>i, ~~'~~i~",~d~:eC'~i':\'li·.·~lr:( 7 Submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY CATHERINE FENNELL 11111111111111111111111111111111111111111111111111 School of Botany &Zoology, Faculty of Science &Agriculture, University of Natal, Pietermaritzburg 2002 Crinum moorei: Propagation and secondary metabolite production in vitro table ofcontents Declaration -i­ Abstract -ii­ Acknowledgements -iv­ List ofabbreviations -v­ List oftables -viii­ List offigures -x­ List ofpublications -xii- Chapter 1 Habitat destruction 21 INTRODUCTION 1 The trade in medicinal plants 21 DISTRIBUTION AND Fodder for animals 22 MORPHOLOGY 1 Conservation for the future 22 HORTICULTURAL USE 5 Problems in the History of cultivation 5 Amaryllidaceae 23 Crinums in the garden 5 PROPAGATION 24 Popular hybrids 6 Conventional propagation 24 Crinums as future floricultural Seeds 26 crops 6 Offsets . 26 ETHNOBOTANY 7 Value of aseptic culture methods 27 PHARMACEUTICAL USE 15 Amaryllidaceae alkaloids 16 Chapter 2 Biological activity 16 TISSUE CULTURE OF Other compounds and their eR/NUM: A REVIEW 30 biological effects 18 CRINUMS IN CULTURE 30 Future phytochemical research 19 FUTURE RESEARCH CONSERVATION STATUS 20 PERSPECTIVES 32 Plant extinction: a global crisis 20 Micropropagation of Crinum: problems and prospects Bulbs in danger 20 33 Potential for long-term in vitro Amaryllidaceae and the genus storage Crinum: threatened species 20 33 Potential for alkaloid production Contributing factors to the 34 species loss 21 RESEARCH OBJECTIVES 34 Aims 34 CONCLUSIONS 51 Production stages 35 The induction ofgrowth & morphogenesis 53 Chapter 3 EXPLANT SELECTION INTRODUCTION 53 AND THE MATERIALS AND METHODS 54 ESTABLISHMENT OF AN Anthers 54 37 ASEPTIC CULTURE Filaments 54 Explant selection and Petals 54 sterilization 37 Base of the perianth tube 54 INTRODUCTION 37 Ovaries 54 Inflorescence explants 38 Pedicels 54 Bulb explants 39 Pedicel-Peduncles 54 Decontamination 40 Peduncles 55 MATERIALS AND METHODS 41 Leaves 55 Collection and cultivation of Bulb scales 55 parent plants 41 Twin-scales 55 Explants 42 RESULTS 55 Decontamination 45 Anthers 55 Preparation of the plant material 45 Filaments 55 Decontamination of Petals inflorescences and leaves 45 55 Decontamination of bulbs 45 Base of the perianth tube 55 Aseptic culture methods 46 Ovaries 55 Statistical analysis of data 46 Pedicels 56 RESULTS 46 Pedicel-Peduncles 56 Decontamination of Peduncles 56 inflorescences and leaves 46 Leaves 56 Decontamination of bulbs 47 Bulb scales 58 DISCUSSION 49 Twin-scales 58 Decontamination of DISCUSSION 61 inflorescences and leaves 49 CONCLUSIONS 67 Decontamination of bulbs 49 Chapter 4 Introduction 78 THE PRODUCTION OF Materials and methods 78 PROPAGULES 68 Results 79 INTRODUCTION 68 Discussion 81 ' Regeneration from floral CONCLUSIONS 82 stem explants: Factors Regeneration from twin- affecting growth and 70 scales: Factors affecting morphogenesis growth and morphogenesis 83 INTRODUCTION 70 INTRODUCTION 83 EXPLANT AGE 70 EXPLANT POSITION 83 Introduction 70 Introduction 83 Materials and methods 70 Materials and methods 84 Results 70 Results 84 Discussion 71 Discussion 85 EXPLANT POSITION 73 EXPLANT SIZE 86 Introduction 73 Introduction 86 Materials and methods 73 Materials and methods 86 Results 73 Results 86 Discussion 73 Discussion 87 EXPLANT POLARITY 76 EXPLANT POLARITY 88 Introduction 76 SUCROSE CONCENTRATION 89 Materials and methods 76 Introduction 89 Results 76 Materials and methods 89 Discussion 77 Results 90 THE CULTURE ENVIRONMENT: LIGHT 77 Discussion 91 introduction 77 PLANT GROWTH REGULATORS 93 Materials and methods 77 Introduction 93 Results 77 Materials and methods 94 Discussion 78 Results 94 MAINTENANCE OF CALLUS GROWTH AND INDIRECT Discussion 96 ORGANOGENESIS 78 CHARCOAL 99 Introduction 99 Chapter 6 Materials and methods 99 PLANT GROWTH AND MORPHOGENESIS IN Results 99 LIQUID CULTURE 129 Discussion 101 INTRODUCTION 129 LIGHT 102 Limitations of micropropagation 129 Introduction 102 Why use a liquid system? 130 Materials and methods 102 Pathways 132 Results 102 Physical and nutritional 132 Discussion 103 requirements TEMPERATURE 105 The problem of hyperhydricity 133 Introduction 105 AIMS 134 Materials and methods 106 A COMPARISON OF DIFFERENT CULTURE Results 106 PROCEDURES FOR THE Discussion 106 MULTIPLlCATION OF PROPAGULES 134 CONCLUSIONS 109 Introduction 134 Multiplication ofthe Materials and methods 135 propagules 111 Adventitious shoot initiation from INTRODUCTION 111 bulblets in different culture conditions 135 MATERIALS AND METHODS 111 Shoot growth and RESULTS 113 morphogenesis in different DISCUSSION 116 culture conditions 135 CONCLUSIONS 121 Results 135 Adventitious shoot initiation from Chapter 5 bulblets in different culture TRANSFER TO THE conditions 135 NATURAL ENVIRONMENT Shoot growth and 122 morphogenesis in different INTRODUCTION 122 culture conditions 137 MATERIALS AND METHODS 123 Discussion 137 RESULTS 123 FACTORS AFFECTING THE MULTIPLlCATION OF DISCUSSION 127 PROPAGULES IN LIQUID CONCLUSIONS 128 CULTURE 141 Introduction 141 Materials and methods 141 Results 158 Results 141 Discussion 159 Discussion 142 Explant effects 159 FACTORS AFFECTING SHOOT The induction of the AND BULBLET INDUCTION embryogenic state: the role of FROM MERISTEMOID auxins and cytokinins 162 CLUSTERS 145 Direct and indirect Introduction 145 embryogenesis 164 Materials and methods 146 MATURATION OF EMBRYOS AND THE DEVELOPMENT OF Results 146 GERMINABILlTY 164 Discussion 147 Introduction 164 CONCLUSIONS 150 Materials and methods 165 Results 165 Chapter 7 SOMATIC Discussion 168 EMBRYOGENESIS 153 The effect of plant growth regulators 168 INTRODUCTION 153 The developmental anatomy of Somatic embryos and plant somatic embryos 170 propagation 153 Abnormalities 171 The advantages of somatic embryogenesis 153 Poor germination response 171 Somatic embryogenesis in CONCLUSIONS 171 ornamentals and bulbs 155 Somatic embryogenesis in the Chapter 8 Amaryllidaceae 155 THE PRODUCTION OF Somatic embryogenesis in eRINUM ALKALOIDS IN Crinum 156 VITRO 174 Somatic embryogenesis: stages 156 INTRODUCTION 174 AIMS 156 Secondary products of economic CALLUS AND EMBRYO importance 174 INDUCTION 157 Sources of secondary 174 Introduction 157 metabolites derived from plants The induction of somatic Natural products in plant tissue embryos 157 and suspension cultures 175 Materials and methods 158 Advantages of using an in vitro system 176 Declaration I hereby declare that this thesis, unless acknowledged to the contrary in the text, is the result of my own investigation under the supervision of Professor J. van Staden, Research Centre for Plant Growth and Development, School of Botany and Zoology, University of Natal, Pietermaritzburg. Catherine W. Fennell April 2002 I certify that the above statement is correct. Profess r J. van Staden (Supervisor) April 2002 -i- Abstract As an alternative to conventional methods of vegetative propagation, micropropagation attracts much attention, because the levels of multiplication are increased, somac\onal variation is limited and disease-free material can be obtained. The technique is invaluable to the conservation of Crinum species belonging to the Amaryllidaceae which, as a group, possesses several biological features that make them particularly vulnerable. This is in addition to other problems relating to their value as horticultural material, traditional medicines and sources of phytochemicals of interest to medical science. Two in vitro systems are widely used for the propagation of amaryllidaceous species; regeneration from young floral stem explants and from twin-scales excised from bulbs. Although plantlet regeneration could be obtained from peduncle explants of Crinum moorei, a complex of factors including: the age of the floral stem; explant position and; hormonal factors, limited growth. Callus production was poor and indirect organogenesis could not be achieved. Twin-scales were used for the induction of somatic embryos. Morphologically these were different depending on the concentrations of 2,4-D and BA used in the induction medium. Although some of them went on to germinate, the use of somatic embryos for large-scale culture is not an efficient micropropagation route, owing to the low frequency ofboth embryo production and germination and to the long culture times. Regeneration of shoots and bulblets could, however, be readily induced from twin-scales using a series of modified MS media, and this despite the fact that explants from the bulb were more difficult to decontaminate than the above ground parts. Shoots arose in the axes of the twin-scales close to the basal plate. Initiation was greatest on a basic Murashige and Skoog medium, containing 4 g Q-1 sucrose, and in the dark. No hormones were required. At high concentrations, the hormones stimulated abnormal organogenesis. BUlbing of the shoots was further enhanced using higher than normal levels of sucrose Le. 6% and 5 g Q-1 activated charcoal. The response was also influenced by the size of the twin-scale and its position in the parent bulb. Greater numbers of bulblets with larger diameters developed in large twin-scales from an intermediate position between the inner and outer scales. Furthermore, light, and a temperature of25°C were required for normal bulblet development. Bulblets grown in this manner were used as a source of secondary explants by splitting them vertically in half. The addition of 10 mg Q-1 BA resulted in mUltiple shoot development. In a -ii- liquid-shake culture