Ex Situ Conservation of Australian Citrus Species: Investigations on Seed Biology, Cryopreservation and in Vitro Culture
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Ex Situ Conservation of Australian Citrus Species: Investigations on Seed Biology, Cryopreservation and in Vitro Culture Author Hamilton, Kim Nicole Published 2007 Thesis Type Thesis (PhD Doctorate) School School of Biomolecular and Biomedical Sciences DOI https://doi.org/10.25904/1912/1593 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/365585 Griffith Research Online https://research-repository.griffith.edu.au EX SITU CONSERVATION OF AUSTRALIAN CITRUS SPECIES: INVESTIGATIONS ON SEED BIOLOGY, CRYOPRESERVATION AND IN VITRO CULTURE By Kim Hamilton BAppSc (Hons) MTeach School of Biomedical and Biomolecular Science Centre for Forestry and Horticultural Research Griffith University, Brisbane, Queensland A thesis submitted in fulfillment of the requirements of the degree of Doctor of Philosophy January, 2007 STATEMENT OF ORIGINALITY This work has not been previously submitted for a degree or diploma in any university. To the best of my knowledge and belief, the thesis contains no material published or written by another person except where due reference is made in the thesis itself. Kim Hamilton January 2007 ii ACKNOWLEDGEMENTS Firstly, I wish to express my gratitude to all those people who have contributed to the production of this thesis. In particular, I thank my supervisors Dr. Sarah Ashmore, Prof. Rod Drew and Dr. Hugh Pritchard. They have provided invaluable guidance, assistance, encouragement and advice throughout this research work. I thank all the staff of the Millennium Seed Bank Project (Royal Botanic Gardens, Kew) for training in seed science research and their friendly support. I am appreciative of the training in Scanning Electron Microscopy to Dr. Deb Stenzel (AEMF QUT) and in Differential Scanning Calorimetry to Dr. Chris Wood (Royal Botanic Gardens, Kew), Dr Wayde Martens (QUT) and Dr. Greg Cash (QUT), as well as Dr. Cameron Mc Conchie (CSIRO) for his help in seed oil extraction. A special thank you to Mr. Malcolm Smith (QDPI Bundaberg) for his assistance, advice and the supply of fruit throughout this project. I also wish to thank Roger Goebel (QDPI), Phil Boyle (Mt Coot-tha Botanic Gardens), Phil Cameron (Mt Coot-tha Botanic Gardens) and John Wrench (QLD Bushfood Association) for also kindly donating fruit. I gratefully thank Dr. Barbara Reed (USDA), Dr. Florent Engelmann (IPGRI), Dr. Paul Forster (QLD Herbarium), Dr. Sue Lee (Griffith University) and Dr. Jacinta Zalucki (Griffith University) for their helpful advice. I acknowledge the generous financial support of this PhD provided by the Student Steering Committee of the Millennium Seed Bank Project (Royal Botanic Gardens, Kew), the International Plant Genetic Recourses/Australian Centre for International Agricultural Research, the Centre for Forestry and Horticultural Research and Griffith University. Finally, I thank Brian Reis for the permission to use copyrighted photos of wild habitats of north Queensland from the Len Webb Ecological Images Collection. iii CONTENTS ACKNOWLEDGEMENTS ..............................................................................................III LIST OF TABLES .......................................................................................................... VII LIST OF FIGURES.......................................................................................................... IX ABSTRACT ...................................................................................................................XIII CHAPTER 1.0 INTRODUCTION......................................................................................1 1.1 Background to sustainable conservation for socio-economically valuable biodiversity……………………………………………………………………….2 1.2 Ex situ conservation approaches……..……………………….…………...….9 . 1.3 Priority species…............................................................................................20 1.4 Citrus biodiversity…………………………………………………….…......21 . 1.5 Ex situ conservation of Citrus.........................................................................23 1.6 Citrus seed biology and storage……………………………………………..36 1.7 In vitro options in Citrus.................................................................................52 1.8 Summary………………………………………………………………….…58 1.9 Aims and objectives…………………………………………………...….…59 CHAPTER 2.0 COMPARATIVE SEED MORPHOLOGY OF CITRUS AUSTRALASICA, C. INODORA AND C. GARRAWAYI. .....…………………………..62 2.1 Introduction .....................................................................................................63 2.2 Materials and Method......................................................................................64 2.3 Results .............................................................................................................65 2.4 Discussion........................................................................................................79 CHAPTER 3.0 EFFECT OF TEMPERATURE ON GERMINATION OF CITRUS AUSTRALASICA, C. INODORA AND C. GARRAWAYI SEEDS.....................................89 3.1 Introduction .....................................................................................................90 3.2 Materials and Method......................................................................................92 iv 3.3 Results .............................................................................................................94 3.4 Discussion......................................................................................................104 CHAPTER 4.0 DISTINGUISHING CHARACTERISTICS OF SEED MATURITY IN CITRUS GARRAWAYI IN RELATION TO GERMINABILITY …………………..109 4.1 Introduction ...................................................................................................110 4.2 Materials and Method....................................................................................111 4.3 Results ...........................................................................................................113 4.4 Discussion......................................................................................................127 CHAPTER 5.0 DESICCATION AND CRYOPRESERVATION OF SEEDS OF CITRUS AUSTRALASICA, C. INODORA AND C. GARRAWAYI..................................130 5.1 Introduction ...................................................................................................131 5.2 Materials and Method....................................................................................133 5.3. Results ..........................................................................................................137 5.3.1 Effect of desiccation and liquid nitrogen exposure on seed germinability in Citrus australasica........................................................137 5.3.2 Effect of desiccation and liquid nitrogen exposure on seed germination in Citrus inodora.................................................................146 5.3.3 Effect of desiccation and liquid nitrogen exposure on seed germination in Citrus garrawayi, including the effect of seed maturity 151 5.3.4 Analysis of responses to cryopreservation in C. australasica, C. inodora and C. garrawayi and the association with climatic range and seed oil thermal properties of each species.......................................162 5.4 Discussion......................................................................................................168 CHAPTER 6.0 IN VITRO CULTURE AND CRYOPRESERVATION OF CITRUS AUSTRALASICA, C. INODORA AND C. GARRWAYI ……………………… .........................................................................................183 6.1 Introduction ...................................................................................................184 6.2 Materials and Method....................................................................................187 v 6.3 Results ...........................................................................................................197 6.3.1 Embryogenesis……………………………………………………197 6.3.2 Micropropagation…………………………………………………220 6.3.3 In vitro seed germination………………………………………….226 6.4 Discussion......................................................................................................234 CHAPTER 7.0 GENERAL DISCUSSION ....................................................................253 7.1 Overview of main findings/outcomes............................................................254 7.2 Future research studies .................................................................................258 7.3 Conservation options in Australian wild Citrus ..........................................260 CHAPTER 8.0 REFERENCES.......................................................................................265 APPENDIX .....................................................................................................................302 Appendix 1: Research dissemination……………………………………………....…….303 vi LIST OF TABLES Table 1.1 List of Queensland rare and threatened edible plants and/or crop wild relatives. Table 1.2 Summary of conservation approaches investigated in cultivated Citrus. Table 1.3 List of major cultivated and wild species of Citrus. Table 1.4 Ex situ collections of Citrus as listed by Bioveristy International. Table 1.5 Distribution, habitat and climatic conditions of Australian wild Citrus species, C. australasica, C. inodora and C. garrawayi. Table 1.6 Summary of reports of effect of desiccation and liquid