Towards Development of a Cryopreservation Protocol for Germplasm of Podocarpus henkelii

Towards Development of a Cryopreservation Protocol for Germplasm of Podocarpus henkelii by Lubaina Essack Submitted in fulfilment of the academic requirements for the degree Master of Science School of Life Sciences College of Agriculture, Engineering and Science University of KwaZulu-Natal Westville Campus Durban 2012

ii Preface The experimental work described in this dissertation was carried out in the School of Life Sciences at the University of KwaZulu-Natal (Westville), Durban, under the supervision of Professor Patricia Berjak and Professor Norman W. Pammenter. These studies represent original work by the author, and have not otherwise been submitted in any form for any degree or diploma to any tertiary institution. Where use has been made of the work of others, it is duly acknowledged in the text. L. Essack January 2012

iii Declaration 1 Plagiarism I, Lubaina Essack, 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 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 dissertation and in the References section Signed: (January 2012)

iv Acknowledgements I am sincerely grateful to my supervisor Prof. Patricia Berjak for her invaluable counsel and unwavering support during the course of this investigation. The time and effort you invested in reviewing this manuscript is much appreciated as is the fact that you made this endeavour an important and formative experience for me. To my cosupervisor, Prof. Norman W. Pammenter, I am truly appreciative of your advice on statistical analyses as well as for helping me to streamline the experimental design and for always reminding me to focus on the primary aims and objectives of the study when conducting the research. I am also grateful to Dr. R. Mackey for her assistance with statistical analyses and to Priscilla Maartens for her help with photography. To the staff of the Pietermaritzburg National Botanic Gardens, I thank you for always going over and above the call of duty to provide me with good-quality seeds as promptly as possible. An earnest thank you is also extended to my friend, and colleague, Cassandra Naidoo for her unfailing willingness to assist with the technical aspects of this study. To my maternal family, my husband, and my friend, Prathiska Rambally, I not only thank you for your support, love and encouragement throughout this undertaking but also for never allowing me to lose sight of the wood for the trees. I am also grateful for the friendships afforded to me by the members of the African Centre for Plant Germplasm Conservation Research (UKZN). The National Research Fund and the Department of Labour are also acknowledged for their financial assistance, without which this project would not have been possible.

v Abstract The trees belonging to the genus Podocarpus, of which only four species are native to South Africa, are renowned for their superior quality timber. Prior to 1880, Podocarpus henkelii, together with P. falcatus and P. latifolius, played a significant role in the development of the country as they were heavily utilised as timber trees for the building of dwellings, furniture and other necessary items. Due to this over-exploitation in the timber trade, all the Podocarpus species in South Africa have been afforded a Protected status on the IUCN red data list of species that are either threatened or in danger of extinction. However, despite the obvious need to conserve the threatened genetic diversity of these species, few attempts (aside from in vitro micropropagation) have been made to explore ex situ Podocarpus germplasm conservation in the longterm. Consequently, the primary aim of this study was to establish a protocol for the long-term conservation of germplasm of Podocarpus henkelii Stapf ex Dallim. Jacks. The seeds of Podocarpus henkelii exhibit recalcitrant behaviour and can therefore not be stored in conventional seed banks. This has necessitated the investigation of alternative methods of germplasm conservation with a focus on cryopreservation which is presently considered the most reliable, efficient and cost-effective means of storing the genetic resources of recalcitrant-seeded species for prolonged periods. The first objective of this study was to investigate the effect of slow (two-step) and ultra-rapid cooling on the post-thaw survival of variously treated P. henkelii embryos. The results of this investigation revealed that the rate of cooling employed had a significant effect on explant viability as none of the precultured, cryoprotected embryos that were slowly cooled survived cryostorage while some of the preconditioned embryos responded to ultra-rapid cooling (i.e. 36% shoot production and 88% callus formation). For ultrarapid cooling, it was found that flash-drying prior to cooling was a prerequisite for survival as osmotic dehydration alone did not effectively prepare the tissues for the stresses imposed during cryostorage. Furthermore, for those flash drying intervals that yielded positive results, preconditioning explants with 10% glycerol proved the most effective pre-cooling treatment. However, due to the low recovery numbers after ultrarapid cooling, a third cryopreservation technique i.e. cryogenic vitrification, was investigated.

vi For cooling by vitrification, data obtained from preliminary experiments showed that precultured explants needed to be initially loaded with 18% sucrose (w/v) + 14% glycerol (v/v) for 20 min and subsequently immersed in Plant Vitrification Solution 3 (PVS3) at 0 C for 10 min prior to cooling. However, relatively low success was achieved for P. henkelii embryos cooled by vitrification as the highest post-cooling survival obtained was only 20% germination, 27% shoot formation and 37% callus formation. Due to the low post-thaw survival obtained despite the rigorous manipulations employed in the development of the slow cooling, ultra-rapid cooling and vitrification protocols, it was decided that an alternative explant should be investigated for the conservation of P. henkelii germplasm. The explant of choice was adventitious buds induced to form on, and subsequently excised from, mature P. henkelii embryos. The first objective was to develop a suitable protocol for the induction of adventitious buds on P. henkelii embryos. The medium that induced in the highest percentage of embryos (85%) to form adventitious buds consisted of Douglas-fir cotyledon revised (DCR) basal medium supplemented with 30 g L -1 sucrose, 0.05 mg L -1 NAA, 0.5 mg L -1 BA and 6 g L -1 agar. This medium also resulted in the highest average number of buds formed per embryo (i.e. 35 ± 3 buds per embryo). Once the adventitious bud induction medium was developed, it was necessary to optimise the size of adventitious bud clumps to be used as explants for cryopreservation. Three bud clump sizes were investigated: ca 3, 5 and 10 buds per clump. However, none of the bud clumps survived excision from the mother-tissue despite the investigation of three different types of bud-break media. The resultant tissue mortality is suggested to have occurred because the adventitious bud clumps were excised prior to bud break and shoot development which could have exacerbated excision-related cellular and sub-cellular damage. It was therefore decided that attempts should be made to induce adventitious buds directly on P. henkelii embryos post-cooling, thereby eliminating the possibility of potentially lethal excision-related damage. The protocols that yielded the best results after ultra-rapid cooling and cooling by vitrification were used in this experiment. For ultra-rapid cooling, embryos were first cryoprotected with 5% followed by 10% glycerol for 1 h in each and subsequently flash dried for 30 min prior to immersion in nitrogen slush. For cooling by vitrification, embryos that were first precultured on 0.3

vii M sucrose for 1 d were loaded with 10% glycerol + 14% sucrose (LS4). The loaded explants were then immersed in ice-cold PVS3 and maintained on ice for 10 min prior to cryostorage. The effect of each pretreatment (either independently or in combination) on adventitious bud production pre-cooling was also investigated. For both protocols the various pretreatments decreased not only the capacity of the embryos to form buds but also the average number of buds formed per embryo (i.e. 7 ± 2 buds per embryo and 14 ± 2 buds per embryo were formed on treated embryos prior to ultrarapid cooling and cooling by vitrification, respectively). Thus, it was predicted that even if the percentage of cryopreserved embryos forming buds was minimal, the number of possible plantlets that could be regenerated from adventitious buds per cryopreserved explant would compensate for the low recovery of embryos post-cooling. However, none of the embryos that were cryopreserved by either ultra-rapid cooling or by vitrification formed adventitious buds after eight weeks in culture. The very restricted success achieved in this study despite the investigation of three cryopreservation techniques and two different explants only serves to reinforce the difficulties associated with the conservation of recalcitrant germplasm. The large size and structural complexity of P. henkelii embryos, coupled with their high water content post-shedding, are just some of the characteristics to which their intractability to the manipulations involved in the development of a successful cryopreservation protocol could be attributed. For future investigations, development of adventitious buds produced on cryopreserved root segments (as opposed to entire roots), and/or use of seedling meristems as explants which might be amenable to cryopreservation are suggested as possible avenues for the long-term conservation of P. henkelii genetic diversity.

viii TABLE OF CONTENTS TITLE PAGE PREFACE DECLARATION 1 PLAGIARISM ACKNOWLEDGEMENTS ABSTRACT TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIONS CHAPTER 1 i ii iii iv v viii xii xiv xv General Introduction 1 1.1. Conservation of plant genetic resources 1 1.2. Ex situ conservation 2 1.3. Seeds 3 1.3.1. Post-harvest seed behaviour and storage 4 1.4. In vitro conservation 6 1.4.1. Developing a micropropagation protocol 8 1.4.2. Plant tissue culture media 9 1.4.3. The tissue culture environment 10 1.4.4. In vitro storage 11 1.5. Cryopreservation 13 1.5.1. The cooling (freezing) process 14 1.5.2. Cryopreservation-induced injury 15 1.5.3. Developing a cryopreservation protocol 17 1.5.3.1. Explant of choice 18 1.5.3.2. Preconditioning 19 1.5.3.3. Dehydration 22 1.6. Podocarpus henkelii and the rationale of this study 24

ix CHAPTER 2 Effects of Pre-treatments, Dehydration and Slow and Ultra-rapid Cooling of Podocarpus henkelii Embryos 28 2.1. Introduction 28 2.1.1. Slow cooling 28 2.1.2. Rapid cooling 30 2.1.3. Thawing and rehydration 32 2.1.4. Onwards development 34 2.1.5. Aims and objectives 35 2.2. Materials and Methods 35 2.2.1. Plant material 35 2.2.2. Embryo excision, decontamination and plating on basal medium 36 2.2.3. Gravimetric determination of water content 37 2.2.4. Preculture 37 2.2.5. Cryoprotection 37 2.2.6. Flash drying 38 2.2.7. Pre-cooling combination treatments 38 2.2.8. Cooling 39 2.2.9. Thawing and regeneration 40 2.2.10. Statistical analyses 40 2.3. Results 41 2.3.1. Preculture 41 2.3.2. Cryoprotection 45 2.3.3. Flash drying 46 2.3.4. Preculture and cryoprotection 48 2.3.5. Cryoprotection and flash drying 50 2.3.6. Preculture, cryoprotection and flash drying 52 2.3.7. Cooling 54 2.4. Discussion 57 2.4.1 Pre-cooling treatments 57 2.4.2. Cooling treatments 63

x CHAPTER 3 Attempted Cryopreservation of Podocarpus henkelii Embryos using Vitrification-based Techniques 67 3.1. Introduction 67 3.1.1. The vitrification protocol 70 3.1.2. Aims and objectives 72 3.2. Materials and Methods 73 3.2.1. Plant Material 73 3.2.2. Vitrification solution 73 3.2.3. Loading solutions 74 3.2.4. Pre-cooling combination treatments 74 3.2.5. Cooling 75 3.2.6. Warming and regeneration 75 3.3. Results 77 3.3.1. Vitrification solution 77 3.3.2. Loading solutions 78 3.3.3 Loading solution and PVS3 79 3.3.4. Preculture and loading solutions 81 3.3.5. Preculture, loading solutions and PVS3 82 3.3.6. Cooling 83 3.4. Discussion 84

xi CHAPTER 4 Induction of Adventitious Buds on P. henkelii Embryos Pre- and Postcooling 90 4.1. Introduction 90 4.1.1. Aims and objectives 94 4.2. Materials and Methods 94 4.2.1. Induction of adventitious buds on P. henkelii embryos 94 4.2.2. Subsequent induction of adventitious buds for experimentation 95 4.2.3. Excision of adventitious buds 96 4.2.4. Bud-break and shoot development 96 4.2.5. Induction of adventitious buds on treated P. henkelii embryos preand 97 ultra-rapid post-cooling 4.2.6. Induction of adventitious buds on treated P. henkelii embryos preand 98 post-cooling by vitrification 4.3. Results 99 4.3.1. Induction of adventitious buds on mature P. henkelii embryos 99 4.3.2. Bud-break and subsequent shoot development 107 4.3.3. Induction of adventitious buds on mature P. henkelii embryos prior 109 to cooling in LN 4.3.4. Induction of adventitious buds on mature P. henkelii embryos after 111 ultra-rapid cooling and cooling by vitrification 4.4. Discussion 111 CHAPTER 5 Concluding Comments 119 Appendix 125 REFERENCES 126

xii List of Figures Figure no. Title Page no. 1.6.1. Immature and mature Podocarpus henkelii seeds. 26 1.6.2. Freshly excised non-germinable and germinable immature and mature 27 Podocarpus henkelii embryos. 2.1. Water contents of immature P. henkelii embryos precultured on 41 standard basal medium supplemented with 0.06, 0.1, 0.3, 0.5, 0.7 and 1.0 M sucrose for 1, 2, 3, 4 and 5 d. 2.2. Percentage germination of immature P. henkelii embryos precultured 42 on standard basal medium supplemented with 0.06, 0.1, 0.3, 0.5, 0.7 and 1.0 M sucrose for 1, 2, 3, 4 and 5 d. 2.3 Water contents and percentage germination of immature P. henkelii 44 embryos precultured on medium containing 0.3 M sucrose or 0.3 M sucrose + 0.1% DMSO for 1 and 5 d. 2.4. Water contents and percentage germination for freshly excised 45 immature and mature P. henkelii embryos cryoprotected with 10% glycerol and 10% glycerol + sucrose. 2.5. Water contents and percentage germination for immature P. henkelii 46 embryos flash dried from 0 240 min; sampled at 30 min intervals. 2.6. Water contents and percentage germination for immature and mature 47 P. henkelii embryos flash dried from 0 60 min; sampled at 10 min intervals. 2.7. Water contents and percentage germination mature P. henkelii 48 embryos precultured on 0.3 M sucrose and 0.3 M sucrose + 0.1% DMSO for 1 d, cryoprotected with 10% glycerol or 10% glycerol + sucrose. 2.8. Water contents and percentage germination for mature P. henkelii 50 embryos cryoprotected with 10% glycerol and 10% glycerol + sucrose and subsequently flash dried for 20, 30 and 40 min. 2.9. Water contents and percentage germination for mature embryos that 52

xiii were precultured on either 0.3 M sucrose or 0.3 M sucrose + 0.1% DMSO, cryoprotected with either 10% glycerol or 10% glycerol + sucrose and subsequently flash dried for 20, 30 and 40 min. 3.1. Water contents and percentage germination of mature P. henkelii embryos immersed in PVS3 at 0 C and 25 C for 10, 20, 30, 40, 50 and 60 min. 3.2. Water contents and percentage germination for mature P. henkelii embryos exposed to different loading solutions for 20 min. 3.3. Water contents and percentage germination for mature P. henkelii embryos treated with different loading solutions for 20 min and subsequently immersed in PVS3 at 0 C for either 10 or 20 min. 3.4. Water contents and percentage germination for mature P. henkelii embryos either freshly excised or first precultured on 0.3 M sucrose for 1 d and subsequently treated with different loading solutions for 20 min. 3.5. Water contents and percentage germination for mature P. henkelii embryos either freshly excised or first precultured on 0.3 M sucrose for 1 d and subsequently treated with different loading solutions and PVS3. 4.1 Effect of BA or TDZ, incorporated in DCR-based Medium 1 and Medium 2, on the percentage of P. henkelii embryos forming adventitious buds 4.2. Effect of BA or TDZ, incorporated in DCR-based Medium 1 and Medium 2, on the number of buds produced per embryo of P. henkelii 4.3. Adventitious buds induced to form on a mature P. henkelii embryo cultured on bud induction Medium 1 + 0.3 mg L -1 BA for 6 weeks. 4.4. Single, magnified adventitious bud attached to a P henkelii embryo cultured on bud induction Medium 1 + 0.3 mg L -1 BA for 6 weeks. 4.5. Green and white callus around the periphery of Clump Size 3 after 2 weeks in culture. 77 78 79 81 82 101 104 106 107 108

xiv List of Tables Table no. Title Page no. 2.1. Percentage shoot production of mature P. henkelii embryos after rapid 54 and slow (two-step) cooling, following various pre-treatments. 2.2. Percentage callus formation on mature P. henkelii embryos after rapid 56 and slow (two-step) cooling, following various pre-treatments. 3.1. Percentage germination, shoot production and callus formation of 83 treated P. henkelii embryos after cooling in liquid nitrogen. 4.1a. Original data reflecting the percentage of mature P. henkelii embryos 100 forming adventitious buds when cultured on the DCR-based media, 1 and 2, containing either BA or TDZ and NAA or IBA 4.1b. Effect of the cytokinins BA and TDZ at a range of concentrations, on 102 adventitious bud production by P. henkelii embryos 4.2a. Original data reflecting the average number of adventitious buds 103 formed per P. henkelii embryo when cultured on the DCR-based media, 1 and 2, containing either BA or TDZ and NAA or IBA. 4.2b. Effect of the cytokinins BA and TDZ at a range of concentrations, on 105 the average number of buds produced per embryo for P. henkelii embryos. 4.3 The effect of different pre-treatments on mature P. henkelii embryos 109 prior to ultra-rapid cooling. 4.4. The effect of different pre-treatments on mature P. henkelii embryos 110 prior to cooling by vitrification. A1 Composition of media used in this study 125

xv List of Abbreviations BA Benzyladenine BAP 6-benzylaminopurine C degrees Celsius CaMg calcium magnesium CaOCl calcium hypochlorite comm. communication d day(s) DCR Douglas-fir cotyledon revised medium dmb dry mass basis DMSO dimethylsulphoxide FDA fluorescein diacetate g gram(s) gly glycerol h hour(s) IAA indole-3-acetic acid IBA indole-3-butyric acid 2iP isopentyl adenine K kinetin L litre(s) LN liquid nitrogen LS loading solution M molar m metres min minute(s) ml millilitre(s) mm millimetre(s) mol mole(s) MS Murashige and Skoog (1962) nutrient formulation NAA α-naphthaleneacetic acid NaOCl sodium hypochlorite no. number

xvi % percent PE phosphatidylethanolamine pers. personal PGR plant growth regulator ph hydrogen ion content pt. point PVS Plant Vitrification Solution ROS Reactive Oxygen Species s second(s) SD standard deviation sucr. sucrose TDZ thidiazuron T g T m TTZ glass transition temperature exothermic melting point triphenyl tetrazolium chloride µ micro µe microeinstein v volume vs. versus w weight WPM Woody Plant Medium