THE ESTABLISHMENT OF EMBRYOGENIC CALLUS CULTURE OF Hyoscyamus niger AND THE DETECTION OF HYOSCYAMINE IN THE CULTURE NHAWAL AMINIE SAIDON UNIVERSITI SAINS MALAYSIA MAY 2008 THE ESTABLISHMENT OF EMBRYOGENIC CALLUS CULTURE OF Hyoscyamus niger AND THE DETECTION OF HYOSCYAMINE IN THE CULTURE by NHAWAL AMINIE SAIDON Thesis submitted in fulfillment of the requirement for the degree of Master of Science MAY 2008 ACKNOWLEDGEMENT First of all, I want express my gratitude to my supervisor, Prof. Chan Lai Keng from the School of Biological Sciences, USM for had been given me the chance to continue my study under her supervision. Her guidance, expertise, advice, words of wisdom and continuous support had helped me to complete this program. For this, I am truly thankful. I wish to thank the School of Biological Sciences and the Dean of Institute of Graduate Studies for giving me the chance further my postgraduate studies. The support of Skim Biasiswa Khas granted by the Institute of Graduate Studies had helped me to do my research. I thank the School of Biological Sciences and the School of Chemical Sciences for the utilisation of facilities. My sincere thank to the staffs of both schools who had given me lots of support throughout my study especially Puan Sabariah, Puan Afida, Encik Bakar and Mr Yee. I wish to extend my gratitude to my friends in the Plant Tissue Culture Laboratory : Poh Liang, Pey Shan, Lay Pin, Marvin, Fung Hui and Vun Hui who had been together during the hard and challenging moments, as well as for the beautiful friendship. A sincere thank to all my senior David, Fung Liang, Wai Fun, Zainah and Chee Leng for the inspiration and advice. Not forgetting also all the member of the Plant Tissue Culture Laboratory. I also want to acknowledge my greatest appreciation to my parents for their support, understanding and encouragement during my research. I wish to express my thank and gratitude also to all my dear family and friends especially Jasreza, Kak Intan ii and ME team who have supported me in my research work in one way or another. Thanks to everyone, for everything. NHAWAL AMINIE BT SAIDON iii TABLE OF CONTENTS No. Content Page Acknowledgement ii Table of Contents iv List of Tables viii List of Figures x List of Abbreviations xiii Abstrak xiv Abstract xvi 1.0 Introduction 1 2.0 Literature Review 5 2.1 History and distribution of Hyoscyamus 5 2.2 Hyoscyamus niger 7 2.2.1 Botany of Hyoscyamus niger 7 2.2.2 Uses of Hyoscyamus niger 7 2.3 Somatic embryogenesis 10 2.4 The Chemical Constituents of Hyoscyamus niger 20 2.4.1 Plant Alkaloids 20 2.4.2 The Tropane Alkaloids of H. niger 21 2.5 Chemical Analysis Techniques 26 2.5.1 Gas Chromatography-Mass Spectrometry 27 3.0 Materials and Methods 31 3.1 Embryogenic Callus Culture 31 iv 3.1.1 Plant Materials 31 3.1.2 Induction of Embryogenic Callus 31 3.1.3 Selection of Best Explant and Best Embryonic Callus 32 Induction Medium. 3.2 Development and Regeneration of Somatic Embryos 33 3.2.1 Determination of Development Stage of Somatic 33 Embryos 3.2.2 Effect of BA on Germination of Somatic Embryos 35 3.2.3 Effect of Solid and Liquid Medium on Development of 35 Somatic Embryos 3.2.4 Effect of Casein Hydrolisate on Development of Somatic 36 Embryos 3.3 Histological Study of Embryogenic Callus and Different Stages 36 of Somatic Embryos 3.4 Propagation of Embryogenic Cells in Liquid Medium 37 3.4.1 Propagation of Embryogenic Cells Using the Callus 37 Induction Medium 3.4.2 Propagation of Embryogenic Cells in MS Medium 38 3.5 Direct Embryogenesis 38 3.5.1 Effect of BA on Induction of Direct Embryogenesis 38 3.5.2 Effect of BA and NAA on Induction of Direct 38 Embryogenesis 3.5.3 Histological Study of Direct Embryogenesis From H. 39 niger petiole explant 3.6 Detection of Hyoscyamine of H. niger 39 3.6.1 Extraction and Sample Preparation 39 3.6.2 Gas Chromatography Mass Spectrometry 40 v 4.0 Results 42 4.1 Embryogenic Callus Culture 42 4.1.1 Plant Materials 42 4.1.2 Induction of Embryogenic Calli 42 a) Leaf Explants 42 b) Petiole Explants 47 c) Root Explants 50 4.1.3 Selection of Best Explant and Best Embryogenic Calli 53 Induction Medium 4.2 Development and Regeneration of Somatic Embryos 56 4.2.1 Establishment of Embryo Maturation Medium 56 4.2.2 Effect of BA on Germination of Somatic Embryos 61 4.2.3 Effect of Solid and Liquid Medium on Development of 64 Somatic Embryos 4.2.4 Effect of Casein Hydrolisate on Development of Somatic 67 Embryos 4.3 Histological Study of Embryogenic Calli on Different Stages of 70 H. niger. 4.4 Propagation of Embryogenic Cells in Liquid Medium 72 4.4.1 Propagation of Embryogenic Cells Using the Callus 72 Induction Medium 4.4.2 Propagation of Embryogenic Cells in MS Medium 72 4.5 Direct Embryogenesis 75 4.5.1 Effect of BA on Induction of Direct Embryogenesis 75 a) Leaf Explants 75 b) Petiole Explants 78 c) Root Explants 78 vi 4.5.2 Effect of BA and NAA on Induction of Direct 82 Embryogenesis a) Leaf Explants 82 b) Petiole Explants 85 c) Root Explants 85 4.5.3 Histological Study of Direct Embryogenesis From H. 86 niger Petiole Explant 4.6 Detection of hyoscyamine of H. niger 90 4.6.1 Gas Chromatography Mass Spectrometry 90 5.0 Discussion 98 5.1 Production of Embryogenic Callus 98 5.2 Maturation, Optimization and Germination of Somatic Embryos 101 5.3 Histological Study for Confirmation of Indirect and Direct 105 Embriogenesis Process in H. niger 5.4 Propagation of Embryogenic Cells 107 5.5 Direct Embryogenesis 107 5.6 Detection of Hyoscyamine in H. niger 109 6.0 Conclusion 111 6.1 Conclusion of Study 111 6.2 Suggestions for Further Research 111 References 112 Publication List 131 Appendix 132 vii LIST OF TABLES Table Title Page Table 4.1 Effects of MS medium supplemented with 2,4-D (0-10.0 45 mg/L) and Picloram (0-10.0 mg/L) on the induction of embryogenic callus (g ± s.d.) from the leaf explants of Hyoscyamus niger after four weeks of culture. Table 4.2 Effects of MS medium supplemented with 2,4-D (0-10.0 48 mg/L) and Picloram (0-10.0 mg/L) on induction of embryogenic callus (g ± s.d.) from the petiole explants of Hyoscyamus niger after four weeks of culture. Table 4.3 Effects of MS medium supplemented with 2,4-D (0-10.0 52 mg/L) and Picloram (0-10.0 mg/L) on induction of embryogenic callus (g ± s.d.) from the root explants of Hyoscyamus niger after four weeks of culture. Table 4.4 Effect of different maturation media on the production and 60 development of somatic embryos after 4 weeks of culture. Table 4.5 Effect of different maturation medium without culturing on 63 MS basic medium for one week on development of somatic embryos after 4 weeks of culture. Table 4.6 Effect of different concentration of BA for germination of 65 somatic embryos after 4 weeks. Table 4.7 Effect of solid and liquid MS medium supplemented with 65 1.0 mg/L BA on development of somatic embryos of H. niger. Table 4.8 Effect of MS medium supplemented with different 69 concentration of casein hydrolysate and 1.0 mg/L BA on development of somatic embryos of H. niger after four weeks of culture. Table 4.9 Effect of BA on percentage of leaf explants of H. niger 76 showing different morphological changes after four weeks of culture. Table 4.10 Effect of BA on percentage of petiole explants of H. niger 80 showing different morphological changes after four weeks of culture. Table 4.11 Effect of BA on percentage of root explants of H. niger 81 viii showing different morphological changes after four weeks of culture. Table 4.12 Effect of MS supplemented with BA and NAA on 83 percentage of leaf explants showing different morphological changes after four weeks of culture. Table 4.13 Effect of MS supplemented with BA and NAA on 87 percentage of petiole explants showing different morphological changes after four weeks of culture. Table 4.14 Effect of MS supplemented with BA and NAA on 88 percentage of root explants showing different morphological changes after four weeks of culture. ix LIST OF FIGURES Figure Title Page Figure 2.1 Tropane Alkaloids (Hughes and Genest, 1973). 21 Figure 2.2 Metabolic pathway of tropane alkaloids in H. niger (Nakajima 25 and Hashimoto, 1999). Figure 3.1 Establishment of embryo maturation process. 34 Figure 4.1 Hyoscyamus niger plantlets derived from seeds. 43 Figure 4.2 Morphology of embryogenic callus derived from the leaf 46 explant of H. niger observed under the light microscope. A Friable embryogenic callus (EC) formed at the cutting edge of leaf explant inoculated on MS + 6.0 mg/L picloram (bar = 1000 µm); B Globular somatic embryos (G) formed on the outer layer of callus from leaf explant after four weeks of culture on MS supplemented with 6.0 mg/L picloram (bar = 500 µm). Figure 4.3 Whitish green friable callus formed at the cutting edge of 49 petiole explant that were cultured on MS supplemented with 4.0 mg/L picloram after four weeks of culture (bar = 1000 µm).