Study of Intergeneric Hybridization in Hippeastrum by Yaowapha Jirakiattikul M.Sc. Kasetsart University, Thailand Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy UNIVERSITY OF TASMANIA gc,r;c HOBART November, 1999 II Declaration I hereby declare that this thesis contains no material which has been accepted for the award of any other degree or diploma in any University, and, to the best of my knowledge, contains no copy or paraphrase of material previously published or written by any other person, except where due reference is made in the text of this thesis. I It . LA, I 9 aowaf In a Y-Ct Yaowapha Jirakiattikul November, 1999 Authority of Access This thesis may be made available for loan and limited copying in accordance with the Copyright Act in 1968. ki aft*, 9a0 toctect Yaowapha Jiralciattikul November, 1999 List of Abbreviations ADP Adenosine-5 '-diphosphate BA Benzyladenine (a cytolcinin) DAP Day after pollination DMSO Dimethylsulphoxide FCR Fluorochromatic reaction FDA Fluorescein diacetate GDH Glutamate dehydrogenase IAA 3-indoleacetic acid (an auxin) Kinetin (a cytolcinin) NAA 2-Naphthyleneacetic acid (an auxin) NADP Nicotinamide-adenine dinucleotide phosphate MIT Tetrazolium thiazolyl blue MS Murashige and Skoog medium NBT Nitro blue tetrazolium PGI Glucosephosphate isomerase PGM Phosphoglucomutase PMS Phenazine methosulfate Relative mobility rpm Revolutions per minute TTC Tetrazolium chloride iv Summary Hippeastrum is a bulbous genus within the family Amaryllidaceae which is of horticultural importance as a cut flower and potted plant crop. The colour range available in commercial hybrids is dominated by white and red. The commercial potential of Hippeastrum as a floriculture crop is significant, but production of the cut flowers is limited to warm climatic regions or glasshouse production in temperate regions. Improved cold tolerance, along with extended colour range and flower fragrance, are key breeding objectives for Hippeastrum hybrids. One breeding strategy which may facilitate the introduction of these characteristics is intergeneric crossing. This strategy has recently been used successfully in other important bulbous genera including Lilium, and has some potential for Hippeastrum breeding as a number of intergeneric hybrids within family Amaryllidaceae have been reported previously. This study therefore aimed to develop methods for intergeneric hybridization of Hippeastrum based on detailed investigation of flowering physiology, reproductive biology and in vitro plant growth techniques. Rapid development of the flower bud and at least two new growth units was observed in bulbs which flowered following planting whereas none or only one growth unit was initiated in bulbs which produced only vegetative growth. A decrease in dry weight suggested that the stored reserves from the bulb were utilized during the flowering process. Starch and fructans were found to be the major storage carbohydrates in H. hybridum bulbs and fructans appeared to be involved in scape elongation and floret growth. The transport carbohydrate sucrose was found in lower concentrations in non-flowering bulbs than in flowering bulbs, implying a lower rate of carbon mobilization in non-flowering bulbs. ' 4C-sucrose translocation studies showed that under glasshouse conditions sucrose originating from the outermost scale tended to be partitioned towards the roots and the emerging flower bud, in contrast to carbon originating from the youngest expanded leaf which was directed predominantly towards the inflorescence. Carbohydrate partitioning from the bulb scales to the emerging flower bud was increased by repotting, a treatment shown to stimulate flower emergence. Pollen viability of H. hybridum, Brunsvigia orientalis and Amaryllis belladonna was high from 0-6, 0-2 and 0-2 days after anthesis respectively. Long term storage of pollen was necessary to overcome lack of synchronisation in flowering for controlled crosses and the results for this study showed that pollen of these three plant genera can be stored for at least 1 year at 2°C, -18°C or -80°C. Pollen viability of H. hybridum decreased rapidly after 64 weeks storage at 2°C but remained high after 104 weeks storage at -18°C and -80°C. The period of maximum receptivity of H. hybridum, B. orientalis and A. belladonna stigmas was two days after anthesis. Slow growth rate of pollen tubes were recorded in all intergeneric crosses with H. hybridum, suggesting a pre-fertilization incompatibility barrier based on rate of pollen tube growth in the style. Despite this barrier, pollen tubes were detected in the base of H. hybridum styles following crosses with A. belladonna and swelling of ovaries was observed in these crosses. The cross-pollinated pods, however, often died 10-18 days after pollination, suggesting that a post-fertilization bather was present. Ovule and ovary culture were used to overcome the post-fertilization incompatibility in the crosses H. hybridum x A. belladonna and H. hybridum x B. orientalis. Excised ovules or ovaries were cultured on MS medium supplemented with 60 g/L sucrose. Small bulblets were obtained from the cross H. hybridum x A. belladonna using ovule and ovary culture. Cut-style pollination, heat treatment of the style prior to pollination and in vitro pollination were used in an attempt to overcome the pre-fertilization barrier in the style. Application of cut-style pollination and heat treatment had no effect on pollen tube growth rate in H. hybridum styles. Small bulblets, however, were obtained from the crosses H. hybridum x B. orientalis, and H. hybridum x A. belladonna using in vitro pollination combined with ovule culture. The hybridity of these bulblets was confirmed using isozyme electrophoresis. The techniques developed in this study thus provide the basis for the incorporation of desirable traits from other genera of family Amaryllidaceae into H. hybridum. vi Acknowledgments I would like to sincerely thank my supervisor Dr Philip Brown for his continual guidance, his enthusiasm to help me at all times and his support over the duration of my study. A special thanks also to Head of the School, Professor Rob Clark for his support throughout my study. I also extend my sincere thanks to Mr Harold Cross and Mr Colin Drake who provided the bulbs used in this project. I would also like to thank all the staff members at the School of Agricultural Science, especially Bill Peterson, Sally Jones, Jane Bailey, Lynne Dow, Laura Maddock and to the staff at Horticultural Research Centre, Philip Andrews and David Wilson. Special thanks are extended to Dr David Ratkowslcy and Dr Steve Wilson for help with statistical analysis. A special thanks to my English teachers, Frank Conrow and Louise Oxley, for language assistance and encouragement. Sincere thanks to Martin Blake, Alistair Gracie, Dr Linda Falzari and Mark Gardner for their assistance with proof reading this manuscript. Many thanks also to Natalie Brown for her suggestion in the aniline blue test, Dr Dean Metcalf for his assistance with electrophoresis technique, Diane Smith, international officer, for her advice and assistance during my time living here, and my friends in the Horticultural research group, Martin Blake, Alistair Gracie and Porntip Sae Jeang for their friendship and lending a helping hand. To all my fellow postgraduate students at School of Agricultural Science and all my Thai friends, who have been called upon for assistance, support and help to make life here enjoyable, thank you. Finally but not least, I would like to sincerely thank all my family members: my grandmother, father, mother, brothers, sisters, sister in law, nephews and niece for their love, giving me all their endless support and encouragement throughout this study. As a special mention, I would like to dedicate this thesis to my grandmother and father who unfortunately passed away before the completion of this thesis. VII Table of Contents Summary iv Acknowledgments vi I. General Introduction 1 II. Literature Review 4 1. Introduction 4 1.1 Anatomy and Morphology of Amaryllidaceae 4 1.2 Hippeast rum 5 The Hippeastrum Bulb Type and Morphology 5 World Hippeastrum Production 8 Cultural Details and Propagation 8 2. Regulation of Flowering 11 2.1 Factors Controlling Flowering 11 2.2 Carbohydrate Metabolism 13 Starch 14 Fructans 14 Sucrose, Glucose and Fructose 15 2.3 Partitioning of Assimilates 17 Assimilate Partitioning and Flower Development 18 3. Reproductive Biology 20 3.1 Pollen 20 Pollen Viability 21 Pollen Viability Tests 21 Long Term Storage of Pollen 23 3.2 Stigma 24 Stigma Receptivity 25 3.3 Pollen Germination and Tube Growth 26 3.4 Incompatibility 28 Classification of Incompatibility 29 Overcoming Incompatibility 30 4. Breeding and Plant Tissue Culture 35 4.1 Plant Tissue Culture of Flower Bulbs in Amaryllidaceae .36 Hippeastrum 36 Other Amaryllidaceae Genera 37 4.2 Plant Tissue Culture in Plant Breeding 38 In Vitro Pollination 38 Ovule and Ovary Culture 40 Embryo Culture 41 III. General Materials and Methods 45 1. Plant Material , 45 Hippeastrum hybridum 45 Brunsvigia orientalis 46 Amaryllis belladonna 46 Nerine 47 2. Cultural Conditions 47 2.1 Glasshouse 47 2.2 Shadehouse 49 2.3 Field Conditions 49 2.4 Propagation Beds 49 3. Pollen Collection and Rehydration 50 4. General Media Preparation, Cultural Conditions, 50 and Surface Sterilization for Tissue Culture Technique 4.1 Media Preparation 51 4.2 Surface Sterilization 51 5. Statistical Analysis 52 IV. Experimental 53 Part A. Flowering