THE DEVELOPMENT OF WASABIA JAPONICA (MIQ.) MATSUMURA IN VITRO Cao Dinh Hung Thesis submitted for degree of Master of Science Department of Environmental Sciences, Faculty of Science, University of Technology, Sydney MSc 2007 CERTIFICATE OF AUTHORSHIP/ORIGINALITY I certify that the work in this thesis has not previously been submitted for a degree nor has it been submitted as part of requirements for a degree except as fully acknowledged within the text. I also certify that the thesis has been written by me. Any help that I have received in my research work and the preparation of the thesis itself has been acknowledged. In addition, I certify that all information sources and literature used are indicated in the thesis. Signature of Student ______________________ January 2007 i ACKNOWLEDGEMENTS I would like to thank my principal supervisor, Dr Krystyna Johnson for her guidance and friendship throughout my whole project, and my co-supervisors, Emeritus Professor Ron Wills and Dr Debbie Shohet (School of Applied Sciences, University of Newcastle), for their guidance and advice during the last two semesters of my candidature. I am really grateful to them for their valuable expertise and support. I wish to thank Gemma Armstrong for her direct help, advice and encouragement throughout my candidature. I also thank Tony Ye, Christine Wojak and Melinda Ellith for their support during my project research in the Biology Annex. A special thank you is given to Professor Margaret Burchett for her comments on my presentation and editorial assistance on my thesis, and to the staff at the ANSTO and Royal North Shore Hospital for their assistance with ionising-irradiation treatments. I thankfully acknowledge Narelle Richardson and Sue Fenech for assisting with the laboratory work at Dunbar Building, and Dr Fraser Torpy for help with the statistical analysis of the experimental results. Countless thanks are given to the Gore Hill campus staff at the Department of Environmental Sciences, Faculty of Science, University of Technology, Sydney, who have helped me during my two-year candidature. Sincere thanks to my colleagues Hoang Van Hung and Pham Thi Thu Nga, and Vietnamese families residing in Australia, for their friendship, encouragement and invaluable support throughout my two academic years at UTS. This thesis would not have been possible without financial assistance from an AusAID scholarship and a grant from the Department of Environmental Sciences. Finally, this project may not have been accomplished without the assistance and emotional support from my institute, my family and my friends in Vietnam. ii TABLE OF CONTENTS Acknowledgements…………………………………………………………………... i Table of Contents……………………………………………………………….......... ii List of Figures………………………………………………..………………………. vi List of Tables………………………………………………………….……………... ix List of Appendices...…………………………………………………………………. x Abbreviations………………………………………………………………………… xii Abstract………………………………………………………………………………. xiv Publication and poster..………………………………………………………………. xvii CHAPTER 1 – INTRODUCTION………………………………….……………... 1 1.1 Background and aims…………….………………………………………………. 1 1.2 Wasabi crop………………………………………………...……………………. 2 1.2.1 Taxonomy, distribution and morphology………………...…………..... 2 1.2.2 Bioactive compounds in wasabi………………………………………... 4 1.2.3 Wasabi cultivation…………………………….………..……………… 4 1.2.3.1 Propagation…………………………………………………... 4 1.2.3.2 Varieties……………………………………………………… 6 1.2.3.3 Production……………………………………………………. 7 1.2.3.4 Yield………………………………………………………….. 8 1.2.3.5 Diseases and pests…………………………….…………….... 9 1.2.3.6 Storage……………………………………………………….. 9 1.2.4 Markets and uses……………………………………………………….. 10 1.2.5 Recent studies on wasabi………………………………………………. 13 1.3 Plant tissue culture……………………………………………………………….. 16 1.4 Plant mutagenesis…………………………………………………………………17 1.5 Experimental objectives…………………………….……………………………. 18 CHAPTER 2 – IN VITRO PROPAGATION STUDIES………………………..... 20 2.1 Introduction ……………………………………………………………………… 20 2.1.1 Rapid clonal propagation……………………...……………………….. 20 2.1.2 Liquid culture for mass propagation…...........…………………………. 23 2.1.3 Callus culture for the production of bioactive compounds ……………. 25 2.2 Materials and methods …………………………………………………………... 26 2.2.1 Clonal propagation……………………………………………………... 26 2.2.1.1 Sterilisation of explants for culture initiation………………... 26 2.2.1.2 Experimental design for shoot proliferation, root induction and plantlet acclimatisation…………………... 26 2.2.1.3 Culture conditions……………………………………………. 30 2.2.1.4 Growth measurement………………………………………… 30 2.2.1.5 Data analysis…………………………………………………. 31 2.2.2 Callus culture…………………………………………………………... 31 iii 2.2.2.1 Selection of explants and culture conditions for callus induction ………………………………………….. 31 2.2.2.2 Selection of PGRs for callus proliferation……………...……. 32 2.3 Results……………………………………………………………………………. 34 2.3.1 Clonal propagation……………………………………………………... 34 2.3.1.1 In vitro culture initiation……………………………………... 34 2.3.1.2 Shoot multiplication………………………………………….. 34 2.3.1.3 In vitro root formation……………………………………….. 44 2.3.1.4 In vivo acclimatisation of plantlets………………..…………. 48 2.3.2 Callus culture…………………………………………………………... 52 2.3.2.1 Callus initiation………..……………………………………... 52 2.3.2.2 Callus proliferation…………………………………………... 55 2.4 Discussion………………….…………………………………………………….. 58 2.4.1 Clonal propagation…………………..………………….…………….... 58 2.4.1.1 Choice of culture basal media………….……..……………… 58 2.4.1.2 Choice of PGRs for shoot multiplication..…………………… 59 2.4.1.3 Choice of PGRs for root formation……………..……………. 60 2.4.1.4 Choice of culture conditions (gelled vs. liquid media) for efficient shoot proliferation………………………………. 61 2.4.1.5 Choice of root origins and substrates for acclimatisation of plantlets……………..……………….... 63 2.4.1.6 Problems in tissue culture of W. japonica……….…………... 64 2.4.2 Callus culture……….………………………...………………………... 66 2.5 Conclusions..……………………………………………………………………... 69 CHAPTER 3 – STUDIES IN MUTAGENESIS…………………………………... 70 3.1 Introduction…...………………………………………………………………….. 70 3.1.1 Chemical mutations……………………………………………………. 70 3.1.2 Physical mutations……………………………………………………... 74 3.2 Materials and methods…………………………………………………………… 78 3.2.1 Plant materials…..……………………………………………………… 78 3.2.2 Treatment methods………………………………….…………………..78 3.2.2.1 Chemical mutagens…………………………………………... 78 3.2.2.1.1 Colchicine………………………………………...... 78 3.2.2.1.2 Oryzalin…………………………………………......78 3.2.2.2 Physical mutagens……………………………………………. 79 3.2.2.2.1 X-irradiation………………………………………... 79 3.2.2.2.2 Gamma irradiation…………………………………. 80 3.2.3 Culture conditions…………………..………………………………….. 80 3.2.4 Growth measurement…………………………………………………... 81 3.2.5 Data analysis…………………………………………………………… 81 3.3 Results…………………….……………………………………………………… 81 3.3.1 Chemical mutagen treatments…………………………...……………... 81 3.3.1.1 Selection of optimal treatments based on time exposure and concentrations of mutagens….… 81 3.3.1.2. Effect of chemical mutations on in vitro growth and morphological variations…………..… 85 3.3.1.2.1 Explant weight……………………………………... 85 3.3.1.2.2 Shoot multiplication………………………………... 86 3.3.1.2.3 Shoot height………………………………………... 87 iv 3.3.1.2.4 Morphological variations of shoots and leaves…….. 89 3.3.1.3 Survival of explants in vitro and in vivo………………..……. 91 3.3.2. Physical mutagen treatments…………………………………………...92 3.3.2.1 Survival of explants in vitro and morphological variations….. 92 3.3.2.2 Effect of physical mutation on in vitro growth………………. 96 3.3.2.2.1 Explant weight……………………………………... 96 3.3.2.2.2 Shoot multiplication……........................................... 98 3.3.2.2.3 Shoot height………………………………………... 99 3.3.2.3 Survival of explants in vivo…………………..……………….100 3.4 Discussion………………………………………………………….……….......... 101 3.4.1 Mutation induction with polyploidy-inducing agents………………..… 101 3.4.2 Mutation induction with ionising-irradiations………………..………... 104 3.5 Conclusions………………………………………………………...…………….. 106 CHAPTER 4 – ANALYSIS OF BIOACTIVE COMPOUNDS – ALLYL ISOTHIOCYANATE..………………….…………….…108 4.1 Introduction ……………………………………………………………………… 108 4.2 Materials and methods.………………………………………………………....... 111 4.2.1 Collection and preparation of W. japonica samples…….…………....... 111 4.2.1.1 Calli………….………………………………………………. 111 4.2.1.2 In vitro mutant explants……………………………………… 111 4.2.1.3 In vitro non-mutant explants…………………………………. 112 4.2.1.4 In vivo non-mutant plants……………………………………..112 4.2.2 Analytical methods…………………………...………………………... 113 4.2.2.1 Extraction procedures………………………………………... 113 4.2.2.2 Gas chromatography…………………………………………. 115 4.2.2.3 Mass spectrometry…………………………………………… 115 4.2.2.4 Quantitative analysis of AITC……………………………….. 115 4.2.2.5 Qualitative analysis of AITC………………………………… 116 4.2.2.6 Experimental design…………………………………………..117 4.2.2.7 Data analysis…………………………………………………. 119 4.3 Results……………………………………………………………………………. 119 4.3.1 AITC content as affected by callus cultures….………………….....….. 119 4.3.2 AITC content as affected by mutagens………………………….……... 120 4.3.3 Investigation of AITC and plant yields from plant organs…………….. 123 4.3.4 Necessary conditions for AITC yield optimisation……………………. 127 4.3.4.1 Effect of particle size...………………………………………. 128 4.3.4.2 Effect of drying temperature…………………………………. 129 4.3.4.3 Effect of storage temperature...………………………………. 129 4.3.4.4 Effect of shaking conditions…………………………………. 131 4.3.4.5 Effect of solvent……………..………………………………. 132 4.4 Discussion………………………………………………………………………... 133 4.4.1 AITC in calli…………..…………………..…….…………..…………. 133 4.4.2 AITC in mutant lines as affected by antimitotic agents………………... 134