase. More- over, the increase in glutathione caused by the several bud-forcing chemi- cals is not proportional to the bud-forcing action. The chemicals seem to induce the synthesis of glutathione rather than cause its accumulation through the hydrolysis of proteins of the tuber, for sulphuric and nitric acids disappear parallel with the increase in glutathione and in approxi- mately the right proportion to account for the sulphur and nitrogen in the glutathione. A fall in acidity accompanies the rise in glutathione. This is accounted for by the use of citric acid in respiration and sulphuric and nitric acids in the synthesis of glutathione. A rise in the reducing power of the juice also accompanies the rise in glutathione. This is accounted for in part, especially in iodine reduction, by the increase in sulphydryl groups in the synthesized glutathione. Many chemical changes are caused in plant organs by bud-forcing chemi- cals, as well as by chemicals that are not bud forcers, and there are no doubt many still to be found. The very multiplicity of changes, together with the fact that none of the changes brought about by the several bud-forcing and other chemicals is parallel with the bud-forcing action, means that the 256 GROWTH OF PLANTS metabolic changes do not throw much Ught on the mechanism of the bud- forcing action. These chemically induced metabolic changes are, however, of great interest because they show in how many ways and to what degree the chemical composition of plants and plant organs can be changed by the introduction of foreign chemicals. Literature Cited 1. Appleman, C. 0., "Biochemical and physiological study of the rest period in the tubers of Solanum tuberosum," Maryland Agric. Exp. Sta. Bull. 183 : 181-226 (1914). 2. Arthur, J. M., and E. K. Harvill, "Flowering in Digitalis purpurea initiated by low temperature and Ught," C. B. T. I., 12 : 111-117 (1941). 3. Astwood, E. B., "Treatment of hyperthyroidism with thiourea and thiouracil," /. Am. Med. Assoc, 122 : 78-81 (1943). 4. Chesney, A. M., T. A. Clawson, and B. Webster, "Endemic goitre in rabbits. I. Incidence and characteristics," Bull. Johns Hopkins Hosp., 43 : 261-277 (1928); Abstr. in Biol. Abstr., 4 : 1379 (1930). 5. Coville, F. V., "The influence of cold in stimulating the growth of plants," J. Agric. Res., 20 : 151-160 (1920). 6. Denny, F. E., "Hastening the sprouting of dormant potato tubers," Am. J. Bot., 13 : 118-125 (1926); also in C. B. T. I., 1 : 59-66 (1926). 7. , "Effect of thiourea upon bud inhibition and apical dominance of potato," Bot. Gaz., 81 : 297-311 (1926); also in C. B. T. I., 1 : 154-168 (1926). 8. — — , "Second report on the use of chemicals for hastening the sprouting of dormant potato tubers," Am. J. Bot., 13 : 386-396 (1926); also in C. B. T. I., 1 : 169-180 (1926). 9. , "Shortening the rest period of gladiolus by treatment with chemicals," Am. J. Bot., 17 : 602-613 (1930); also in C. B. T. I., 2 : 523-534 (1930). 10. , "Sucrose and starch changes in potatoes treated with chemicals that break the rest period," Am. J. Bot., 17 : 806-817 (1930); also in C. B. T. I., 2 : 580-591 (1930). 11. , "Direct versus indirect effects upon potato amylase by chemicals which induce sprouting of dormant tubers," C. B. T. I., 4 : 53-63 (1932). 12. , "Effect of ethylene chlorhydrin vapors upon the chemical composition of gladiolus corms," C. B. T. I., 5 : 435-440 (1933). 13. , "Efifect of potassium thiocyanate and ethylene chlorhydrin upon amylase activity," C. B. T. I., 5 : 441-450 (1933). 14. , "Thiourea prevents browning of plant tissues and juices," C. B. T. I., 7 : 55-61 (1935). 15. , "Storage temperatures for shortening the rest period of gladiolus corms," C. B. T. I., 8 : 137-140 (1936). 16. , " Spring- treatment of autumn-harvested gladiolus cormels," C.B.T.I., 8 : 351-353 (1937). 17. , "A retrial of the ethylene chlorhydrin method for hastening the germination of freshly-harvested gladiolus corms," C. B. T. I., 8 : 473-478 (1937). 18. , "Prolonging, then breaking, the rest f)eriod of gladiolus corms," C. B. T. I., 9 : 403-408 (1938). 19. , "Respiration of gladiolus corms during prolonged dormancy," C.B.T.I., 10 : 453-460 (1939). DORMANCY IN BUDS 257 20. Denny, F. E., "Inactivation of the browning system in frozen-stored fruit tissue," C. B. T. I., 12 : 30&-320 (1942). 21. , "Effect of a few hours of chiUing upon the germination of gladiolus oorms subjected to an artificially prolonged rest period," C. B. T. /., 12 : 375-386 (1942). 22. , "The use of methyl ester of a-naphthaleneacetic acid for inhibiting sprouting of potato tubers, and an estimate of the amount of chemical retained by tubers," C. B. T. I., 12: 387-403 (1942). 23. , "Treatment of gladiolus bulblets to stimulate germination," Flor. Exch., 98(15) : 10, 11 (April 11, 1942). 24. , "Inactivation of the browning system in dried apples," C. B. T. I., 13 : 57-63 (1943). 25. , "Suggestions on inducing early germination of potato tubers in greenhouse tests for virus," Am. Potato J., 20 : 171-176 (1943); also in B. T. I. Prof. Pap., 2 : 7-12 (1943). 26. — — , and L. P. Miller, "Effect of ethylene chlorhydrin vapors upon dormant lilac tissues," C. B. T. I., 4 : 513-528 (1932). 27. , , "Hastening the germination of dormant gladiolus cormels with vapors of ethylene chlorhydrin," C. B. T. I., 6 : 31-38 (1934). 28. , , "Storage temperatures and chemical treatments for shortening the rest period of small corms and cormels of gladiolus," C. B. T. I., 7 : 257-265 (1935). 29. , , and J. D. Guthrie, "Enzym activities of juices from potatoes treated wnth chemicals that break the rest period," Am. J. Bat., 17:483-509(1930); also in C. B. T. I., 2 : 417-443 (1930). 30. , and E. N. Stanton, "Chemical treatments for shortening the rest period of pot-grown woody plants," Am. J. Bot., 15 : 327-336 (1928); also in C. B. T. I., 1 : 355-364 (1928). 31. , , "Localization of response of woody tissues to chemical treatments that break the rest period," Am. J. Bot., 15:337-344 (1928); also in C.B.T.I., 1 : 365-372 (1928). 32. Fhnn, F. B., and J. M. Geary, "Feeding tests with thiourea (thiocarbamide)," C. B. T. /., 11 : 241-247 (1940). 33. Gookel, H., "Stable vitamin C and process for preparing the same," U. S. Patent No. 2,297,212 (1942). 34. Gordon, A. S., E. D. Goldsmith, and H. A. Charipper, "Thiourea and resistance to low atmospheric pressures (high altitudes)," Science, 99 : 104—105 (1944). 35. Guthrie, J. D., "The effect of various chemical treatments of dormant potato tubers on the peroxidase, catalase, pH, and reducing properties of the e.xpressed juice," C. B. T. I., 3 : 499-507 (1931). 36. , "Effect of chemical treatments of dormant potato tubers on the conductivity of the tissue and on the leaching of electrolytes from the tissue," C. B. T. I., 5 : 83-94 (1933). 37. , "Change in the glutathione content of p>otato tubers treated with chemicals that break the rest period," C. B. T. I., 5 : 331-350 (1933). 38. , "Metabolism of citric, sulphuric, and nitric acid in the potato tuber. An explanation for the high pH of the juice of tubers treated with ethylene chlor- hydrin," C. B. T.I.,6: 247-268 (1934). 39. , "Effect of light and of ethylene chlorhydrin on the citric acid content of Bryophyllum leaves," C. B. T. /., 8 : 283-288 (1936). 40. , "Factors influencing the development of ascorbic acid and glutathione in potato tubers following treatment with ethylene chlorhydrin. I," C. B. T. I., 9: 17-39 (1937). 41. , "The utilization of sulphate in the synthesis of glutathione by potato tubers following treatment with ethylene chlorohydrin," C. B. T. I., 9 : 233-238 (1938). 258 GROWTH OF PLANTS 42. Guthrie, J. D., "Effect of ethylene thiocyanohydrin, ethyl carbylamine, and in- doleacetic acid on the sprouting of potato tubers," C. B. T. I., 9 : 265-272 (1938). 43. , "Inhibition of the growth of buds of potato tubers with the vapor of the methyl ester of naphthaleneacetic acid," C. B. T. /., 10 : 325-328 (1939). 44. , "Control of bud growth and initiation of roots at the cut surface of potato tubers with growth-regulating substances," C. B. T. I., 11(1939) : 29-53 (1940). 45. , "Role of glutathione in the breaking of the rest period of buds by ethylene chlorohydrin," C. B. T. I., 11 : 261-270 (1940). 46. , "A preparation from yeast that is active in breaking the rest period of buds," C. B. T. I., 12 : 195-201 (1941). 47. , F. E. Denny, and L. P. Miller, "Effect of ethylene chlorhydrin treatments on the catalase, peroxidase, pH, and sulphydryl content of gladiolus corms," C. B. T. I., 4 : 131-140 (1932). 48. Hartzell, A., "Adult Ufe span animal feeding experiments with thiourea (thiocar- bamide)," C. B. T. I., 12 : 471-480 (1942). 49. Kennedy, T. H., "Thio-ureas as goitrogenic substances," Nature [London], 160 : 233- 234 (1942). 50. Leblond, C. P., "Increased resistance to anoxia after thyroidectomy and after treat- ment with thiourea," Proc. Soc. Exp. Biol. Med., 55 : 114-116 (1914). 51. Loomis, W. E., "Temperature and other factors affecting the rest period of potato tubers," Plant Physiol, 2 : 287-302 (1927). 52. McCallum, W. B., "Physiological." In Arizona Agric. Exp. Sta. Ann. Kept., 20(1908/09) : 584-586 (1909). 53. Miller, L. P., "The effect of thiocyanates upon amylase activity. II. SaUvary amylase," C. B. T. L, 3 : 287-296 (1931). 54. , "The influence of sulphur compounds in breaking the dormancy of potato tubers. PreUminary report," C. B. T. /., 3 : 309-312 (1931). 55. , "Effect of sulphur compounds in breaking the dormancy of potato tubers and in inducing changes in the enzyme activities of the treated tubers," C. B. T. I., 5 : 29-81 (1933). 66. , "Effect of various chemicals on the sugar content, respiratory rate, and dormancy of potato tubers," C. B. T. I., 5 : 213-234 (1933). 57. , "Time relations in effect of ethylene chlorhydrin in increasing and of ethyl alcohol in decreasing the respiration of potato tubers," C. B. T. I., 6 : 123-128 (1934). 58. , "Further experiments on the effect of halogenated ahphatic compounds on the respiration of potato tubers," C. B. T. I., 7 : 1-17 (1935). 59. , "Evidence that plant tissue forms a chlorine-containing /3-glucoside from ethylene chlorhydrin," C. B. T. I., 9 : 213-221 (1938). 60. , " Formation of i3-(2-chloroethyl)-(/-glucoside by gladiolus corms from absorbed ethylene chlorohydrin," C. B. T. I., 9 : 425-429 (1938). 61. , "Synthesis of /3-(2-chloroethyl)-(i-glucoside by potato tubers treated with ethylene chlorohydrin," C. B. T. L, 10 : 139-141 (1939). 62. , "Formation of /3-o-chlorophenyl-gentiobioside in gladiolus corms from ab- sorbed o-chlorophenol," C. B. T. I., 11 : 271-279 (1940). 63. , "Induced formation of a /3-gentiobioside in tomato roots," C.B.T. I., 11 : 387-391 (1941). 64. , "Formation of /3-2,2,2-trichloroethyl-gentiobioside in tomato plants grown in media containing chloral hydrate, trichloroethyl alcohol, or chloral cyanohydrin," . C. B. T. I., 12 : 15-23 (1941). 65. , "Synthesis of ^-2-chloroethyl-d-glucoside by wheat plants grown with ethylene chlorohydrin added to the nutrient medium," C. B. T. /., 12 : 25-28 (1941). DORMANCY IN BUDS 259 66. Miller, L. P., "Simultaneous formation of a /3-gentiobiosidc and a /3-glucoside in gladiolus corms treated with chemicals," C. B. T. I., 12 : 163-166 (1941). 67. , J. D. Guthrie, and F. E. Denny, "Induced changes in respiration rates and time relations in the changes in internal factors," C. B. T. I., 8 : 41-61 (1936). 68. Rosa, J. T., "Abbreviation of the dormant p>eriod in potato tubers," Proc. Am. Soc. Hort. Sci., 20(1923) : 180-187. 69. , "Shortening the rest period of potatoes with ethylene gas," Potato News Bull., 2 : 363-365 (1925). 70. Stuart, W., "The role of anesthetics and other agents in plant forcing," Vermon, Agric. Exp. Sta. Bull. 150 : 449-480 (1910). 71. Thornton, N. C, "Carbon dioxide storage. XIII. Relationship of oxygen to carbon dioxide in breaking dormancy of potato tubers," C. B. T. I., 10 : 201-204 (1939). 72. , "Oxygen regulates the dormancy of the potato," C.B. T.I., 10:339-361 (1939). 73. , and F. E. Denny, "Oxygen intake and carbon dioxide output of gladiolus corms after storage under conditions which prolong the rest period," C. B. T. I., H : 421-430 (1941). CHAPTER 8 Plant Cell Membranes Wanda K. Farr The studies of the formation and structure of plant cell membranes, which were carried out at Boyce Thompson Institute for Plant Research, Inc. over a ten-year period (1930-1940), had their origin in the earlier work of Clifford H. Farr and Wanda K. Farr on the cell divisions of pollen 'mother cells,^- ^ and the growth of root hairs in solutions.^- ^ In the con- sideration of these two aspects of growth, i.e., cell division and cell enlarge- ment, the problems relating to the elaboration of cell membrane materials in the living protoplasm, as well as those relating to the methods of forma- tion and microscopic structure of mature membranes, were constantly in evidence. In 1929 the Division of Cotton Marketing, United States Depart- ment of Agriculture, provided the facilities for an intensive study of the third major aspect of growth, cell differentiation. The project outlined dealt specifically with the cotton fiber, and particular emphasis was placed upon the study of the formation and structure of its cell wall. The thin, colloidal cell membrane of root hairs, the more or less general absence of cellulose, and the lack of pronounced microscopic structural differentiation in the walls of many of them had rendered them of limited value for the study of the so-called "cellulose" fibers of industrial importance. The cotton fiber membrane, with its large accumulations of doubly refractive cellulose and microscopically visible fibrillar structure, promised greater advantages. For more than a year the work was carried out in Washington, D. C. and at Clemson College, S. C. Late in 1930 the headquarters for this research was transferred from Washington to Boyce Thompson Institute. Labora- tory space and facilities were provided by the Institute for Mrs. Farr and .one assistant. Continued cooperation Avith Clemson College afforded field- grown cotton fibers of various stages of development. Methods of growing the cotton plants to maturity in the Institute's greenhouses were soon worked out; they provided one of the most important steps in the detailed studies of cotton fiber development which followed. At this same time a cooperative arrangement was made with the x-ray laboratory of the Depart- ment of Chemistry, University of Illinois, in order that this more recently developed technique might be added to the microscopic and chemical tech- niques already in use. 260 PLANT CELL MEMBRANES 261 Figure 97. Median cross-sections of bolls of Gossypium hirsutum L. showing daily- increase in size from the date of flowering to the twenty-first day of development (1 H X). General Studies of Cotton Fiber Growth The results of the earhest work were of a more general nature, dealing with the origin and early stages of elongation of the cotton (Gossypium hirsutum L.) fiber ^^ (Fig. 97) ; cell divisions in the epidermal layer of the ovule subsequent to fertilization " (Fig. 98) ; structural features of the wall suggested by x-ray diffraction analyses and observations in ordinary and plane-polarized light ^^ (Fig. 99) ; and fiber abnormalities as related to varietal differences and to the density of the fiber mass within the boll.^^ Large numbers of dividing cells in the epidermal layer from the date of flowering to the twelfth day follomng showed that cell enlargement is not alone responsible for the tangential extension of the epidermal layer of the Figure 98. Late stages in cell division and certain appearances in early stages of fiber formation. D, a, late stage of nuclear reorganization; D, b, early stages of nuclear reorganization; E, a, early stage of fiber formation in two adjacent cells whose nuclei appear to be incompletely reorganized; F, o, b, c, and d, lateral wall relationships in the basal portions of fiber-forming cells; F, e, young fiber showing dense nucleus and basal vacuole. (D and E, 1170 X; F, 660 X-) 262 PLANT CELL MEMBRANES 263 seed coat. Early stages of fiber elongation from epidermal cells which were apparently daughter cells of these recent divisions furnished direct evidence that fibers may originate from cells which are not yet formed upon the date of flowering. The absence of Hquid substance in the boll cavity which had been com- monly designated "boll sap" removed the possibility of nutrition of the developing fiber through such a medium, and indicated that the materials for its growth are transported through its basal connection Avith the seed. The study of the formation of abnormalities in developing fibers showed that while cell enlargement and the formation of a thick cell membrane are two of the most conspicuous phases of cotton fiber growth, the tendency to enlarge in an approximately linear direction is inherent. If obstructed in one region of the boll cavity, however, the portion of the fiber concerned appropriates any available space in the immediate vicinity for enlargement. This may result in change in diameter, change in direction of growth, or any one of many types of abnormality. In the three varieties of cotton, Pima, Super Seven, and Acala, the measured densities of the fiber masses were found to increase in the order named. Fiber abnormalities were observed to increase in the same direction and a definite relationship was suggested between these two factors. Broadening of the Experimental Approach During the progress of these studies it became evident that the advan- tage of the cotton fiber, with respect to the large quantity of cell membrane material, was not furnishing, under the experimental conditions employed, additional information concerning the fine structure of the cell membrane and the formation of membrane-building materials in the living protoplasm. From the viewpoint of plant cells in general, the cotton fiber is one of the most highly differentiated. Even among those cell membranes which contain cellulose — and many do not — it would be classed as a specialized and not a primitive type. The failure to observe finer details of membrane formation and structure indicated a possible need for a broader attack upon the problem, involving cells of various types throughout the plant kingdom. Facts gleaned from such sources might then be used in attempting to under- stand the development and structural problems of the cotton fiber. This task was undertaken by Mrs. Farr and Dr. Sophia H. Eckerson of the Institute staff. Their first studies were made with the cellulose-forming bacterium, Acetohacter xylinus. All the microscopic observations were made without the use of the usual bacteriological methods of staining; however, certain microchemical reactions and polarized light were used to assist in the examination of individual organisms and chains of organisms (Fig. 100). The cellulose layer surrounding the protoplast and the non- cellulosic exterior layer of the bacterial organism were thus differentiated. These procedures represented a, more direct approach to the physical and 264 GROWTH OF PLANTS chemical properties of a bacterial membrane and one less likely to affect its natural properties than many of the staining techniques in current use. They necessitated some changes in microscopic technique, however, the most important of which consisted in the readjustments of the illuminating system. This involved mainly the lowering of the intensity of the artificial light by suitable rheostat control, so that the structural differentiations in the diminutive organisms would not be obscured by a flood of bright light. The results of these bacterial studies were of interest in the light of current attempts to "synthesize" cellulose membranes by growing A. xyli- nus in glucose solutions.^^ In these contemporary Canadian experiments the process of cellulose formation was considered to be intercellular, the cellulose molecules having been synthesized directly from the sugar mole- cules in the nutrient medium and deposited in long, well-oriented chains of cellulose unit-cells. At the request of and from cultures furnished by Dr. H. L. Hibbert, the entire "membrane" was found by Farr and Ecker- son 2" to consist of bacterial organisms with no true intercellular substance. The single bacterium is composed of a protoplast surrounded by a cellulose membrane which, in turn, is covered with a layer of gelatinous material which reacts positively with the ruthenium red test for pectic substance and negatively to the H2SO4 and I2KI test for cellulose. Studies in both ordinary and polarized light at low and high magnifications produced no evidence of continuous, long chains of cellulose in the membranes. They did indicate, however, extreme regularity in directional arrangement of the strands of the aerobic organisms as they grew in thin layers upon the sur- face of the nutrient media. The development of microscopic techniques by means of which such bacterial membranes could be analyzed was of even greater importance. The optical systems, thus illuminated, were then available for use in exam- ining other types of plant cells. One of the first of these which was studied was the developing conidiophore of Aspergillus niger. Dr. Charles Thom had called to Mrs. Farr's attention structures in the wall of the mature stalk of a certain strain of this fungus which closely resembled the spiral fibrils in the wall of the cotton fiber. In the protoplasm of the very young stalks tiny granules were found which gave the characteristic cellulose reaction with sulphuric acid and iodine. As the stalks developed they were carefully mounted for observation in both ordinary and polarized light. It was thus found that, within the limits of one sporangiophore, the successive Figure 99. Hand-colored photomicrographs of portions of fibers in plane-polarized light: a to r from Upland cotton, s and t from Jungle cotton; long axes of portions oriented at 45° with reference to the plane of vibration of the light; d, n, o, and r slightly swollen in a solution of ammonium thiocyanate; a, b, c, I, m, s, and t photographed with the analyzer; d, n, 0, and r without the analyzer; c and m colored with the selenite plate (red of the first order) ; a, b, d, I, n, o, r, s, and t without the selenite plate; a, b, c, I, and m, 690 X ; d and n, 810 X',o,r, s, and t, 1200 X . (Coloring of photomicrographs was done by Miss Flora White.) Figure 99. Portions of fibers in plane-polarized light. (For description see legend on page 26^.) c m e *%- ■;;ji?;5^^irn-iiijiiiii I jijiir!iyivu!)|iT)tin^wiTiCCii)miiiili ■?- ^^•— '!> F & W Figure 100. (For description see legend on page 265.) PLANT CELL MEMBRANES 265 stages of development of the single fibrils could be observed. Each fibril was formed by the arrangement, in single rows, of the diminutive cellulose particles. In the mature conidiophore these fibrils were in spiral arrange- ment in the secondary lamellae. The primary wall in this strain of Asper- gillus was found to be comparatively thin, non-doubly refractive, and largely pectic in composition, as indicated (Fig. 100) by its staining with ruthenium red. By similar reactions to ruthenium red the individual fibrils and even the individual cellulose particles were found to be coated with a non-cellulosic substance, one component of which is pectic. The deep yellow coloration of this colloidal coating with iodine solution ex- plained, at the time, Strasburger's earlier observations of protein "micro- somes" which behave similarly in building up the cell wall lamellae. The cellulose ''microsome" is coated with a non-crystalline film containing both protein and pectic material. However, the developmental importance of this characteristic protein reaction was not recognized until later when, in the course of studies of cellulose formation, it was found to come into exist- ence in a protoplasmic matrix rich in protein. With these observations of bacteria and fungi as a basis, a comparison of young cotton fibers in various stages of development showed the presence of similar cellulose particles and similar stages of fibril formation. In the cotton fiber, however, the quantity of colloidal material is greater than in the conidiophore of A . niger and had previously obscured the brightness of the cellulose particles in polarized light. The larger amounts of colloidal material were associated in the cotton fiber with strength and flexibility; the smaller amounts in the conidiophore with weakness and brittleness. These results were published by Farr and Eckerson under the title "For- mation of cellulose membranes by microscopic particles of uniform size in linear arrangement." ^o The broader botanical attack upon cell-membrane Figure 100. a, Single bacteria from cultures of Acelobader xylinus in positions of extinction and brightness in polarized light with selenite screen (2700 X). b, Mount shown in a without selenite screen (2700 X). c, Sulphuric acid-iodine reaction in A. xylinus (1950 X). d, Pectic coating upon the surface of A. xylinus stained with ruthenium red (1950 X). e. Cellulose particles separate and in chains in young spor- angiophore of Aspergillus niger. Polarized hght (700 X). /, Portion of young spor- angiophore of A. niger showing original pectic membrane and pectic coating upon the individual cellulose particles stained with ruthenium red (1150 X). g, Cellulose particles in the process of fibril formation in sporangiophore of A. niger (1900 X). h, Tip and base of young sporangiophore of A. niger showing earhest cellulose membrane formation near base of stalk. Polarized light (1700 X). k, Developing sporangiophore of A. niger showing increasing thickness of cellulose membrane in lower portion. Polarized light (1700 X). m, Portion of stalk of mature sporangiophore of A. niger showing crossed spiral arrangement and reversal area of cellulose fibrils. Polarized light (1150 X). n, Another portion of a mature sporangiophore of A. niger showing more frequent areas of reversal. Polarized Hght (1150 X). o, Mature sporangiophore of A. niger showing thick cellulose membrane throughout its entire length. Polarized light (500 X). p, Por- tion of fiber of Hibiscus spathecus stained with ruthenium red to indicate its outer pectic layer (700 X). r, Portion of intact fiber of H. spathecus showing longitudinal areas of coloration in polarized light (200 X). s, Base of H. spathecus fiber shghtly crushed to bring out crossed spiral arrangement of fibrils (550 X). 266 GROWTH OF PLANTS Figure 101. a, Portions of fibers from the residues after treatment for 23^ hours with HCl. The fibril and particle structure of the membranes are shown as well as the cross-sectional rupturing of the fiber (350 X) '•n? u^'r-wo^^'''''! '^^^''^^ ''^ ^^^^ dissociation after 18 hours' treatment with HCl (350 X). c, Extreme degree of fiber dissociation after 5 days' treatment with HCl (350 X). PLANT CELL MEMBRANES 267 |f#^' if^-#4 Figure 102. X-ray diffraction patterns of cellulose, a, Cellulose particles from cotton fibers separated by 18-hour treatment with HCl; b, pulverized cotton fibers; c, paralleled cotton fibers; d, paralleled ramie fibers. formation and structure would seem to have been justified by the fact that the particulate structure of the cellulose fibril was first observed in a fungus as a result of optical improvements which had been made to meet the microscopic requirements of unstained bacteria. These observations of fibril formation recalled the earlier reports of similar phenomena by Strasburger ^^ and Wiesner.'^ In addition to report- ing the existence of "microsomes" or " dermatosomes " in the living proto- plasm and their behavior in building up the cell membranes, Wiesner had sho^\^l that the granular dermatosomes maintain their identity in the fibrils of the mature membrane and had demonstrated the fact by disintegrating the membrane of the cotton fiber into lamellae, the lamellae in turn into fibrils, and the fibrils into dermatosomes by treatment with hydrochloric 268 GROWTH OF PLANTS acid. Farr and Eckerson ^^ published a similar result in 1934 under the title "Separation of cellulose particles in membranes of cotton fibers by treatment with hydrochloric acid" (Fig. 101). X-ray diagrams of these preparations (Fig. 102) were pubhshed by Farr and Sisson ^^ during the same year. The controlled treatment with hydrochloric acid had removed Figure 103. 1. Cell membranes of Spirogyra sp. show the presence of crystalline material through their double refraction in polarized light (480 X). 2. Spiral fibrils in the cell membrane of a cotton fiber {Gossypium hirsutum) in polarized hght (775 X). 3. Fibrils in the cell membrane of the cotton fiber are arranged parallel to the axis at intervals throughout its length and produce "extinction areas" in polarized Ught (775 X). Figure 104. Cotton fibers, partially disintegrated by bacteria. A, From 24-day culture (675 X, enlarged to 1000). B, From 8-week culture (900 X, enlarged to 1350). C, From 9-week culture (900 X, enlarged to 1350). D, From 24-day culture (675 X, enlarged to 1000). 269 270 GROWTH OF PLANTS the greater part of the relatively small amounts of non-cellulosic material from the fiber membrane, had reduced the physical state of the fibers to a fine white powder, but had not changed the native cellulose pattern. Three years later Sisson,^^ by means of x-ray diffraction analysis, corroborated the earlier work of Farr and Eckerson ^o on the presence of crystalline cellulose in young cotton fibers. These botanical studies were of sufficient chemical interest to bring a contribution from The Chemical Foundation, Inc. in 1936 for the increase in staff and facilities for plant cell-membrane research. The work was continued at Boyce Thompson Institute. Drs. Florence L. Barrows, Jack Compton, Stanton A. Harris, Florence E. Hooper, Richard E. Reeves, and Wayne A. Sisson were added to the research staff and the numbers of laboratory assistants increased correspondingly. Studies of various types which were undertaken resulted in the publica- tion of papers dealing with certain colloidal reactions of cell membranes to treatment with sulphuric, hydrochloric, and phosphoric acids; ^^ the isola- tion of pectic acid from the cotton fiber; ^^ the effect of certain non-cellu- losic constituents upon the x-ray diagram of cellulose; ^'^ orientation in young cotton fibers, as indicated by x-ray diffraction analysis; ^° a consider- ation of the microscopic structure of plant cell membranes (Fig. 103) from various parts of the plant kingdom in relation to the micellar hypothesis of Nageli; ^^ x-ray diffraction behavior of cellulose derivatives; ^^ the dis- integration of the cell membrane (Fig. 104) of the cotton fiber by a pure culture of bacteria; ^^ microscopic analyses of additional cell membranes from various parts of the plant kingdom; ^ x-ray analyses of textile fibers; ^^ x-ray diffraction analysis and its application to the study of plant constitu- ents (Fig. 105) ; ^1 the behavior of the cell membrane of the cotton fiber in cuprammonium hydroxide solutions with particular reference to their dis- persion, electrokinetic, and coagulation behavior; 4- 1^. 32 ^^^q lamellate structure (Fig. 106) of certain plant cell membranes; ^ and the structural relationship of rayon to natural cellulosic fiber materials, as sho^\^l through a study of the viscose process.^ The studies of the behavior of cell mem- brane materials in cuprammonium hydroxide and m the carbon disulphide used in the viscose process showed that in both instances the cellulose par- ticles are dispersed, not dissolved, in the medium, and that the non-cellu- losic materials present in small quantities in the cotton fiber membrane play a part in bringing about this dispersion and in producing the final viscosities of the mixtures. These observations are in keeping with the fact that highly purified cell membranes of cotton fibers lose their viscosity- producing power in many reagents, although the nature of their cellulosic component remains relatively unchanged as observed microscopically and examined by means of x-ray diffraction (Fig. 107). Observations on the membranes of epidermal cells of the Avena coleop- tile revealed another different inter-particle relationship. ^^ In the cotton fiber and wood fiber, where fibrils constitute a unit of structure, the end-to- PLANT CELL MEMBRANES 271 Figure 105. Detection and estimation of plant constituents: presence of cellulose in lignin, A, lignin; B, cellulose from lignin; C, cotton cellulose; detection of cellulose in young cotton fibers, D, 10; E, 25; F, 30; G, 35; H, 50-day-old fibers; I, same as D after extraction; presence of cellulose in Valonia cytoplasm, J, original cytoplasm; K, cellulose obtained from cytoplasm by extraction; L, cellulose from cell wall. Figure 106. (See legend on opposite page.) 272 PLANT CELL MEMBRANES 273 end bondage of the cellulose particles is strong. In the cells of the Avena coleoptile, where rapid cell elongation takes place, these end-to-end bond- ages are weak enough to be broken during this growth process; in the mature, elongated membrane, the single rows of particles are arranged side by side along the long axis of the cell in "barrel-hoop" fashion. In this connection it may be pomted out that in all the cells studied to date, the burden of swelling, plasticity, and elasticity has rested upon the inter- crystalline material. When extreme plasticity and absorptive properties are desired, and strength is not at a premium, as in the root hab, the cellu- lose is often excluded entirely from the construction of the cell membrane. When strength as well as flexibility is desired, the colloidal materials, such as pectin and protein, are reduced m quantity; in some instances they are rendered more impervious to swelling agents by compound formation {e.g., calcium or sodium pectates), and are intimately blended with cellulose particles to produce the desired physical and chemical combination. Formation of the Cellulose Particles The findings up to this point had furnished no clue to the method of elaboration of cell membrane materials in the living protoplasm. Small cells, such as the cotton fibers, required that the observer look through the membrane in order to see protoplasm during its period of synthetic activity. Protoplasm expressed from such cells tends to produce artifacts by coagula- tion and other physicochemical changes; optical sectioning of carefully mounted fibers revealed nothing definite in these cells as long as an entirely unknown process was involved. The large, one-celled alga, Valonia ventri- cosa, had been exammed and reported upon at one of the meetings of the American Chemical Society.^' Earlier workers had been attracted to the study of Valonia because of its highly crystalline, well-oriented membrane. ^ We were particularly interested in studying it because of the ease with which its protoplasm could be removed and maintained in good condition for microscopic examination. Arrangements were made, through the cour- tesy of the Carnegie Institution of Washington, to obtain fresh samples of Valonia ventricosa, V. macrophysa, and a closely related form, Halicystis osterhoutii from their Dry Tortugas laboratory and the Bermuda Biologi- cal Station for Research, Inc. Preserved samples of Halicystis ovalis were furnished from the beaches of Pacific Grove, California, by Dr. George J. Hollenberg. The mature membrane of Halicystis was found to contain a high propor- tion of non-cellulosic material. Through this colloidal matrix the cellulose Figure 106. Cotton fibers grown in constant light. Lamellae about 1 /z wide. A, 15 days, 1 lamella, 2nd forming (920 X). B, 49-day cross section; 6 cellulose particles with micrometer scale (1 space equals 2.35 m measured from center to centerof adjacent Unes). C, E, F, 49-day cross sections at middle. D, 60-day dry, cut in cork (460 X, enlarged to 890). G, X-ray diffraction pattern of fibers matured in constant Ught. H, In daylight. 274 GROWTH OF PLANTS Figure 107. A, X-ray diffraction pattern of cellulose particles from cotton fibers after treatment for 48 hours with the standard solution {American Chemical Society) of cuprammonium hydroxide. B, A similar sample after three months' treatment with the same reagent. C, 3 g of cellulose particles separated by treatment with HCl (sp gr 1.19) produce a mixed mercerized and native cellulose x-ray diffraction pattern after treat- ment for 18 hours in cuprammonium hydroxide. D, 7 g of cellulose particles similar to those used in C produce a native cellulose x-ray diffraction pattern after treatment for 18 hours in cuprammonium hydroxide. particles are distributed at random, with no tendency toward fibril forma- tion. The x-ray diagram of the cellulose in this membrane (Fig. 108) shows that it is in a "mercerized" state, comparable to that produced in the cellulose of a cotton fiber when it is treated with strong (17 to 18 per cent NaOH) alkah.^s In the protoplasmic mounts of Halicystis were found the structures which had long been the object for which we had searched — either the PLANT CELL MEMBRANES 275 B FiGtTRE 108. X-ray diffraction diagrams of Halicystis. A, untreated; B, stretched 75 per cent (fiber axis vertical); C, after purification treatments; D, after treatment with hot glycerin; E, after treatment with hot 0.1 N hydrochloric acid and 1 percent sodium hydroxide; F, for comparison, the x-ray diagram of mercerized cotton treated as in E, 276 GROWTH OF PLANTS cellular organs or regions in the protoplasm which elaborate cellulose to be used in the construction of the cell membrane. These proved to be cellu- lose-forming plastids. The mechanism of formation of cellulose in the chloroplasts of Halicystis has no apparent points in common \vith the mechanisms of starch formation as it has been observed in many types of plant cells. It operates by the successive formation of cellulose rings of uniform thickness and increasing diameter, as the plastid enlarges. These rings, at first in a liquid state, then gel-like, and then solid, finally fragment to form mercerized cellulose particles. When the period of cellulose synthesis comes to a close, the plastid membrane disintegrates and the cellulose particles, with their coating of colloidal plastid plasma, are set free in the outer regions of the cytoplasm of the cell. They are later deposited directly, with their associated materials of plastid and cyto- plasmic origin, to form a new lamella of the cell membrane. The newly deposited lamellae are green due to the presence of the chlorophyll of the plastid plasma (see Fig. 109). After observing this phenomenon of cellulose formation in the chloroplasts of Halicystis, a careful study of the protoplasm of the cotton fiber was resumed, with the result that colorless plastids, which were performing a similar function of cellulose formation by a similar process of ring forma- tion and fragmentation, were found. These plastids had been seen and photographed previously in many living cotton fibers. Their function in the cell had been obscured by the slight differences in refractive indices of the plastid plasma and the cell plasma which surrounds them (Fig. 110). They had been observed and considered to be vacuoles in the young fibers. Once removed from the fiber, however, the stages of cellulose ring formation and fragmentation (Fig. Ill) were clearly visible.^'' The chloroplasts of Valonia were found to be engaged in the elaboration of cellulose by a different process from that observed in the chloroplasts of Halicystis and in the colorless plastids of the cotton fiber. Following the formation of a single cellulose ring in the very young plastid, a cellulose fibril begins to form in the plastid plasma. These fibrils attain great length and, in the mature plastid, are coiled tightly within the plastid membrane. When the membrane of the mature plastid disintegrates, the coiled cellulose fibril is freed and straightens. At this stage it can be disintegrated into cellulose particles with slight pressure. Continued study of the early stages of cell membrane formation has indicated, however, that in the living cell the fibril is deposited directly in the lamella of the membrane after it has uncoiled from the plastid. Observations show that it is deposited in close Figure 109. Column A, Stages in development of the chloroplast of Halicystis showing cellulose ring and cellulose particle formation (1540 X). Column B, Stages in starch formation in the chloroplasts of the cotton plant (1540 X). Column C, Cellulose particle formation takes place in the colorless plastids of the cotton fiber by a process of successive ring formation and fragmentation essentially similar to the mechanism of cellulose formation in Halicystis (1540 X). .(FiGUKE 109) 277 Figure 110. A, Cross section of a young cotton seed showing the cells of the epidermal layer in the process of elongation to form cotton fibers and certain cells of the outer integument of the seed filled with starch (420 X). B, Starch-forming plastid from an integument cell and a ceUulose-forming plastid from a cotton fiber photographed together to show their comparative visibihty (1100 X). 278 Figure 111. A, In the protoplasm of young cotton fibers, disc-shaix,>d cellulose- forming plastids, varying over a wide range in diameter, are barely visible. The relative refractive indices of the plastids and of the cytoplasm in which they are float- ing obscure their contents and cause them to resemble vacuoles (1000 X). B, Early stage of fibril formation in the young cotton fiber (1320 X). C, Cellulose particles and a cellulose ring in a plastid removed from a young cotton fiber (1650 X). D, Frag- mentation of a cellulose ring, taken from a plastid, to form the cellulose particles (1650 X). E, Cell membrane of the cotton fiber after the deposition of cellulose fibrils in spiral arrangements (1254 X). 279 280 GROWTH OF PLANTS proximity to and in precise alignment wdth the adjacent fibrils, thus exclud- ing, in large measure, the colloidal, non-cellulosic materials from the cell membrane. This procedure will account for the high degree of crystallinity and the regular orientation of the mature Valonia membrane. ^^ Detailed accounts of the visible aspects of cellulose and cell membrane formation in both Halicystis and Valonia (Fig. 112) are being prepared for publication in Contributions from Boyce Thompson Institute. These studies of bacteria, fungi, algae, and other types of cells from vari- ous parts of the plant kingdom, represent the experimental procedures in the attempt to understand the chemical and physical variations mvolved in the formation and structure of plant cell membranes. When the cell membrane studies at the Institute terminated m 1940, sufficient evidence had been accumulated to indicate the chemical hetero- geneity and physical complexity of plant cell membranes in general; the particulate state of the cellulose in many membranes from various parts of the plant kingdom; the importance of non-cellulosic materials ui mem- brane formation and function; the intimate colloidal associations of the membrane components, which render their identification extremely diffi- cult; the importance of using fresh (undried) material, whenever possible, for experimental purposes; and the need to develop more precise methods of fractionation and identification in order to determine the roles played by the various membrane components in both native and processed mate- rials. The physical aspects of cellulose synthesis in the living plastid, as well as the behavior of membrane-forming materials during their period of organization in the outer layers of the protoplasm, suggest a new field of experimental approach to the study of the formation and structure of plant cell membranes. The forces mvolved are frequently neither of a simple molecular nor of a gross physical type. They fall rather into the colloidal field of molecular aggregates of various dimensions and degrees of purity. Surface coatings sometimes mask the chemical identity of such molecular aggregates and determine their behavior in an electrical field. As data accumulate, it becomes more evident that the properties of both living and processed plant cell membranes must be interpreted in terms of the colloidal systems which they represent, and not in terms of the molecular behavior of any one component. The skill with which nature synthesizes and manipulates the plant cell membrane materials will become more and more impressive as a clearer understanding of the chemical and physical forces involved is obtained. Until the accumulated information is more extensive than what we have at present, we shall not be able to understand fully, e.g., the observed similarities of properties of membranes of cells of very different types and the differences in the make-up of membranes of closely related cells. To future research is left the task of finding and establishing basic principles of structure and composition common to all types of plant cell membranes, PLANT CELL MEMBRANES 281 Figure 112. A-D, Cellulose ring and cellulose fibril formation in the developing chloroplasts of Valonia ventricosa (500 X). E, Removal of plastid membranes from two plastids and washing away green plastid plasma makes rings and fibrils more clearly visible (625 X). F, After removal of plastid membranes mature fibrils straighten and readily break into cellulose particles (700 X). G, H, Fibrils uncoil from plastids on inner surface of membrane and align themselves with fibrils in developing membrane (G, 625 X; H, 700 X). J, Orientation of fibrils in mature membrane as shown by microscope and X-ray diffraction (700 X). 282 GROWTH OF PLANTS and of explaining the procedures by means of which every given type of plant cell produces the membrane suited to its vital needs. Summary (1) Problems relating to the formation and structure of plant cell mem- branes have been approached, in the Boyce Thompson Institute for Plant Research, through microscopic, chemical, and x-ray diffraction analyses of cell membranes from various parts of the plant kingdom. (2) The first experiments dealt exclusively with the phenomenon of cell enlargement in root hairs, in the membranes of which cellulose is either absent or only sparingly present. Their plastic properties are determined by the colloidal mixtures of non-cellulosic materials. (3) Cotton fiber studies began with the more general aspects of their development from epidermal cells of the seed coat and resulted in additional information concerning their origin from cells which had divided subse- quent to fertilization; their nutrition through the basal connection with the seed and not from "boll sap"; and the spiral fibrillar structure of their secondary lamellae, as shown in polarized light and by means of x-ray diffraction. The latter studies were made possible by the presence of crys- talline cellulose in the fiber membranes in addition to colloidal non-cellulosic materials. (4) Failure to obtain information concerning the fine structure of the fibrils of the secondary lamellae led to studies of less highly differentiated cellulose-forming cells including bacteria, fungi, and algae. (5) As a result of improved microscopic techniques, designed to observe the cellulosic and non-cellulosic portions of Acetohacter xylinus, crystalline cellulose was identified for the first time in the protoplasm of young cotton fibers. It was in the form of ellipsoids, approximately 1.0 X 1.5 ^i in size, which were named "cellulose particles" and were identified with Stras- burger's "microsomes" and Wiesner's "dermatosomes." (6) Single rows of these cellulose particles are arranged end to end to form the fibrils of the cotton fiber. The process takes place in the outer regions of the living protoplasm and is followed by the deposition of spirally arranged fibrils in a matrix of non-cellulosic colloidal material, to form the secondary lamellae. Both pectic material and protein are present in this matrix. Although the lamellate structure of the secondary wall indicates the periodic deposition of wall materials, evidence was obtained to indicate that this is not a daily periodicity; hence the lamellae do not represent "daily growth rings." (7) In cells such as the green alga (Halicystis) fibrils are not formed and cellulose particles in the successive lamellae are in more or less random arrangement. In the Avena coleoptile, end-to-end bondage of cellulose particles is not strong enough to prevent separation during cell elongation. The stretching of the non-cellulosic membrane materials leaves the cellu- lose in ring-like bands along the extended membrane. PLANT CELL MEMBRANES 283 (8) The process of cellulose formation in living protoplasm was first observed in Halicystis. The mechanism involved is one of successive cellu- lose ring formation in the green chloroplasts, followed by ring fragmentation to form the cellulose particles. (9) Cellulose formation by a similar method was later observed in color- less plastids of the cotton fiber and in cells of the leaf and stem tissues of the cotton plant. In the latter cells starch and cellulose are formed simul- taneously in chloroplasts and colorless plastids respectively. (10) A conspicuous variation in the process of cellulose formation in the chloroplast of Valonia consists in the formation of one closed cellulose ring followed by the formation of a coiled cellulose fibril. (11) All mature cell membranes containing cellulose particles in various proportions and types of arrangement were found to contain non-cellulosic materials of various types and in different proportions. These combina- tions of crystalline and non-crystalline components in intimate colloidal association constitute a basis for understanding the relative properties of the membranes of highly differentiated fibrous cells, such as cotton and ramie, as well as those of the more primitive bladder-like cells, Halicystis and Valonia. Considerations of the function of non-cellulosic materials in controlling the phenomena of plasticity and deformability during the period of cell enlargement, and the function of cellulose in establishing strength and rigidity in the process of cell differentiation, constitute a new analytical approach to the study of these two aspects of plant growth. Literature Cited 1. Astbiiry, W. T., T. C. Marwick, and J. D. Bernal, "X-ray analysis of the structure of the wall of Valonia ventricosa," Proc. Roy. Soc. [Land.], B109 : 443-450 (1932). 2. Barrows, F. L., "Cellulose membranes from various parts of the plant kingdom," C. B. T. L, 11(1939) : 61-82 (1940). 3. , "Lamellate structure of cellulose membranes in cotton fibers," C. B. T. /., 11 : 161-179 (1940). 4. Compton, J., "On the behavior of plant fibers dispersed in cuprammonium hydroxide solution," C. B. T. I., 10 : 57-70 (1938). 5. , "Structural relation of rayon to natural collulosic fibers. Study of the viscose process," Ind. Eng. Chem., 31 : 1250-1259 (1939). 6. Farr, C. H., "Cytokinesis in the pollen-mother-cells of certain dicotyledons," Mem. New York Bot. Gard., 6 : 253-317 (1916). 7. , "Root hairs and growth," Quart. Rev. Biol, 3 : 343-376 (1928). 8. Farr, W. K., "Cell-division of the pollen-mother-cell of Coboea scandens alba," Bull. Torrey Bot. Club, 47 : 325-338 (1920). 9. , "Studies on the growth of root hairs in solutions. The pH molar-rate relation for Brassica oleracea in calcium sulphate," Proc. Nat. Acad. Set., 15 : 464-470 (1929). 10. , "Cotton fibers. I. Origin and early stages of elongation," C. B. T. I., 3 : 441- 458 (1931). 11. , "Cotton fibers. III. Cell divisions in the epidermal layer of the ovule sub- sequent to fertiUzation," C. B. T. I., 5 : 167-172 (1933). 284 GROWTH OF PLANTS 12. Farr, W. K., "Cotton fibers. IV. Fiber abnormalities and density of the fiber mass within the boll," C. B. T. I., 6 : 471-478 (1934). 13. , "The membrane structure of Valonia," Amer. Chem. Soc, Div. Cellulose Chem. Absts. papers. 93rd meeting, Chapel Hill, N. C. Apr., 1937, p. C2. 14. , "Certain colloidal reactions of cellulose membranes," /. Phys. Chem., 41 : 987-995 (1937). 15. , "Behavior of the cell membrane of the cotton fiber in cuprammonium hy- droxide solution," C. B. T. I., 10 : 71-112 (1938). 16. , "The microscopic structure of plant cell membranes in relation to the micellar hypothesis," /. Phys. Chem., 42 : 1113-1147 (1938). 17. , "Formation of microscopic cellulose particles in colorless plastids of the cotton fiber," C. B. T. I., 12 : 181-194 (1941). 18. , "Plant cell membranes," in Alexander, Jerome, "Colloid Chemistry," Vol. V : 610-667, Reinhold Publishing Corp., New York, 1944. 19. , and G. L. Clark, "Cotton fibers. II. Structural features of the wall suggested by X-ray diffraction analyses and observations in ordinary and plane-polarized fight," C. B. T. I., 4 : 273-295 (1932). 20. , and S. H. Eckerson, "Formation of cellulose membranes by microscopic particles of uniform size in linear arrangement," C. B. T. I., 6 : 189-203 (1934). 21. , , "Separation of cellulose particles in membranes of cotton fibers by treatment with hydrochloric acid," C. B. T. I., 6 : 309-313 (1934). 22. , and W. A. Sisson, "X-ray diffraction patterns of cellulose particles and interpretations of cellulose diffraction data," C. B. T. I., 6 : 315-321 (1934). 23. , , "Observations on the membranes of epidermal cells of the Avena col'eoptile," C. B. T. I., 10 : 127-137 (1939). 24. Harris, S. A., and H. J. Thompson, "Pectic acid from the cotton fiber," C. B. T. I., 9 : 1-5 (1937). 25. Hibbert, H. L., and J. Barsha, "Studies on reactions relating to carbohydrates and polysaccharides. XXXIX. Structure of the cellulose synthesized by the action of Acetobacter xylinus on glucose," Can. J. Res., 6 : 580-591 (1931). 26. Hooper, F. E., "Disintegration of the cell membrane of the cotton fiber by a pure culture of bacteria," C. B. T. /., 10 : 267-275 (1939). 27. Sisson, W. A., "The effect of certain non-cellulosic constituents on the X-ray diagram of cellulose," Am. Chem. Soc, Div. Cellulose Chem. Absts. papers, Pittsburgh, Pa., Sept. 7-11, 1936, p. 3. 28. , "Identification of crystalline cellulose in young cotton fibers by X-ray diffrac- tion analysis," C. B. T. I., 8 : 389-400 (1937). 29. , "X-ray analysis of textile fibers. Part V. Relation of orientation to tensile strength of raw cotton," Textile Res., 7 : 425-431 (1937). 30. , "Orientation in young cotton fibers as indicated by X-ray diffraction studies," C.B.T. I., 9 : 239-248 (1938). 31. , "X-ray diffraction analysis and its application to the study of plant con- stituents," C. B. T. I., 9 : 381-395 (1938). 32. , "Some observations upon the dispersion, electrokinetic and coagulation be- havior of cotton fibers in cuprammonium hydroxide solution," C. B. T. I., 10:113-126(1938). 33. , "The existence of mercerized cellulose and its orientation in Halicystis as indicated by X-ray diffraction analysis," Science, 87 : 350 (1938). 34. , "X-ray diffraction behavior of cellulose derivatives," Ind. Eng. Chem., 30 : 530-537 (1938). 35. Strasburger, E., "Ueber den Bau und das Wachsthum der ZellhJiute," Gustav Fischer, Jena, 1882. 36. Wiesner, J., " Untersuchungen uber die Organisation der vegetabilischen Zellhaut," Sitzungsber. Akad. Wiss. Wien. Math. Naturmss. Kl. Abt. I., 93 : 17-80 (1886). CHAPTER 9 Plants Grown under Controlled Environmental Conditions Just previous to the planning and building of the Institute there were several new developments in plant and animal physiology that indicated the desirability of equipment for growing plants on a fairly large scale and under a wide range of conditions as to light (quality, intensity and daily duration), concentration of carbon dioxide, temperature, humidity, nitrate supply, etc. The following may be mentioned as the more prominent of these researches. In 1920 Garner and Allard " had published their monu- mental discovery that day length initiated flowering in various kinds of plants. Kraus and Kraybill 2" gave a new slant to the problem of reproduc- tion in plants when they published their paper on the importance of the proper balance between carbohydrate and nitrogenous substance in plants as a determiner of fruit set and development. This paper focussed much attention on the significance of the C/N ratio in plants. This ratio is of course determined by the relative amount of photosynthesis on one hand, and the amount of nitrogen compounds absorbed on the other; therefore, all factors that affect these two processes in plants need consideration. Schanz ^"^ ^^ found evidence that ultraviolet rays may cause cataract by coagulating the proteins of the crystalline lens of the eye, and that these rays are of great importance in determining the form of plants. The injuri- ous action ^° of ultraviolet was receiving much consideration. Before the Institute was formally opened, Steenbock,^^ and Hess and Weinstock,i^ had found simultaneously that ultraviolet rays impart antirachitic action to various vegetable and animal materials. It turned out later that the action of the ultraviolet rays was on one of the sterols, i.e., ergosterol. It has long been kno^vn that the carbon dioxide content of the atmos- phere is too low to give maximum photosynthesis when other conditions are at or near the optimum for this process. The experiments of H. Fischer, Riedel, Jesse and others,^'*' p-^^^ just previous to 1920, indicated that crop yields could be increased markedly in greenhouses and even outside by CO2 enrichment of the air; this of course increases the amount of photosynthe- sis, as does longer daily illumination. Would it induce the flowering of long-day plants as does the long day in accordance with the C/N ratio conception, or is flower induction by day length due to a specific effect of light quite independent of photosynthesis? 285 286 GROWTH OF PLANTS Three rather large installations -^ for modifying and controlling gro^i^h conditions for plants were built as a part of the Institute : (A) the constant- condition dark and light rooms where plants could be gro^^'n under artificial light exclusively with day length, temperature, humidity, and carbon dioxide concentration of the air regulated; (B) the gantry crane house where plants were gro^vn during the day in daylight and 6 or 12 hours during the night under light from tungsten lamps attached to a gantry crane; (C) the spectral glass houses in which plants were gro^^Ti under dif- ferent regions of the solar spectrum, as determmed by the solar transmis- sion of the glasses on the several houses. Later an insulated greenliouse was built which was Ughted by sunlight through a southern ex-posure of glass during the day and wliich was further illuminated by tungsten lights attached to a thermostat. These Hghts were the sole heat source of the house, and at the same time they supplemented the sunlight, largely at night or on cloudy days, for when the sun was shining little additional heat was required. Besides these larger pieces of equipment, much other appara- tus was purchased or made at the Institute for regulating, measuring, and recording various growth conditions. The several pieces of equipment will be briefly described later m connection with the experunents carried out with them. Plants Grown Entirely in Artificial Light To attempt to grow plants under artificial light in competition \dth sun- light is very expensive. Arthur '" figured the value of the radiant energy falUng on an acre of land at Yonkers, New York, during 1936 under the assumption that it could be converted quantitatively to electricity and the electricity sold at two cents a kilowatt hour. The figures in Table 29 are calculated from New York meteorological data. On this basis the annual value of sunlight falling on an acre at Yonkers is about S106,000. This makes it very clear why artificial light cannot compete ^^'ith sunlight in growing plants, and why men like Abbott of the Smithsonian Institution spend their time working up apparatus for trapping sunlight as a source of energy. "\Mio ever heard of a crop being worth a considerable fraction of -1106,000 per acre year? Constant-condition rooms. The two constant-condition rooms ^' ^ (Fig. 113) were built in the basement under the greenhouses, each having a floor space of 11 X 11 feet, or 121 square feet. Both rooms were attached to the same air-conditioning and carbon dioxide-enriching systems so that during any given period both had the same temperature, humidity, and CO2 concentration in the air. One room was dark and gave the plants their night ; the other was iUuminated by 25 1000- or 1500- watt Mazda lights. The lighting made it difficult to air-condition the room. This was accomplished by placing a plate-glass ceiling between the lights and growing room, over which a wen-regulated layer of water flowed. A large fan also aided in cool- CONTROLLED ENVIRONMENTAL CONDITIONS 287 Table 29. With Electricity at 2c per Kilowatt Hour, What Was Sunlight Worth per Acre for Each Average Day During the Year 1936? {The figures are calculated from New York Meteorological data) Month Total gram calories/sq cm /mo Average kw hours/acre/day Average net worth /acre /day of sunUght May July June August April September March October February November January December 16,935 15,824 13,510 12,672 10,752 9,479 8,520 7,135 5,659 4,968 4,030 3,771 25,689 23,995 21,173 19,243 16,844 14,868 12,939 10,822 9,175 7,810 6,117 5,740 $514.00 480.00 423.00 385.00 337.00 297.00 259.00 216.00 184.00 156.00 122.00 115.00 Average for year 9,438 14,534 $291.00 ing by giving a rapid exchange of air about the lamps. WTiile Mazda lights have about 90 per cent of their energy in the infrared, this cooling reduced the heat ray to about 50 per cent, about the proportion of heat to light ray existing in sunlight. The light reaching the plants, however, was dominantly red-yellow, so mercury-vapor lights in glass tubes were burned along the walls of the growing chamber to increase the blue-\aolet rays. Even ^^'ith this enrichment, the light in this room was dominantly red-yellow as compared with daylight. With all 25 1500-watt lights burning, the intensity of the light in the gro^^^ng room was about 900 foot-candles. This is a low inten- sity compared with the maximum of sunlight at Yonkers at noon in June, which is about 10,000 foot candles. The light in the room, however, was constant for 24 hours of the day, whereas that in nature is markedly variable during the day, and is non-existent at night. ^\Tien 25 1000-watt lamps were used or when only a portion of the 25 lamps were on, the intensity of the light was lower. The same was true after the lamps began to age. To avoid the reduction in intensity due to aging of the lights, they were changed after 40 to 45 days of constant burning, or the experiments were limited to this period. During some of the experiments the intensity of the light in this room was as low as 350 foot-candles. In most of the experiments the temperature of the room was held at 78° F (26° C), but runs were also made at 68° F (20° C). This proved for- tunate in the study of the potato, as we shall see later. The CO2 in these rooms was run at ten times normal, or 0.3 per cent * and the relative * The CO2 enrichment of the air in the constant-condition rooms was always accom- phshed by use of tanks of Uquid CO2. Mostly the same was true of the greenhouses. For two years the source of CO2 for one of the greenhouses was scrubbed flue gas from the Institute boilers. To free the flue gases of all toxic or tarry materials, and at the same 288 GROWTH OF PLANTS FiGUKE 113. Constant condition light room. humidity largely at 80 per cent. All plants except those groAvn under con- tinuous illumination were kept on wheeled benches so they could be rolled from the light to the dark room and vice versa to get the light day and night desired. The plants were grown under 5, 7^ 12, 17, 19, and 24 hour daily time leave the OO2 in the gas, required a complex system of scrabbers. Planning and building the scrubbers and determining the chemicals and other materials to be used in the several units was a research problem in itself. Because of lack of space the reader ia referred to the original article for details.® CONTROLLED ENVIRONMENTAL CONDITIONS 289 S o o M c 03 CO a o Ci • vH CO >, ^ -o^ ___o^ o ®v, ^ "o n •V, ©"^ — s- ® ^^>" ^-.♦..,___ 13 — ■ • • • -.1 1 . 1 1 - t . T . 10 16 20 24 MILLIGRAMS SULPHUR PER SLIDE 32 100 T- Z o 5 5 u O z u u cc LU Q. 60 20 ® o NIAGARA 200 © ANSBACHER-SIE&LE | • NATIONAL 300-7 — 1 1 L ® 1 1 ' 1 1 0 2 8 0 32 NUMBER PARTICLES PER 100 MICRON SQUARE Figure 143. (Above) Toxicity curves, for three unmodified sulfur dusts of signifi- cantly differing mean particle size, plotted on a weight basis and showing a significant difference in toxicity. Germination of conidia of Sclerotinia fntdicola. (Below) The three upper curves plotted on the basis of particle numbers become one curve showing that the distribution or number of sulfur*particles determines the toxicity. most important copper fungicide. Bordeaux is a complex mixture of copper sulfate and hydrated lime, and despite much research its chemistry is still not entirely understood, though careful work by Martin -^ indicates that it is cupric hydroxide stabilized by adsorbed sulfate ions. The copper is present 368 GROWTH OF PLANTS in an insoluble form, and various theories have been advanced to explain its fungicidal action.^ These may be divided into three groups: (a) that atmospheric agents such as CO2 and rain liberate soluble copper; (6) that the fungus itself secretes materials which free the copper; and (c) that excre- tions from the host plant act on the insoluble copper. Studies were made of the action of fungus spores on Bordeaux ^^ and of the weathering of Bordeaux mixture. ^^ The amount of copper that goes into solution in distilled water above a dried deposit of Bordeaux was determined to be less than 0.3 ppm, an amount insufficient to affect mate- rially the germination of most fungus spores. Action of spores. Fungus spores were obtained by a vacuum technique which prevented their contamination by the nutrient medium.-^ These were suspended in water, allowed to stand for several hours, and filtered. The filtrates were then placed over dried Bordeaux mixture and agitated overnight. The amount of copper rendered soluble varied with the seven different species tested and was directly proportional to the number of spores. For example, the water extracts from 100,000,000 spores of the most active fungi, Uromyces caryophyllinus and Sclerotinia fructicola, brought into solution 1.01 and 0.76 mg of copper respectively, whereas the least active fungus, Alternaria solani, liberated 0.013 mg of copper. Determinations of total solids excreted by the spores showed that those species excreting the greatest amount of solids were also the most active in bringing copper into solution. It was also found that those spores most sensitive to the toxicity of Bordeaux mixture were in general the ones most active in solubilizing copper. By means of ultrafiltration tests it was further found that the active solubilizing material in the spore excretions is all in true solution. Also, practically all of it may be removed in the first washing of the spores. A quantity of spore excretion was obtained from approximately 360,000,000,- 000 spores of Neurospora sitophila, and an analysis indicated 3.1 per cent of malic acid present in the solid matter. There was also found 0.75 per cent of amino nitrogen, thus indicating the presence of amino acids. Since the spore excretions are practically neutral, their action cannot be due to any acidic properties. It is well known that certain amino acids form soluble complexes with copper oxide, and it was found that glycine and aspartic acid as well as neutral sodium malate will dissolve large amounts of copper from Bordeaux mixture. Finally, comparative toxicity tests of sodium cuprimalate and of a copper glycine derivative showed that these forms of copper exert substantially the same action as copper sulfate. Thus, in view of these findings, it is believed that the salts of hydroxy acids such as malic and perhaps others, as well as of amino acids present in the spore excretions, act on Bordeaux mixture to form soluble toxic copper hydroxy and copper amino salts.^^ Weathering action. The action of the spores in liberating soluble copper from Bordeaux mixture was of course demonstrated on glass in the labora- FUNGICIDES 369 tory; under actual field conditions other agencies such as rain may also be a factor. Although considerable research had been done on the action of weathering, possible changes which may occur in the copper-lime-sulfate ratio of Bordeaux under the influence of rain had not been studied. It was found that when glass plates are sprayed with an 8-8-100 Bor- deaux mixture and exposed outdoors, the sprayed film undergoes a change in composition under the leaching influence of rain and dew, leading to a mixture relatively richer in copper. ^^ This change in composition is accompanied by an increase in soluble copper. The highest amount ob- served was 0.45 mg per plate (225 sq cm), when the plate was agitated with 50 cc of water. Carbonation of the excess lime was complete in a few hours, as judged by pH measurements, but the increases in soluble copper did not occur until much later. The results could be duplicated in the laboratory using artificial rain, but only if a sprayed, dried film of Bor- deaux was used. Washing the Bordeaux precipitate in bulk by centrifuging, or on a Biichner funnel, did not lead to substantial increases in soluble copper. When Bordeaux mbctures low in lime were subjected to the leach- ing action of rain, soluble copper appeared sooner than with an 8-8-100 mixture. Treatment of the sprayed films "\\ith CO2, either wet or dry, did not lead to much increase in soluble copper. It is considered that the in- creases in soluble copper observed can be best explained by assuming that the weathered Bordeaux precipitate is an adsorption complex, or a solid solution containing copper, lime, and sulfate, the copper of which is soluble in water to an extent which varies with its composition. The appearance of small amounts of soluble copper must be considered as a factor in con- nection w^th foliage injury, as well as in fungicidal action where it may supplement the solvent action of spore excretions. The Laboratory Slide-Germination Method of Evaluating Fungicides Although the ultimate evaluation of fungicides must depend on their effi- ciency under actual field conditions, such tests are costly and time-consum- ing. It is most desirable to be able to test fungicides under the rapid and simple conditions of the laboratory and greenhouse and considerable effort has been devoted to developing such methods and to a clarification of the important factors mvolved.^- ^°' "■ i^' ^^- ^^- ^^- ^°' '^' 2"- 25. 26. 29. 30, 31, 32. 39 Theoretical principles. In the beginning of laboratory testing "^ little or no attention was paid to such factors as concentration or deposition and variability of results, while statistical interpretation was unheard of. Pioneering studies were undertaken in these matters. The relative preci- sion of spore germination tests was demonstrated,-" and it was pointed out that the errors arise from two sources, namely, faulty technique and random sampling; the former may be reduced but the latter cannot be avoided. Toxicity surface. When tests are performed with three variables of con- centration, time, and response, i.e., per cent germination, it is possible to 370 GROWTH OF PLANTS show the results in the form of a three-dimensional solid model, as illustrated in Fig. 144. The concept of a "toxicity surface," as is seen in the figure, was thus introduced.-^ It was further sho\vn that the form of the toxicity surface determines the precision of a toxicity experiment. Whether com- parisons of toxic agents are made on the basis of the times required for an equal percentage response, or on the basis of percentage responding in equal times is largely a matter of convenience, for at a given point on the toxicity surface both methods are capable of equal precision. Figure 144. The toxicity surface for the action of copper sulfate on conidia of Sclerotinia fr u cticola. The dosage-response curve. The germination of fimgus spores in the presence of toxic chemicals was sho\\Ti to be similar to that of toxicity curves in other fields and in general to give a normal distribution when plotted against the logarithm of the dose or concentration.^^ In making these calculations the newly devised "normal equivalent deviations" of Gaddum ^ were at first employed ^^ and then succeeded by the later "probits" of Bliss ^ which are fundamentally similar and now more gen- erally used. However, it was further sho^vn ^^' ^^ that in most cases rapid graphic methods of calculation will suffice and for this purpose the now widely used logarithmic probability paper (as may be seen in Fig. 145) was introduced into the fungicide field. Here spore germination or toxicity curves usually plot as straight lines and comparisons may be readily made, ordinarily at the most precise midpoint of 50 per cent response, that is, the LD50. Extensive studies have been made on the factors causing variation in FUNGICIDES 371 spore germination tests of fimgicides-^^- ". 26, 29 j^ analysis of 718 indi- vidual toxicity or dosage-response curves was undertaken.^^ Six different fungi and 20 different chemicals, both soluble and insoluble, were used. Four different types of slopes were observed on logarithmic probability paper: (a) orthodox simple straight lines, (6) double slope with left hand "break" in lower values giving a curve concave upwards, (c) double slope A^th right hand "break" in upper values or curve convex upwards, and (d) triple slope or sigmoid curve. The first three types are illustrated in 99.9 99 ^ 95 2 i tiJ O 90 80 ti. 70 O 60 2: O 50 40 30 ZO- IC 5 z UJ o UJ a. — , — p^T 1 r-. 1 ^ — ,-^^-,., , , , — ''''■■ Aq ; / / / / / / 1 1 1 / / f /Cd if / /"^ .^ Zn 1 1 1 1 1 / 1 ^lA' Mocrosporium sorcinaeforme ' 0.1 01 I 10 109 1000 R P. M. METALLIC ION Figure 145. Toxicity curves of heavy metals on Mocrosporium sarcinaeforme. Note that relative toxicity of the metals varies at the different inhibition levels due to differ- ences in slope of the curves. Each point mean of six repUcate tests. Fig. 145. The type and steepness of slope are determined more by the chemical than by the fungus. Differences in the slope of the dosage-response curve for two fungicides ^vill cause changes in their relative effectiveness at different levels of inhibition or control; this is especially noticeable if the curves cross each other. Since fungi vary in their sensitivity from day to day or season to season, not only in the laboratory but also in the field, conmionly observed differences in the relative rating of two fungicides at different times can be expected and explained. Error of replicate tests. It has long been observed that spores germinated at different times, though produced under seemingly standard conditions, vary -^ddely in their response. A portion of this variation was found to be due to the presence of variable amounts of water-soluble nutrient derived from the cultures when procuring the spores.^^ If the spores are obtained by a vacuum technique or are washed and centrifuged, they may be rid of the contaminating stimulant. However, in many cases it will be necessary 372 GROWTH OF PLANTS to substitute a known quantity of stimulant, such as ultra-filtered orange juice,^^' ^® in order to obtain a consistent and high percentage of germina- tion. Under these conditions fungicide tests replicated on the same day using the same lot of spores in general do not vary more than is to be ex- pected from their internal error, whereas tests replicated at different times with different lots of spores vary considerably more than is to be expected.^^ This is believed to indicate that the replicate test variation in the main is due to the use of different lots of spores, rather than to errors of technique in applying the chemical. These results have led to the conclusion that when comparing fungicides the replicate count or internal error should not be used as the error term but rather the compound X replicate test inter- action. Time and temperature. The effect of time and temperature has been determined for the germination of spores of Sclerotinia fructicola, Alternaria solani, Glomerella cingulata, and Macrosporium sarcinaeforme in water and in the presence of various chemicals.^^ No significant difference in precision could be shown between counts made at 6, 12, 24, 48, or 96 hours. A linear relation was found to hold between the reciprocal of elapsed time and germination expressed as probits for the spores of all the fungi when ger- minating in water at all temperatures from 10° to 35° C (50° to 95° F). The results with Sclerotinia fructicola are showTi in Fig. 146A. A similar relation held for germination in the presence of a given concentration of chemical, provided that concentration permitted germination, as shoAvn in Fig. 146B. A linear relation was also demonstrated for LD50 values when the logarithm of concentration was plotted against reciprocal of elapsed time. This curve is important in the estimation of the potency of a fungi- static agent, since compounds are rated differently at various times on the same fungus as in Fig. 146C; also, fungi may differ in their relative suscep- tibility to a single compound, depending on the elapsed time before counts are made, as may be seen in Fig. 146D. No significant difference in LD50 values could be demonstrated at 15°, 21°, or 27° C (59°, 70°, or 81° F) but there was a temperature effect at 10° and 35° C (50° and 95° F), where compounds were not rated in the same order. This temperature effect may in part explain differences in field performance of fungicides in dif- ferent seasons. Fungicidal vs. fungistatic. The fungicidal property of a material may be defined in general terms as the ability to kill or inhibit the development of fungus spores or mycelium. This is the sense in which it is commonly used and has been employed previously in this chapter ; however, in the restricted sense, "fungicidal" means the property of killing fungi, and ''fungistatic" the property of inhibitmg. A clarification and comparison were under- taken of the distinction between fungicidal (restricted) and fungistatic. ^^ A series of water-soluble chemicals was compared as to their relative action on four different species of fungi. Fungistatic action was determined by the usual slide-germination tests where the fungus remains throughout in con- FUNGICIDES 373 TIME IN HOURS - RECIPROCAL SCALE FiGTTRE 146. Time, temperature, and fungicide dosage curves for spore germination. A, Time-germination curves at different temperatures for Sderotinia frudicola spores in water. B, As A except in presence of fungicides, dosage in micrograms Cu per sq cm, 21° C (70° F). C, Time-LD50 curves for different fungicides against Alternaria solani showing difference of slope. D, Time-LD50 curves for Bordeaux mixture on different fungi. 374 GROWTH OF PLANTS tact with the chemical. For fungicidal tests the fungus spores were allowed to remain in contact with the chemical for different periods of time, after which the chemical was removed by centrifuging and decanting and the spores were washed and allowed to germinate in water. As fungicidal activity cannot exceed fungistatic activity, three kinds of response are possible. Thus certain chemicals such as silver nitrate and copper sulfate show both high fungistatic and fungicidal activity; others exhibit high fungistatic but low fungicidal, such as formaldehyde and phenol; and stUl others are low in both properties. Hence the correlation between fungicidal and fungistatic properties is high except for the second group. The fungi- cidal dosage-response curves, like the fungistatic, tend to plot as straight lines on logarithmic probability paper. However, in many cases the fungi- cidal curves are decidedly flatter. These fundamental studies on spore germination tests of fungicides, espe- cially the dosage-response curve, have not only encouraged and advanced laboratory testing but they are also stimulating comparable studies under actual field conditions and are resulting in new concepts regarding the com- parison of fungicides in the field.® The standard method. The slide-germination method as now developed has been accepted as a standard by the American Phytopathological Society.^ The method is designed to evaluate the fungistatic properties of protectant fungicides. Spores of certain fungi are placed on glass shdes in water in the presence of the chemical to be tested. All conditions are rigidly controlled and, except for the chemical, are favorable for spore germination. The chemical may be applied either by the test-tube dilution teclmique or by the horizontal sprayer or settling-tower technique.'® The results are expressed as concentration of chemical necessary to inhibit the germination of 50 per cent of the spores, i.e., the LD50, or as the LD95; the former is more precise. Settling tower. Prior to the development of the standard method, a special study was made of the method of applying the fungicide, and the settling tower as finally developed constitutes the most precise method of applying insoluble fungicides to glass slides. The tower is illustrated in Fig. 147. Greenhouse Methods of Evaluating Fungicides 'Recently, attention has been devoted to developing greenhouse methods of testing as an intermediate between the laboratory and field tests. This has seemed particularly desirable because of the specificity of the newer fungicides, especially among the organic chemicals. Tomato foliage diseases. The tomato foliage diseases,^® early and late blights, caused respectively by Alternaria solani and Phytophthora infestans, have been found particularly suitable for this purpose, in addition to repre- senting important economic diseases caused by members of the Moniliales and Phycomycetes. The leaf spot (Septoria lycopersici) has also been used but has not been found as desirable. The tomato plants are sprayed with FUNGICIDES 375 Figure 147. Permanently installed stainless steel settling tower for the pre- cision application of fungicides to glass slides. A, Ringstand atomizer apparatus on steel runway; B, reducing valve on air pressure line; C, tray of slides; D, self- closing door through which tray is inserted; E, sliding glass window for draft; F, levers for opening and shutting top of settling chamber; G, upper window for adjustments on top, sprinkler nozzles, etc.; H, laboratory exhaust duct; I, water line to sprinkler nozzles for washing out; J, drain valve. 376 GROWTH OF PLANTS Figure 148. A, Spraying systems. Upper left: low pressure for inoculation; upper right: high pressure for applying fungicide. Note swinging arm mounting spray gun, height adjustment, attached stirrer, and rimmed plate holding beaker containing fungi- cide. B, Spraying tomato plants on compound turntable in hood, with fungicide at 40 lbs pressure. FUNGICIDES 377 the chemical under controlled conditions by means of a paint spray gun, as shown in Fig. 148. After drying they are inoculated with a known concen- tration of fungus spores produced under standard conditions and placed m high humidity infection chambers at controlled temperatures (see Fig. 149) for 24 hours, then removed to the greenhouse. Necrotic lesions develop in Figure 149. Insulated, temperature-controlled humidity infection chambers. Atomiz- ing jet nozzles with water and air pressure line for maintaining high humidity enter at upper sides of each chamber, two may be seen above the chamber letters B and C. Refrigeration machinery for B at lower left. The chambers are fitted with a movable shelf in center, plate glass false ceiling below nozzles to prevent direct water spray on plants, and fluorescent Hghts on top. several days, are counted, and expressed as per cent of the number of lesions on the control plants. Prior to inoculation, the plants may be sub- jected to several cycles of growth, high humidity, and laboratory "rain" in order to determine the tenacity of the chemical being tested. ^^ ^Yhe details of this method are well worked out and for a greenhouse procedure are considered precise. Special attention is required in order to ensure an adequate supply of pathogenic Alternaria solani spores; this is accomplished by scraping the Petri dish cultures and irradiating for 20 seconds under ultraviolet lamps transmitting to about 250 mju.^^ When the number of lesions is expressed as a per cent of the check, the dosage results may be plotted on logarithmic probability paper and, like the spore germination curves, they tend to give straight lines; however, the former are much flatter. Perhaps this may indicate that when fungi- 378 GROWTH OF PLANTS cides are applied to the foliage of plants they act in a fungicidal rather than a fungistatic manner. For the first time an absolute and direct com- parison was made of the action of the same fungicides on the same organ- ism, Alternaria solani, at the same spore concentration on glass slides and on the leaves of a growing plant. ^'^ The results of this interesting compari- son are shown in Fig. 150. It will be seen that the LD50 levels are about equal, but as noted above, the greenhouse or leaf curves are much flatter; however, the same fungicide Thiosan (tetramethylthiuram disulfide) was the more toxic in both methods. is; 0.1 .0? /- X o 25 - 20 - 15 10 - - 1 0 1 1 ) 1 1 1 1 1 1 1 1 0 y t '—<'>— ^""Vnf -—Ui / / ; - / f - ~ J 1 f 1 . 1 1 . ,..! 1 1 1 1 1 1 1 16 24 HOURS 32 40 48 Figure 161. Influence of CO2 on the rate of oxygen uptake by dormant Irish Cobbler potatoes at 25° C (77° F). Efifect of carbon dioxide in the air on the ascorbic acid (vitamin C) con- tent of fruits and vegetables. Carbon dioxide in the surrounding air induces a great fall in ascorbic acid in some fruits and vegetables. Fig. 162 shows the effect of various percentages of CO 2 in the atmosphere at 22° C (72° F) for 24 hours on the ascorbic acid content of asparagus sprouts. ^^ The loss of ascorbic acid rises as the concentration of CO 2 rises until in 60 per cent CO2 about one-half the ascorbic acid is destroyed. It will also be noted that the pH of the tissue rises from about 6.2 to about 7.1 pH as the CO 2 concentration rises from 0 to 60 per cent. There probably is a causal relation between rise in pH and fall in ascorbic acid, since the latter is protected by acids and is readily oxidized in basic solutions. Upon MISCELLANEOUS 407 removal to air the sprouts return to the original pH, but there is no increase in the ascorbic acid content. The CO 2 has a similar effect at all tempera- tures from 2° to 27° C (36° to 81° F) and the fall is greater m the buds than in the sprouts. Since, as shonii by the curve, even low concentrations of CO2 cause a marked destruction of ascorbic acid, no doubt the accumulation of CO 2 due to respiration in the packages during shipping and marketing leads to a great loss in the vitamin C content of the sprouts by the time 10 I4. O o c UJ Q. z UJ o >- X o a o < m a o o 10 < ii. o eb 2 PERCEJMTAGE OF CARBON DIOXIDE Figure 162. The effect of CO2 on the ascorbic acid content, rate of respiration, and pH of asparagus tissue during storage of 24 hours at 22° C (72° F). The oxygen uptake is mg of O2 per 100 g of tissue. they are sold. This curve shows that the oxygen absorption rate (mg of O2 per 100 g of tissue) of asparagus sprouts is reduced by CO 2, a fact mentioned above. Carbon dioxide ^" causes marked reduction in the ascorbic acid content of green bananas with four days' storage at 19° C (66° F); 60 per cent CO2 gives 66 to 85 per cent reduction, even 16, 24, and 40 per cent give great reductions and concentrations of 3 to 5 per cent cause 10 to 20 per cent reduction. Carbon dioxide has little effect on the ascorbic acid content of fruit in the yellow ripe stage. At any stage of ripening where CO2 causes a reduction, the ascorbic acid rises back to normal some time after the fruit is removed from the CO2; also fruit kept in CO2 from the green stage to complete ripening ended up with about the same ascorbic acid content as fruit brought to the same stage of maturity in air bearing no CO2. Carbon dioxide caused the juice of bananas to become more alkaline by 0.2 408 GROWTH OF PLANTS to 0.5 of a pH, but the juice is always acid, pH 5.9 being the highest recorded after four days in 60 per cent CO2. Ethylene, which is a ripening hormone produced by bananas during maturity and is also sometimes added to the air to hasten ripening, did not modify the ascorbic acid content. Carbon dioxide ^^ does not modify the ascorbic acid content of apples or green snap beans but does reduce the ascorbic acid somewhat in freshly harvested potatoes rich in this acid and in green pod peas, but has no effect on the ascorbic acid in potatoes after a long period of storage. It is interesting to find CO 2, a naturally existing chemical environment of plants, having such marked effects upon certain metabolic processes here studied. It is likely that CO2 modifies many other metabolic processes not included in these investigations. The effects produced by CO 2 are strikingly like those produced by ethylene chlorhydrin which are discussed in Chapter 7. Diurnal and Autumn Changes in Leaves of Deciduous Plants Diurnal changes. Denny "• i^. 20 j^ade a study of the diurnal changes in leaves and examined critically the several methods that might be used for accurately determining these changes. Offhand, so far as methods are concerned, the problem looks simple. If one wants to find out what changes occur in leaves during the night he can take a sample of leaves at dusk, a similar sample at da^\^l, and analyze the two for the various organic con- stituents making up the two samples. For the day changes, the first sample could be taken at dsivm and the second one at dusk. The main problem is to make sure that the two samples to be compared are comparable except for the changes the 12 hours of darkness or light bring about. Denny's work shows that getting comparable samples and a correct basis for calcu- lation of the results are far from simple. He describes four different methods that give reliable data, which, of course, give agreement in the changes obtained by the four methods. He perfected the twin-leaf or, in compound leaves, twin-leaflet method. His later studies with this method were made on mature leaves or leaflets in order to avoid changes in weight involved in growth. In appljdng this method, kinds of plants were selected in which the opposite leaves are of approximately the same size. A sufficient number of the leaves are used for each sample to give a low error due to possible]variation in size (25 to 300 per sample), and care was taken that on the average the two leaves of the pair received equal light exposure. One leaf of each pair was used for the first sample and the other was collected at a later hour for determining the changes brought about by the day or night exposure. Since, as the data show, the two samples were practically identical except for the changes caused by the day or night exposure, the changes in each constituent are reported in amount for the whole sample mthout reference to any measure- ment that might vary with exposure, such as wet or dry weight, or area. MISCELLANEOUS 409 The t^^Tll-leaf method — as do the other three methods, as we shall see later — shows that the mam changes m mature leaves with night and day- are changes in assimilable carbohydrates, soluble sugars, and starch (deter- mined by the acid hydrolysis method). The acid hydrolysis undoubtedly breaks dowm some other polysaccharides besides starch. During the day photos;yTitliesis leads to the accumulation of starch and sugars in the leaves, which increases the dry weight of the leaves. At night the starch is partly or wholly digested and the sugars and starch in part used up by respiration of the leaf and in part transported to other regions of the plant. Dermy's data show that the total nitrogen of the leaf did not change measur- ably from day to night. He finds that a modification of Sachs' half-leaf method gives results that check ^^•ith the twin-leaf method. Sachs' method consisted in cutting a given area from one-half of many leaves for the first sample and a like sur- face from the other half of the leaves for the later comparable sample. Two main criticisms of this method have been offered: the mutilation of the leaf due to removal of the first sample modifies the processes going on in the other half of the leaf, and the two halves of the leaves are not sym- metrical. Consequently, equivalent tissue is not used in the two cases. Another error m this method has been pointed out: the water content of the leaf generally increases during the night and with it the area of the leaf; consequently, the morning sample, while it has the same area, has less tissue. Dermy's modified half-leaf method consists in selecting plants that have symmetrical leaves and cutting off one-half of the several leaves close to the midrib for the first sample and using the other half, discarding the midrib, for the later comparable sample. In sampling, the right and left halves of the leaves were taken alternately for the first and, of course, the remaining halves for the later sample. Here, as in the twin-leaf method, the several constituents were determined on the total amounts in the samples and need not be related to surface or to wet or dry weight, all of which are variables. Unlike the twin-leaf method, tliis method can be used on plants with alternate as well as opposite leaves. Denny concludes that when the half-leaf method is used with proper precautions it gives reliable results. Apparently the disturbance due to mutilation is not as great as has been assumed. As stated above, the dry and wet w^eights, the assimilable carbohydrates, and the total leaf surface in mature leaves vary with day and night. There are, however, tw^o fractions that remain constant in mature leaves during day and night, according to Denny's findings: residual dry weight (the total dry weight minus the assimilable carbohydrates) and the total nitro- gen. Mason and Maskell ^^ had already used the residual dry weight as a basis for calculating changes; and Denny confirms the constancy of this fraction and justifies its use as a basis for calculating leaf changes. While Denny finds in the leaves that he studied that the total nitrogen is a con- stant, he warns that w^orkers ought to determine that it is a constant in 410 GROWTH OF PLANTS the particular leaves being studied before they use it as a basis for calcu- lating leaf changes. If one has complete analyses of two similar samples of leaves taken, say at dusk and dawn, he can calculate the percentage change in any constituent during the night on the basis of either of these fractions and get a correct picture. In this case -the absolute amount of change in like samples will not be compared, but rather the percentage change based on one or the other of the two constants. In using these two methods of calculation one is not limited to twin-leaf or half-leaf sampling, but can compare any tw^o similar samples of leaves taken at dusk and at dawn or for any other period studied. Table 47. Comparison of DitTerent Methods of Computing Changes Period Per cent loss Plant Twin-leaf basis Residual-dry- weight basis Total nitrogen basis Total carbohjdr. Total sugar Total carbohydr. Total sugar Total carbohydr. Total sugar Stringless bean 5:40 P.M. to 9:20 P.M. 6:30 P.M. to 2:00 A.M. 7:20 P.M. to 5:45 A.M. 23.0 43.3 58.3 15.1 14.3 30.2 22.9 42.7 57.5 15.1 13.8 28.5 23.9 42.8 56.9 16.1 13.6 27.6 Cutshort bean 5:50 P.M. to 9:30 P.M. 6:40 P.M. to 2:20 A.M. 7:30 P.M. to 6:00 A.M. 24.8 49.7 57.2 22.5 19.6 44.6 26.6 50.3 58.8 24.2 20.5 45.6 24.3 50.7 58.8 21.6 21.2 45.4 Peanut 6:00 P.M. to 9:40 P.M. 6:50 P.M. to 2:40 A.M. 7:40 P.M. to 6:40 A.M. 6.3 12.4 17.2 13.9 14.0 15.8 6.3 13.1 18.0 13.7 14.6 16.7 5.6 12.7 18.0 12.9 14.0 16.3 We have already said that the twin- and half-leaf methods give com- parable results. Table 47 shows that the twin-leaf, the residual dry weight, and the total nitrogen methods give comparable results. These analyses are made on the basis of the twin-leaf method, and the results are then calculated on the basis of residual dry weight and the total nitrogen. For each of the three kinds of leaves reported in the table, three different com- MISCELLANEOUS 411 parable pairs of samples were taken: one to show the change taking place from evening until early in the night; another to show the change taking place from evening until late in the night; and a third to show the changes occurring during the whole night. In the first column under each method of calculation is shown the percentage loss in total carbohydrate (starch by acid hydrolysis method plus soluble sugars) and in the second column the loss in soluble sugars. One will see by following along the several lines that the three methods of calculation give comparable results both as to the total carbohydrate and soluble sugars. Followmg down the columns for each kind of plant makes it evident that the percentage of loss in both total carbohydrates and sugars increases as the night progresses. If one compares the two beans with the peanut, he will see that in the beans a much larger percentage of both constituents is lost during the night than in the peanut. An examination of the original analytical data, not here given, shows that the starch in the several leaves in the evening exceeds by five- to ten-fold the amount of soluble sugars, and that peanut leaves are richer in both starch and sugar than the bean leaves. All these leaves are starch storers. In leaves that do not store starch, as is the case with onion, the change, of course, is in the sugars. in V) o \- z u u q: ui Q. 10 - 20 - 30 - 40 • 50 60 70 - ^\r^ 1 1 1 " x^ - • ^ ^ - - ^^ ■^■^s,.^ - ® Peanut • Stringless Bean ^--...^ O Cutshort Bean 1 1 1 .. -1 1 1 p 1 • 6 PM. 10 II 12 TIME 3 A.M. Figure 163. Per cent loss of polysaccharides (alcohol insoluble acid hydrolyzable substances, including starch) of peanuts and beans at intervals from night to morning; percentage computed on the basis of the amount present in the initial sample taken in the evening. Fig. 163 shows the percentage loss in the polysaccharides in the bean and the peanut leaves during the three periods of the night, and Fig. 164 shows the amount of total carbohydrates in the same leaves at various times during the night calculated on the basis of residual dry weight and total nitrogen. 412 GROWTH OF PLANTS 200 (- 150 X o ui > O _l < 9 250 10 uJ - I o m < too 200 150 100 425 375 325 Sfringless Be- I o m < u Figure 164. Amounts of total carbohydrates found in peanut and bean leaves at intervals during the night. Comparison of residual dry weight and total nitrogen as bases for calculating the change in total carbohydrates. Fig. 165 shows the amount of sokible sugars found in the bean and peanut leaves at various periods during the night. All these are plotted on the basis of data given in Table 47. The diurnal changes in leaves of several other plants (tobacco, salvia, sunflower, hawthorn, redbud, lilac, Virginia creeper, peach, soybean, cotton, grape, etc.) are reported using the twin- or half-leaf method of determination. In general, herbaceous plants show much greater diurnal changes than woody forms. The night changes in lilac are not measurable, those in hawthorn small, those in Virginia creeper and grape considerably larger, while in some herbaceous plants, as the data reported on the two kinds of beans show, more than 50 per cent of the starch and sugars dis- appears from the leaf during the night. In the peanut under similar condi- tions the loss in these two constituents is less than 20 per cent. Autumn or pre-leaf fall changes in deciduous leaves. Denny ^i used the twin-leaf method of sampling to study the chemical changes occurring in MISCELLANEOUS 413 35 30 25 20 h I o ui > Q _J < g 30 UJ IT 25 20 O d K UJ a. < o D -J ? 10 40 35 30 Strin|lts9 inn Cutihort B»n • Rtsid Dry Wr. iivi O Tot-al Nitrogen Bisii O Pfanut 3 AM. TIME 065 0.55 045 035 0 50 0 40 030 0 20 0 75 O IT I- < I- O 1- o UJ 0. < o _J < O O Figure 165. Amounts of total sugar found in peanut and bean leaves at intervals during the night. Comparison of residual dry weight and total nitrogen as bases for calculating the change in total sugars. deciduous leaves during a period of 41 days previous to autumn leaf fall. His methods, results, and conclusions are stated by him as follows: p-^" "Samples of 50 pairs of opposite leaves of Viburnum dentatum and Syringa vulgaris were selected to measure the changes in leaves at intervals of three to five days from September 24 to November 4, at which time the experi- ment was ended because of frost. One leaf of each pair was taken at the beginning and the other was left on the plant until the end of each interval, there being ten such intervals during the experimental period. "Dry weight, sugars, polysaccharides (alcohol-insoluble substances hydrolyzable \\'ith dilute acid), and nitrogen were determined, and calcu- lations were made upon three bases: per cent of the dry weight, per cent of the residual dry weight (obtained by subtracting from the dry weight the sum of the carbohydrates and 6.25 times the nitrogen), and the total amounts of constituents in 50 leaves. "The dry weight of the leaves was nearly constant throughout the period 414 GROWTH OF PLANTS of sampling, and no important change was observed in total carbohydrate (sum of sugar and polysaccharide) . Previous reports of extensive losses of substances from leaves during the interval preceding frost (autumnal migration) were confirmed only for the nitrogenous substances in these species, and, even in this case, only the Viburnimi results should be empha- sized, as the nitrogen losses from lilac were small and of doubtful signifi- cance." Concerning the Determination of the Isoelectric Point OF Protoplasm Attempts had been made to determine the isoelectric point of living plant tissue by placing pieces of the tissue in buffer solutions of low con- centration but of various H-ion concentrations. The buffer solution in which the tissue did not change the pH was interpreted as the isoelectric point of the tissue. It was assumed that in this pH the amphoteric substances (especially proteins) of the protoplasm did not react differentially mth either basic or acid ions of the buffer solution and that consequently the pH of the buffer solution was not changed. It was assumed, on the other hand, that if the buffer solution had either a higher or lower pH than the living tissue the amphoteric substances reacted differentially with basic ions in the first case and acid ions in the second, moving the pH of the buffer toward that of the tissue. Youden and Denny ^^^ find that it is the substances that leach out of plant tissues when soaked in the buffer solutions (phosphate, phthalate, and borate) that mainly determine the change m the pH of the solutions, and not the ion absorption from the solutions by the insoluble amphoteric substances of the protoplasm. Tissue soaked in distilled water gave solu- tions of the same pH as the buffer solution that was not changed in pH when the tissue was soaked in it. The substances that leach from the tissue and mainly determine the pH of water or buffer solutions are heat-stable, diffuse through collodion membranes, and are soluble in acid alcohol. This shows that proteins or other colloidal substances do not play an impor- tant role in causing the change in pH. Fig. 166 shows how several plant tissues soaked in buffer solutions of a considerable range of pH values change the pH of the solutions in the direction of the pH of water m which the tissue has been soaked for the same length of time. One exception is mentioned in the description of the figure. The authors make the following statements: ^^^^ ^^^^^ °^ p-^^^'" ^■^- ^- ^ "Most of the effect upon the buffer solution was not due to absorption of ions from the buffer by the tissue, but was caused by substances leaching out of the tissue into the buffer. On the acid side of the isoelectric point only about 5 per cent of the change in reaction undergone by the buffer was caused by the tissue itself; on the alkaline side the tissue was more effective, causing about 25 per cent of the change." ^'''-'''^ - p.286-287 in "It is not our purpose to claim that plant tissue does not contain C. B. T. I. MISCELLANEOUS 415 3\0 I 4.|0 I 5|3 ' dp ' 7.\o ' 3^ ' ?lo /"/y Apple Tissue - Thin Slices I Hour J • ■H • ♦— — — — • • 5=5. • » Cor/7 Seeds - /Mr Dry l/l- Hrs. Corn - Seed Powder Monilia Sitophilia "* l^at of H\/phae - >? Mrs. ^ tgU ^ Potato Tuber -Thin Slices I 4- /yr5. 4 /Vr5. _, Potato Tuber Thin Slices 3 Hrs. Rye Seeds ■• /I/r Dry - 3 Hrs. Wheat Seeds "• /J/V- Dry -5 Hrs. Potato Roots 3 Hrs. _, Bar/ey Roots 3/2 Hrs. 3\0 I 4|0 .1 5\0 I 6\0 I 7|0 8\0 9.\0 pH Figure 166. The relation between the pH equilibrium of tissues in a series of buffers and the pH of the water extracts of these tissues. The arrows show the pH of the buffer in which the sample of tissue was placed, the direction of change in pH, and the final pH attained. The vertical lines show the pH of the water extracts of the tissues. It is seen that the isoelectric point of the tissue as determined by this method coincides with the pH of the water extract in all tissues except in the case of corn seeds, air-dry whole seeds. substances with isoelectric points, nor that these substances are not of great importance in the Ufe processes of plants. Nor do we claim to have shown that the tissue itself does not have an isoelectric point. Robbins and his co-workers have brought evidence by other methods of experimentation (water absorption, toxicity of ions, staining of tissues, etc.) regarding the existence of such a point. "Our objection is mainly to the method of determining tissue isoelectric points by immersing the tissue in a series of buffers and assuming that the 416 GROWTH OF PLANTS pH value at which no change in reaction is shown is the isoelectric point of the tissue. "Furthermore, it is unlikely that the equilibrium point of a tissue in a series of buffers represents the isoelectric point of the proteins of the tissue. Thus the equilibrium point for potato tissue is about pH 6.4; but the iso- electric point for tuberin, the principal protein of potato, is about pH 4.0 according to Cohn, Gross, and Johnson. Pearsall and Ewing find that when the tissue is made as acid as, or more acid than, the isoelectric point of the principal protein in the tissue there is a rapid exosmosis of ions, indicating a serious injury to the tissue. The point for potato at which rapid exosmosis of chlorids took place was not at pH 6.4 but at about pH 4.4. "Chibnall found that the pH of the cell contents and the isoelectric point of the cytoplasmic proteins are not identical in any tissue he studied, and points out the probability that any change in the reaction of the cell Avhich brings the proteins to their isoelectric points Anil result in the death of the cell. The buffer capacity of the cell contents protects the cell against injury by tending to prevent the [H+] from reaching the isoelectric point of the proteins of the cytoplasm." Rudolfs ^^ finds that when any given kind of seed is soaked in solutions of various salts and organic and mineral acids, the solutions (except for very dilute solutions) all change to the same pH, which he terms the iso- electric point of the particular seed. He believes that the change is brought about by ion absorption by the amphoteric substances, mainly proteins of the living portions of the seeds, and not by substances leaching out of the seeds. In grains it is chiefly due to the embryo rich in proteins rather than to the endosperm, which is mostly carbohydrates. The pH equilib- rium point for corn grains is at 3.9 to 4.1 pH, for Phaseolus vulgaris seed at 5.5 pH, and for Lupinus albus seed 4.7 pH. Scott ''* finds similar results by soaking living mycelium of Fusarium lycopersici in unbuffered and buff- ered salt solutions with the equilibrium point at 5.4 pH. Dead mycelium did not give a definite equilibrium point. He interprets this as the iso- electric point of the main proteins of the mycelium. Denny and Youden -^ placed various plant tissues (thin slices of potato tubers, carrot roots, and apple fruit, whole seeds of corn, rye, and wheat, and corn seed-powder) in solutions of various salts ranging in concentra- tions from O.lilf to O.OOlilf . Samples of the salt solutions were removed after various periods of soaking and the pH determined. In all cases where a change in pH occurs, the change is in the acid direction. Salts of the monovalent cations, sodium and potassium, give slight or no shift in acidity. Salts of the bivalent cations, calcium, magnesium, and strontium, give decided shifts toward the acid; those of the bivalent cation, zinc, still greater shifts; and finally those of the bivalent cation, copper, and of the trivalent cation, lanthanum, give very large shifts toward the acid. The amount of acidulation increases with the concentration of the solutions, especially in the lower ranges of concentration. The final pH equilibrium MISCELLANEOUS 417 Figure 167. Ordinates show the pH values of the external solution when 25 grams of tissue were immersed in 25 cc of various salt solutions of the concentrations shown as abscissae. In the graph for rye seeds the salts listed in the shaded areas gave similar results, all the data for these salts falhng within the limits shown. Note that each tissue did not bring the external solution to definite pH values, but that the values obtained varied with the salt used, with the concentration of the salt, and especially with the nature of the cation. reached varies with both the concentration of the solution and the nature of the cation, which means that pH was not constant and could not repre- sent the isoelectric points of the living cells. All these findings are shown by Fig. 167 for potato tissue and rye seeds. It will be noted that high 418 GROWTH OF PLANTS concentrations of copper salts for potato tissue and of copper salts and LaCls for rye seeds reduced the pH to 3, far below any value that is claimed for the plant protems of these plant organs. Salt solutions are similarly acidified when water extracts of plant tissue are added to them; and this occurs even when the extracts are boiled or dialyzed to precipitate proteins and remove all colloids from the extracts. When salts were added to organic acid solutions (malice, oxalic, succinic, aspartic, etc.) there is an increase in hydrogen-ion. Additions of CaCl2 to salts of organic acids increased the acidity in all pH values from 2 to 7.5. The increase in hydrogen-ion appears whether the salts of the organic acid were or were not precipitated. Additions of CaCl2 to phosphates and pectin increased the hydrogen-ion. The addition of CaCl2 to that portion of potato extract containing the protem tuberin shows that the change in hydrogen-ion is not analogous to the change caused by addition of potato tissues. From a consideration of the results of all their experiments, the authors draw the following conclusions : ''- ^■'''- ^'^ ""'' ^° ^- ^- '"■'■■ "Although our results do not show that the tissue itself or the proteins take no part whatever m these changes m pH, they indicate that the solu- ble, non-protein, non-colloidal substances which diffuse out of the tissue into the salt solution and which then react with it are important factors in the acidifications that are produced in the external solution. "The observed changes in hydrogen-ion concentration, therefore, can not be interpreted as indicating an isoelectric point for the tissue as a whole, nor furnish proof that reaction has occurred between the ions of the salt solution and proteins with characteristic isoelectric points." Growth Substances and Vitamin Bi for Seed Treatment One commercial firm has been vigorously promoting the use of plant hormones for seed treatment, claiming that the treatment greatly mcreases crop yield. Investigators at this Institute and most, though not all, of many investigators of the subject elsewhere have found no advantage in hormone treatment of seeds. Barton ^ soaked non-dormant seeds of twenty-nine different species and varieties of farm, garden, and flowering plants and grasses in a wide range of concentrations (320.0 to 1.2 mg per liter of water) of three different growth substances (|3-indoleacetic, /3-indolebutyric, and o;-naphthalene- acetic acids) and grew them to maturity in soil. Except for higher concen- trations which ui some cases inhibit growth or cause malformations, the treatments had no effects, either on germination or final yield. She also treated seeds with several proprietary plant hormone preparations, mainly talc dust mixtures, with similar results. Dormant American elm seeds i" soaked in various concentrations of potassium a-naphthaleneacetate showed some improvement in germina- MISCELLANEOUS 419 tion, but this treatment was ineffective compared with low-temperature stratification or ilkmiination of the seeds while under water. Treatment with growth substances had no beneficial effect on the germination of dor- mant seeds of a variety of domestic apple and a species of crabapple. Low- temperature after-ripened seeds of Cornus and Pyrus were thro^^•n back into dormancy by treatment with growth substances. Growth substances did not force the growth of the naturally dormant epicotyls of seeds of Paeonia, Viburnum, or lily., Youden ^^- p--^^ describes a set of experiments in which he treated wheat and soybean seeds with growth substances. ''WTieat and soybean seeds were treated in the dry state with indoleacetic acid, naphthaleneacetic acid, and indolebutyric acid, talc, and Rootone (commercial preparation) and gro\vn in sand and soil in the greenhouse and in the field. The con- centration of the organic compounds in the talc preparations, as well as 'the proportion of powder to seeds, was adjusted to cover the range 0.5 to 240 parts per million of active substance by weight of the seeds. In a series of ten experiments observations were made of the germination, seedling height, wet weight of tops, yield of grain, and root systems, and no significant case was found in which the germination and growth of the treated lots exceeded the controls. On the contrary the aggregate of evi- dence points to slightly lower values for the treated lots, and indicates that these are the result of the presence of the talc. Nineteen contrasts of talc-treated seeds with controls gave fifteen cases in w^hich the controls were superior and one tie. On the average, the excess weight of the con- trol plants was about 5 per cent." An experiment was conducted to test the report that treatment of grass seeds with plant hormones stimulated the growth of lawns. Fig. 168 shows that there were no beneficial effects on any of the 14 plots tested. In 1939, Better Homes and Gardens published an article announcing the remarkable stimulative effect of vitamin Bi on green plants. It was said to produce veritable giants out of garden and house plants and to make trans- planting entirely safe when plants were m full bloom. The article would lead one to think that the greatest discovery of modern horticulture had broken upon an unexpecting world. Investigators ^^' ^'^^ at this Institute as well as most of many investigators elsewhere who have worked upon the problem have found that vitamin Bi has no such remarkable effects upon growth of higher green plants — indeed that it has very minor, if any, effects. The B group of vitamins is an important accessory factor in the growth of various non-chlorophyllous plants, such as yeasts and fungi. If vitamin Bi is important for the growth of higher green plants it is likely that these plants synthesize an adequate supply to meet their needs. Zimmerman ^°^' p-^'* says: "The preponderance of evidence from scien- tists is opposed to the use of vitamin Bi for practical purposes. My own convictions, based on our experience at the Boyce Thompson Institute and 420 GROWTH OF PLANTS 12 3 4 5 6 yT^'T'^rm'y»^'''V''aii'j^iW»t»f«|>qywCT^^ ■ i^v^tr^^^ Figure 168. Seed (Scott's lawn mixture) treatment involving three of the most effec- tive synthetic "plant hormones," a commercial preparation (Rootone), and talc. The chemicals were mixed with talcum powder and the seed dusted with the preparations. The concentrations were at the rate of 1, 2, and 3 mg of the substance per gram of talcum powder. Top rows: 1-3, three different concentrations of indoleacetic acid; 4, talc control; 5-7, three different concentrations of naphthaleneacetic acid. Lower rows: 8, non-treated control; 9-11, three concentrations of indolebutyric acid; 12, talc control; 13, "Rootone," commercial preparation; 14, non-treated control. the published results of other scientific laboratories, are that vitamin Bi has been hadly exploited, and that it has no value as recommended for horti- cultural practice." Importance of the Mother-Tuber in the Growth of the Potato Plant Denny i^- ^® undertook to determine the importance of the mother-tuber to the development of the potato, also how long it must be retained on the vine to insure maximum growth of vine. He developed a clever technique for removing the mother-tuber from the plant at any stage of develop- ment without seriously disturbing the soil or interfering with the root sys- tem that had been established up to the time of removal. This method is shown in Fig. 169. The mother-tuber was removed at four different stages of development MISCELLANEOUS 421 Figure 169. Method of amputating mother-tuber from sprout. Left: plant grown in pot buried in soil; right: plant inverted, placed upon board with slot from edge to center used for support; pot then discarded. Note mother tissue being removed piece by piece with scalpel; plant placed in soil after removal of mother-tuber. of the plant: (stage 1) when the plant was just emergmg from the soil; (stage 2) when the germination was complete and the leaves were expanded; (stage 3) when the plant was 10 mches above ground and tubers w^ere set; and (stage 4) when the plants were at nearly maximum height and flower- ing. The various stages of development when the mother-tubers were removed are shown in Fig. 170. Removing the mother-tubers at stages 1 and 2 reduces the yield of both Irish Cobbler and Bliss Triumph plants, and the reduction is greater witb 56- than with 14-grara seed-pieces. Removing mother-tubers of Irish Cobbler at stage 3 reduces the yield when 56-gram seed-pieces are used by 14 per cent, but not significantly when 14-gram seed-pieces are used. Bliss Triumph plants are not affected significantly when the mother- tubers are removed at this stage whether the seed-piece is 56 or 14 grams. Removal of mother-tuber at stage 4 did not reduce the yield of either variety for either size of seed-piece. In fact, a slight increase is shown which Denny, in agreement with previous mvestigators, attributes to toxic substances from some of the seed-pieces that rot late in the season. Chemical analyses were made of the mother-tuber seed-pieces at the beginning and at the other stages of development. Table 48 shows these 422 GROWTH OF PLANTS Figure 170. Conditions of plants at four different stages at which mother-tubers were amputated: A, stage 1; B, stage 2; C, stage 3; D, stage 4. analyses. The data in this table are given on the basis of the percentage of the original total amount of each constituent remaining in the seed- piece at each stage of development. Large and rapid losses in dry weight, starch, and nitrogenous substances are shown. The losses are greater and earlier in Bliss Triumph than in Irish Cobbler, also greater in 14-gram seed- pieces than in 56-gram seed-pieces. Since starch constitutes a large per cent of the original dry weight, a large loss of starch — 89 per cent in Cobbler and 97 per cent in Triumph — means a large loss in dry weight, 75 and 90 per cent respectively. Moisture and soluble sugars increase in Cobbler mother-tubers throughout the growth period; but with Triumph, increase in the early season is followed by a later decrease. What does the mother-tuber furnish the growing plant that is necessary for maximum yield? As is seen from the chemical data in Table 48, the mother-tuber ceases to be of importance about the time the main nutrients, especially starch, are exhausted. In Irish Cobbler mother-tuber, the starch is exhausted later than in Bliss Triumph; also in the former the mother-tuber has a beneficial effect on the growth of the plant for a longer period. This at least indicates that the mother-tuber may be o'f importance as a source of nutrients. Appleman ^ thinks growth-promoting substances MISCELLANEOUS Table 48. Changes in Amounts of Substances in Mother-Tubers 423 Variety Approx. size of seed pieces (srams) Stage of development Per cent of amount originally present H2O Dry matter Starch Total sugar Total nitrogen Cobbler 14 14 14 14 Start Stage 2 Stage 3 Stage 4 100 126 131 143 100 59 30 18 100 53 12 4 100 167 181 114 100 71 43 29 Cobbler 56 56 56 56 Start Stage 2 Stage 3 Stage 4 100 118 125 139 100 74 39 25 100 80 27 11 100 118 169 187 100 79 41 24 Triumph 14 14 14 Start Stage 2 Stage 3 100 162 130 100 43 10 100 19 2 100 200 45 100 50 17 Triumph 56 56 56 56 Start Stage 2 Stage 3 Stage 4 100 135 145 131 100 53 23 9 100 37 6 3 100 161 133 50 100 67 25 17 from the mother-tuber are of importance in promoting the growth of the daughter plant and that the mother-tuber must be of a certain mmimum size in order to furnish enough of these substances. Denny's work does not disprove this claim. It is also claimed that the mother-tuber serves as a water-storage organ. Denny observes wilting in some cases after mother- tuber removal, but in the later stages of growth this did not reduce the jdeld. His plants were gro^vn with adequate water. Water storage may have some significance in dry seasons. Flower Color of Hydrangea macrophylla The amount of aluminimi available determines the color of the flowers of the common hydrangea, Hydrangea macrophylla DC. Many gardeners and a number of scientists have debated the question, WTiy are the flowers of our common hydrangea (H. macrophijlla) some- times pink and at other times blue, and how is it that some flowers on cer- tain plants the same year are pink while others are blue? Allen ^ gives a critical review of the investigations on this question and describes a set of experiments that appear to give the answer. He states that several explanations have been offered, but that recently the question has resolved itself into whether it is an abundant supply of aluminum or iron in the flower that makes it blue, a medium supply that produces a mauve flower, and low supply that gives a pink flower. His answer is in favor of aluminum salts. Part of his evidence is given in Fig. 171. In the upper left corner is a 424 GROWTH OF PLANTS branch showing a pink corymb and a blue corymb. Both were pink at the beginning but the one at the right was changed to blue by spraying it with 0.5 per cent solution of aluminum ammonium citrate. The flower at upper right was produced when the nutrient solution for the plant bore 1.34 ppm or less of aluminum. The middle left flower (mauve) was pro- duced on a plant receiving 13.4 ppm of aluminum in the nutrient, and the middle right on a plant furnished a nutrient bearing 134 ppm of aluminum. The plant at the bottom of the plate was grown in a pot with the root sys- tem and lower part of the stem divided with a plate of glass, so the right- hand side has a lower supply of available aluminum than the left. In this way both pink and blue flowers are produced on the same plant. It is common knowledge that when this plant is gro\vn in nearly neutral or alkaline soil the flowers are generally pink, while those gro\\Ti in acid soils are generally blue. Neutral or alkaline soils bear little iron or alumi- num in solution, while acid soils bear much more. The following statements by the author ^' p -240-241 ^^^^ further evidence for his conclusions and bring out other interesting facts about the problem : "Blue flowers from plants grown in sand cultures had an aluminum con- tent of more than 250 ppm; pink flowers contained less than 150 ppm, while mauve flowers varied from approximately 150 to 250 ppm. Bright blue flowers from plants growing in the field usually contained 800 to 900 ppm of aluminum, although the amount varied widely depending upon the conditions under which the plants were growing. Aluminum com- pounds added to the soil increased the aluminum content of the flowers. "The iron content showed less relationship to the flower color than did the aluminum. Blue flowers from plants growing in soil had a higher iron content than pink ones, but in sand cultures where the amount of iron in the nutrient solution was controlled, some of the blue flowers contained less iron than the pink. "Flowers produced on plants grown from cuttings in soil at pH 5.5 or below were blue. Between pH 5.5 and 6.25 the flowers were intermediate between pink and blue. Above pH 6.7 the flowers normally showed no trace of blue. Blue flowers were produced on plants growing in alkaline soil at pH 7.5 or above. There was evidence that aluminum could be accu- mulated in the tissue and bring about mauve or blue color when the plants were transferred to neutral or alkaline soil. "Single plants were caused to produce flowers varying from clear blue to clear pink by dividing the root system and growing the two halves in different types of soil. Wherever there was a vascular connection between the roots growing in acid or aluminum sulphate treated soil, the flowers were blue. "Some varieties failed to produce clear blue flowers in soil made acid by the addition of aluminum sulphate. Flowers of the same varieties turned blue when aluminum compounds were sprayed on mature sepals or absorbed through a slit in the stem." Figure 171. (See text for descriptions.) MISCELLANEOUS 425 Soil Studies Peats and composts. McCool ^^ made a study of widely different classes of peats as substitutes for the ever-diminishing supply of manure. The peats varied in mineral content from 3.45 to 53.75 per cent, in nitrogen from 1.78 to 3.35 per cent and in pH from 3.39 to 6.85. Under storage they showed considerable but variable nitrate formation. When added in large amounts along with complete fertilizers, and lime for the more acid peats, they brought non-productive mineral soils to a high level of fertility. Use of peats ^^ in composts increased nitrification and plant growth, but differ- ent peats varied considerably in effectiveness. A partly decomposed, sedi- mentary, fibrous peat, when composted Avith fertilizer salts, Hme, and a small amount of manure, proved to be a valuable soil improver. It was superior to the same material mthout composting. Straw disintegrated readily when composted ^\ith the peat mentioned above and with proper amounts of fertilizer salts and lime ; it was more effective for soil improve- ment than equal amounts of commercial manures. A number of factors ^^ were found to modify the pH of peats. In some peats increasing the water content increased the pH by 1, while in others adding water had little effect. Oven-drying of peats had little effect upon the pH when they were again soaked, although the effect was measurable in some cases. Air-drying increased the soluble salt content of some peats and oven-drying was more effective. Very acid peats were rendered far less acid by leaching with large volumes of distilled water. The less acid peats showed slighter changes. Addition of salts to peats including fertilizers increased the acidity of peats. Salts with bivalent cations were more effective than salts of monovalent cations and less acid peats were more modified than more acid ones. It took heavy applications of super phosphate and complete fertilizers to change the pH noticeably. A study *^ was made of various methods of composting several kinds of plant materials (leaves of oak, Norway maple and sugar maple, pine leaves, salt-marsh hay, straw and cat-tail plants) and the value and best methods of applying the several composts to soils. Additions of cyanamide greatly hastened the decomposition of leaves, pine needles, and cat-tail composts. The acidity of leaf composts decreased rapidly upon standing, and salt- marsh hay composts were alkaline. Adding the uncomposted materials to soil decreased crop growth; oak leaves and some other composts also decreased crop growth if added to soils alone, but proved very beneficial if added with complete fertilizers. A domestic peat composted with cyana- mide alone proved injurious; but when composted with cyanamide plus a still greater amount of acid phosphate it was beneficial to the growth of rye. Soil solutions. Following the suggestion of Knudson and Ginsburg that the density of juice pressed out of plant tissue varied Avith the amount of pressure used, McCool and Youden ^' determined the acidity of successive samples of water pressed out of ground tissue of several plant organs and 426 GROWTH OF PLANTS several peat soils as the pressure rose. The pressure varied from that of a screw hand press to 30,000 pounds per square inch exerted by a hydrauhc press and rose in steps of 1000 or several thousand pounds per square inch. The acidity fell as the pressure to squeeze out the successive water fraction rose. In the more acid peats the acidity fell as much as 3 pH, from 3.64 pH at low pressures to 6.77 pH at high pressures. The most marked fall was from minimal pressures to 1000 to 4000 pounds, but there were sUght falls above 4000 pounds. In peat soils the phosphorus content of the water fractions pressed out by increasing pressures fell markedly. In a brown sphagnum peat, phosphorus content fell from 50 to 55 ppm with slight pressure to 3 to 4 ppm with 20,000 to 30,000 pounds. The phosphorus content of expressed water continued to fall noticeably even above 4000 pounds. Even a sandy loam showed similar changes in phos- phorus content of water fractions as the pressure increased. The supply of acids and phosphorus was not exhausted by the pressure; for on rewetting these materials give fractions similar to those initially observed as far as the hydrogen-ion concentration and phosphorus content are concerned. The authors suggest that high pressures may form semi-permeable membranes in the materials and mention Bouyoucos' suggestion that the small capillary spaces may contam more dilute solutions. They feel, however, that the real explanation of the phenomenon is still to be learned. Insoluble organic sources of nitrogen. In mixed fertilizers insoluble organic nitrogen sources that gradually become available during the crop season are supposed by some to have an advantage over nitrates and other soluble compounds which are subject to leaching in light soils under heavy rainfall. Such nitrogen sources also add to organic content of soil. McCool *■* has made a study of several insoluble organic nitrogen com- pounds as to rate of ammonification and nitrification in soils and as to their relative values as nitrogen sources for crop growth. A patented mixture of fermented molasses concentrate, after distilling off alcohol, and calcium cyanamide, bearmg about 55 per cent soluble nitro- gen, was compared as such and after leaching out the soluble nitrogen with two grades of tankage. While there was considerable variation in the relative rate of ammonification of the four materials in Gloucester loam and Norfolk fine sand the two tankages early showed most rapid nitrifica- tion in both soils; but later the nitrification of tankage was no more rapid in Gloucester loam than that of the molasses mixture. The molasses mixture was equal to high-grade tankage for production of snap beans, tobacco, and corn, and superior to tankage for cotton, rye grass, and millet. Also the slower rate of nitrification of the molasses mixture should lead to lower rate of loss of nitrogen by leaching in soils. McCool also made a similar study ^'' of the du Pont Urea- Ammonia Liquor-37 (UAL-37) which, when mixed with acid phosphate, potash, and other materials, forms an insoluble nitrogenous material. This proved a little less effective for millet growth ui Norfolk fine sandy loam than the insoluble nitrogen of MISCELLANEOUS 427 cottonseed meal. It was, however, as valuable as cottonseed meal for the growth of millet, corn, and tomatoes in Gloucester loam. It was also supe- rior to processed tankage. The residual (or carry-over) effect of insoluble nitrogen in UAL-37 base was greater than it was in cottonseed meal, as evidenced by the yield of a second crop of millet grown in Gloucester loam. A ground plastic molding resin bearing nitrogen proved of little value as a N-source for crops, while the nitrogen in a resin scrap proved effective. The nitrogen in garbage tankage had low value. The readiness ^^ of nitrifi- cation of three organic sources of nitrogen in Sassafras sandy loam was in the order named: urea, cottonseed meal, and UAL-37. Cottonseed meal and UAL-37 base gave greater crop yields if added to the soil 68 days be- fore planting rather than at planting time. In greenhouse tests ground samples of kitchen wastes or garbage ^^ were less effective in crop growth than tankages with equal nitrogen content but superior to cow manure and shredded stockyard manure on the same basis. The residual, or second crop, effects were greater than the manures or tankage. Incubatmg the wastes at 40° to 55° C (104° to 131° F) did not increase the availability of the nitrogen but it did improve the texture and disposed of obnoxious volatile materials. In field tests mixed kitchen waste proved superior to stockyard manure but both showed similar residual effects. Placing the ground wastes in the soil 30 days before plant- ing the crop increased their value for the early growth of the crop. Kitchen wastes ^^ were improved somewhat by liming, inoculating with active cultures of decomposing bacteria, and incubation at 40° C (104° F) but incubation should be discontinued when nitrogen reaches the maximum percentage and before it begins to fall. It is evident that with proper treatment and usage a number of insoluble organic nitrogen compounds can be used both as nitrogen and as a source of organic material for soils. Sulfur dioxide from cities. Attempts ^^' ^- were made to determine the effect of SO2 produced in burning coal in our cities on the soils and plants about the cities. St. Louis, Mo. was selected as a city producing much SO 2 due to burning high-sulfur soft coals and Philadelphia, Pa. as a city producing much less due to burning low-sulfur hard coals. Samples of soil and of various species of plants were taken from each of several soil types. The stations from which the soil and plant samples were taken ranged from the centers of the cities to localities 30 to 40 miles away. For the soils, the pH values, titratable acidity, base exchange capacity, replace- able calcium and magnesium, and sulfur content were determined and for the plants the total sulfur, sulfate, and nitrogen content. The authors conclude that soils about centers of considerable SO2 production, like St. Louis, have not been noticeably altered in spite of many years of exposure, and that the acidulation, if any, is much less than that due to natural processes in the soil. It can be overcome by slight increases in the rate of lime application. With few exceptions the total sulfur, sulfate, 428 GROWTH OF PLANTS and nitrogen content of the leaves of plants did not decrease significantly with distances from the source of SO 2. Manganese injury. Certain steam-sterilized soils'*- used at the Institute proved toxic to plants because of the high content of soluble manganese salts. Soils steamed at 240° F (116° C) showed a manganese content rang- ing from 384 to 22.9 ppm. High organic content seemed to increase the amount of manganese rendered soluble. Other factors that affect the solu- bility of manganese in soils were studied and the data obtained were dis- cussed in relation to previous literature on the subject. Soybeans, snap beans, and tobacco *^ were grown in untreated soil and in soil to which 400 and 600 ppm of manganese sulfate were added under four different intensities of light: full sunlight, 78, 58, and 35 per cent of full sunlight. In general, the injury to the leaves by manganese salts, as shown by brown-spotting, chlorosis, and the amount of manganese salt absorbed by the leaves, decreased as the light intensity decreased. The salt decreased the yield of plants in all light intensities, except perhaps tobacco under heaviest shading. The amount of manganese absorbed by roots and stems was much less affected by light intensity than was that of the leaves. Colloidal phosphate. Florida pond or colloidal phosphates^® were no more effective for crop production than finely ground Tennessee brown rock phosphate. Fungi for determining soil fertility. Mehlich ^ (joint contribution of Boyce Thompson Institute and Tennessee Agricultural Experiment Station) extended the earlier research by himself and associates and by other investigators on the use of fungi to determine deficiency of potash, nitrogen, and phosphorus in soils for the growth of crops. Use of these organisms requires much less space and less time than the growth of crop plants. Mehlich considers Aspergillus niger to be well adapted to deter- mine potash deficiency, and within limits Cunninghamella blakesleeana is of value in detecting phosphorus and nitrogen deficiencies. Thallium effects. It is established that use of thallium sulfate for rodent control leaves patches of soil where it is applied incapable of pro- ducing plants for a considerable period. McCool '^^ carried out a series of researches to put the knowledge in this field on a quantitative basis. He found that it was not possible to render thallium sulfate-treated soils capable of growing plants even by leaching with large quantities of water, partly because of the base exchange of thallium with calcium, aluminum, and other bases. Commercial fertilizers and lime did not lower measurably the toxicity of thallium in the soil. Crop plants were badly injured by con- centrations of thallium in the soil that were too low to affect nitrification; hence it is more toxic to green plants than to nitrifying microorganisms. The nature of the soil determines to a degree the amount of thallium salt that must be added to injure plants; sandy loam requires less than silt loam and silt loam less than fibrous sedimentary peat. Very low concen- MISCELLANEOUS 429 trations of the salt in the soil injure plants growing in it; 2.1 ppm of the sul- fate in the sandy loam slightly retarded the growth of roots and tops of soybean, wheat, alfalfa, buckwheat, rye grass, corn, tobacco, and tomato, while 8.5 ppm was very injurious to these plants. The first dosage would amount to 4.2 pounds per acre, considering plow depth of soil; the latter would be 17 pounds per acre, considering the thallous sulfate, and much less considering only the thallium. Wax beans were somewhat more resist- ant. The type of injury also varied with the kind of plant. In corn the tissue between veins of the leaves was killed; in soybeans, wax beans, and buckwheat tissue along the veins was damaged ; in rye and wheat this was true all over the plant; and in alfalfa it was limited to basal tissue. Studies on Lilium, Gladiolus, and Dahlia Storage of pollen and hybridization. In breeding work it is often desir- able to keep pollen in a fully viable condition for considerable periods in order that crosses may be made between plants flowering at different times. It is also sometimes desirable to ship pollen to distant points for breeding work. Following the earlier experiments on the effect of humidity and temperature on the life span of pollen, Pfeiffer ^^' *'•*• ^'^ in order to facilitate breeding experiments on Lilium, hybrid Amaryllis, and Gladiolus attempted to determine good conditions for pollen storage in these genera. She later made a study of Cinchona pollen. ^^ The viability of the pollen was tested on a synthetic medium when possi- ble and in some cases on stigmas of living plants. In the latter cases the development of fruits and set of seeds were the criterion of viability. The pollen was stored at 10° C (50° F) with various constant humidities of the atmosphere, at sub-freezing temperatures in gelatin capsules, and in gelatin capsules wrapped in paraffined paper without humidity control. Also the pollen was sealed in evacuated tubes and stored at various temperatures. Humidity controls were obtained by storing in desiccators over saturated solutions of various salts, as well as by using various concentrations of H2SO4 as well as CaClo. The following are the saturated solutions used with the relative humidities produced by each: Magnesium chloride: 35 per cent humidity at 10° C (50° F); 33 per cent at 20° C (68° F). Calcium chloride: 38 per cent humidity at 10° C (50° F); 32.3 per cent at 20° C (68° F). Potassium carbonate: 44 per cent humidity at 18.5° C (65° F); 43 per cent at 24.5° C (76° F). Potassium thiocyanate: 47 per cent humidity at 20° C (68° F). Sodium dichromate: 52 per cent humidity at 20° C (68° F). Magnesium nitrate: 56 per cent humidity at 18.5° C (65° F). Ammonium nitrate: ca. 68 per cent humidity at 10° C (50° F); 64 per cent at 20° C (68° F). For all pollens studied, thorough desiccation over concentrated H2SO4 or CaCla was injurious. In general, proper humidity of the atmosphere in the 10° C (50° F) storage greatly lengthened the longevity of the pollen. 430 GROWTH OF PLANTS Cinchona pollen at 10° C (50° F) lived a year with relative humidity of 35 to 50, gave 3 per cent germination in 3 months with 65 per cent R,. H., and 2 to 3 per cent after 6 weeks with 25 per cent R. H. Gladiolus pollen kept best at 10° C (50° F) with 40 to 50 per cent R. H., but degenerated rapidly in open air storage at room temperatures. Pollen of most varieties used kept 8 to 10 weeks under this condition and pollen of a few varieties showed live grains after 102 days. Lilium pollen kept as long as 7 months when stored at 10° C (50° F) with controlled humidity; L. auratum best at 35 per cent R. H. ; L. speciosum best at 50 per cent R. H. ; and L. longiflorum at 65 per cent R. H. Under the same conditions the Amaryllis hybrid pollen lived for 5 months. Under similar conditions Lilium pollen in gelatin capsules wrapped in paraffin paper at —10° or — 11°C (14° or 12° F) kept well. Sealed vacuum storage at 5° or -5° C (41° or 23° F) was unfavorable for Cinchona but favorable for Lilium pollen. As with seeds, the longevity of pollen can be increased greatly by improving storage conditions. Constant humidity is important in both, but in many seeds much lower humidities are favorable. Likewise, lowering the temperature is beneficial in both. Absence of oxygen is beneficial in dry seeds. The evidence for this on pollen is not clear. It would be interesting to know the optimum of all three of these factors for several pollens and to learn how much the life span could be increased by putting all these factors at the optimum. In hybridization several species of lilies show maternal characters. This is true of L. superhum L. and L. auratum Lindley. In spite of this, Pfeiffer ^^ in her lily hybridization work got some hybrids with these two species as maternal parents that combined the characteristics of both parents. This was true of L. auratum 9 with both L. ruhellum Baker and L. japonicum Thunberg as pollen sources. The same was true of L. superhum 9 with L. canadense as the pollen source. Intermediate forms ^^ were also obtained with L. sulphureum Baker as seed parent and L. Henryi Baker as the pollen source. McLean and McLean ^^ got reciprocal crosses between Lilium tigrinum Ker-Gawl., noted for its sterility, and two different lily hybrids, L. X um- hellatum and L. X elegans. The new hybrids showed characteristics of both parents and produced viable seeds. McLean ^^ produced a bigeneric hybrid by using the pollen of the garden hybrid gladiolus "Byron L. Smith" on Antholyza revoluta Burm., the Rood Jvapje of South Africa. The hybrids showed strongly the vegetative and flowering characters of the pollen parent, but the seed parent habit of winter growth during short daily illu- mination and low temperatures. The hybrids produced no viable pollen and did not produce seed when pollinated from either South African or American gladioli. McLean ^^ produced a scented hybrid gladiolus ap- proaching in stature and flower size our summer flowering gladioli. He designates it as Z-36 and describes it as "being derived on the one hand from a garden hybrid gladiolus, 'Gretchen Zang,' as pollen parent, and MISCELLANEOUS 431 from two clones of vrAA species, Gladiolus tristis L. concolor I and G. recurvus L. bronze as great grandparents on the other." Dormancy in Easter lily bulbs. Thornton and Imle ^^' ^^ point out that for many years commercial growers of Easter lilies have been faced with the problem of dwarfed plants, or even no growth whatever, from bulbs which at the time of planting appeared to be perfect specimens free from mosaic. Immaturity at time of harvest or unfavorable storage condi- tions before forcing may induce this bulb dormancy. Bulbs harvested before the flowers open remained alive and later produced roots but no tops. Those harvested just after the flowers had withered produced about 50 per cent stand in the forcing house, while the other half remained dor- mant but sound. Bulbs harvested when fully mature (when the plants were almost dead) gave full stands when forced after storage. Storing the bulbs in conditions that led to the exhaustion of oxygen or to the accumu- lation of high concentrations of carbon dioxide also induced dormancy. These injurious conditions were especially detrimental on immature bulbs. A short drying period after harvest and low-temperature storage up to forc- ing time were necessary to avoid dormancy, the latter especially in the case of immature bulbs. Pfeiffer ^^ studied the effect of varying the cold storage period and time of planting the two lilies {Lilium longiflorum Thumb, var. eximium Nichols [L. eximium Courtois] produced in Bermuda, and var. giganteum, Hort. produced in Japan) formerly used in United States for forced Easter lilies upon the date and rate of development of flower primordia. In the first kind the purely vegetative stage seen in August persisted until the middle of October in cold storage at 10° to 13° C (50° to 55° F). If continued in cold storage until November 26 there was a broadening of the apex and slow elongation of the axis indicating the pre-differentiation stage of the floral axis. In bulbs of the second kind, first obtained in early December, there was already a broadening of the apex and in cold storage at 3° C (37° F) there was slow elongation of the axis. Potting the bulbs even without change of temperature hastened the development of flower primordia. The order of development of the floral organs was acropetal. Lily diseases and pests. Because of lack of knowledge of lily diseases and pests, a fellowship was established in the fall of 1927 by The Horti- cultural Society of New York, New York Botanical Garden, Cornell University, and Boyce Thompson Institute. This fellowship was continued for fourteen years, or up to 1941. The laboratory work was done mainly at the last two institutions mentioned above, and during the last five years the feUowship was supported entirely by these institutions. The fellowship was held in succession by the following plant pathologists, C. E. F. Guter- man, 1927 to 1930, Keith O'Leary, 1931 to 1937, and E. P. Imle, 1937 to 1941. The work was done in close cooperation with commercial and pri- vate growers and with lily breeders in eastern United States. In 1930 Guterman ^i summarized his work and that of other investigators 432 GROWTH OF PLANTS on lily troubles. The author describes and suggests methods for control of two virus diseases (mosaic and yellow flat) ; five fungal diseases or groups of diseases (Botrytis blight, bulb rots, rust, stump rot, and foot rot); four physiological troubles (limber neck, frost injury, non-infectious chloroses, and brown-tip of leaves) ; and one insect pest (bulb mite) . He also men- tions several other minor diseases described in the hterature. Because it is the most destructive disease of lilies, except for species and varieties that are tolerant, mosaic deserves special mention. When it attacks lily plants outside it causes complete degeneration in a year or two in some species. The disease is transmitted from diseased to healthy plants by Aphis gossypii Glover and spreads rapidly under conditions favorable for this insect. It can also be transmitted by intergraftiag between diseased and healthy plants and with some difficulty by scratch- ing juice from diseased plants into tissue of healthy plants. Once the disease enters a plant, there is no cure for it since it is carried by the bulb. The main sjonptoms are yellow mottling of younger leaves, showing three types in different species, deformation and stunting of flowers and more or less stunting of the whole plant. The main remedy is roguing out and destruction of all plants showing the disease. This must be done as soon as the disease appears on a plant in order to prevent spreading by the vector. Also the foliage takes on a deeper color when the plant sets flowers or when the temperature is high, thereby masking the mottling. Control of the insect vector by sprays of course slows the spread of the disease. It spreads faster in some species than in others, because the insect prefers these as food. It is not carried by seeds; consequently seedlings are disease- free until infected from an external source. Although of less economic importance than the mosaic, the yellow flat or lily rosette disease of L. longifloruin is of interest. It is carried by the same vector as mosaic but cannot be transmitted mechanically. It causes general chlorosis of the foliage rather than mottling. It also causes marked stunting of the whole plant and rolling or t\visting of the outer portions of the leaves, reduces or prevents flower set, and distorts the flower and modi- fies the shape and size of the bulb. Later work '^ indicates that this disease is of considerable economic importance in Easter lilies and can be trans- mitted by the vector to several species and varieties. The symptoms of the disease vary with the species, which makes it difficult to diagnose in some cases. Botrytis blight is a very destructive disease, sometimes under favorable conditions ruining an entire crop in a few days. There is considerable variation between species and varieties as to susceptibility to the disease. It may attack the top of the young plant and stop further growth, and even in some cases destroy the bulb. It may merely spot the leaves. The spots may increase in size in more susceptible forms under favorable condi- tions until the whole leaf is destroyed and plants are without live foliage. When the disease attacks the plants in late stages, the flowers are spotted, MISCELLANEOUS 433 deformed, and may rot completely. The best remedial measure for stop- ping spread of the disease is removal and burning of all diseased portions of plants. In greenhouses disease-free soil should be used and plants grown at low humidities and without over-watermg. Sulfur fungicides, copper lime dust, and Bordeaux are effective if the plants are sprayed before the disease starts and frequently enough thereafter to maintain a protective cover. Besides the lily pests mentioned in Guterman's summary, the follo\\ing were later studied and reported upon under this fellowship : Penicillium rot of lily bulbs,^^ a cecidomyid larva of lily stems, ^^ bulb rots of lilies, ^^ and some insect pests of Ulies.^^ Some other work done has not yet been pub- Ushed. Dahlia mosaic. A survey showed that dahlia mosaic "-^^ jg prevalent throughout Connecticut, New York, and New Jersey. It is probably dis- tributed widely. All members of the genus are susceptible, but there are no suspects outside the genus. Mosaic is not synonymous with "stunt " which is applied to the less tolerant varieties that are stunted by mosaic and by certain insect injuries. The symptoms of the disease on the less tolerant varieties are chlorosis, leaf distortion, shortening of internodes and flower stems, and vein necrosis. The more tolerant varieties that are not stunted by mosaic had been overlooked as carriers of the disease by the earlier American work on dalilia stunt. The mosaic is carried in the tuber and other vegetative parts of the plant but is not transmitted through the seed ; also it has not been transmitted mechanically. It can be transmitted to all varieties by grafting and manifests itself with marked or shght symp- toms on the various varieties in line ^A-ith their tolerance. Myzus persicae seemed to be the specific insect vector of the disease. Other insects tested did not transmit the disease. Its symptoms appear in the plants four weeks or longer after insect transmission. Since dahlia is not a preferred food of the insect, the disease spread is at the rate of 10 to 25 per cent of the crop per year. The disease is controlled by selection and isolation of disease-free plants along with control of aphids during early growth and roguing during later growth. Tolerant varieties, if growTi at all, should be isolated from those showing marked s^inptoms of the disease. Seedlings are free from the mosaic untU infected by the insect. The infected tuber, of course, carries the disease to the plant gro^^•n from it. Ring spot, yellow ring spot, and oak leaf are three other virus diseases of the dahlia studied by Brierley. The researches indicate that they too are specific dahha diseases. Propagation of Trailing Arbutus and Lycopodium Abundant moisture, protection from excessive sunlight and probably the presence of a mycorrhizal fungus in the soil are necessary for rooting trailing arbutus.^ Vigorously growing plants bore well-developed coils of the endophytic fungus in the roots; stunted plants showed little or no fungus 434 GROWTH OF PLANTS in the roots. Cuttings taken in August or September and including current and previous year wood rooted readily; those taken in the spring root less readily. Live sphagnum, native soil, peat moss, and mixtures of peat moss and good sand were all good rooting media. There was found to be a great difference in the percentage of germination in seeds taken from different fruits, the germination ranging from 0.3 to 87.3 per cent. Best germination occurred when the seeds were planted soon after harvest. In open air storage the vitality of the seeds fell rapidly. Batches of seeds that gave 45 per cent germination in July gave only 2 per cent in the 5 out of 80 lots which showed any germination after storage for six and one-half months. Arbutus is dimorphous as to style and stamens and subdioecious and hand pollination mcreased the average set of seeds per fruit from 241 to 403. Plants of trailing arbutus thrive over a wide range of pH; plants did well in soils with pH anywhere between 4.65 and 7.67. No observations were made in lower or higher acidity ranges. A later study ^ emphasized again the significance of the endophytic fun- gus in the life of trailing arbutus. This fungus appears in the roots, stems, leaves, and fruit and on the pollen, seed, and ovule. If the fungus is not present, the gro's^ii.h of the plant is improved by the addition of soil from around vigorously growing plants. Seedlings develop normally if fungus is present. The root tip of this plant furnishes excellent material for study- ing living chromosomes. The increasing use of several species of Lycopodiums (mainly collected in the \vild) for Christmas decorations threatens the depletion of these plants. Barrows ^' ^' ^ made a study of the propagation of several species of the genus, hoping that commercial growing might protect the wald stands. Her work with the germination of spores makes it quite evident that on the basis of present knowledge the spores cannot be used as a commercial means of propagation. After 30 months with the best cultural conditions used, 15.8 per cent of the spores of L. complanatum var. flabelliforme ger- minated. In 28 months L. ohscurum spores gave 20 per cent germination. In no case did the gametophyte, prothallium, develop beyond the 10-cell stage. The problem of securing more advanced development on artificial media remains unsolved. The 10-cell stage is far short of a mature prothal- lium with archegonia and antheridia, and finally with the sporophyte resulting from the fertilization of the egg. While apparently some previous investigators working with other species produced mature gametophytes and sporophytes from spores, most workers have met difficulties similar to or even greater than Barrows in spore germination and growth of the pro- thallium. In fact, most of our knowledge of the gametophyte of Lycopodium species has been gained from material collected in nature. The gametophytes are not abundant in nature but they have been found repeatedly in various stages of maturity. It is a challenge to the botanist to learn how to grow a species of Lycopodium from spores with the same assurance he now grows MISCELLANEOUS 435 ferns from spores. This ■^ill be of scientific interest rather than interest in commercial propagation. Barrows discusses the structure and chemical composition of the spores and the ability to adapt them to such commercial uses as toilet powder and powder to prevent castings from sticking to the sand in moulding. She found an endophytic fungus generally present in gametophytes collected in nature, and agrees with Treub, Bruchmann and others on the necessity of the fungus for gametophyte growth beyond an early stage. She grew many cultures of the fungus but was unable to identify it. The spore coats seem to offer resistance to the germination of the spores, but other unknown factors present even greater difficulties in producing mature gametophytes. Apparently gametophytes sometimes produce antheridia but no archegonia. These of course cannot produce sporophytes. The sporophytes of Lycopodiinn can be propagated by cuttings or bul- bils. Spring cuttings of L. clavatum L., L. complanatum var. flahelliforine Fernald, and L. obscurum L. did better than fall cuttings. Fall cuttings of L. lucidulum Michx. rooted well. L. lucidulum can be propagated from bulbils, two crops of which are produced annually under greenhouse con- ditions. Young apical growth of rhizomes roots readily; older, hgnified material roots poorly. Lycopodium needs well-watered but well-drained soil and, except for L. complanatum, some protection from direct simlight. They grow in soils that range from 5.28, or perhaps lower, to 8 pH. An endophytic fungus is found generally distributed in the roots and old parts of the rhizome of all species studied. The fungus was cultured from several species but not identified. Two Studies on Physiology and Cytology of Fungi After having found that when opposite sex strains of Neurospora sitophila (Mont.) Shear & Dodge are gro^^^l in opposite arms of U-tubes filled with nutrient agar no fusion of the two strains occurs to form perithecia until the agar dries and cracks to supply oxygen at the base of the tube. Dr. B. O. Dodge suggested to Denny ^^ that he determine the minimum oxygen pressure necessary for filament fusion, perithecia formation, and growth of the mycelia. At room temperature perithecia formed in 1 to 2 per cent oxygen by volume, but reducing the oxygen to 0.5 per cent by volume inhibited perithecia formation for at least 30 days, the duration of the experiments. At 10° C (50° F) no perithecia form in any oxygen percentage after 30 days, and only an occasional one at 15° C (59° F). Perithecia are formed somewhat less readily at 31° C (88° F) than at room temperature. Growth of the mycelivmi occurs under much lower oxygen pressure; 0.3 per cent or higher gives rapid growth of the mycelium, but the rate of growth diminishes as the oxygen percentage falls below this. At 0.01 per cent oxygen the growth was very slow, but complete inhibition of growth occurs only when the atmosphere over the culture is held free of oxygen by alka- 436 GROWTH OF PLANTS line pyrogallol. In 0.3 per cent oxygen 32 per cent CO2 retards the growth of the mycehum only slightly. Backus^ studied Coccomyces hiemalis Higgins, the cherry leaf-spot pathogen, throughout its life history in the cherry leaf, and also in artificial culture. Special emphasis was placed on the mechanism by which the ascocarp originated, whether by heterothallism or homothallism. Micro- conidia were found attached to trichogyne-ends but fusion was not ob- served, nor could microconidia be germinated. The disposition and behav- ior of the structures led to the conclusion that the ascocarp is homothallic. The results are fully discussed in relation to the work of many investiga- tions of autonomous Ascomycetes, lichens, Rhodophyceae, and various fungi in which similar problems appear. Effect of Nitrogenous and Carbohydra.te Reserves on Growth OF Seedlings Reid ^^' ""^^ '''• '^2 varied the relative amovmt of nitrogenous and carbohy- drate material supplied to growing seedlings first by selecting high and low protein and high and low carbohydrate seeds, by removing CO2 from the air supplied to growing seedlings, or by raising the CO2 in the air more than ten-fold to 0.4 per cent, and by withholding nitrates from or adding them to the nutrient solutions. High oil reserves in seeds were aligned with high carbohydrate reserves. In the case of wheat and corn it was possible to get high and low protein and high and low carbohydrate varieties within the same species. Other seedlings used in the studies were: sunflower, tomatoes, barley, peas, beans, soybeans, and muskmelon. In general, when the reserve available to the growing seedling was high in carbohydrates in proportion to nitrogenous material, the roots were large in proportion to the tops; and when the nitrogenous reserves were high in proportion to the carbohydrates, the tops were large in proportion to the roots. High CO 2 concentration in the air increased the carbohydrate supply, and nitrates in the nutrient solution increased the nitrogenous reserve. Seedlings from seeds with high carbohydrate but low nitrogen reserves give more increase in growth with nitrate addition than with extra CO2 in the air; and the reverse is true of seedlings from seeds with high nitrogen in proportion to carbohydrate reserves. Seedlings with low nitro- gen reserves and without nitrates added to the nutrient solution tend to accumulate much carbohydrate and have thicker-walled xylem vessels and greater lignification of bast fibers. Contrary to other investigators, the author finds that excessive amounts of carbohydrates in seedlings limit growth and chlorophyll development unless nitrates are added to the nutrient solution. Analytical Methods Ashing plant tissue. Ashing plant tissue ''^ in a combustion tube fur- nace under one pound oxygen pressure at 450° C (842° F) for 8 to 16 hours MISCELLANEOUS 437 gives higher ash content and better checking dupHcates than ashing in a muffle furnace for the same time at 650° C (1202° F). Tliis method of ashing leaves no residual carbon and volatilizes none of the chlorides except magnesium chloride, whereas the higher temperatures necessary in the muffle furnace volatilize chlorides of potassium and calcium and lead to loss of the metals themselves by sublimation. This method also eliminates the necessity of adding chemicals to further the ashing, such as sulfuric acid, which gives too high an ash value by changing the chlorides and car- bonates to sulfates. The authors describe the method in detail and give the precautions necessary for avoiding explosions. Determination of reducing sugars. The permanganate method for estimating reducing sugars is used extensivel}^ in biological work. It was considered necessary to standardize the potassium permanganate against cuprous oxide produced by a known quantity of pure glucose, since it was claimed that the use of oxalic acid, sodium oxalate, or iron as a standard gave results which were too low. Kraybill, Youden, and Sullivan ^^ standardized the permanganate with Bureau of Standards sodium oxalate in acid solutions and also with weighed samples of cuprous oxide prepared by reduction with invert sugar, and obtained excellent agreement. The amounts of copper found by the per- manganate method were closely checked by direct electrolytic determina- tions of the copper. Capillary glass electrode. The complex and unstable nature of many biological fluids, such as plant sap, milk, and blood, complicate the task of determining the hydrogen ion concentration of these liquids. In many cases it is a great convenience to use extremely small samples of Uquid. This is true for work with insects or small localized regions in plants. The glass electrode possesses the important advantage of eliminating the possibility of altering the sample, as may happen by bubbling hydrogen through the liquid with the hydrogen electrode or adding quinhydrone for the quinhydrone electrode. The action of the glass electrode depends on separating the test solution by a thin glass membrane from a reference solution. A glass electrode ^°- in the form of a fine-bore capillary tube ^^^th very thin walls was found to possess, besides the usual desirable features of the glass electrode, the further advantages of employing less than 0.01 cc of sample and exposing only a minute surface of the liquid to the air. Experimental Planning and Statistics The variability of biological material has always been a source of diffi- culty in biological experiments. In chemical and physical experimentation the materials are usually more homogeneous and the environment more easily controlled and reproduced. Various methods are open to the biolo- gist in order to deal with this variability, which in itself is often a charac- teristic of considerable interest. Among these methods the first to be used was the employment of extensive replication of the samples and of the 438 GROWTH OF PLANTS measurements made on these samples. A second method is to take great pains to secure homogeneous material by selecting from a large supply a relatively homogeneous portion and discarding the remainder. At the same time much effort may also be expended in the control and regulation of the environment, thus providing uniform conditions in the relatively large spaces often required. Finally, in the last two decades there has been a rapid development in experimental planning and statistical methods. The essence of this approach lies in the division of the experimental material into parts, each relatively homogeneous within itself. As a consequence, the parts now differ substantially among themselves. In the simplest form each part constitutes a complete experiment, there being as many replications as there are parts. It has been found that the marked differ- ences which exist between the parts do not contribute to the experimental error. Subject to appropriate randomization of the comparisons within the parts, statistical techniques exist w'hich provide for a valid estimate of the precision attained. The precision attained by this technique is substantially that which would have been possible if all the material had been as homogeneous as the material within the parts into which the initially heterogeneous supply was divided. This procedure, and more or less complex modifications of it, has been very widely employed in recent years, and it is evident that some understanding of these developments in experimental planning is becoming of increasing importance in many fields of work. The papers mentioned in this section, while usually concerned with a specific inquuy, also constitute examples of the application of experimental design and statistical analysis. A field trial ^"^ of fiber pots which could be buried and allowed to disintegrate and possibly give the seedling an added advantage over the usual practice was arranged on a somewhat complex plan. In all, there were 40 different combinations of pot type, pot size, soil, and variety of tomato. Based upon early field technique, considerable difficulties would be expected from employing so many treatments. The use of a split-plot Latin Square arrangement in the field made possible the detection of an effect of pot diameter on the yield of tomatoes which was of the order of 6 per cent. The plants in the most successful type of pot showed a large gain over the plants grown in flats. From one point of view, a field trial may be regarded as a sampling process. The field is divided into plots which may be thought of as samples of the field and which it is hoped wall be closely similar except as treatments are deliberately applied. In consequence the same statistical processes are valid in appraising a sampling technique and they provide an approach to determine the number of samples needed for a specified precision. Youden and Mehlich ^°^ tested the efficiency of different spacings in soil sampling and found that, for surveying large areas, intervals as small as 10 or even 100 feet between replicate samples were inefficient. For a given MISCELLANEOUS 439 number of samples larger spacings would give more information about the area. Another example of sampling technique is discussed in a paper ^- on the determination of arsenic spray residues on apples which concludes that the sampling variation of the apples was in excess of the analytical errors of the Gutzeit analytical method. The carrj^ing over of the identical experimental designs used in field studies of crops to experiments in pathology is reported in a series of papers. ^'*' ^^' ^^' '"*' A method for measuring the concentration of mosaic virus makes use of the appearance of local lesions on Nicotiana glutinosa L. leaves. In a given batch of plants the top leaves form a class which has an average susceptibility which is different from the average for the leaves in the second (or some other) position on the plant. Cutting across these classes, the leaves on a given plant form a family such that all the leaves on it are more (or less) susceptible than leaves in corresponding positions on some other plant. Thus the whole assembly of leaves can be divided into regions or parts in which a certain degree of homogeneity of response exists; by planning the experiments with this in mind a three-fold increase in the precision of comparisons w^as attained. ^^ A statistical study ^°° of the existing literature on the relationship between \'irus concentration and the number of lesions produced on the leaves led to the formulation of a mathematical relationship between these quantities. Further experiments ^^ undertaken with a view to getting improved data to test this equation led to a novel experimental design — a Latin Square ^\'ith missing rows.^'* This arrangement (also called Youden Square) was found especially adapted to greenhouse trials/^ since fewer replications are required than in the Latin Square design. Two other papers illustrate applications of statistics. A statistical analysis ^^ of a considerable body of data obtained in seed germination tests showed how the Chi-square test can be used to check the sources of variation in seed-testing methods. In connection with, studies of the effect of SO2 on plants, a record of the SO2 concentration in the atmosphere was available at half-hour intervals for a year. The concentration of the SO2 showed wide fluctuations,^^ with pronounced weekly and annual cycles. By means of a linear regression technique the SO2 concentration was found to be related to the wind direction and velocity, the temperature, and the rainfall. Literature Cited 1. Allen, R. C, "Influence of aluminum on the flower color of Hydrangea macrophylla DC," C. B. T. I., 13 : 221-242 (1943). 2. Appleman, C. O., "Special growth-promoting substances and correlation," Science, 48 : 31&-320 (1918). 3. Backus, M. P., "Initiation of the ascocarp and associated phenomena in Coccomyces hiemalis," C. B. 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For the lily disease investigation fellow- ship," Hort. Soc. New York Yearbook, 1930:51-102 (1931); also in B.T.I. Prof. Pap., 1 : 146-197 (1931). 32. Hitchcock, A. E., and P. W. Zimmerman, "Further tests with vitamin Bi on estab- lished plants and on cuttings," C. B. T. I., 12 : 143-156 (1941). 33. Imle, E. P., "Bulb rot diseases of lilies," Am. Lily Year Book, 1942 : 30-41. 34. , and A. Hartzell, "A cecidomyid larva infesting flowering stems of lihes," C. B. T. /., 10 : 277-279 (1939). 35. , , "Some insect pests of lilies," Am. Lily Year Book, 1942 : 42-47. 36. Kraybill, H. R., W. J. Youden, and J. T. Sullivan, "Notes on the permanganate method of estimating reduced copper in the determination of reducing sugars," J. Assoc. Off. Agric. Chem.., 19 : 125-130 (1936). 37. Laing, H. E., "The composition of the internal atmosphere of Nuphar advenuvi and other water plants," Am. J. BoL, 27 : 861-868 (1940). 38. McCool, M. M., "Value of peats for mineral soil improvement," C. B. T. 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I., 11 : 393-401 (1941). 48. , "Effect of temperature on the availability of insoluble nitrogen," C. B. T. I., 12 : 213-216 (1941). 49. , "Some conditions which affect rate of decomposition of kitchen waste," C. B. T. I., 12 : 269-276 (1942). 50. — , "Agronomic value of kitchen waste," C. B. T. I., 12 : 345-358 (1942). 51. , and A. N. Johnson, "Nitrogen and sulphur content of leaves of plants within and at different distances from industrial centers," C. B. T. I., 9 : 371-380 (1938). 52. , and A. Mehhch, "Soil characteristics in relation to distance from industrial centers," C. B. T. I., 9 : 353-369 (1938). 53. , and W. J. Youden, "The pH and the phosphorus content of the expressed liquids from soils and plant tissues," C. B. T. I., 3 : 267-275 (1931). 54. McLean, A. C, and F. T. McLean, "A new race of lily hybrids," C. B. T. I., 11 : 119-121 (1940). 55. McLean, F. T., "A bigeneric gladiolus hybrid," C. B. T. /.. 10 : 189-190 (1939). 56. , "A new fragrant gladiolus hybrid," C. B. T. I., 10 : 377-380 (1939). 57. Magness, J. R., "Composition of gases in intercellular spaces of apples and pota- toes," Bot. Gaz., 70 : 308-316 (1920). 58. , and W. S. Ballard, "The respiration of Bartlett pears," J. Agric. Re^., 32 : 801-832 (1926). 442 GROWTH OF PLANTS 59. Mason, T. G., and E. J. Maskell, "Studies on the transport of carbohydrates in the cotton plant. I. A study of diurnal variation in the carbohydrates of leaf, bark, and wood, and of the effects of ringing," Ann. BoL, 42 : 189-253 (1928). 60. M(5hUch, A., "Use of Cunninghamella blakesleeana and Aspergillus niger for measur- ing the manurial requirements of plants," Soil Sci. Soc. Proc, 1937 : 279-288. 61 O'Leary, Keith, and C. E. F. Guterman, " Penicilliimi rot of lily bulbs and its con- trol by calcium hypochlorite," C. B. T. I., 8 : 361-374 (1937). 62. Pfeiffer, N. E., "Development of the floral axis and new bud in imported Easter lilies," C. B. T. I., 7 : 311-321 (1935). 63. J "Longevity of pollen of Liliiim and hybrid Amaryllis" C. B. T. I., 8 : 141- 150 (1936). 64. , "Viability of stored Lilium pollen," C. B. T. I., 9 : 199-211 (1938). 65. , "Life of Gladiolus pollen prolonged by controlled conditions of storage," c'b. T. I., 10 : 429-440 (1939). 66. , "New hybrids of Lilium auratum and L. superhum as seed parents," Am. Lily Year Book, 1942 : 50-53; also in B. T. I. Prof. Pap., 1 : 297-301 (1942). 67. , "Recent hybrids of Lilium sulphvreum, X L. Hevryi," Am. Lily Year Book, 1942 : 54-56; also in B. T. L Prof. Pap., 1 : 303-305 (1942). 68 , "Prolonging the life of Cinchona pollen by storage under controlled condi- tions of temperature and humidity," C. B. T. L, 13 : 281-293 (1944). 69. Reid, M. E., "Growth of seedling in relation to composition of seed," Bot. Gaz., 8l': 196-203 (1926); also in C. B. T. /., 1 : 115-122 (1926). 70 , "Growth of seedlings in light and in darkness in relation to available nitrogen and carbon," Bot. Gaz., 87 : 81-118 (1929); also in C. B. T. I., 2 : 1-38 (1929). 71 , "Relation of composition of seed and the effects of hght to growth of seed- Ungs," Am. J. Bot, 16 : 747-769 (1929); also in C. B. T. I., 2 : 251-273 (1929). 72. , "Effect of variations in the amounts of available carbon and nitrogen on the growth of wheat seedlings," Am. J. Bot., 16 : 770-779 (1929); also in C. B. T. L, 2 : 274-284 (1929). 73. Rudolfs, W., "Effect of seeds upon hydrogen-ion concentration equihbrium in solution," J. Agric. Res., 30 : 1021-1026 (1925). 74. Scott, I. T., "Some protein analogies of the mycelium of Fusarium lycopersici," Missouri Agric. Exp. Sta. Res. Bull. 92, 44 pp., 1926. 75. Stewart, W. D., and J. M. Arthur, "An improved method for ashing of plant material," C. B. T. L, 8 : 199-215 (1936). 76. Thornton, N. C., "Carbon dioxide storage of fruits, vegetables and flowers," Ind. Eng. Chem., 22 : 1186-1189 (1930); also in B. T. L Prof. Pap., 1 : 137-144 (1930). 77. , "The effect of carbon dioxide on fruits and vegetables in storage," C. B. T. I., 3 : 219-244 (1931). 78 , "Carbon dioxide storage. III. The influence of carbon dioxide on oxygen uptake by fruits and vegetables," C. B. T. I., 5 : 371-402 (1933). 79. , "Carbon dioxide storage. IV. The influence of carbon dioxide on the acidity of 'plant tissue," C. B. T. L, 5 : 403-418 (1933). 80. , "Carbon dioxide storage. V. Breaking the dormancy of potato tubers," C. B. T. I., 5 : 471-481 (1933). 81. , "Carbon dioxide storage. VI. Lowering the acidity of fungal hyphae by treatment with carbonic acid," C. B. T. I., 6 : 395-402 (1934). 82. , "Carbon dioxide storage. VII. Changes in flower color as evidence of the effectiveness of carbon dioxide in reducing the acidity of plant tissue," C. B. T. /., 6 : 403-405 (1934). 83. ," Carbon dioxide storage. VIII. Chemical changes in potato tubers resultmg from exposure to carbon dioxide," C. B. T. I., 7 : 113-118 (1935). MISCELLANEOUS 443 84. Thornton, N. C, "Carbon dioxide storage. IX. Germinal ion of lettuce seeds at high temperatures in both light and darkness," C. B. T. J., 8 : 25-40 (1930). 85. , "Carbon dioxide storage. X. The effect of carbon dioxide on the ascorbic acid content, respiration, and pH of asparagus tips," C. B. T. L, 9 : 137-148 (1937). 86. , "Carbon dioxide storage. XL The effect of carbon dioxide on the ascorbic acid (vitamin C) content of some fruits and vegetables," Proc. Am. Soc. Hort. Sci., 35(1937) : 200-201 (1938). 87. , "Carbon dioxitie storage. XIII. Relationship of oxygen to carbon dioxide in breaking dormancy of potato tubers," C. B. T. /., 10 : 201-204 (1939). 88. , "Development of dormancy in lily bulbs," C. B. T. /., 10 : 381-388 (1939). 89. , "Dark brown color of potato chips caused by high reducing sugar (not by sucrose nor total sugar) content," Am. Chem. Soc, Div. Agric. A: Food Chem. Abstr. papers, 99th meeting, Cincinnati, Ohio. Apr., 1940. p. A18-19. 90. , "Carbon dioxide storage. XIV. The influence of carbon dioxide, oxygen, and ethylene on the vitamin C content of rip>ening bananas," C. B. T. /., 13 : 201-220 (1943). 91. , and E. P. Imle, "Why a dwarf Easter lily?" Flor. Exch., 94(15) : 9 (April 13, - 1940). 92. Youden, W. J., "A nomogram for use in connection with Gutzeit arsenic determi- nations on apples," C. B. T. /., 3 : 363-373 (1931). 93. , "Statistical analysis of seed germination data through the use of the Chi- square test," C. B. T. L, 4 : 219-232 (1932). 94. , "Use of incomplete block replications in estimating tobacco-mosaic virus," C.B.T. J., 9:41-48 (1937). 95. , "Dilution curve of tobacco-mosaic virus," C. B. T. I., 9 : 49-58 (1937). 96. , "Seed treatments with talc and root-inducing substances," C. B. T. /., 11 : 207-218 (1940). 97. , "Experimental designs to increase accuracy of greenhouse studies," C. B. T. I., 11 : 219-228 (1940). 98. , "Fluctuations of atmospheric sulphur dioxide," C.B.T.L, 11:473-484 (1941). 99. , and H. P. Beale, "A statistical study of the local lesion method for estimating tobacco-mosaic virus," C. B. T. L, 6 : 437-454 (1934). 100. , , and J. D. Guthrie, "Relation of virus concentration to the number of lesions produced," C. B. T. /., 7 : 37-53 (1935). 101. , and F. E. Denny, "Factors influencing the pH equilibrium known as the isoelectric point of plant tissue," Am. J. Bol., 13 : 743-753 (1926); also in C. B. T. /., 1 : 278-288 (1926). 102. , and I. D. Dobroscky, "A capillary glass electrode," C. B. T. I., 3 : 347-362 (1931). 103. , and A. Mehlich, "Selection of efficient methods for soil sampling," C. B. T. /., 9 : 59-70 (1937). 104. , and P. W. Zimmerman, "Field trials with fibre pots," C. B. T. /., 8 : 317-331 (1936). 105. Zimmerman, P. W., "The present status of vitamin Bi," Am. Rose Ann., 26(1941) : 87-94 (1941). Author Index Abott, 286 Afanasiev, Michel, 73, 98 Agati, J. A., 1G4 Aldrich, W. \V., 398 Allard, H. A., 285, 309 Allen, R. C, 423, 424 Allers, 51 Alway, F. J., 101 Ames, R. W., 180 Appleman, C. O., 230, 422 Arthur, J. C, 80-81 Arthur, John M., 5, 0, 230, 286, 288, 203, 205, , 307, 300, 311, 318, 320, 321, 322, 323, 328, 332, 334, 337, 351, 354, 359, 436 Asmaev, P. G., 164 Astbury, W. T., 273 Astwood, E. B., 236 Atterberg, Albert, 125 Atwood, W. M., 30, 84, 125 Avery, A. G., 40, 62 Backus, M. P., 436 Bacon, Raymond Foss, 3, 5 Ballard, AV. S., 398 Barker, J., 160 Barrows, Florence L., 270, 433, 434, 435 Barsha, J., 264 Barton, Lela V., 31, 32, 39, 41. 52, 53, 55, 56, 89, 101, 102, 111, 113, 118, 119, 120, 122, 127, 196, 418 Batjer, L. P., 215 Baj-field, E. G., 164 Beal, 34, 36, 38, 39 Beale, Helen Purdy, 14, 16, 439 Becker, 84 Becquerel, Paul, 28, 29, 30, 39, 52, 65 Bell, Frank H., 22 Berg, R. O., 194, 195 Berger, L. B., 147 Berkeley, C. H., 12 Bernal, J. D., 273 Bier, August, 51 Blakeslee, A. F., 40, 62 Blauvelt, W.E..357 Bliss, C. I., 370 Borriss, Heinrich, 44 Bourn, W. S., 15. 16, 20-22, 23, 24-26 Boussingault, 183 Boysen-Jensen, P., 205, 206 Bradford, 312 Bredeman, G., 199 Brenner, M.W., 165 Brierley, P., 432, 433 Brown, E., 68, 70, 74 Brown, H. T., 329 Brown, R., 79 Brown, Robert, 29, 30 Bruchmann, 435 Burrell, G. A., 179 Busse, W. F., 32. 73 Byrnes, J. Wise, 166 126, Candolle, de, 28, 33 Carter, J. Bailey, 155 Cation, D., 12 Chace, E. M.j, 150 Chan, Shuk Yee, 369, 377 Chang, S. C, 70 Charipper, H. A., 236 Chase, \V. W., 398 Chesney, A. M., 236 Chibnall, 416 Chipman, C. N., 156 304, Cholodny, N., 206 329, Christensen, B. E., 166 Clare, T. S., 102 Clark, G. L., 261 Clawson, T. A., 236 Clay, Edythe I., 22 Cohn, 416 Collier, T. R., 186, 191 Collins, D. L., 357 Compton, Jack, 270 Corey, William E., 16 Corl, C. S., 350 Coulter, John M., 5 Coville, F. v., 230 Cress, M., 99 Crocker, William, 5, 44, 45, 61, 67, 74, 79, 81-83, 87, 101, 123, 125, 120, 140, 141, 142, 143, 144, 147, 148, 151, 152, 158, 162, 164, 167, 173, 175, 176, 179, 180, 181, 182, 189, 190, 191, 206 Daines, R. H., 182 Darlington, H. T., 38 Darwin, C, 205 Davenport, Eugene, 5 Davies, P. A., 73 Davis, W. E., 46, 47, 48, 49, 51, 79, 84, 87, 96, 101, 123, 129 Deiman, 182 Delavan, 32 Deming, G. W., 70 Denny, F. E., 16, 39, 41, 131, 143, 147, 148, 158. 160, 165, 167, 216, 231-232, 233, 234, 235, 239, 240, 241, 242, 243, 245, 246, 248, 249, 250, 251, 386, 387, 389, 393, 395, 408, 409, 412-414, 416, 418, 420, 421, 423, 435 Dillman, 56 Dills, L. E., 346 Dimitriewicz, 129 Dinnis, E. R., 73 Dobroscky, Irene D., 14, 437 Doby, Tom, 2 Dodge, B. O., 435 Dolk, H. E., 206 Dorph-Petersen, K., 40, 43 Doubt, S.L., 142, 150 Dow, R. B., 73 Dowd, O. J., 308 Duncan, Robert Kennedy, 2, 4 DuvpI, 28, 31, 35 Dykins, F. A., 160 445 I 446 AUTHOR INDEX Eckerson, Sophia H., 87, 97, 263, 264, 265, 268, 270 Elmer, O. H., 164 Englis, D. T., 160 Erxleben, H., 206 Escombe, F., 329 Esdorn, I., 73 Evenari, Michael, 44 Ewart, AUred J., 28, 29, 30, 33, 34, 39, 40, 55, 61 Ewing, 416 Eyster, W. H., 68 Faraday, 185 Farr, Clifford H., 260 Farr, Wanda K., 260-283, 261, 263, 264, 265, 268, 270, 273, 276, 280 Finch, A. H., 150 Fischer, H., 285 Fisher, D. F., 148 • Flemion, F., 87-89, 92, 93, 96, 98-99, 104, 105, 107, 108, 109-111, 118, 123, 125, 128, 129-131 Flinn, F. B., 236 Flint, L. H., 77 Frear, D. E. H., 383 Froschel, P., 44 Fuller, H. J., 185 Funke. H., 78 Furr, J. R., 398 Gabunva, M. K., 160 Gadd, I., 73, 126 Gaddum, J. H., 370 Gailey, W. R., 166 Galang, F. G., 164 Gane, R., US, 164-165 Gardner, F. E., 215 Garner, W. W., 285, 309 Gassner, G., 109 Geary, J. M., 236 Gersdorff, Charles E. F., 60 Giersbach, J., Ill, 118 Giese, A. C, 185 Gimingham, C. T., 350 Ginsburg, 425 Gnadinger, C. B., 346, 350 Gockel, H., 236 Goldsmith, E. D., 236 Goodspeed, 62 Gordon, A. S., 236 Goss, W. L., 36, 51 Goulart, M. D., 44 Graham, J. J. T., 350 Graham, S. A., 343 Gray, N. E., 185 Grimm, K., 73 Gross, 416 Groves, 61 Gtimbel, Hermann, 41 Guillaumin, 55 o Gustafsson, Ake, 62 Guterman, C. E. F., 431-433 Guthrie, D., 155 Guthrie, J. D., 212, 216, 231, 234, 239, 240, 248, 249, 250, 251, 252, 286, 288, 311, 328, 330, 439 Haagen-Smit, A. J., 206 Hagedorn, Hermann, 1, 3 Hale, W. S., 164 Hamly, D. H., 72 HanIein,H.,41,43 Hansen, E., 160, 165, 166 Harrington, G. T., 70, 71, 73, 84, 85, 102, 125, 129, 185 Harrington, J. B., 70 Harris, Stanton A., 270 Hartman, H., K)6 Hartzell, Albert, 12, 14, 191, 192-193. 196,236,343- 359, 344, 346, 348, 351, 352, 353, 354, 355, 356, 357, 366, 433 Harvey, E. M., 142, 148, 159, 160, 172, 176 Harvey, R. B., 159 Harvill, E. K., 223, 230, 304, 307, 334, 350, 351, 354, 358, 359 Hendricks, R. H., 186, 191 Henry, B. S., 166 Herbert, D. A., 164 Hess, A. F., 285 Hibbard, R. P., 159, 160 Hibbert, H. L., 264 Hildebrand, E. M., 12 Hill, George R., 186, 188, 190, 191 Hiltner, L., 51 Hitchcock, A. E., 142, 143, 144, 147, 152, 158, 160, 162, 164, 173, 175, 176, 179, 206, 211, 215, 216, 218, 220, 223, 225, 226, 309, 323, 419 Hobson, R. P., 350 Hohnel, F. von, 72 Holbrook, W. T., 147 Hollenberg, George J., 273 Hobnes, F. O., U Hooker, 312 Hooper, F. E., 270 Horsfall, J. G., 365, 374, 383 Hottes, Chas. F., 139 Hubner, R., 71 Huelin, F. E., 160 Humbert, 29 Hutton, Mary Erne-Jean, 72 lelpo, M., 44 Imle, E. P., 431, 433 Ivanov, N. N., 160 Ivie, J. O., 186 Jensen, C, 78 Jesse, 2S5 Johnson, 416 Johnson, A. N., 427 Johnson, L. P. V., 84 Johnstone, G. R., 102 Jones, D. Breese, 31, 60 Jones, G. W., 147 Jones, H. A., 97 Jones, J. P., 73 Jones, L. R., 10 Jordan, S., 73 Joseph, Hilda C, 55 Juel, Inger, 60 Kellev, J. N., 165 Kennedy, T. H., 236 Kerensky, 3 Kidd, Franklin, 33, 45, 400 Kiesselbach, T. A., 55 Kincaid, R. R., 185 Kinzel, 45, 75, 76 Kirkpatrick, H., Jr., 215 Kittredge, George Lyman, 4 Kjaer, Arne, 40 Klinger, H., 348 Knight, Henry G., 158 Knight, L. I., 140, 141, 142, 147, 151, 167 Knop, 75 Knott, 309 Knowles, P. F., 70 Knudson, 425 Kockemann, Alfons, 44 Kogl, F., 206 AUTHOR IXDEX 447 Kommerell, E., 77 Kondo, Mantaro, 51 Konis, E., 44 Kraus, Ezra J., 5, 285, 311 Kraybill. H. R., 285, 311, 437 Kroeger, G. S., 123-125 Kriiger, F., 348 Kunkel, L. O., 6, 10, 11, 12, 14, 15 Laibach, F., 206 Laing, H. E., 398 Lakon, G., 129 Lammerts, W. E., 94 Langdon, S. C, 166 Lathrop, F. H., 353 Lauwerenburgh, 182 Leblond, C. P., 236 Lehmann, 84 Leonard, C. S., 323 Lesage, 129 Lewcock, H. K., 164 Lewis, D., 155 Livingstone, H. M., 155 Lojkin, Mary E., 15, 321, 334 Loomis, W. E., 230, 231 Luckhardt, A. B., 155 Lumsden, D. V., 166 Lundegardh, H., 285 Lundy, J. S., 155 Lute, A. M., 73 Lynch, L. J., 160, 164 Lyssenko, 334, 340 McAlister, E. D., 77 McAtee, W. L., 15-16, 22-23 McCaUan, S. E. A., 193, 196, 362-383, 362, 363, 365, 366, 368, 369, 370, 371, 372, 374, 377, 378, 379, 380, 381 McCallum, W. B., 230, 234 McCool, M. M., 425, 426, 427, 428 Mcllvaine, H. R. C., 78 McKeever, D. G., 72 McKenna, G. F., 357 MacLachlan, P. L., 78 McLean, F. T., 430 Magness, J. R., 398 Mangelsdorf, P C., 68 Manns, T. F., 12 Marsh, R. W., 363 Marth, P. C.. 215 Martin, H., 367, 383 Martin, J. N., 128 Marwick, T. C, 273 Maskell, E. J., 409 Mason, T. G., 409 Mehhch, A., 427, 428, 438 Mellon, A. W., 3 Mellor, J. W., 181 Menusan, H., Jr., 346 Metman, 29 Meyer, Bernard S., 22 Middleton, G. K., 73 Midgley, A. R., 73 Milbrath, J. A., 166 Millardet, 306 Miller, E. C, 329 Miller, E. V., 148 Miller, L. P., 148, 165, 231, 234, 240, 241, 246, 247, 248, 249, 250, 251, 252 Moore, W., 343 Moreland, C. F., 77 Morinaga, T., 80 Muller, A., 206 Miiller, G., 112 Muenscher, W. C., 78 Murakami, R., 78 Murphy, D. F., 351 N-igpli, 270 Nakajima, 32 Navashin, 62 Neljubow, D., 140, 151 Nelson, R. C, 165 Newell, Arthur Cotton, 4 Newell, J. M., 286, 288, 311, 322 Niederl, J. B., 165 Nobbe, F., 41, 43, 51 Nord, F. F., 160 Oberfell, G. G., 179 O'Gara, 186 Ohga, Ichiro, 34, 50-51 O'Leary, Keith, 431, 433 Oortwijn Botjes, Je, 148, 164 Orth, R., 77 Ozorio de Almeida, A., 44 PadI, A., 205 Paats, 182 Pack.D. A., 92, 97, 98, 101 Parker, E., 108 Pascal, 185 Pearsall, 416 Peet. C. H., 351 Penhallow, 10 Peter, 34 Peters, Richard, 10 Peto, 62 Petre, A. W., 14 Pfeiffer, N. E., 107, 112, 331, 332, 429, 430, 431 Pietruszczyiiski, Z., 123 Pinto, A. Vieira, 44 Poe, J. G., 155 Pollacci, 363 Pope, Fred J., 4, 5 Pope, M. N., 68, 70 Popp, H. W., 73, 78, 314, 316, 320 Porter, L. C., 295, 304 Porter, R. H., 72, 74 Price, W. C., 14 Priode, C. N., 15 Prokoshev, S. M., 160 Pryor, D. A., 182 Purdy, H. A., 206 RadelofT, H., 199 Raleigh, G. J., 71, 77 Rancken, M., 73 Ratliffe, G. T., 97 Ratsek, J. C, 180-181 Rees, Bertha, 51 Reeves, Richard E., 270 Reid, M. E., 436 Riedel, 285 Rigg, G. B., 166 Rivera, R., 73 Robertson, D. W., 70 Robins, Raymond, 3, 4 Rodriguez, A. G., 104 Rosa, J. T., 230 Rose, R. Catlin, 87, 96, 148, 172, 176 Rostrup, O., 74 Rudolfs, W., 416 Ruzicka, L., 350 Sachs, 409 Saulescu, N., 73 Schiifer, W., 206 Schaible, F., 78 Schaller, Johnny, 2 448 AUTHOR INDEX Schanz, F., 285 Sehaumann, K., 86 Schjelderup-Ebbe, Thorleif, 30 SchoUenberger, C. J., 180 Schreiner, Olive, 4 Schroeder, Eltora M., 122, 127 Schwemmle, J., 61, 62 Schwendiman, A., 102 Schwimmer, S., 164 Scott, I. T., 416 Scudder, H. I., 348 Seegal, Beatrice Carrier, 15 Seil, H. A., 350 Setterstrom, C, 186, 190, 191, 192-193, 196, 199, 366 Seubert, E., 206 Shands, H. L„ 102 Shaw, M.F., 71, 72 Sherman, 39 Sherman-Pappenheimer, 321 Shirlev, H. L., 320, 323, 328, 331. 332, 333 Shuck, A. L., 44, 125 Shull, C. A., 82, 83 Sissoii. Wayne A., 268, 270, 274 Slogteren, 337 Smith, Erwin F., 10 Smith, F. F., 432 Smork, R. M., 166 Soding, H.,206 Solheim, W.G., 180 Sorber, D. G., 150 Spaeth, J. N., 84 Spencer, Herbert, 4 Speyer, E. R., 352 Sprague, V.G., 78 Stahmann, M. A., 182 Stalfelt, M. G., 79 Stanley, W. L., 15 Stanton, E. N., 245 Stark, P., 205, 206 Staudinger, H.,350 Steenbock,H., 285 Stevenson, T. M., 71 Stewart, W. D., 293, 295, 322, 328, 329, 436 Stier, H. L., 84 Stout, Myron, 44 Strasburger.E., 267 Strong, M., 352 Stuart, W., 230 Stubbe, H., 61 Stiitz, H., 73 Sullivan, J. T., 437 Takahashi, T., 70 Tattersfield, F., 350 Taylor, D. L., 79 Taylor, Major General H., 23-24 Thimann, K. V., 206 Thorn. Charles, 264 Thomas. Moyer D., 186, 188. 190, 191 Thompson, H. C, 333 Thompson, H. J., 270 Thompson. Lawrence C, 22 Thompson, William Boyce, 1-6 Thornber, J. J., 51 Thornberry,H.H., 15 Thornton, Norwood C, 48, 82, 83, 84, 85, 196, 199, 216, 230, 234, 235, 241, 386, 387, 389, 393, 395, 398, 399, 400, 401, 402. 403. 404, 406, 407, 408, 431 Tolman, Bion, 44 Toole, E. H., 56, 128 Toole, V.K.. 73, 102, 128 Treub, 435 Tufts, 4 Turner, J. H., 30 Ullman, Salomon Baruch, 44 Van-Troostwyck, 182 Verret, 33 Verschaffelt, E., 72 Vilmorin, 28 Vinson, C. G., 14, 15 Waggoner, H. D., 55 Walker, J. C, 182 WaUace, R. H., 150 Waiti, A.,44 Waterbury,E., 96 Webster. B., 236 Webster. C.B., 97 Weedon, F. R., 191, 192-193. 196, 366 Weinstock, M., 285 WeUman, R. H., 369. 370. 371. 372, 374, 377, 378, 379, 381 Went, F. W., 206 Wentworth, George, 4 Whetzel, H. H., 6 White, J., 71 Whiteman, T. M., 166 , Wiesehuegel, E. G., 51 Wieser, 76 Wiesner. J.. 78, 267 Wilcoxon, F.. 193. 206. 216, 344, 346, 348, 350, 351, 352, 353, 354, 355, 356, 357, 358, 362, 363, 365, 366, 368, 369, 370. 371. 372, 374, 380, 381 Wilcoxon. F. (Mrs.), 354 Windisch, W., 125 Winston, J. R.. 148 Witte. H. 73 Woo. 312 Woodhouse. K. W., 155 Woods, M.W., 15 Wright, R. €., 166 Youden, W. J., 353, 355, 414, 416, 418, 419, 425, 437, 438, 439 Young, H. C, 362 Zimmerman. P. W., 142, 143, 144. 147. 152. 158, 160. 162, 164. 173, 175, 176, 179, 180, 181, 182, 186. 189. 190, 191, 194, 195, 204-226. 206. 211, 215, 216, 218, 220, 223, 225, 226, 309, 323, 419, 438 I Subject Index Abies, 90 Abies arizonica, 102 Abscission: ethylene effects, 150 Abutilon, 30, 35, 51 Acacia, 30 Acenaphthene-(5)-acetic acid, 207 Acer, 89, 90 Acer saccharinum: vitality of seeds, 31 Acetohacter xylinus: formation of cellulose mem- branes, 263-264 Acetylene: effects on roots, 173 physiological effects on plants, 142-143, 152, 163 Acids, fatty: contact insecticides, 346 Acrocomia , 33 After-ripening of seeds: butternut, hickory, wal- nut, 32 Cotoneaster, 111 Crataegus, 109-111 Impatiens bahamina, 123-126 Symphoricarpos racemosus, 107-lOS temperature effects, 48-49, 86-123 After-ripening of seeds, epicotyl: temperature effects, 113-123 Aging of seeds: causes, 60-63 Agrostemma, 36, 38 Albizzia, 30, 51 AHsma. 86. 90 Alisma Plantago, 79, 101 Allium, 75 Alsine, 36 Alsophila pometaria: {See Cankerworm) Alternaria solani: comparison of greenhouse and laboratory spore tests of fungicides on to- mato, 378 induced sporulation in culture, 377 time and temperature effect on germination of spores, 372 use in greenhouse tests of fungicides on tomato, 374-378 Amarardhus: 36, 37, 38, 39, 40, 129, 312 dormancy and water-absorption of seeds, 126 Amaranthus retroflexus: 39, 41 dormancy and germination of seeds, 127-128 Amaryllis: pollen storage, 429-430 Ambrosia, 35, 36, 37, 45, 46, 47, 84, 85 Ambrosia trifida: after-ripening and development of secondary dormancy in embryos, 4t>-47 Amelanchier , 90 2-Amino-5-chlorobenzoic acid, 223 2-Amino-3,5-diiodobenzoic acid, 223 Ammonia effects: on plants and animals, 196-200 Amorpha, 72 Amylase: affected by ethylene chlorohydrin, potas- sium thiocyanate, 250 Anagallis, 34 Analytical methods: capillary glass electrode, 437 for ashing plant tissue, 436-437 for determination of reducing sugars, 437 Anatomical studies of effect of light on plants, 331- 333 Anesthesia: induction by gases, 150-158 Anesthetic effects on animals: of ethylene,^155-1.58 Anesthetic effects on man: of ethylene, 155-158 Anesthetic effects on plants: of growth substances, 22G Anthemis, 36 Anthocyanin: effect of low temperature on devel- opment, 320-321 Antholyza revoluta, 430 Anthraceneacetic acid, 207 Anthriscus, 43 Anthyllis, 30 Aphis gossypii: transmits lily mosaic, 432 Aphis rumicis: nicotine toxicity, 344 organic thiocyanates toxicity, 351 pyrethrum toxicity, 350-351 Apium, 35, 40 Apple: arsenic residues from spraying, 355 red pigment production by artificial light, 320-321 Aquilegia, 42 Aralia, 90 Aralia racemosa, 112 Arbutus, 90 Arbutus, trailing: propagation, 433-434 Arceuthobium, 75 Arceuthobium oxycedri: dormancy and germination in seeds, 123 Arctium, 35 Arctostaphylos Uva-ursi, 112 Arsenic: determination in sprayed apples, 355 sampling method of residue on sprayed apples, 439 Artificial light: effects on growth of plants, 285-340 Asarum, canadense, 113 Ascorbic acid: carbon dioxide effect on fruits and vegetables, 406—408 production in potatoes treated with ethylene chlorohydrin, 252 Ash, plant: effect of ultraviolet, 322 Ashing plant tissue: analytical methods, 436-437 Asimina, 90 Aspergillus niger: value in determining potash de. ficiency in soils, 428 Aspidium filix-mas: as an insecticide, 354 Aster yellows: studies, 10-12 symptoms, 11 Astragalus, 29, 30, 51 Attalea, 33 Auxin: (See Growth substances) Avena: 39, 205, 206 cellulose in coleoptile epidermis, 270-273 Avena coleoptile: test object fur growth promoting substances, 206 Azalea nudiflora: 244 shortening rest period, 245 Ballota, 43 Belamcanda, 90 Benzoic acid derivatives: growth substance activity as affected by their str\icture, 223 Benzoin, 90 Beta, 35 Betula, 90 Boea, 33 Boehmeria, 35 Bordeaux mixture: effects on spore germination. 366-368 fungus spores solubilize copper, 368-369 weathering, 368-369 Botrychium lariceolatum, 122 449 450 SUBJECT INDEX Botryiis: 197 fungicidal action of pentathionio acid, sulphuric acid and hydrogen sulphide on spores, 303- 366 Boyce Thompson Institute for Plant Research: aims and scope of work, 6-8 development and organization, 1-S founder, 1-6 Brassica, 35, 36, 37 Brassica rapa, 332 Brauneria sp.: as insecticide, 354 2-Bromo-3-nitrobenzoic acid, 223 Bromus, 36, 38 Bryophyllum: 255 effects of ethylene chlorohydrin, light, on citric acid content, 250 Buckwheat: studies of artificial light effects on dry weight, 293-309 Bud inhibition: delayed flowering of fruit trees, 216 of potato tubers by methjl a-naphthaleneacetate, 216 Buds: dormancy, 230-256 Buffers: factors influencing pH equilibrium of plant tissue, 414-118 Bunt of wheat: method for evaluating fungicides, 379 Buried seed project: Beal's, 36-39 of U.S.D.A., 34-36 Bursa, 36 Butane: injury to plants, 179-180 Butomus, 90 Butomus umbeUatus, 101 Butylene: physiological effects on plants, 142-143 Butylene effects: on roots, 173 Calceolaria, 308, 309, 338 CaUistcmon, 33 Calochortus macrocarpus: dormancj- and germina- tion of seeds, 127 Camassia Leichtlinii: dormancy and germination of seeds, 127 Camellia: as affected by growth substances, 212,214 Campanula, 42, 43 Canavalia, 30 Cankerworm: control by banding, wind dispersal, 355-356 Capillary glass electrode: determination of pH, 437 Capsella, 42, 43 Carbohydrate nitrogen ratio: 285 effect of artificial light on plant composition, 311- 312 Carbohydrates: changes in leaves during night, 409, 412 effects of artificial light on plant composition, 311-312 effects of carbohydrate reserves on growth of seedlings, 436 Carbon dioxide: physiological and biochemical effects on fruits, seeds, vegetables and flowers, 398-408 Carbon dioxide concentrations: effects on plant growth, 286-290 Carbon dioxide effects on: acidity of plant tissue, 400-403 aquatic plants, 23 ascorbic acid content of fruits and vegetables, 400-408 flower color, 403 fungal hyphae, 401-403 germination of lettuce seeds, 399-400 potato tuber dormancy, 400 respiration rate of plant tissue, 403—106 vitality: (See Vitality of seeds) Carbon dioxide storage effects on potato tubers: reducing sugar, 395-397 Carbon disulphide effects on: dormant potato tubers, 234 Carbon monoxide: physiological effects on plants, 142-143, 152-154, 160-164 Carduus, 35 Carica, 44 Carya, 90 Cassia, 29, 35 Catalase: changes in relation to respiration rate, 249 Catalpa, 148 Caulophyllum thalictroides: dormancy and germina- tion of seeds, 120-121 Cdastrus, 90 Cell membrane studies, 260-283 Cellulose: bacterial"studies, 263-264 detection in fibers, 271 formation of membranes, 264-273 formation of particles, 273-282 in Avena coleoptile epidermis, 270-273 microscopic techniques used for analyzing bac- terial membranes, 264-270 separation of particles by hydrochloric acid, 268- 270 studies, 260-283 Celtis, 90 Ceratophyllum, demersum, 15 Ceratostomella ulmi, 197 Cercis, 73 Chaerophyllum, 42 Chaetochloa, 35, 36 Chamaesyce, 36, 38 Chara, 21, 22, 24, 25 Chelidonium. , 42 Chemicals: as forcing agents for dormant seeds, 131 effect of, on permeability of dormant potato tubers, 251 Chenopodium, 34, 35, 40, 42, 43 Cherry: effects of illuminating gas on roots, 173 Chionanthus virginiana, 118 Chlorinated water effects on: goldfish, 194-196 plants, 194-196 Chlorine effects on: plants and animals, 194-200 Chloris, 75, 76, 109 2-Chloro-3,5-diiodobenzoic acid, 223 2-Chloro-5-nitrobenzoic acid, 223 2-Chlorophenoxyacetic acid, 209, 218 3-Chlorophenoxyacetic acid, 209 4-Chlorophenoxyacetic acid, 209, 218 a-(2-Chlorophenoxy)-?i-butyrio acid, 209, 218 a-(3-Chlorophenoxy)-n-butyric acid, 209, 218 a-(4-Chlorophenoxy)-n-butyric acid, 209, 218 a-(2-Chlorophenoxy)-propionic acid, 209, 218 a-(3-Chlorophenoxy)-propionic acid, 209, 218 a-(4-Chlorophenoxy)-propionic acid, 209, 218 Chlorophyll: artificial light effects, 295 effect of light on concentration, 328 Chlorophyll pigments: effect of environment, 330- 331 Chrysanthemum, 35 Chrysanthemum cinerariaefolium: {See Pyrethrum) Chrysanthemum leucanthemum, 11 Cicadula sexnotata: aster yellows carrier, 1 1 hosts, 11 Cinchona: 52 pollen storage, 429-430 Cinnamic acid, 207 Cinnamic acid, irradiated: applied as a vapor pro- duces epinasty in plants, 226 Cirsium, 40 Cissus aerial roots: inhibition by a-naphthalene- acetic acid, 216 Citric acid in potato tubers: changes in relation to respiration rate, 249 effects of ethylene chlorohydrin on, 248-249 Citrus: moisture effects on vitality of seeds, 31-32 Coccomyces hiemalis: origin of ascocarp, 436 Cocklebur: primary and secondary dormancy, 47-48 SUBJECT INDEX 451 Cocos, 33 Coffea, 33 Coleus, 1.30 Color in flowers: effects of carbon dioxide, 403 Color in hydrangea: acidity of soil, aluminum, iron effects, 423-424 Comespenna, 30 Composts: improved by addition of peats, 425 Constant^condition rooms: for studying effect on grow-th of plants, 2S6-290 Convallaria majalis: dormancy and germination of seeds, 120-123 Convolvulus, 35 Copper: in Bordeaux mixture solubilized by fun- gus spores, 368-369 Copper fungicides: action, 366-369 Coral-berry: (See Sijmphoricarpos orbiculatiis) Corethra larvae: effects on nerves, muscles and hy- podermis by insecticides, 348-350 Cornus, 90, 419 Cornus ca7iadensis, 112 Cotoneaster : 106 after-ripening, dormancv, germination of seeds, 111 Cotoneaster acutifolia. 111 Cotoneaster apiculata. 111 Cotoneaster Dielsiana, 111 Cotoneaster divaricata, 111, 112 Cotoneaster horizontalis, 111, 112 Cotoneaster lucida. 111 Cotoneaster Zabelii, 111 Cotton: (See Gossypium hirsutum) Crassula arborescens, 148, 150, 151 Crassula rubicunda: 337, 340 low temperature effects on dormancy of buds, 230 Crataegus: 89, 90, 92, 96, 97, 106 after-ripening, dormancy, germination of seeds, 109-111 Crataegus arnoldiana , 111 Crataegus carrierei, 111 Crataegus coccinea, 109 Crataegus cordata, 109 Crataegus Crus-gaUi, 109, 112, 1.30 Crataegus flara, 109, 110, 112 Crataegus mollis. 111 Crataegus Oxyacantha, 112 Crataegus punctata, 109, 112 Crataegus rotundifolia, 109, 110, 112 Crataegus sanguinea. 111 Crataegus tomentosa. 111 Crepis tectorum, 62 Cucurbita, 79 Culex Quinquefasciatus: larvae as test objects for insecticides, 354 Cumulative error terms: in fungicide tests, 379 Cunniiigliamella blakesleeana: vahie in detecting phosphorus and nitrogen deficiencies in soils, 428 Cupressus, 90 Cuscuta, 3.5 Cyanamide: effect on composts, 425 Cynoglossum, 43 Cyperus, 35 Cytisus, 29, 30, 31 Dahlia: 11 mosaic diseases, 433 Dandelion: effect of storage on seeds, 57 Datura, 35, 40, 43, G2 Daucus, 40 Day-length, effect on flowering: constant-condition rooms for study, 286-290 Delphinium: effects of storage on seeds, 56-57 Deutzia, 245 » Diagnosis of tree and shrub injury by artificial il- luminating gas, 178-179 Diehloroethyl ether, /S,/3'-: fumigation, 353, 356-3.57 Dichloroethylene: effects on dormant potato tubers, 233-234 2,4-Dichlorophenoxyacetic acid, 209, 218 2,5-Dichlorophenoxyacetic acid, 218, 222 a-f2,4-Dichlorophenoxy)-n-butyric acid, 209, 218 a-(2,4-Dichlorophenoxy)-propionic acid, 209, 218 Dictamnus, 90 Digitalis, 42, 307, 323, 336, 339 Digitalis purpurea: light, low temperature effects on flowering. 334-337 2,4-Diiodobenzoic acid, 223 3,.5-Diiodobenzoic acid, 223 2,4-Dimethylphenoxyacetic acid, 222 3,4-Dimethylphenoxyacetic acid, 218, 222 3,5-Dimethylphenoxyacetic acid, 222 o:-(2,4-Dimethylphenoxj-)-n-butyric acid, 222 a-(2,5-Dimethylphenoxy)-«-butyric acid, 218, 222 a-(3,4-Dimethylphenoxy)-n-butyric acid, 222 a-(3,5-Dimethylphenoxy)-n-butyric acid, 222 /3-(3,.5-Dimethylphenoxy)-n-butyric acid, 222 a-(3,5-Dimethylphenoxy)-malonic acid, 222 a-(2,4-Dimethylphenoxy)-propionic acid, 222 a-(2,5-Dimethylphenoxy)-propionic acid, 218, 222 a-(3,4-Dimethylphenoxy)-propionic acid, 218, 222 a-(3,5-Dimethylphenoxy)-propionic acid, 222 Dioclea, 29 Diospyros, 90 Diurnal changes in leaves: 408-412 comparison of methods of computing, 410-411 Dormancy: chemical treatments for shortening rest period of deciduous trees and shrubs, 243- 246 effects of carbon dioxide on potato tubers, 400 effects of ethylene chlorohydrin on lilac buds, 245 in potato buds, 2.50-251 Dormancy in buds: 230-2,50 low temperature effects, 230 Dormancy in gladiolus: respiration rate, 39 temperature effects, 41 Dormancy in gladiolus cormels: effects of ethylene chlorohydrin, 241-243 Dormancy in gladiolus corms: as affected by ethyl- ene chlorohydrin, temperature, 241 effects of ethjlene chlorohydrin, 240 maintenance, 241 Dormancy in imbibed seeds: causes, 43—45 Dormancy in lily bulbs: effects of oxygen, 431 effects of temperature, 431 Dormancy in potato buds: 230-240 effects of methyl ester of a-naphthaleneacetic acid vapor, 239-240 effects of oxygen, 230-231 metabolic changes induced by chemicals, 246 use of anesthetics, 230 Dormancy in potato tubers: as affected by gluta- thione, 251-2.52 chemical effects, 231-238 growth substances effects, 231-238 sulphur compounds effects, 234 thiourea effects, 234-235 Dormancy in seeds: 67-132 advantage to man, 68-70 after-ripening in dry storage, 123-127 Amaranthus retrojlexus, 127-128 Arceuthobium oxycedri, 123 Calochortus macrocarpus, 127 Camassia Leichtlinii, 127 Caulophyllum thalictroides, 120-121 chemicals as forcing agents, 131 Convallaria majalis, 120-123 Cotoneaster, 111 Crataegus, 109-111 dormant embryos, 86-101 drving effects, 32 epicotyl, 113-119 452 SUBJECT INDEX Dormancy in seeds — continued Impaliens balsainina, 123-12t) inconveniences toman, 70-71 lettuce, 399-400 Leu'isia rediinva, 127 light effects on germination, 7-t-78 low temperatuie eftects, 86-123 oxygen requirement for germination, 78-85 Polygonatum commutatum, 120-121 quick vitality tests, 128-131 Sanguinaria canadensis, 120-121 seed coat effects, 46-47, 71-74 seeds requiring two low-temperature exposures, 119-123 significance, 67—68 Sinilacina racemosa, 120-123 sulplmric acid effects, 106-112 Symphoricarpns orbiculatus, 108-109 temperature effects, 48-49 temperature effects on wild plants, 41-43 temperature requirements for germination, 127- 128 Trillium erectum, 120-121 Trillium grandiflorum, 119-121 types, 71-128 Viscum, album, 123 water absorption in Amaranthus seeds, lettuce seeds, Rumex seeds, 126 Dormancy in seeds, epicotyl: in Chionanthus rir- giniana, 118 in Symplocos paniculata, 118 in tree peony, 113-117 in yibur««m, 113, 117-118 Dormancy in seeds, primary and secondary: in cocklebur, 47-48 Dormancy in seeds, secondary: in Ambrosia trifida, 46-47 in Nigella saliva, 45 Dormancy in tubers, 230-256 Drip oils: effects on growth of tomato plants, 178 Duck-food plants: ecological and physiological studies on certain aquatic plants, 15-26 economic loss due to destruction of certain aquatic plants, 25 restoration of aquatic plants, 25-26 sea-water tolerance, 20 Duck-food problem: study of aquatic plants, 15- 26 Dwarf seedlings: grown from plants with dormant embryos, 93-97 Electrodes: capillary glass electrode, 437 Emanations from soil treated with mercury com- pounds: effects on plants, 180-185 Embryos, dormant: chemical and physiological changes during low-temperature after-ripen- ing, 97-101 physiological studies, 86-101 Embryos, non-dormant: germination, 101-105 Energy: received and lost by leaves, 329 Entelea, 30, 31 Enzymes: (See name of enzyme) Epicotyl dormancy in: seeds, 113-119 Epilachna variveslis: black pepper toxicity, 354 Epilobium, 75, 76 Epinasty: gas effects on leaves, 143-148 plant tissue emanations effects, 164-166 volatile chemicals effects, 143 Epinasty produced: by capping of plants, 226 by growth substances, 209-210 by irradiated cinnamic acid, 226 Erechtites, 36, 38 Ervum, 29 Escherichia coli, 197 Ethane: injury to plants, 179-180 Ethyl bromide: effects on dormant potato tubers, 243 Ethylene: anesthetic effects on plants, 1.50-158 detection by plant response, 141-143 physiological effects on plants, 139-168 production by plant tissues, 164-166 Ethylene chlorohydrin: hastening the sprouting of dormant potato tubers, 231-233 Ethylene chlorohydrin effects on : amylase activity, 250 ascorbic acid production in potatoes, 252 citric acid content of Bryophyllum leaves, 250 dormancy in gladiolus cormels, 241-243 dormant gladiolus corms, 240 enzyme activities in potato tubers, 250-251 /3-glucoside formation in living tubers, 246-247 metabolism of dormant potato tubers, 246-249 Ethylene dichloride effects on: dormant potato tubers, 234 Ethylene effects: abscission, 150 epinasty of leaves, 143-148 on carnations, 139-140 on celery, 159 on color and ripening of fruits, 158-159 on metabolism of plant tissues, 1.59-160 on rooting, 163 on roots, 173 proliferation of tissues, 148-150 Eucalyptus, 33 Euphorbia, 30 Eutaxia, 30 Evonymous: as affected by growth substances, 213 response to a-indolebutyric acid, 210-211 Experimental planning and statistics, 437-439 ricus, 14 Field trials with fibre pots, 438 Flowering: artificial light effects, 300-.309 effects of gases, 164 Flowers: carlion dioxide storage effects, 399 Fluoreneacetic acid, 207 Fruit drop, apple: as affected by growth substances, 215 Fruit ripening: ethylene effects, 15,8-159 Fruit set: as affected by growth substances, 218-220 Fruits: carbon dioxide storage effects, 399 Froghopper: (See Philaenus leucopht halnius) Fztchsia, 144 Fumigation: /3,/3'-dichloroethyl ether, 353 gases, 353 naphthalene, 352-353 Fumigation chambers: to study sulphur dioxide effects on animals, 192-193 Fungi: for determining soil fertility, 428 oxygen requirements for formation of perithecia and mycelial growth, 435-436 Fungicidal: definition, 372 Fungicidal action: of copper, 366-369 of sulphur, 362-366 r Fungicides: causes of variation in spore germination tests, 371-372 comparative toxicity of sulphur, selenium and tellurium, 365-366 correlations between laboratory and greenhouse tests, 379-380 cumulative error terms, 379 glyoxalidine derivatives, 381-383 greenhouse methods of evaluation, 374-379 organic compounds, 381-383 settUng tower method of applying fungicides, 374 spore germination tests, 369-374 standard slide-germination method of evaluating, 374 toxicity as related to position of elements of com- pounds in periodic system, 380 toxicity curves, 370-371 toxicity surface, 370 Fungistatic: definition, 372 SUBJECT INDEX 453 Fungus spores: induced in Alternaria solani cultures, 377 Fzisarium lycopersici, 416 Cralega, 30 Galeopsis, 43 Galinsoga, 325, 327 Gantry crane: greenhouse lighting, 297, 300-304 Gas residues in soil: toxicity to tomato plants, 176- 177 Gas, artificial illuminating: diagnosis of tree and shrub injury, 178-179 Gas, illuminating, effects: on plants, 139-168 on roots, 172-176 toxic action in soil, 148-150, 172-180 {See also Ethylene) Gases: fumigation, 353 Gases effects: epinasty of leaves, 142-148 on coloring and ripening of fruits, 158-159 on growth inhibition, 150-155 on rooting of plants, 160-164 responses of plants to treatment, (See Ethylene) Gases effects on plants: unsaturated carbon-con- taining gases, 139-168 Gases, lethal: effects on plants and animals, 172-201 Gases, natural: constituents, 179-180 toxicity to plants, 179-180 Gases, scrubbed: effects on plants, 175 Gases, toxic: effects on animals, 197-200 effects on fungi and bacteria, 196-199 effects on green plants, 196-199 effects on plants and animals, 196-200 effects on seeds, 196-199 Gases, unscrubbed: effects on plants, 17') GauUheria, 90 Gentiana, 90 Gentiobioside, P-: formed in gladiolus corms treated with ethylene chloroh\drin, 247 Geranium, 43 Germination: dormant gladiolus corms, 240-241 gladiolus cormels, 241-243 laboratory slide-germination method of evaluat- ing fungicides, 369-374 use of chemicals for hastening sprouting of dor- mant potato tubers, 231-240 Germination of seeds: after burial for various peri- ods, 37-38 carbon dioxide effects on lettuce seeds, 399-400 Cotoneaster, 111 Crataegus, 109-111 growth substances effects, 418-420 Impatiens balsamina, 123-126 inhiVjitors, 44-45 light effects on dormancy in seeds, 74-78 oxygen effects, 47-48 quick vitality test, 128-131 seed coat effects, 46-47, 106-112 stratification effects on seeds with non-dormant embryos, 101-105 Symphoricarpos orbiculatus, 108-109 temperature effects, 86-123 temperature effects on wild plants, 41-43 trailing arbutus, 433—434 tree peony, 113-117 Viburnum, 113 wild plants, 41-43 with dormant embryos, 86-101 with non-dormant embryos, 101-105 Germination of spores: Lycopodium, 434-435 (See also Spore germination) Germination, delayed: advantages to plants, 68 Geum, 325, 327, 340 Gladiolus: 212, 429, 430 bigeneric hybrid, 430—431 new fragrant hybrid, 430-4.31 pollen storage, 429-430 temperature effects on dormancy, 41 Gladiolus corms: as affected by growth substances, 212 Gladiolus dormant cormels: ethylene chlorohydrin effects on germination, 241-243 Gladiolus dormant corms: effects of ethylene chloro- hydrin, 240 Gladiolus recurvus, 431 Gladiolus tristis, 431 Glass: ultraviolet transmitting, 319 Glomerella cingulata: 197 effects of pentathionic acid, sulphuric acid and hydrogen sulphide on germination of spores, 363-366 time and temperature effects on germination of spores, 372 /3-Glucoside: formation by ethylene chlorohydrin, 246-247 formed in gladiolus corms treated with ethylene chlorohydrin, 247 Glutathione: changes in dormant potato tubers treated with ethylene chlorohydrin, 251-252 changes in relation to respiration rate, 249 Glycine soja, 332 Gompholobium , 30 Goodia, 29, 30 Gossypium hirsutum: cell divisions in the epidermal layer of the ovule subsequent to fertilization, 261-263 cell wall structure, 261-263 density of fiber mass, 261-263 general studies of fiber growth, 261-263 origin and sarly stages of elongation of fiber, 201- 263 Greenhouse: artificial lighting, 297, 300-309 CO2 supply in, 306, 307 controls for intermittent light, 305-306 lighting costs, 304, 309 Greenhouse, insulated: heated by Mazda lamps, 304-310 Growth: importance of mother-tuber in growth of potato plant, 420-423 Growth inhibiting substances: germination of seeds, 125 Growth inhibition: by growth substances, 216 gases effects, 150-155 methyl ester of a-naphthaleneacetic acid vapor, 239-240 Growth inhibition in potato tubers: methyl ester of a-naphthaleneacetic acid effects, 397-398 Growth of plants: under artificial light, 293-340 Growth promoting substances: growing plants as test objects, 206-209 Growth promotion: (See Dormancy; Epinasty; Germination; Growth substances; Hor- mones; Rooting; Roots; etc.) Growth regulators: (See Growth substances) Growth substances: absorption and movement, 225 aerosol method of application, 220 as bud inhibitors, 216 comparative effectiveness of acids, esters and salts 226 for leaf modification, 209-211, 218 for producing epinasty, 209-210 formative effects, 220-225 formative effects on tobacco plants, 220-221 induction of adventitious roots, 209-215 light and dark effects, 225 list of physiologically active acids, 207, 209, 218, 222, 223 methods of application to plants, 206-209 methods of upijlicalion to tomato flowers, 218- 220 phenoxy acids, 200-215 vitamin Bi, 419-420 Growth substances effects: of mother-tuber on daughter plant, 423 on plants, 204-226 454 SUBJECT INDEX Growth substances effects — continued on plants under varying light conditions, 205-208 on preharvest apple drop, 215 on seeds, 418-420 Halesia: effects of illuminating gas on roots, 173 Halesia Carolina, 112 Half-leaf method of studying changes in leaves, 409 Halicijstis: formation of the cellulose particles, 273- 2S2 Halicyslis osterhoutii, 273 Halicystis ovalis, 273 Hamamelis 90 Hardenbergia, 30 Helianthus cucumerif alius, 316, 332 Helianthus tuberosus: as affected by growth sub- stances, 212 Heptane: injury to plants, 179-180 Hermannia, 30 Herpetomonas hancrofti, 14 Hevea, 33 Hexane: injury to plants, 179-180 Hibiscus, 30, 35, 148, 149 Ilippeastrurn, 14 Holly leaf miner: control, 357 (See also Phytomyza ilicicola) Hormones: {See Growth substances) Hormones, natural: produced by gravity and shad- ing, 220 Houseflies: (See Musca domestica) Hovea, 29, 30 Humidity effects on: moisture content of seeds, 53-55 stored pollen, 429-430 H^■l)ridization: bigenerio gladiolus, 430-431 Liliuni, 430 new fragrant gladiolus, 430-43 1 Hydrangea, 78 Hydrangea macrophylla: acidity of soil, aluminum, iron effects on color, 423-424 Hydrocyanic acid in illuminating gas: toxicity to plants, 175-178 Hydrogen cyanide gas: effects on plants and ani- mals, 197-200 Hydrogen sulphide: effects on plants and animals, 197-200 injury to plants, 179-lSO, 193-194 role in fungicidal action of sulphur, 363-366 toxicity to fungus spores, 363-366 Hydroids: on aquatic plants, 20-21 Hypericum, 34, 42 Hypodermis of insects: effects of insecticides, 348- 350 Ilex: as affected by growth substances, 213 Illuminating gas: (See Ethylene) Impatiens, 90, 126 Impatiens balsamina: dormancy and germination in seeds, 123-126 dry storage effects, 123-125 Indigofera, 30, 31 /3-Indoleaeetic acid effects: on germination of seeds, 418-419 on plants, 206-215 /3-Indolebutyric acid, 218 |3-Indolebutyric acid effects: on germination of seeds, 418-419 on plants, 200-215 Indolepropionic acid: effects on plants, 206-21 o Infra-red effects: on transpiration in plants, 328- 330 Inhibition of growth: (See Growth inhibition) Insect vectors: of yellows diseases, 10-15 Insecticides: ^./S'-dichloroethyl ether fumigation, 356-357 effects of wetting agents on efficiency, 357 effects on nerves and muscles of houseflies, 348- 350 factors affecting efficiency and mode of action of contact insecticides, 343-352 research on synthetic products for, 354 survey of plant products for, 354 thiocyanogen compounds, 351 Insecticides, contact: fatty acids and soaps, 344-346 studies, 343-359 2-Iodobenzoic acid, 223 3-Iodobenzoic acid, 223 4-Iodobenzoic acid, 223 Ipomoea, 30, 35 Iris, 90 Iris, Japanese: carbon dioxide effects on flower color, 403 Isoelectric point: acidification of salt solutions in relation to plant tissue isoelectric points, 416-418 concerning the determination of the isoelectric point of protoplasm, 414-418 Jacksonia, 30 Japanese beetle: (See Popillia japonica) Jasione, 42 Juglans, 90 Juncus, 34 Junipfrrus, 89, 90, 92, 97, 98 Kalanchoe: responses to growth substances, 211 Kennedya, 30 L/ahurnum, 51 Lactuca, 77 Lactuca sativa: carbon dioxide effects on dormancy, 399-400 dormancy and germination in seeds, 125 Lathyrus, 73 Lavatera, 29 Leafhopper: (See Cicadula sexnolata; Macropsis trimaculata) Leaves: changes during night, 410-412 changes in nitrogen, 410-411 changes in residual dry weight, 410-411 chemical changes during period preceding frost, 412-414 diurnal changes, 408-412 effect of transpiration on, 329 half-leaf method of studying changes in, 409 twin-leaf method of studying changes in, 408-414 Length of day: (See Photoperiodism) Lepidium, 36, 37, 38 Leptospermum, 33 Lesvedeza, 35, 72, 73 Lettuce : (See Lactuca sativa) Leucaena, 29 Lewisia rediviva: dormancy and germination of seeds, 127 Libocedrus, 90 Life span of seeds: (See Seeds; life span) Life span of seeds in dry storage, 39-40 Life span of seeds in soil: 33-41 factors affecting, 38-39 Light duration: effects on plants, 304-309 Light effects: as related to growth substances, 205 on chlorophyll pigments, 330-331 on flowering of Digitalis purpurea, 334-337 on plant anatomy, 331-333 on plant microchemistry, 331-333 on plants' response to growth promoting sub- stances, 225 on seed germination, 74-78, 123 on transpiration in tobacco, 328-330 Light effects on plants: constant-condition rooms for study, 286-290 SUBJECT INDEX 455 Light intensity effects: on duck-food plants, 21-22 on green plants, 323-328 on manganese content of plants, 428 Light measurement: development of thermoelectric radiometer, 328 Light, artificial: effects on growth of plants, 28.5-340 Light, intermittent: effect.s on plants, 304-310 Lisus^r!/7n.' effects of illuminating gases on roots. 173 response to a-naphthaleneacetic acid, 210-211 Lilac: effects of ethylene chlorohydrin, 245 Lilies; diseases and pests, 431-433 Lilium: germination of seeds, 118 hybrids, 430 pollen storage, 429-430 Lilium auratum, 113, 430 LUium canadense, 113, 430 Lilium eximium, 4.31 Lilium giganteum: development of floral axis, 431 Lilium harrisii, 308 Lilium Henryi, 430 Lilium japonicum,, 113, 430 Lilium. I ongiflorum: 430, 432 development of floral axis, 431 Lilium rubellum, 113, 430 Lilium speciosum, 430 Lilium sulphureum, 430 Lilium, superbum, 430 Lilium szovitsiartum, 113 Lilium tigrinum, 430 Lily mosaic methods of transmission, 432 Liquidambar , 90 Liriodendron 90 Lit hospermum, 42 Lobelia, 78 Longevity: pollen, 429-430 Longevity of seeds: {See Seeds: causes of degenera- tion; Seeds: longevity) Lotus, 30 Lotus, Indian: (See Nelumho nucifera) Lupinus albus, 416 Lycopodium: propagation, 434—43.5 Lycopodium claratum, 433 Lycopodium complanatum, 434, 435 Lycopodium lucidulum,. 43.5 Lycopodium obscurum, 434, 43.5 Lysimachia, 42 Lythrum, 7.5, 76 Macropsis trimaculala: peach yellows carrier, 12 Macrosporium sarcinae forme: 197 effects of pentathionic acid, sulphuric acid and hydrogen sulphide on germination of spores, 363-366 time and temperature effects on germination of spores, 372 Malva, 36, 43 Manganese: solubility in soils, 428 Manganese in plants: effects of light intensity, 428 Maple: effects of illuminating gas on roots, 173 Mauritia, 33 Mazda lamps: effects on plant growth, 285-340 Medicago, 30 Melilotus, 29, 30, 31, 72 Mercuric chloride: toxicity to plants, 180-185 Mercurous chloride: toxicity to plants, 181-182 Mercury compounds: effects on germination of seeds, 185 Mercury effects: on roses, 183 Mercury vapor: toxicity to plants, 180-185 Mercury vapor lamps: effects on buckwheat seed- lings, 29.3-295 Metabolism: chemical effects, 253 effects of ethylene chlorohydrin on dormant po- tato tubers, 246-249 Metabolism of plant tissues: ethylene effects, 159- 160 Methane: injury to plants, 179-180 Methods: of applying growth substances, (See Growth substances) o:-(2-Methylphenoxy)-propionic acid, 218 Microchemical studies of effect of light on plants, 331-333 Mimosa, 29, 30, 151 Mimosa pudica, 1.54 Mirabilis jalapa, 332 Mitchella, 90 Modiola, 30 Monilia albicans, 197 Morphology: development of floral axis in Easter lilies, 431 Mosaic disease: dahlia, 433 lily, 432-433 Musca domestica: insecticides effects on nerves and muscles, 348-3.50 piperine effects on nervous system, 352 toxic effects of synthetic compounds, 3.54 Muscles of houseflies : effects of insecticides, 348-3.50 Muscles of insects: effects of insecticides, 348-352 MyceUum: oxygen requirements effects on growth, 435 oxj'gen requirements for formation, 435 MyosuTus, 42 Myrica, 90 Myzus persicae: transmits dahlia mosaic, 433 IVaias flexilis, 15 Naphthalene: fumigation, 352-353 a-Naphthaleneacetic acid effects: on germination of seeds, 418-419 on plants, 206-215 /S-Naphthoxyacetic acid, 218 /3-Naphthoxypropionic acid, 218 Nelumbium, 29, 30 Nelumbo, 34, 38, .50. 71 Nelumbo nucifera: seed coat effects on germination, 60-51 vitahty of seeds, 50-51 Xeon lamps: effects on plant growth, 293-295 Nereocystis leulkeana, 166 Nerves of houseflies: insecticides effects, 348-350 Nerves of insects: as affected by insecticides, 348- 352 Nicotiajia, 35, 40 Nicotiana glutinosa, 197, 439 Nicotiana tabacum, 162 Nicotine: toxicity to Aphis rumicis, 344 toxicity to Phlegethontius quinguemaculata, 344 Nigdla, 45, 75, 76 Nigella sativa: development of dormancy in seeds, 45 Nitella, 21, 22, 24, 25 Nitrate formation by peats: in composts, 425 Nitrogen: changes in leaves, 410—111 changes in leaves during night, 409—110 effect of nitrogenous reserves on growth of seed- lings, 436 insoluble organic sources, 426—127 Nyssa, 90 Octane: injury to plants, 179-180 Oenothera, 35, 36, 37, 40, 61, 75, 76 Onopordon, 35 Oreodoxa, 33 Organic compounds: as fungicides, 381-383 Organic sulphur compounds: injury to plants, 179- 180 Oxalis, 42 Oxygen effects on: aquatic plants, 23 dormancy, (See Dormancy) lily bulb dormancy, 431 Neurospora sitophiJa, 43.5 Oxygen requirement: for germination in dormant seeds, 78-85 Oxylobium, 30 456 SUBJECT INDEX Paeonia: 419 seedling production, 113-117 Paleacrita vernata: (See Canl£erworm) Panicum sanguinale: dormancy and germination of, 128 Papaver, 42 I'arthenocarpy: growth substances effects, 218-220 I'article size of fungicides: determination, 366 Pea seed decay: method for evaluating seed treat- ments, .379 Peach yellows: incubation period, 12 not transmitted by pollen, 14 studies, 12-14 symptoms, 10 Peats: pH, 425-426 phosphorus content, 426 to improve composts, 425 Pentane: injury to plants, 179-180 Pentathionic acid: role in fungicidal action of sul- phur, 362-363 Peroxidase: changes in relation to respiration rate, 249 Pesfalotia stellala: 197 effect of sulphuric acid and hydrogen sulphide on germination of spores, 363-366 Petunia, 80 Phacelia, 75, 76 Phalaris, 35, 205 Phaseolus vulgaris, 416 Phenols: effects on growth of tomato plants, 178 toxic effects on houseflies, 354 Phenoxy compounds: (See Growth substances) Phenoxyacetic acid, 209 a-(Phenoxy)-n-butyric acid, 209, 218 |3-(Phenoxy)-propionic acid, 209, 218 Phenyl compounds: ultraviolet effects, 323 Phenylacetic acid, 207 Phenylacrylic acid, 207 a-(Phenyl)-propionic acid, 207 Pbilaenus leucophthalmus: peach yellows carrier, 12 Phlegethontius quinquemaculala: nicotine toxicity, 344 Phleum, 35 Phosphate, colloidal: fertilizer value, 428 Phosphorus: content in peat, 426 Photoperiodism: effects on plants, 286-309 Photosjnthesis: increased by CO2 enrichment of air, 285 Physocarpus, 00 Phyteuma, 41, 42 Phytohormones: (See Growth substances) Phytolacca, 35 Phytomyza ilicicola: control, 357 Phytophthora iiifestans: use in greenhouse tests of fungicides on tomato, 374-378 Ph}i;otoxicity: determination of, in fungicide testa, 379 Picea, 90, 102 Pigment production: La apples, 320, 321 Pinus, 90-91, 102 Pinua caribaea, 102 Pinus ecldnata, 102 Pinus palustris, 102 Pinus rigida, 102, 103, 130 Pinus Taeda, 102 Piper cubeba: as an insecticide, 354 Piper nigrum: as an insecticide, 354 Piperine: as an insecticide, 351-352 effects on muscles of houseflies, 352 Plantago, 34, 35, 30, 37, 40, 42 Plantago major, 11 Plants grown under controlled environmental con- ditions, 285-340 Poa, 35, 40, 75, 70, 78 Pollen: germination, storage, 429-430 Pollen storage: Amaryllis, 429^30 Cinchona, 429-430 Gladiolus, 429-430 Lilium, 429-430 Polygonatum commutatum: dormancy and germina- tion of seeds, 120-121 Polygonum, 34, 35, 36, 37, 40, 42, 91 Polysaccharides: diurnal changes in leaves, 411-412 Popillia japonica: larvae control by /3,/3'-dichloro- ethyl ether fumigation, 256-257 Populus sp.: as an insecticide, 354 Porthetria dispar: pyrethrum toxicity, 348 Portulaca, 35, 36, 37, 40 Portulaca oleracea: dormancy and germination of, 128 Potamogeton, 21, 43 Potamogeton foliosus: 15 sea-water tolerance, 20 Potamogeton pectinatus: 15, 20, 21, 22, 23, 26 sea- water tolerance, 20 Potamogeton perfoliatus: 15, 20, 26 sea-water tolerance, 20 Potassium thiocyanate effects on: amylase activity, 250 Potato: (See Solanum tuberosum) Potato chips: color related to sugar content, 386- 398 effect of frying temperature on color, 388 factors for color, 386-398 Potato tubers: as affected by growthsubstances, 212 (See also Dormancy) Potentilla, 35, 42 Primula, 41, 42, 43 Privet: (See Ligustrum) Proliferation of plant tissue: ethylene effects, 14S- 150 Propagation: Lycopodium, 434-435 trailing arbutus, 433—434 (See also Growth substances) Propagation of plants (vegetative) : effects of growth substances, 211-215 Propane: injury to plants, 179-lSO Propylene: effects on roots, 173 physiological effects on plants, 142-143, 152, 163 Prunus, 14, 91 Prunus americana, 92, 130 Pseudayitltus, 30 Psoralea, 30 Ptelea, 91 Puccinia antirrhini: effect of sulphuric acid and hydrogen sulphide on germination of spores, 3(33-360 use in greenhouse tests of fungicides, 379 Pultenaea, 30 Pyrethrin I : improvement in method of determina- tion, 350 Pyrethrin I and II: relative toxicity to insects, 350- 351 Pyrethrum: effects on muscles, nerves of houseflies, 348-350 toxicity, 346-351 toxicity to Tenebrio molitor, 346-347 Pyrus, 91, 419 Quality of radiation effects: study of plants grown under glasses transmitting light of various ranges of wave lengths, 313-320 Radiant energy: (See Infra-red effects) Radiation: amount per acre, 286-287 Radiation and water loss of plants: (See Transpira- tion) Radiation effects: on plant growth, 285-390 Radiation, solar: required for plant growth, 286 Ranunculus, 75, 70 Red spider: (See Tetranychus bimaculatus; Tetramj- chus telarius) SUBJECT INDEX 457 Respiration: effects of carbon dioxide on plant tissue, 403-406 effects of chemicals on dormant potato tubers, 249-2.-)0, 251 ethylene effects, 160 of dormant gladiolus corms, 39 of dormant seeds, 39 Rest period: {See Dormancy) Rhizoclotiia tuHparum , 190, 197 Rhizopus Jiigricayts, 165 Rhodotypos. 89, 92, 93, 94, 98 Rhodotypos kerrioides, 93, 95, 98, 112, 130 Ribes, b\ Ripening: ethylene effects on fruits, 158-159 River maple: (See Acer saccharinum) Robinia, 35, 38 Rooting: carbon monoxide effects, 161-164 effects of growth substances, 209-215 gases effects, 160-164 methods of treating cuttings with growth sub- stances, 213-215 Rootone effects on: germination of seeds, 419 Roots: ethylene effects, 172-176 growth substances effects, 209-215 Rom, 89, 91, 159 Rosa rubiginosa, 89 Rose: carbon dioxide effects on flower color, 403 niercurj^ effects, 183 Rudberkia, 35 Rumex: 35, 36, 37 dormancy and water-absorption of seeds, 126 Ruppia marHima: 15 sea-water tolerance, 20 Sabal, 33 Saint paulia: as affected by growth substances, 215 Salix, 32 Salvia, 150, 151 Salvia officinalis: as an insecticide, 354 Sanguinaria canadensis: 120, 121 dormancy and germination of seeds, 120-121 Scirpus, 91 Scirpus americanus, 101 Scirpus campestris, 101 Sclerotinia fructicola: 197 effects of carbon dioxide on pH of hyphae, 401- 403 ■ effects of pentathionic acid, sulphuric acid and hydrogen sulphide on spore germination, 363-366 time and temperature effects on germination of spores, 372 Sclerotium delphinii, 197 Scrophularia, 42, 76 Sea-water concentration: titration studies, 16-20 Sea-water effects: on aquatic plants, 15-26 Sea- water tolerance: by aquatic plants, 20 Seed coats: effect on dormancy, 71-74 effect on seed vitality, 51 restricting oxygen supply to embryo, 80-85 Seed storage: citrus, 31-32 dandelion, 57 delphinium, 56-57 drying effects, 32 economic importance, 52 humidity effects on vitality, 53-55 importance of air conditioning, 60 moisture effects, 31-33, 57-59 optimum conditions, 28-29 oxygen effects, 32, 56 temperature effects, 31, 32, 55 under controlled conditions, 52-60 Seed testing: quick vitality test, 128-131 Seedless fruit: as produced by growth substances, 218-220 Seedling growth: effect of nitrogenous and carbo- hydrate reserves, 436 Seeds: after-ripening in dry storage, 12^5-127 causes of degeneration, 60-63 dormancy: {See Dormancy in seeds) factors influencing pH equilibrium, 416 germination: {See Germination of seeds) life span, 28-63 longevity, 28-63 quick vitality tests, 128-131 respiration: {See Respiration of seeds) temperature effects on vitality, 55 two-year seeds, 106 vitality, 28-63 vitality of old seeds, 30-31 water absorption capacity, 5.3-55 with dormant embryos, St)-101 with non-dormant embryos, 101-105 with resistant coats and dormant embryos, 106- 112 Seeds in soil: life span, 33-41 Seeds of long life span: germination tests, 29-31 Seeds of .short life span: vitality, 31-33 Seeds of wild plants: dormancy and delayed ger- mination, 41-43 Seeds, macrobiotic: germination tests, 20-31 Selenium: toxicity compared to sulphur, 365-366 Seploria lycopersici: use in greenhouse tests of fun- gicides on tomato, 374-378 Sequoia, 91 Sequoia gigantea, 102 Setaria, 102 Settling tower: method of applying fungicides, 374 Shade: effects on plants. 323-328 Shrubs: diagnosis of artificial illuminating ga.s in- jury, 178-179 effect of illuminating gas, 172-176 Sida, 30 Silver bell: (See Halesia) Sinapis, 34, 40, 43 Sisymbrium, 43 Smilacina, 91 Smilacina racemosa: dormancy and germination of seeds, 120-123 Snapdragon rust: greenhouse method of testing fungicides, 379 Snowberry: (See Symphoricarpos racemosus) Soaps, potassium: contact insecticides, 346 Sodium thiocyanate eft'ects: on enzyme activities in potato tubers, 250-251 Sodium vapor lamps: effects on plant growth, 293- 296 Soil studies, 425-429 Soils: as affected by sulphur dioxide, 427-428 sampling method, 438—139 Solanum, 35 Solanum tuberosum: effects of carbon dioxide on dormancy, 400 importance of mother-tuber in growth of potato plant, 420-423 Solanum tuberostim tubers: critical storage tempera- tures for sugaring and sprouting, 394 effects of carbon dioxide on respiration, 403-406 low temperature storage effects on sugars, 388- 389 storage temperature effects on cane sugar con- tent, 389, 390, 393 storage temperature effects on de-sugaring, 389- 392 storage temperature effects on reducing sugar. 392-394 sugar content at time of harvest, 388-389 varieties classified by sugar content, 388-389 Sorfrus, 89, 91,92, 93, 99 Sorbus aucuparia, 86, 87, 88, 99, 100, 130 Spectral glasses, effects: (See Quality of radiation effects) Spore germination: effect of Bordeaux mixture, 366- 368 458 SUBJECT INDEX Spore germination — continued effect of hydrogen sulphide, pentathionic acid, sulphuric acid, 362-366 of Lycopodium, 434-435 temperature and time effects, 372 Spores, fungus: induced in Alternaria solani cul- tures, 377 Sporobolus, 35 Spray residues: on apples, 355 Sprouting: of dormant potato tubers, 230-240 Stachys, 29, 30, 34 Statistical analysis of germination data: spores, 369-374 Statistical methods: experimental design, 437-439 Stellaria, 34, 43 Storage effects: (See Carbon dioxide storage effects) Storage of potato tubers: effects on starch and sugar content, 240 Storage of seeds: {See Seed storage) Stratification effects on germination: (See Germina- tion of seeds: stratification effects) Strawberry bvish: (See Evoyiymus) Sucrose: changes in potatoes treated with chemi- cals that break rest period, 251 storage temperature effects on potato tubers, 392-394 Sucrose in potato tubers: carbon dioxide effects, 395-397 storage temperature effects, 3SS-389 Sugar: in potato tubers, 251 Sugar, cane: storage temperature efTects, 389, 390 Sugar, cane, in potato tubers: storage temperature effects on de-sugaring, 392 Sugar, reducing: analytical methods for determina- tion, 437 storage temperature effects on potato tubers, 392-394 Sugar, reducing, in potato tubers: amount afTects color in potato chips, 3S6-394 carbon dioxide effects, 395-397 storage temperature effects, 388-389 storage temperature effects on de-sugaring, 389- 392 Sugars: changes in relation to respiration rate, 249- 251 (See also Sucrose; Sugar, reducing; Sugar, cane) Sulphur: method of making colloidal sulphur by distilling flowers of sulphur, 353 Sulphur compounds effects on: dormant potato tubers, 234 Sulphur dioxide: apparatus for studying effects on plants, 186-187 environmental factors affecting injury to plants, 189-191 in air at Boyce Thompson Institute, 190-191 Sulphur dioxide effects: injury to plants from smelt- ers, 186-188 nutrition of plants, 191 on animals, 191-193 on plants and animals, 197-200 on soils, 427-428 toxicity to plants, 185-191 Sulpliur fungicides: action, 362-366 Sulphuric acid effects on: dormancy in seeds, (See Dormancy in seeds) Sunlight: energy value per acre, 286-287 Sweet pea, 217 Symphoricarpos, 106, 112, 129 Symphoricarpos orbiculatus: 122 embryo dormancy, 109 germination of seeds, 108-109 Symphoricarpos racemosus, 112 Symphoricarpos racemosus seeds: after-ripenmg, 107-108 dormancy, 107-108 germination, 107-108 morphology, 107-108 Symplocos paniculala, 181 Syringa vulgaris: chemical changes in leaves during period preceding frost, 412-414 Xalc effects: on germination of seeds, 419 Taxodium, 91 Taiodium distichum, 102 Taxus cuspidata, 112 Telluriuin: toxicity compared to sulphur, 365-366 Temperature: {See Thermoperiodism effects) Temperature effects on: after-ripening of epicotyl, 113-123 anthocyanin development, 320-321 dormancy, (See Dormancy) lily bulb dormancy, 431 plants, 328-330 stored pollen, 429-430 transpiration of plants in infra-red, light, 328-330 yarovization of .seeds, 333-334 Temperature, low: on flowering of biennials, 333-337 on flowering of bulbous plants, 337 Tenehrio molitor: pyrethrum toxicity, 346-347 Tergitol 7 penetrant with j3./3'-dichloroethyl ether: Japanese beetle larvae control, 356-357 Tetranychus bimaculatus, 351 Telraiiychus telarius: naphthalene toxicity, 352-353 Thallium sulphate: toxicity in soils, 428—429 Thea, 33 Thermoperiodism effects: on flowering of Digitalis purpurea, 334-337 Thiocyanates: hastening sprouting of dormant po- tato tubers, 231-233 Thiocyanogen compounds: as insecticides, 351 Thiourea: as an antioxidant, 235-238 hastening sprouting of dormant potato tubers, 234-235 prevents browning of plant tissues and juices, 235-238 Thiourea effects: on enzyme activities in potato tubers, 250-251 toxicity to mammals, 236-238 Thlaspi, 34, 35, 40, 42, 43 Thrinax, 33 Thuja, 91, 102 Tilia americana, 112 Tissues: acidification of salt solutions by plant tis- sue, in relation to question of tissue isoelec- tric points, 416-418 Tobacco mosaic: inactivation by ultraviolet, 323 Tomato: as affected by growth substances, 210-212 effects of illuminating gas on roots, 173 Tomato foliage diseases: early blight, late blight and leaf spot, 374-378 greenhouse method of testing fungicides, 374-378 Toxicity: pyrethrum, 346-351 spore determination method, 369-370 Toxicity of fungicides: as related to the periodic system of the elements in compounds, 380 slide-germination method, 369-370 toxicity curves, 370-371 toxicity surface, 370 Toxicity of insecticides: fatty acids and soaps, 344- 346 thiocyanogen compounds, 351 Toxicity to plants: illuminating gas, 172-180 mercury and mercury compounds, 180-185 sulphur dioxide, 185-191 Tracheae: penetration of contact insecticides, 343- 345 Translocation: twin-leaf method of studying changes in leaves, 408-414 Transpiration: effects of light and infra-red on to- bacco, 328-330 effects of radiation on, 328-330 Tree peony: seedling production, 113-117 Trees: diagnosis of artificial illuminating gas injury, 178-179 effect of illuminating gas, 172-176 SUBJECT INDEX 459 Trichloroethylene: effects on dormant potato tu- bers, 233-234 2,4,5-Trichlorophenoxyacetic acid, 209, 218 2,4,6-Trichlorophenoxyacetic acid, 218 a-(2,4,5-Trichloropiienoxy)-n-butyric acid, 209, 218 /3-(2,4,6-Trichlorophenoxy)-/3'-chlorodiethyl ether, 218 a-(2,4,.5-Trichlorophenoxy)-propionic acid, 209, 218 Trifolium, 29, 30, 35, 36, 38 2,3,.5-Triiodobenzoic acid, 223 Trillium erectum: dormancy and germination of seeds, 120-121 Trillium grandiflorum: dormancy and germination of seeds, 119-121 2,4,5-Trimetli.\lpbenoxyacetic acid, 218 Tropisms: efTects of growth substances, 225-226 Tuber, mother-: chemical analyses of mother-tuber seed-pieces during development, 421-423 importance of mother-tuber in growth of potato plant, 420-423 method of removing mother-tuber from plant at any stage of development, 421 Tubers: dormancy in, 230-256 Twin-leaf method of studying changes in leaves, 408-414 Typha, 91 Ultraviolet: antirachitic action, 285 induction of antirachitic effect in plants, 321 Ultraviolet effects: irradiated cinnamic acid pro- duces epinasty in plants, 220 on ash of plants, 322 on mineral composition of plants, 322 on phenyl compounds, 323 on plants, 2So, 320-323 on tobacco mosaic virus, 323 on vitamin D, 321-322 red pigment production in apples, 320-321 (See also Quality of radiation effects) Ultroil: effects on efficiency of insecticides, 357 Urea-Ammonia Liquor-37: comparison with other insoluble nitrogen compounds as fertilizer, 42(1-427 Uromyces caryophyllinus : effects of pentathionic acid, sulphuric acid and hydrogen sulphide on germination of spores, 363, 366 imUisne'iia spiralis: 15, 20, 22, 26 sea-water tolerance, 20 Valonia: formation of the cellulose particles, 273- 282 Valonia macrophysa, 273 Valonia ventricosa, 273 Vegetables: carbon dioxide storage efTects, 399 Venturia inaequalis: effects of sulphuric acid and hydrogen sulphide on germination of spores, 363-366 Verbascum, 35, 36, 37, 38, 40, 41, 42, 76 Verbena, 35 Verbena phlogiflnra: carbon dioxide effects on flower color, 403 Veronica, 42 Viburnum: 414, 419 dormancy, germination. 113 seedling production, 113, 117-118 Viburnum acerifolium, 113, 116, 117 Viburnum dentatum: 113 chemical changes in leaves during period pre- ceding frost, 412—414 Viburnum dil at at um, 113, 116, 117 Viburnum nudum, 118 Viburnum opulus, 113 Viburnum prunifolium, 113, 116, 117 Viburnum scabrellum, 118 Viburnum tomentosum, 245 Vicia, 40, 51, 73 Virus diseases: statistical method for estimating virus infectivity, 439 (See also Aster yellows; Peach yellows) Viruses: studies on yellows, 10-15 Viscum, 75, 78 Viscum album: dormancy and germination in seeds, 123 Vitality of seeds: carbon dioxide effects, 33 impermeable coat effects, 51 Nelumbo nucifera, 50-51 seed coat efTects, 52 under controlled storage conditions, 52-60 (See also Seeds: vitality) Vitality tests: chemical methods, 131 embryo methods, 129-131 Vitamin Bi: as growth substances, 419—420 Vitamin D: ultraviolet effects, 321-322 Vitis, 91 Watsonia, 30 Wheat: yarovization, 334 Wild rice: (See Zizania arjjiaticn) Willow: effects of illuminating gas on roots, 173 Xanthium, 46, 82, 84, 85 X-ray diffraction patterns of cellulose particles, 267, 271, 274, 275 Xylene: effects on dormant potato tubers, 234 Xylenoxy acids: growth substance activity as af- fected by their structure, 222 Yarovization: wheat, 334 Yellows diseases: (See Aster yellows; Peach yellows ; Viruses) JCea mays, 60, 75 Zizania, 91 Zizania aquatica: 101 moisture effects on life span, 31 I