MICROPROPAGATION OF GLORIOSA SPECIES AND THEIR POTENTIAL AS A SOURCE OF COLCHICINE A thesis submitted for the degree of Doctor of Philosophy by J ayasiri Ranatunga School of Biochemistry and Molecular Genetics The University of New South Wales December, 1994 CERTIFICATE OF ORIGINALITY I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of a university or other institute of higher learning, except where due acknowledgement is made in the text. (Signed) CONTENTS SUMMARY i ACKNOWLEDGEMENTS iii ABBREVIATIONS iv CHAPTER 1 - INTRODUCTION 1 1. 1 Genus Gloriosa 1 1. 2 Plant Tissue Culture 6 1. 2.1 Botanical Aspects of In Vitro Propagation 9 1.2.2 Methods of In Vitro Propagation 9 1.2.2.1 Callusing 9 1.2.2.2 Adventitious Bud Fonnation 10 1.2.2.3 Axillary Bud Fonnation 11 1. 2. 3 General In Vitro Procedure for Micropropagation 11 1.2.4 In Vitro Propagation of Monocotyledonous Plants 13 1.2.5 In Vitro Propagation of Gloriosa species 17 1. 3 Secondary Metabolites 19 1. 3. 1 Colchicine 26 1. 3. 1. 1 Occurrence in Plants 28 1. 3. 1. 2 Isolation, Purification and Quantitation of Colchicine From Plants 29 1.3.1.3 Biosynthesis of colchicine 31 1. 4 Project Aims 34 CHAPTER 2 - MATERIALS AND METHODS 35 2.1 Plant Materials 35 2.2 Chemicals 35 2.3 Sterile Technique 37 2.4 Aseptic Seed Gennination 37 2.5 Aseptic Tuber Sprouting 37 2.6 Callus Initiation 38 2.7 Proliferation and Maintenance of Callus 38 2.8 Root Cultures 39 2.9 Growth Rate of Callus 39 2.10 Initiation of Shoots from Tuber Explants 39 2.11 Initiation of Shoots from Shoot Bases 40 2.12 Initiation of Shoots from Root-Coleoptile Axis of Seedlings 41 2.13 Initiation of Shoots from Callus 42 2.14 Maintenance of Stock Cultures 42 2.15 Multiplication of Shoots 42 2.16 Replacement of NAA by IAA in Shoot Multiplication Medium 43 2.17 Alteration of Ammonium and Nitrate Concentration in Shoot Multiplication Medium 43 2.18 Alteration of Sucrose Concentration in Shoot Multiplication Medium 44 2.19 Development of Roots and Tubers 44 2.20 Effect of Sucrose on Development of Roots and Tubers 44 2.21 Effect of ABA and GA3 on Development of Roots and Tubers 45 2.22 Deflasking and Transferring to Soil 45 2.23 Micropropagation Growth Measurements 45 2.24 Statistical Analysis of Data 46 2.25 Extraction of Plant Tissues 46 2.26 Partial Purification of Colchicine 46 2.27 HPLC Analysis of Extracts 47 2.28 Mass Spectrometry 47 2.29 Thin Layer Chromatography 47 2.30 Counting of Radioactivity 48 2.31 Dry Weight Determination 48 2.32 Addition of Putative Colchicine Precursors in Liquid Shoot Multiplication Medium 48 2.33 Distribution of Colchicine in Mature Gloriosa simplex Tissues 48 2.34 Screening of High Yielding Cell Lines for Production of Colchicine and Conservation through Plant Regeneration 49 CHAPTER 3 - RESULTS - CALLUS 50 3. 1 Callus Cultures Derived From Mature Tuber Explants 50 3. 2 Callus Cultures Derived From Mature Seedling Ex plants 52 3. 3 Proliferation and Maintenance of Undifferentiated Callus 55 3.3.1 G. superba 55 3.3.2 G. simplex 57 3.4 Proliferation and Maintenance of Differentiated Callus 57 CHAPTER 4 - RESULTS - INITIATION OF SHOOTS 59 4.0 Introduction 59 4.1 Initiation of Shoots 60 4.1.1 Initiation of Shoots From Tuber Explants 60 4.1. 2 Initiation of Shoots From Shoot Base Explants 67 4.1.3 Initiation of Shoots From Root-Coleoptile Axes of Seedlings 71 4.2 Tuber Initiation From Tuber Explants 75 4.3 Tuber Initiation from Root-Coleoptile Axis Explants 80 CHAPTER 5 - RESULTS - SHOOT AND TUBER MULTIPLICATION 83 5. 0 Shoot and Tuber Multiplication 83 5. 1 G. rothschildiana 83 5. 1.1 Multiplication of Cultures Derived From Tuber Explants 83 5. 1. 2 The Effect of Altered Inorganic Nitrogen Concentration and Composition on Shoot and Tuber Multiplication 88 5. 1. 3 The Effect of Altered Sucrose Concentration on Shoot and Tuber Multiplication 91 5 .1.4 Multiplication of Cultures Derived From Shoot Base Explants 91 5 .1. 5 Multiplication of Cultures Derived From Root-Coleoptile Axes of Seedlings 92 5. 2 G. superba 94 5. 2.1 Multiplication of Cultures Derived From Tuber Explants 94 5.2.2 Multiplication of Cultures Derived From Shoot Base Ex plants 95 5.2.3 Multiplication of Culturres Derived From Root-Coleoptile Axes of Seedlings 96 5. 3. G. simplex 98 5.3.1 Multiplication of Cultures Derived From Tuber Explants 98 5.3.2 Multiplication of Cultures Derived From Shoot Base Explants 99 5.3.3 Multiplication of Cultures Derived From Root-Coleoptile Axis of Seedlings 100 5.3.4 Multiplication of Shoots Derived From Undifferentiated Callus 100 5 .4 G. verschuurii 102 5.4.1 Multiplication of Cultures Derived From Tuber Explants 102 5.5 G. richmondensis 103 5.5.1 Multiplication of Cultures Derived from Tuber Explants 103 CHAPTER 6 - RESULTS - ROOT AND TUBER DEVELOPMENT 104 6.0 Root and Tuber Development 104 6.1 Root and Tuber Development of Gloriosa species 104 6.1.1 The Effect of Sucrose Concentration on Root andTuber Development 106 6.1.2 The Effect of ABA and GA3 on Root and Tuber Development of G. rothschildiana 110 CHAPTER 7 - RESULTS - ESTABLISHMENT IN SOIL 111 7. 0 Establishment in Soil 111 7. 1 Survival Rates of In Vitro Generated Gloriosa Species in Soil 111 7. 2 Yield of Tubers After the First Growth Cycle under Glasshouse Conditions 112 7. 3 Yield of Tubers After Second and Third Growth Cycles under Glasshouse Conditions 113 7.4 The Effect of Potting Medium on Tuber Growth of G. superba. 115 7 .5 Micropropagation Protocol For Gloriosa Species 116 CHAPTER 8 - RESULTS - QUANTITATION OF COLCHICINE:DEVELOPMENT OF METHODS 118 8. 0 Extraction, Purification and Quantitation of Colchicine from Gloriosa 118 8. 1 Extraction and Purification from Plant Tissue 118 8. 2 Quantitation of Colchicine 123 8. 3 Purity of the Internal Standard ([3H]-Colchicine) 123 CHAPTER 9 - RESULTS - QUANTITATION OF COLCHICINE IN IN VITRO AND IN VIVO TISSUES 126 9.0 Colchicine Content of in viva and in vitro Tissues of Gloriosa species and in viva Tissues of Sandersonia aurantiaca 126 t 9 .1 Colchicine Con4ent of in viva Tissue 126 9 .1.1 Colchicine Content of Glasshouse Grown Gloriosa and Sandersonia aurantiaca 126 9 .1. 2 Distribution of Colchicine within a Mature G. simplex Vine and a Tuber 128 9.2 Colchicine Content of In Vitro Cultures 130 9.2.1 Colchicine Content of Shoot Initiation Cultures (Stage I) 130 9.2.2 Colchicine Content of Shoot Multiplication Cultures (Stage II) 131 d 9. 2. 3 Manipulation of Multiplication Medium for Increaset_Yield of Colchicine From G. rothschildiana Shoot Cultures 137 9.2.4 The Effect of Putative Colchicine Precursors on the Colchicine Content of Shoots and Tubers of G. superba 140 9. 2. 5 Colchicine Content of Root and Tuber Development Cultures (Stage III) 141 9. 3 Colchicine From In Vitro Generated Plants When Grown Under Glasshouse Conditions 145 9. 4 Colchicine from Undifferentiated and Redifferentiated Callus 14 7 9 .4.1 Colchicine Concentration of G. rothschildiana and G. simplex Differentiated Callus 147 9.4.2 Colchicine Concentrations of G. simplex Undifferentiated Cells 148 9. 5 A Protocol to Screen and Recover Cell Lines Highly Productive for Colchicine 149 CHAPTER 10 - DISCUSSION 151 Initiation and Maintenance of Callus Cultures 151 Micropropagation 154 Colchicine 168 Concluding Remarks 175 REFERENCES 177 APPENDICES 192 (i) SUMMARY Gloriosa species are increasing in popularity in the cut flower industry and they are also a potential source of the phannaceutical, colchicine. However their propagation on a commercial scale is hampered by the slow rate of vegetative multiplication. Furthermore, little information is available on the distribution of colchicine in the genus, except G. superba. This thesis describes studies on the in vitro micropropagation of G. rothschildiana, G. superba, G. simplex, G. verschuurii and the cultivar G. richmondensis. In addition colchicine was quantitated from in vivo and in vitro generated tissues of the above species, growing under identical conditions. A successful protocol for the rapid micropropagation of these species was developed which combined good shoot initiation, a high rate of multiplication and tuber initiation, followed by root and tuber development. Plantlets could then be directly transferred to soil without acclimatization. The effects of different explants, various auxins and cytokinins, and basal media on shoot and tuber initiation and subsequent multiplication were also determined. Juvenile tuber tissues made the best explants and Woody Plant medium was superior to that of Murashige and Skoog,. The best plant growth regulator (PGR) combination was 10 µM NAA and either 20µM or 40 µM BAP for shoot initiation and either 10 µM IAA and 40 µM BAP or 1 µM IAA and 20 µM BAP for multiplication. Root and tuber development was best in the absence of plant growth regulators. The in vitro cloned plants grew normally and produced flowers during the third growth cycle in soil. Increasing the sucrose concentration in root and tuber development medium from 2% to 4%, doubled the number of tubers per replicate for G. superba, while that for G. rothschildiana increased about 25%. Tuber weight per replicate doubled for both species. The optimum concentration of nitrogen was 30mM at a 1 :2 ratio of NH4+/NO3-, for both shoot and tuber multiplication of G.