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Thesis Rests with Its Author University of Bath PHD Micropropagation of juvenile and mature Hevea brasiliensis Seneviratne, Priyani Award date: 1991 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 Micropropagation of Juvenile and Mature Hevea brasiliensis submitted by Priyani Seneviratne for the degree of PhD of the University of Bath 1991 COPYRIGHT ’Attention is drawn to the fact that copyright of this thesis rests with its author. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with its author and that no quotation from the thesis and no information derived from it may be published without the prior written concent of the author*. This thesis may be made available for consultation within the University library and may be photocopied or lent to other libraries for the purpose of consultation’. signature UMI Number: U029428 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U029428 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 UNIVERSITY OP BATH UEflASY — ■ — — II ■ i ■ ii in rnmimtmmmmuHmmm* 2 0 AU6I991 j j Pk‘ ?!•■■■ fo$*X.o5 Contents Page Abstract v Acknowledgements vi List of Tables vii List of Figures viii List of Plates xii Abbreviations x '*i CHAPTER 1 - INTRODUCTION 1.1 Heveabrasilicnsis 2 1.1.1 History and Origin 2 1.12 Distribution and Production 2 1.1.3 Morphology and Taxonomy 4 1.1.4 Exploitation and Improvement 5 1.1.5 Propagation 6 12 Micropropagation of Trees 8 12.1 Problems Associated with Tree Micropropagation 10 d (a). Physiological State 10 d. (b). Phenolic Browning 11 (c). Disinfection 12 1 2 2 Micropropagation Techniques for Trees 13 (a). Axillary Budding 14 (b). I Adventitious Budding 14 2 Somatic Embryogenesis 15 13 Hevea brasiliensis In Vitro 16 1.3.1 Seed and Embryo Culture 17 1.3.2 Axillary Budding 18 (a). Shoot Tip Culture 18 (b). Node Culture 18 1.3.3 Haploidy and Somatic Embryogenesis 20 (a). Haploidy I. Anther Culture 20 II. Ovule Culture 21 (b). Somatic Embryogenesis 21 1.3.4 Callus Culture 22 1.3.5 Cell Suspension Culture 23 1.3.6 Protoplast Culture 23 1.4 Natural Rubber in Hevea brasiliensis and other Species 24 1.4.1 Structure and Occurence 24 1.4.2 Biosynthesis of Rubber and its Regulation 26 1.4.3 Determination of Rubber in Plant Materials 29 1.4.4 Studies of Incorporation of Rubber Precursors 32 1.5 Aims of the Work 35 i CHAPTER 2 - MATERIALS AND METHODS 2.1 Axillary Shoot Proliferation and Somatic Embryogenesis 38 2.1.1 Plant Material 38 (a). Juvenile Plant Material 38 (b). Mature Plant Material 38 (c). Cotyledons and Young Leaves 40 2.1.2 Media 40 (a). Chemicals 40 (b). Stock Solutions 40 (c). Preparation o f Media 41 2.13 Culture Vessels 42 (a). Petri Dishes, Jars and Square Wells 42 (b). Glass Tubes 42 2.1.4 Sterilization 42 (a). Plant Material 42 (b). Media, Water and Instruments 43 2.13 Culture Procedure 43 2.1.6 Culture Conditions 44 2.1.7 Statistics and Calculating of Shoot Doubling Time 44 2 2 Incorporation of 14C-acetate and 14C-mevalooate into Rubber in Stem Slices of Hevea 45 22.1 Plant Material 45 2 2 2 Chemicals 45 2 2 3 Sample Preparation 45 22.4 Incubation Medium 46 2 2 5 Incubation 46 22.6 Extraction and Sampling 46 22.7 Determination of 14C Incorporation 47 22.8 Treatment ofErrors 47 CHAPTER 3 - MICROPROPAGATION OF JUVENILE MATERIALS OF HEVEA 3.1 Results 7 49 3.1.1 Axillary Shoot Production Potential of Shoot Tip Explants 49 (a). Seed Embryo Derived Shoot Tips 49 (b). Root Stock Derived Shoot Tips 51 3.13 The Effect of Explant Type on Axillary Shoot Production 52 3.13 Optimizing the Medium for Axillary Shoot Proliferation of Nodal Explants 55 3.1.4 The Effect of Basic Medium and the Sucrose Level in the Medium for Axillary Shoot Growth 61 3.1.5 The Effect of Thidiazuron as a Cytokinin 69 3.1.6 Selection of Nodal Explants 76 (a). The Effect o f the Growth Stage of the Shoot, from which the Nodes were Harvested 76 (b). The Effect of the Position of the Node in the Shoot on Axillary Shoot Production 79 3.1.7 The Effect of GA3 and Rooting on Elongation of Secondary Axillary Shoots 82 (a). The Effect of GA3 83 (b). The Effect of Rooting 84 3.1.8 Rooting of Shoot Tips 85 3.1.9 Weaning of Plantlets 90 3 2 Discussion 91 i ii CHAPTER 4 - AXILLARY^ SHOOT PRODUCTION POTENTIAL OF NATURE MATERIALS OF HEVEA 4.1 Results 109 4,1*1 Disinfection, Phenolic Browning and Culture Establishment of Shoot Tips and Nodes 109 (a). Sterilization with NaOCl 109 (b). Phenolic Browning 111 (i). The strength o f the Basic Medium 111 (ii). Incubation of Cultures at Low Temperatures 111 (iii), Use o fAntioxidents 112 (iv). Use o f Activated Charcoal in the Medium 113 (v). Frequent Transferres o f Cultures onto Fresh Media 114 (vi). UseofPVP 115 (c). Sterilization with HgCh 117 (d). The Effect o f the Explant Type, the Physiologicl State and the Clone on Phenolic Browning 118 4.1.2 Choice of Explants 120 (a). Comparison o f Shoot Tips and Nodes 120 (b). The Effect o f the Type o fNode 121 (c). The Effect o f the Position ofthe Node 124 4.13 The Effect of Basic Medium (WPM and MS) 128 (a). With Two Types ofNodes 128 (b). With Three Levels o f Sucrose 129 (c). With Two types o f Cytokinins 131 4.1.4 The Effect of Sucrose Level 134 4.13 The Effect of Exogenous Hormones 136 (a). BAP as the Only Cytotinin 136 (b). Combinations of Kinetin and BAP 137 (c). 2iP Containing Media 138 (d). Thidiazuron as a Cytotinin 140 (e). Soating Explants in Hormone Solutions 141 4.2 Discussion 1 4 3 CHAPTER 5 - EXPERIMENTS ON INDUCTION OF SOMATIC EMBRYOS OF HEVEA 5.1 Results 155 5.1.1 With Young Leaf Lobes 155 (a). Different Levels of 2,4-D 155 (b). Combinations of 2,4-D and BAP 156 (c). Combinations of 2,4-D and Kinetin 157 (d). Combinations of 2,4-D and 2iP 158 5.13 With Cotyledons 158 5.1.3 With Roots and Stems 159 5.2 Discussion 163 iii CHAPTER 6 - INCORPORATION OF 14C-ACETATE AND 14C-MEVALONATE INTO RUBBER IN STEM SLICES OF HEVEA 6.1 Preliminary Experiments 169 6.1.1 The Effect of Length of Extraction Time 170 6.1.2 The Effect of Type of Thimble 173 6 2 Bftsnlts 176 62.1 Comparison of 14C-acetate and 14C-mevalonate as Precursors 176 6 2 2 Daily Variation of 14C-acetate Incorporation 178 6 2 3 The Effect of the Physiological State of the Experimental Tissues on 14C-acetate Incorporation 180 62.4 Variation of 14C-acetate Incorporation within Clones 183 6 2 5 Comparison of Clones 185 63 Discussion 188 CHAPTER 7 -GENERAL DISCUSSION 196 LITERATURE CITED 20 2 APPENDICES 228 iv Abstract With the aim of establishing a continuous long term micropropagation system for Hevea brasiliensis, experiments were carried out with shoot tips and nodes of seedling and rootstock origin (juvenile material) and with similar explants derived from selected clones (mature stock). When this work was started in 1988, no published protocol existed for the micropropagation of Hevea even for juvenile plant material; this problem was therefore addressed first. Contrary to expectations, nodes were found superior as explants compared with shoot tips. The medium suggested by Lloyd and McCown (1980) was found to be superior to that of Murashige and Skoog (1962). As regards plant growth regulators, thidiazuron proved more useful than a mixture of BAP and kinetin. By combining these findings, the outline of a protocol for the continuous micropropagation of juvenile Hevea material has been produced. Explants of mature origin posed problems both of establishment in culture and of reluctance to proliferate in vitro. The first problem was overcome by substituting mercuric chloride for sodium hypoclorite as a sterilant and by the incorporation of polyvinyl polypyrrolidon into the culture medium.
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