A STUDY OF THE INFLUENCE OF GIBBERELLIC ACID ON DIGITALIS PURPUREA L. AND FAGOP1RUM ESCULENTUM MOEMCH Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By MAHMOUD DARWISH SATED, B. Ph.Ch., M. Pharm. The Ohio State University 1958 Approved by ^7 Adviser College of Pharmacy ACKNOWLEDGMENTS I wish to express my sincere appreciation and deep gratitude to Dr. Jack L. Beal, for his guidance, counsel and encouragement without which this work could not have been ful­ filled. To the following persons who have served on the advisory committee or reading committee and/or given invaluable classroom instructions, I offer my gratitude and obligation: G.W. Blaydes, R.H. Bohning, E.P. Guth, L.E. Harris, J.W. Nelson, L.M. Parks, R.S. Piatt. I wish also to acknowledge Dr. W.B. Mcintosh's assist­ ance in the statistical evaluation of the data in this dissertation. To the friends and colleagues who have shown interest in this work and who have offered valuable suggestions, I express my thanks. The Author ii TABLE OF CONTENTS PAGE ACKNOWLEDGMENT ii LIST OF TABLES v LIST OF FIGURES vii INTRODUCTION 1 Review of the Literature 1 Statement of the Problem 16 Chosen Experimental Plants 17 Purpose of the Research 17 EXPERIMENTAL Preparation of Gibberellic Acid Solution 19 Digitalis purpurea L. 19 Raising Digitalis Plants 19 Treatment of Digitalis Plants with Gibberellic Acid Solution 19 Harvesting and Sampling of Digitalis 19 Morphology and Histology of Treated and Control Plants 20 Influence on the Dry Weight of the Leaves 20 Evaluation: Preparation of Standard Curve of Digitalis Glycosides 33 Assay of Samples for Total Glycosidal Content 36 Determination of Total Sugar Contents 39 Determination of Digitoxose Sugar 40 Preparation of Standard Curve of Digitoxose Sugar 40 Determination of Crude Fiber Content 46 Determination of Chlorophyll 47 Fagopyrum esculentum Moench 54 Raising Buckwheat plants 54 Treatment of Buckwheat Plants with Gibberellic Acid Solution 54 Harvesting and Sampling of Buckwheat Samples 54 Influence of Gibberellic Acid on the Linear Growth and Dry Weight of the Plant 54 Evaluation: Preparation of Standard Curve of Rutin Glycoside 5& Assay of Buckwheat Samples for Rutin Content 57 iii TABLE OF CONTENTS (Continued) PAGE Determination of Total Sugar Content 57 Separation of Rhamnose Sugar Using Paper Chromatography Technique 67 Localization of Rhamnose on Chromatograms 69 Preparation of Standard Curve of Rhamnose 71 Per Cent Recovery of Rhamnose from Chromatograms 72 Quantitative Determination of the Measurable Rhamnose in Treated and Control Samples of Buckwheat 75 Determination for Quercetin using Paper Chromatography - 77 Determination of Crude Fiber Content 79 Determination of Chlorophyll Content 83 DISCUSSION 84 SUMMARY 93 CONCLUSIONS 96 BIBLIOGRAPHY 98 AUTOBIOGRAPHY 105 iv LIST OF TABLES TABLE PAGE I Fresh and Dry Weight of Digitalis Samples 21 II Length of the Petioles and Dimensions of Treated Control Digitalis Leaves 31 III Surface Area of Treated and Control Digitalis Leaves 32 IV Palisade Ratio and Vein-islet Number of Treated . and Control Digitalis Leaves :~ 33 V Data for. Standard Curve of Digitalis Glycosides —— 34 VI Total Glycosides in Digitalis Samples 36 VII Percentage of Total Sugar in Digitalis Samples 41 VIII Data for Standard Curve of Digitoxin 44 IX Percentage of Digitoxose in Digitalis Samples 46 X Percentage of Crude Fiber in Digitalis Samples 48 XI Percentage of Chlorophyll in Digitalis Samples 53 XII Data on Length of Stems, Dry Weights of Stems and Leaves of Buckwheat Samples 55 XIII Data on Number of Leaves Per Plant 56 XIV Data for Rutin Standard Curve 58 XV Percentage of Rutin in Buckwheat Samples 60 XVI Per Cent of Total Sugar in Buckwheat Samples 63 XVII Rf and R„ Values of Rhamnose and Separated Sugars — 69 XVIII Data of Rhamnose Standard Curve 73 XIX Percentage Recovery of Rhamnose 75 XX Percentage of Rhamnose in. Buckwheat Samples 77 LIST OF TABLES (Continued) TABLE PAGE XXI R of Rutin and Quercetin 79 XXII Per Cent Crude Fiber in Buckwheat 81 XXIII Per Cent of Chlorophyll in Buckwheat Samples 83 XXIV Rutin Content Calculated on Dry Weight Basis of Leaves Per Plant 91 XXV Average Determination of Dry Weight, Measurements of Lamina and Surface Area of Digitalis Leaves 9^ XXVI Average Per Cent of Evaluation of Digitalis Samples 9k XXVII Average Per Cent of Evaluation of Buckwheat Samples 95 vi LIST OF FIGURES FIGURE PAGE 1 A Control Digitalis Plant 22 2 A Treated Bolting Digitalis Plant 22 3 A Treated Digitalis Plant 23 4 Diagram of Treated, Control Digitalis Leaf and Stem 24 5 Diagramatic Transverse Section of the Stem 26 6 Transverse Section in the Stem 27 7 Isolated Elements of the Stem 28 8 Standard Curve for Total Glycosides of Digitalis 35 9 Analysis of Total Glycosides of Digitalis Samples 37 10 Variation of Glycosidal Percentage of Digitalis Samples with Period of Harvest 38 11 Per Cent of Total Sugar in Digitalis Samples 42 12 Variation of Total Sugar Percentage of Digitalis Samples with Period of Harvest 43 13 Standard Curve Used for Determination of Digitoxose - 45 14 Per Cent of Crude Fiber in Digitalis Samples • 49 15 Standard Curve for Rutin 59 16 Per Cent of Rutin in Buckwheat Samples 61 17 Variation of Glycosidal Percentage of Buckwheat Samples with Period of Harvest 62 18 Per Cent of Total Sugar in Buckwheat Samples 64 19 Variation of Total Sugar Percentage of Buckwheat Samples with Period of Harvest 65 20, Illustrations of Rj of Glucose, Rhamnose and Sugars 24 Separated from Buckwheat Extract 70 vii LIST OF FIGURES (Continued) FIGURE PAGE 25 Standard Curve for Rhamnose 74 2.6- Illustrations of Attempts of Separation of 30 Quercetin from Buckwheat Extract 80 31 Per Cent of Crude Fiber in Buckwheat Samples 82 viii INTRODUCTION Review of the Literature During the past thirty years a number of Japanese botanists have been working on a group of plant growth substances which were first shown to be produced by a rice disease fungus, Gibberella Fu.jikuroi, by Kurosawa (1), a Japanese plant pathologist working in Formosa. This group named Gibberellin after the fungus by investi­ gators at the University of Tokyo, notably Yabuta and Sumiki (2). It was these men who were responsible for the isolation of the Gibberillins and the first studies of their properties. Around 1952, their work came to the attention, apparently independently, of a group at the Imperial Chemical Industries in Britain, and another group at the United States Department of Agriculture. These two groups confirmed the Japanese work and contributed their own find­ ings, bringing their results to a much wider audience. History and Identifi cat ion of the Fungus Producing the Gibberellins Ito and Kimura (3) attributed the earliest known description of what is now called the "bakanae disease" to Konishi (4), a semi- literate farmer who dictated an agricultural book in 1809. The most characteristic symptom of the disease is the appearance of tall thin plants, markedly overgrowing their uninfected neighbors. The first description of the disease with its causal fungus is attributed to Hori (5,7). The most complete monographs on this 1 2 disease are those of Ito and Kimura (3) which covers aspects of the history, symptoms, methods of infection, treatment and recom­ mended agricultural practices to reduce its incidence. The disease has since been described in India (8), in the Philippines (9)i in China (10),in British Guiana, where it is known as "man rice" (11), and in Ceylon where it is called "Wanda peedema" (sterile ripen­ ing) (12). Hori (5) identified the agent of the disease as an imper­ fect fungus, Fusarium heterosporum Nees. Later, Fujikuro discovered the perfect stage and this was described as Lisea Fujikuroi by Sawada (13). Noting the similarities of this micro-organism to Giberella moniliformis Winel, Ito (3) suggested Gibberella Fu.jikuroi as a more generic designation. Today, this is the most accepted nomenclature. The agreement on the identify of the bakanae organism has left one question unexplained. Why does a broadly distributed fungus apparently cause its overgrowth effect naturally only in rice? No natural occurrence of an overgrowth in plants other than rice seems to have been reported, although artificial infections have led to overgrowth in mait^ '1^-17), barley (14-), sugar cane (16), wheat (17), and oats (17). A conclusive answer to this paradox has not yet been given, but the following clues are available. Ito and Kimura (3) and later Stol (18) reported that once effective strains maintained in artificial culture lost their bakanae-inducing ability after a few months, and 3 this could not be re-established by inoculation on rice and re- isolation. However, Stol did restore the overgrowth symptom when he grew the fungus on a rice grain medium. It is thus possible that some nutritional factor supplied by rice promotes the over­ growth. There is also some evidence of an effect of temperature on the infected plant. Many workers have shown that the optimum temper­ ature for the growth of the fungus is about 27°C, but for the infection, 30°C. is optimal (19), whereas Seto (20) stated that 35°C. is best for the bakanae effect. Although Seto (21) and Hemmi (22) have indicated that different strains were not identical in temper­ ature response, it seems clear that the optimum temperature for the increased linear growth of the plant was always about 5°C. above that which is optimum for the in vitro culture of the fungus. Another variable is the soil moisture. Sawada and Kurosawa (23) and Taka- hashi (2*0 reported less disease in well-watered soils.