SOME ASPECTS OF THE TISSUE CULTURE AND MICROPROPAGATION OF BORONIA MEGASTIGMA NEES by 5„ 2 Alan G. A. Luckman, B. Agr. Sc. Submitted in partial fulfilment of the requirements for the degree of Master of Agricultural ScfenCe. r. University of Tasmania Hobart February 1989 This thesis contains no material which has been accepted for the award of any other degree or diploma in any university. To the best of my knowledge, it contains no material previously published or written by another person, except where due reference is made in the text of the thesis. G. A. Luckman University of Tasmania Hobart I. Some Aspects of the Tissue Culture and Micropropagation of Boronia megastigma. CONTENTS Abstract V Acknowledgements VII I. INTRODUCTION 1 II. LITERATURE REVIEW 3 The Uses of Plant Tissue Culture and Micropropagation 3 Clonal Propagation 4 Difficulties Associated With Micropropagation 6 Plant Health and Germplasm Storage 7 Germplasm Storage 8 Genetic Improvement 9 Protoplast Technology 10 Somaclonal Variation 11 Knowledge Of Plant Tissue Culture 13 Micropropagation of Boronia species 14 Growth in Tissue Cultures and its Measurement 14 Length of the Culture Period 15 Measurement of Relative Growth 15 The Use of Relative Growth as a Measure of Culture Growth 16 Difficulties of Measuring Relative Growth 17 Alternative Measures of Development in Cultures 18 Subjective Measurement of Cultures 20 pH of Tissue Culture Media 21 Measurement and Adjustment of pH 23 Control of pH in Media 25 pH Change During Culture 26 Effects of pH on Organogenesis 27 Nutrition of Plant Tissue Cultures 29 The Development of Media 29 Macronutrients 30 Micronutrients, Vitamins and Carbohydrates . 31 Other Media Constituents 31 Plant Growth Regulators 32 Nitrogen Nutrition of Cultures 33 Nitrogen Nutrition and Media pH 34 Controlling pH Change in Media 35 Effects of Nitrogen on Differentiation 38 Ammonium Levels and Vitrification 41 Nitrogen Nutrition in Boronia 42 Adenine In Culture Media 42 Possible Roles For Adenine 44 Vitamins 46 Sucrose and its Role in Tissue Culture 49 Sucrose and Osmotic Factors 51 Morphogenetic Effects Of Sucrose 52 Root Initiation and Post-Culture Survival 54 Root Initiation in Non-sterile Media 54 Factors affecting Root Initiation in Culture 56 Nutritional Factors 56 Time in Culture 58 Culture Conditions 58 Post Culture Survival 59 Vitrification 60 The Role of Humidity in Vitrification 61 Root Initiation in Boronia 63 Summary of the Literature Review 64 III. MATERIALS AND METHODS 66 Media 66 Agar 67 Flasks 68 Autoclaving of Media 68 Origins of Cultures 69 Selection of Hormone Levels 70 Pretreatments 70 Growth Conditions 70 Measurement 71 pH Measurement 71 Methods - Growth and Conditions 72 Comparison of Autoclaves 72 Source of pH Variation 73 pH Response 74 G5 Medium 74 G8 Medium 74 Growth Period 75 Explant Trimming 75 Explant Orientation 76 Explant Size 77 Methods - Media Constituents 77 Nitrogen Level 77 Clone Trial 80 Nitrogen Source 80 Buffered Media 81 Adenine 83 Vitamins 83 Sucrose 84 Methods - Root Initiation Experiments 84 Measurement and Handling of Plants 84 Nitrogen Experiment 87 Growth Conditions 87 Root Initiation Time 88 Adenine 89 Initiation Media and Sucrose 89 IV. RESULTS AND DISCUSSION 90 Autoclave Comparison 90 Source of pH Variation 91 pH Response Experiments 93 G5 Medium 93 G8 Medium 96 Effects of Initial pH 100 Growth Period 101 Trimming 107 Explant Orientation 110 Explant Size 112 Media Constituents 116 Nitrogen Trial 116 Clone Trial 126 Nitrogen Source 129 Buffered Media 133 pH Changes 133 Growth Patterns 137 Adenine 142 Vitamins 146 Sucrose 147 Root Initiation Experiments 151 Nitrogen Effects 151 Root Initiation 151 Root Development 153 Drawbacks of the Design 158 Root Initiation Time 160 Root Initiation Conditions 162 Adenine in Rooting Media 164 Sucrose Effects on Root initiation 164 V I. V. GENERAL DISCUSSION 167 Growth conditions and Measurement ....... 167 pH Variation 167 Other Factors Increasing Within Treatment Variation 171 Accurate Growth Measurement 171 Selection of Measurement Parameters 177 Nutrition 178 Effects of Nitrogen on Growth 179 Effects of Nitrogen on Differrent Clones 180 Nitrogen Sources and the Influence of Succinate Buffer 183 Interactions Between Sucrose and Nitrate Concentrations 186 Definition of Culture Media 187 Root Initiation and Post Culture Survival 189 Effects of Adenine 190 Effects of Sucrose and its Interaction With Nitrogen 190 Effects of Nitrogen 191 Effects of Culture Conditions 193 VI. CONCLUSIONS 195 VII. REFERENCES 197 APPENDIX V ABSTRACT This study investigates the effects of a number of factors on the growth and development of Boronia megastigma Nees in 'in vitro ° culture. A number of non-nutrient factors were found to be influencing the growth and development of cultures. The pH of the medium was found to have an effect on the subsequent growth of the cultures, as did the orientation of explants on the medium and the mode of trimming the explants. Relative Growth was found to be an inappropriate measure of the growth of the cultures. Nitrogen nutrition was by far the most important factor affecting growth in the nutrient media. The concentration of nitrogen was found to be influencing the amount of growth and the balance between shoot initiation and shoot extension. The cultures grew poorly in the presence of either ammonium or nitrate as a sole source of nitrogen Changes in the pH of the medium were found to be the cause of the poor growth on media containing ammonium as the sole nitrogen source. Changes in the pH of the medium were also associated with the use of nitrate as the nitrogen source, however correction of the pH change using succinate buffer did not improve growth on this medium. Adenine hemi-sulphate and vitamins had little influence on the growth and development of the cultures. Sucrose VI concentration was found to influence growth, possibly by affecting the osmotic potential of the medium. The rate of root initiation in culture was also substantially affected by the level of nitrogen in the medium. Development of cultures after root initiation was found to be substantially affected by both the level of nitrogen in the root initiation medium and the level of nitrogen in the proliferation medium, prior to root initiation. Root initiation was also affected by the concentration of sucrose in the medium, while adenine hemi-sulphate had no effect. Survival of plantlets after planting in soil was substantially affected by the length of the time spent on root medium. The effect on plantlet survival of temperature conditions during the root initiation phase was also Investigated. The results obtained from this research should assist In the commercial micropropagation of Boronia megastigma and provide a basis for future research on the in vitro culture of this species. VII. ACKNOWLEDGEMENTS I am grateful to my supervisor, Dr. R. C. Menary, Reader in Horticultural Science, for his advice and support during the extended life of this project. I also gratefully acknowledge his support in obtaining funding for the research from the Tasmanian Development Authority. I thank all the staff and post-graduate students at the Agricultural Science Department, University of Tasmania, for their assistance and encouragement over the duration of this project. I also thank Margaret Baikie for her support and assistance during the writing of this thesis, particularly for her assistance in proof reading. I. INTRODUCTION Boronia megastigma Nees (referred to hereafter as boronia) is a plant, native to Western Australia, which is harvested for the essential oil that can be extracted from its flowers. In its native area the flowers are harvested by hand from wild stands of the plant, making the harvesting and processing of this low yielding oil particularly expensive. The oil extracted from boronia flowers is highly prized and highly priced (in excess of $3000 per kilogram). It is used primarily in high class perfumes. The major components extracted from the boronia flowers are dodecanol, dodecyl acetate, tetradecyl acetate and beta-ionone (Legget and Plenary, 1980). Boronia has been cultivated in Tasmania for many years for use by florists and as a garden shrub. In the last few years research has been undertaken to establish plantations of boronia, in Tasmania, for the extraction of the oil. In order to make the cultivation of boronia a commercially attractive proposition, a system for mechanical harvest of the flowers has been devised. Mechanical harvesting is made more efficient if the harvester is able to harvest from bushes of uniform height and maturity. It is also important to ensure that the plants have a suitable growth habit, to allow the flowers to be removed from the bush without causing excessive damage to the plant. Careful selection based on yield, oil and harvest characteristics has given a number of plants that have these harvest characteristics as well as good oil quality and yield. It is necessary to propagate the selected clones to provide large numbers of identical plants suitable for. mechanical harvesting. Micropropagation offers the possibility of propagating very large numbers of cloned plants in quite short periods to allow the rapid expansion of the industry. It also offers the opportunity to propagate clones that are not readily propagated through normal vegetative methods. In the longer term, the techniques now being developed In plant tissue culture offer exciting and important possibilities in the field of plant genetic improvement, germplasm storage and cell suspension culture. In order for these techniques to be successfully applied there needs to be a sound knowledge of the requirements of boronia in tissue culture. As this species has not been reported to have been cultured before there are a wide range of techniques and information that need to be acquired regarding its growth and culture in vitro. This thesis is intended to provide a starting point, in terms of providing basic information on the in vitro culture of boronia and on its successful micropropagation.