Genetic Diversity and Interspecific Relationships in Banksia L.f., (Proteaceae). Tina Louise Maguire B. Ag. Sci. (Hons) Submitted in fulfrlment of the requirements for the degree of Doctor of Philosophy Deparrnent of Horticulnue, Viticulture and Oenology University of Adelaide August 1996 Baril<sia coccínea Table of Contents Table of Contents Abstract i Declaration and authority of access to copying iv Acknowldgments v List of Tables vi List of Figures ix Chapter One: General inroduction 1 The genus Banksía 1 Thesis aims 3 Chapter Two: Literature review 5 Introduction 5 Taxonomy 5 Conservation 8 Plant population management 10 Breeding Systems 11 B anl<s ia flower morPhologY t2 Floral initiation 13 Protandry T4 Pollination t4 Consequences of Different Reproductive Modes 15 Self incompatibility 16 Stigma T7 Stylar inhibition 18 Ovary inhibition 18 Evolution of self incompatibility 18 The self incompatibility mechanism 19 Interspecific Incomp atibilitY L9 Reproductive EcologY 20 Reproductive isolation 22 Natural Hybúdisation 22 Hybrid fitness 24 Pattems of hybridisation 25 Chromosome Studies 25 Propagation 25 Commercial Importance and Improvement of B attksia 26 Interspecifrc Hybridis ation 29 Table of Contents Alternative methods of hybridisation 30 Hybrid Verification 31 Pollen Collection and Uses 32 Factors Controlling Pollen Availability 32 Genetic controls 32 Extemal controls JJ Pollen Storage 33 Relative humidity (RH.) 34 Temperature 34 Gas atmosphere and oxygen pressure 34 Causes of decreased viability during storage 35 Viability tests 35 In vitro assays 36 Non germination assays 36 Recording data 37 Comparison of viability tests 37 Molecular Techniques to Study Relationships in Plants 38 The poly'rnerase chain reaction (PCR) technique 38 Random amplified polymorphic DNA (RAPD) 39 DNA sequencing 4T Molecular Approaches to Plant Systematics 43 Chloroplast DNA M PCR analysis of chloroplast DNA 45 Analysis of DNA sequence data 46 Conclusions 46 Chapter Three: Viability testing of Banl<sia menziesü pollen after storage at different temperatures 48 Abstract 48 Introduction 48 Materials and methods 49 Plant material 49 Pollen collection 49 Pollen Storage 50 Fluorescein diacetate (FDA) test 50 Invitro germination 51 Statistical analysis 51 Results 51 Invitro germination 51 Table of Contents Effect of position on the inflorescence and time during the flowering season 52 Pollen storage 52 Discussion 60 Chapter Four: Interspecific and intergeneric pollination with Banksia coccinea R.Bt (Proteaceae) 62 Abstract 62 lntroduction 63 Materials and methods 66 Plantmaterial 66 Pollinations for assessment of pollen tube growth 66 Resuits 67 Effect on pollen tube growth of site, month and year 67 Reciprocal crosses 67 Interspecific and intergeneric pollination with,B. cocc[nea as male parent 68 Pollen tube abnormaliúes 69 Discussion 81 Chapter Fíve Banksi¿ sect. Coccinea (4.S. George) T.Maguire et aI., (Proteaceae). A new section 86 Abstract 86 Introduction 86 Materials and methods 88 B anl<s ia flower morphology 88 Interspecific pollination 88 Results 89 Discussion 92 Taxonomy 93 Chapter Six: Seed set following interspecific pollination with B. coccinea R.Br. 95 Abstract 95 Introduction 95 Materials and methods 96 Plant material 96 Interspecific seed set 97 Germination trials 97 Hybrid verification 98 Table of Contents Results 98 Interspecif,rc seed set 98 Seed germination of B. coccinea, B. ericiþlia and B. coccinea x B. ericiþIia 99 . Seedling survival and vigour 99 Hybrid verification and early seedling cha¡acters 99 Discussion rt2 Chapter Seven: DNA isolation methods for Banksia and other members of the hoteaceae lr4 Abstract tL4 Introduction 114 Materials and methods 115 Results 116 Protocol 1: DNA extraction from mature leaves 116 Protocol 2: DNA extraction from seedling leaves and other material high in phenolics and polysaccha¡ides 118 hotocol 3: Isolation of toul genomic DNA from seed material (slightly modified "miniprep" method of Weining and Langridge, 1991) lzl Gel electrophoresis I22 Discussion L22 Chapter Eight: Genetic diversity of Banksia and Dryandra (Proteaceae) using RAPD markers. 123 Abstract L23 Introduction I23 Materials and methods 125 Plant material 125 DNA extraction I25 DNA amplification and documentation 126 Data analysis lZ7 Results l2g Discussion I32 Chapter Nine: RAPD variation within and between populations of Bøn*sia cuneata A.S. George (Proteaceae), a rare and endangered species. 134 Abstract 134 Introduction I34 Table of Contents Mate¡ials and methods 136 Population sampling 136 DNA isolation 136 DNA amplification and documentation r37 . Statistical analysis 138 Results r39 The RAPD profile t39 Estimate of genetic diversity r39 Discussion 148 Chapter Ten: Use of RAPD ma¡kers to analyse phylogenetic relationships in Banksta (Proteaceae) 153 Abstract 153 Inroduction 153 Materials and methods 155 Plant material 155 DNA extraction 155 DNA amplification and documentation 156 Data analysis I57 Genetic distance analysis I57 Phylogenetic analysis using parsimony (PAUP) I57 Results 158 Discussion 166 Chapter Eleven: Apptication of non-coding chloroplast DNA sequences to Banksia (Proteaceae) phylogeny 168 Abstract 168 Inroduction 168 Materials a¡d methods 171 Plantmaterial I7I DNA extraction l7I DNA amplification I72 DNA sequencing 172 Data analysis I73 Results 173 Discussion 176 Chapter Twelve: General discussion 179 Banlcsia breeding 179 Table of Contents Genetic diversity 181 Species relationships 184 References cited 187 Appendix 219 Abstract Abstract Banksias are amongst the best known Australian wild flowers. They are used in ornamental horticulture and last well as fresh cut flowers, or indefinitely as dried arangements. Breeding and selection of new cultiva¡s for the cut flower industry is currently underway. This thesis aims to increase knowledge essential for conservation biology and for focused and efficient breeding of banksias. Pollen storage and viability testing are important adjuncts to a plant breeding prog¡am. 0C Banksia menziesii pollen was stored at 20 0C, 40C, -20 0C, -80 0C and -196 and assessed using a semi solid medium of IVo agar, L57o sucrose, 0.0l%o boric acid, 0-037o calcium nitrate, 0.02V0 magnesium sulphate, 0.017o potassium nitrate, and an incubation temperature o125 0C. Germination remained constant at afoundTÙVo in all treatments except room remperature 20 0C, which after six months had only 257o getmination. Pollen viability was assessed using fluorescein diacetate (FDA), but the results did not reflect the loss of germinability at 20 0C. There was no effect of floret position on the inflorescence on germination; but pollen viability va¡ied over the flowering period with maximum germination mid season. Interspecific hybridisation is assessed as a potential breeding tool, and for the assessment of species relationships within the genus. Pollen tube growth was investigated using controlled hand pollination of the commercialty significant species Banl<sia coccinea,to species of Banlcsia, and the related genus, Dryandra. Currently, the relationship between B. coccinea and the other species groups within Banksia is unclear. It has been found previously that success of pollen tube growth in the pistil following interspecifrc pollination was largely related to trxonomic distance between the species (Sedgley et al. lg94). Thus, interspecific hybridisation is a suitable technique to determine the compatibility relationships of the problematic species B. coccíneø. Some species supported no germination of B. coccinea pollen. Others produced pollen tube 1 Absnact abnormalities including thickened walls, bulbous swellings, non-directional growth, burst tubes and. branched tubes. Control of pollen tube growth in the pistil was imposed in the pollen presenter, a specialised region of the style for pollen presentation to foraging fauna, and in the upper style. There was no significant reciprocal effect on pollination success in the lower style. The results of pollen tube compatibility in the lower style indicated that B. coccinea had a closer affinity to the section OncosryIis, than to secúon Bant<sia where it is currently placed. Given the distinct morphology and close pollen - pisúl relationship to section Oncosrylis,it is proposed to move B. coccinea out of secúon Banksía to a new section Coccínea, the sister section to Oncosrylls. Intergeneric crosses of B. coccínea with Dryandra species resulted in some compatibility, with one cross having low numbers of polien tubes in the pollen presenter and upper style region. These results indicate a close relationship between Banksia and Dryandr¿, which a¡e sister genera in the ttbe Banksiae,farnily Proteaceae. Species relationships within Banksia were also assessed using molecula¡ techniques. Random amplified polymorphic DNA (RAPD) markers were assessed for their usefulness at various taxonomic levels within the genus. It was found that RAPDs a¡e informative at the close species level, but not at more distant levels, such as between distantly related series, sections, and subgenera. In addition, species relationships at higher levels were investigated using direct polymerase chain reaction (PCR) sequencing of chloroplast DNA (cpDNA) spacer regions between the trnL and trnF exons. These regions are thought to be universal for plant species and informative at the intra and interspecific level of plants. Using the region be¡ween trnL and nnF, relationships within Banl<sia, and between Banksia and Dryandra were investigated. It was found that this