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POST-HARVEST SEED PHYSIOLOGY and CONSERVATION of the GERMPLASM of Syzygium Cordatum Hochst

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POST-HARVEST SEED PHYSIOLOGY and CONSERVATION of the GERMPLASM of Syzygium Cordatum Hochst POST-HARVEST SEED PHYSIOLOGY AND CONSERVATION OF THE GERMPLASM OF Syzygium cordatum Hochst. by Anastacia Chepkorir Cheruiyot Submitted in partial fulfillment of the requirements of the degree of Master in Science in the School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, South Africa 2013 DECLARATION 1 – PLAGIARISM I, Anastacia Chepkorir Cheruiyot declare that 1. The research reported in this thesis, except where otherwise indicated, is my original research. 2. This thesis has not been submitted for any degree or examination at any other university 3. This thesis does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons. 4. This thesis does not contain other persons’ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a. Their words have been re-written but the general information attributed to them has been referenced. b. Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 5. This thesis does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the thesis and in the reference sections. Signed: 2 DECLARATION 2 – PUBLICATIONS DETAILS OF CONTRIBUTION TO PUBLICATIONS that form part and /or include research presented in this thesis (include publications in preparation, submitted, in press and published and give details of the contributions of each author to the experimental work and writing of each publication). Publication 1. Cheruiyot, C., Wesley-Smith, J., Berjak, P. and Kioko, J.I. (2004). Citric acid as a pre-fixation treatment of phenol laden shoot axes of Syzygium cordatum. Microscopy Society of Southern Africa – Proceedings 34: 62. The experimental work and writing of publication was done by Anastacia Cheruiyot. However, the process of both the experimental work and writing was supervised by Prof. Pat Berjak and Dr Joseph Kioko. Publication 2. Cheruiyot, C., Kioko, J.I., Berjak, P. and Wesley-Smith, J. (2007). In vitro regeneration of recalcitrant embryonic axes: Effects on the biomass characteristics of resulting plants. South African Journal of Botany 73: 483. The experimental work was done by Anastacia Cheruiyot and supervised by Prof. Pat Berjak and Dr Joseph Kioko. The writing was done by Dr Joseph Kioko. Signed: 3 PREFACE The experimental work described in this thesis was carried out in the School of Life and Environmental Sciences, University of Natal, which later changed to the School of Biological and Conservation Sciences, University of KwaZulu-Natal, from February 2003 to November 2011, under the supervision of Prof. Patricia Berjak and Dr Joseph Kioko. Since January 1st, 2012, the School is now known as School of Life Sciences. These studies represent original work by the author and have not been submitted in any form to another university. Where use was made of work of other people, it has been duly acknowledged in the text. Anastacia Chepkorir Cheruiyot 2013 4 ACKNOWLEDGEMENTS I would like to express my gratitude to Prof. Berjak for her encouragement, support, guidance and patience! Thank you for not giving up on me. My sincere thanks to Dr Kioko for his supervision, guidance and patience. You have never given up on me as a scientist and for that I sincerely thank you. I would like to thank Dr James Wesley- Smith for his guidance and supervision while I was at CSIR. I would like to thank Pricilla Maartens for guiding me through electron microscopy, Prof. Berjak’s research team for their valued contribution at all stages of this study, and Prof. Paula Watt for sharing with me the strategy on how to work and write up a thesis at the same time. The strategy worked! Harmony, thanks my friend… thanks. I would like to thank my family, especially my Dad and Mum for tirelessly supporting me. Dad, your continual enquiry into my progress propelled me to finish what I had started. Thank you! Beaty, Cossy and Vero, thank you for being the best sisters in the whole wide world. Kibet, Kiptoo and Beaty, thank you for making special trips to South Africa. Your presence made a whole world of a difference. Kibiwott and Kiyeng’, thanks. Thanks to all my family members who harvested seeds in the forests in Kenya! My darling husband Richard, thanks for the final push! Your contribution at the end made all the difference and for that reason I will be forever grateful. Above all, thank you to my God! You truly are the best. This thesis is dedicated to that little child in this world somewhere who marvels at nature and wonders why plants do things the way they do. You are where I was many years ago. May you get to know the God who created them. May your journey of discovery take you to places beyond your imagination and may you gain knowledge to nurture the very plants at which you marvel. 5 Table of Contents DECLARATION 1 – PLAGIARISM ............................................................................................. 2 DECLARATION 2 – PUBLICATIONS ......................................................................................... 3 PREFACE ............................................................................................................................... 4 ACKNOWLEDGEMENTS ......................................................................................................... 5 ABSTRACT ............................................................................................................................. 8 LIST OF ABBREVIATIONS ..................................................................................................... 11 CHAPTER 1. INTRODUCTION ............................................................................................... 14 Germplasm conservation ....................................................................................................... 16 Ex situ conservation of plant germplasm ............................................................................... 20 Seed banking .......................................................................................................................... 21 Seed Recalcitrance ................................................................................................................. 23 Cryopreservation of plant germplasm.................................................................................... 27 Objectives of this study .......................................................................................................... 39 CHAPTER 2. EFFECTS OF SEED STORAGE TEMPERATURE AND RELATIVE HUMIDITY ON THE VIABILITY AND VIGOUR OF Syzygium cordatum .................................................................. 41 2.1 Introduction ...................................................................................................................... 41 2.2 Materials and Methods .................................................................................................... 43 2.2.1 Seed procurement and handling ............................................................................... 43 2.2.2 Seed storage treatment and sampling ...................................................................... 44 2.2.3 Water content determination ................................................................................... 44 2.2.4 Vigour and Viability assessment ................................................................................ 45 2.2.5 Biomass allocation ..................................................................................................... 46 2.2.6 Statistical analysis ...................................................................................................... 46 2.2.7 Assessment of contamination status: fluorescence microscopy (adapted from Rohringer et al., 1977) ........................................................................................................ 46 2.3 Results and Discussion ...................................................................................................... 48 2.3.1 Seed characteristics and seed‐associated fungi ........................................................ 48 2.3.2 Effects of storage conditions and storage period on the water content of cotyledons and embryonic axes ............................................................................................................ 51 6 2.3.3 Effects of storage conditions and storage period on vigour and viability ................. 54 2.3.4 Effects of storage conditions and storage period on plant growth ........................... 63 2.3.5 Shoot:root ratio ......................................................................................................... 68 2.3.6 Effects of storage conditions on microbial contamination ....................................... 69 2.4 Conclusion: Short‐term storage of Syzygium cordatum seeds......................................... 75 CHAPTER 3. TOWARDS THE CRYOPRESERVATION OF Syzygium cordatum EMBRYONIC AXES ........................................................................................................................................... 77 3.1 Introduction ...................................................................................................................... 77 3.2 Materials and methods ...................................................................................................
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