DEVELOPMENTAL BIOLOGY OF EXACUM STYER GROUP POLLEN AS RELATED TO HAPLOID INDUCTION by TSAN-YU CHIU BSc. (Horticultural Science), Taiwan, 2003 A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The FACULTY OF GRADUATE STUDIES (Plant Science) The University of British Columbia August, 2006 © Tsan-Yu Chiu, 2006 ABSTRACT Exacum Styer Group, a group of interspecific hybrids, possesses several valuable traits for ornamental use. However, current production techniques are limited to asexual reproduction of selected genotypes. Typically, inbred line development is highly desirable for commercial introduction of Fl hybrids as well as to facilitate genetic research. Unfortunately, this group suffers from severe inbreeding depression, precluding sexually derived inbred lines. In order to avoid inbreeding depression, double haploid plant development is one alternative approach. Therefore, this research developed fundamental information related to the successful application of this technology. Initially, fourteen genotypes were evaluated for pollen viability ranging from 0% to 80.5%. Three genotypes, with different levels of pollen viability, were selected and characterized for microsporogenesis, microgametogenesis, pollen morphology, and chromosome number. Microsporogenesis was normal among all three selected genotypes. However, the low fertility genotype and sterile genotype did not complete normal microgametogenesis and were found to have abnormal exine structure, perhaps indicating a dysfunctional tapetum layer. In contrast, the fertile genotype completed microgametogenesis, produced normal exine, and produced functional pollen grains. At anthesis, pollen was shed at the bi-nucleate stage. The chromosome numbers of the three genotypes evaluated ranged from 50 (fertile genotype) to 66 (low pollen viability). The individual chromosomes ranged in size from 0.3 um (dot-shaped) to 2.65 um (rod-shaped) with all three genotypes containing each form. Following characterization of pollen development, reprogramming treatments (e.g., temperature, mannitol, media composition and plant growth regulators) to induce haploid embryogenesis were applied at the mid uninucleate to early binucleate stage. After cold treatment (10°C) for 7 days, microspores maintained normal nuclei development without symmetrical divisions observed, suggesting that no androgenic switch occurred. Furthermore, heat treatment (35°C) for 4 days induced nuclei degradation and microspore non-viability. In addition to temperature treatments, mannitol treatments did not induce symmetrical divisions. When anthers were cultured under common temperature treatments, regardless of media composition, nuclei displayed a similar response indicating temperature was more effective in influencing nuclei development than media ii composition. Three different auxin and cytokinin combinations were evaluated on androgenic calli/embryo induction. However, none of the combinations successfully induced calli/embryo formation. iii TABLE OF CONTENTS TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES viii LIST OF ACRONYMS xii ACKNOWLEDGMENTS xiii Chapter 1. General introduction and literature review 1 1.1 General introduction 1 1.2.1 Exacum in Sri Lanka 3 1.2.2 Exacum species and horticultural potential 4 1.3 Pollen Biology 5 1.3.1 Male gametophyte development 5 1.3.2 Male sterility 6 1.3.3 Androgenesis 7 1.4 Factors related to successful androgenic induction 8 1.4.1 The natural attributes of the donor plant 8 1.4.1.1 Genotype 8 1.4.1.2 Pollen developmental stage 8 1.4.2 Appropriate microspore reprogramming treatments 9 1.4.2.1 Effect of temperature shock on androgenesis 9 1.4.2.2 Effect of mannitol stress on androgenesis 9 1.4.3 Culture environment 10 1.5 Justification and objectives 11 Chapter 2. Characteristics of pollen development in Exacum Styer Group 19 2.1 Introduction 19 2.2 Material and Methods 20 2.2.1 Plant material 20 2.2.2 In vivo pollen viability 21 2.2.3 Microsporogenesis with acetocarmine stain 21 2.2.4 Microgametogenesis with DAP I stain 22 2.2.5 Pollen morphology by scanning electron microscopy (SEM) 22 2.2.6 In vitro pollen hydration 23 2.2.7 Chromosome counts with Giemsa stain 23 2.3 Results 23 2.3.1 Pollen viability 23 2.3.2 Microsporogenesis and microgametogenesis 24 2.3.3 Pollen characteristics by scanning electron microscopy (SEM) 25 2.3.4 In vitro pollen hydration 25 2.3.5 Chromosome counts 26 2.4 Discussion 26 Chapter 3. Stressor effects on in vitro development of microspores from a fertile iv genotype of Exacum Styer Group 44 3.1 Introduction 44 3.2 Material and Methods 47 3.2.1 Plant materials and tissue collection 47 3.2.2 Media preparation 48 3.2.3 Disinfestation and culture procedures 48 3.2.4 Reprogramming treatments 48 3.2.5 Cytological examinations 49 3.2.6 Statistics 49 3.3 Results 50 3.3.1 Effects of temperature and media composition on microspore development 50 3.3.2 Effects of temperature and plant growth regulator combinations on colli formation 50 3.4 Discussion 51 Chapter 4. Concluding remarks and future research 61 Appendix I Characteristics of pollen development and chromosome numbers from additional genotypes in Exacum Styer Group 66 AL 1 Introduction 66 Al. 2 Material and Methods 66 Al. 1.2.1 Plant materials 66 Al. 1.2.2 Microsporogenesis and microgametogenesis with acetocarmine stain 67 ALL2.3 Chromosome counts with Giemsa stain 67 Al. 3 Results and Discussion 68 Appendix II Stressor effects on in-vitro development of microspores from a sterile genotype of Exacum Styer Group 73 AIL 1 Introduction 73 AH. 2 Material and Methods 73 AIL 2.1.1 Plant materials and tissue collection 73 AH. 2.1.2 Media preparation 74 AH. 2.1.3 Disinfestation and culture procedures 74 AIL 2.1.4 Reprogramming treatments 75 All. 2.1.5 Cytological examinations 75 AIL 2.1.6 Statistics 75 AH. 3 Results and Discussion 76 References 78 LIST OF TABLES Table 1.1 Summary of taxonomic treatments for Sri Lankan Exacum 12 Table 1.2 Plant growth regulator combinations used in anther/microspore culture induction media 13 Table 2.1 Parentage of 14 Exacum Styer Group genotypes; by conversion, pistillate parent is listed first followed by the staminate parent 31 Table 2.2 Pollen viability (percent) of 14 Exacum Styer Group genotypes based on acetocarmine stain 32 Table 2.3 Chromosome numbers of three Exacum Styer group genotypes. Chromosome number and frequency of observations (in parentheses) are shown. Most frequent counts are highlighted in bold 33 Table 3.1 Plant growth regulator treatments applied to anthers of Exacum Styer Group genotype 01-09-01 56 Table 3.2 Cold treatment and media effects on pollen development and calli formation from the Exacum Styer Group genotype 01-09-01 57 Table 3.3 Heat treatment and media effects on Exacum Styer Group genotype 01-09-01 pollen development and calli formation 58 Table 3.4 Cold treatment and plant growth regulator combination effects (2,4-D and BA or 2,4-D and NAA) on calli formation of Exacum Styer Group genotype 01-09-01 59 Table 3.5 Cold treatment and plant growth regulator combination (2,4-D and Kinetin) effects on calli formation of Exacum Styer Group genotype 01-09-01 60 vi Table Al. 1 Chromosome numbers of two additional Exacum Styer Group genotypes. Chromosome number and frequency of observations (in parentheses) are shown 69 Table AIL 1 Cold treatment and media effects on pollen development and calli formation from Exacum Styer Group genotype 01-37-37 77 Table All.2 The effect of various BA and 2,4-D combinations on calli formation of isolated anthers of Exacum Styer Group genotype 01-37-37 77 vii LIST OF FIGURES Fig. 1.1 Map displaying the distribution of Exacum species around the Indian Ocean Basin and their proposed dispersal routes (Yuan et al, 2005) 14 Fig. 1.2 Taxonomic treatment of the Genus Exacum and of the 8 taxa native to Sri Lanka (Sumanasinghe, 1986) 15 Fig. 1.3 Simplified schematic of microgametophyte formation in plants 16 Fig. 1.4 Simplified schematic chart of alternative pathways of androgenesis in plants 17 Fig. 1.5 Flowchart of procedures used in this study 18 Fig. 2.1 Pollen development in Exacum Styer Group genotype 01-09-01 by acetocarmine stain. (A) Prophase I, anther length 0.5 cm; (B) Telophase I, anther length 0.5 cm; (C) Prophase II, anther length 0.5 cm; (D) Metaphase II to telophase II, anther length 0.5 cm; (E) Tetrad stage, anther length 0.6 cm; (F) Mature pollen at anthesis, anther length 1.2cm. Scale bar = 10 um 34 Fig. 2.2 Pollen development in Exacum Styer Group genotype 01-42-03 by acetocarmine stain. (A) Prophase I to metaphase I, anther length 0.4 cm; (B) Telophase I to prophase II, anther length 0.4 cm; (C) Prophase II to metaphase II, anther length 0.4 cm; (D) Tetrad stage, anther length 0.5 cm; (E) Microspores released from tetrads, anther length 0.5 cm; (F) Mature pollen at anthesis (darkly stained viable pollen and translucent non viable pollen), anther length 1.1 cm. Scale bar = 10 urn 35 viii Fig. 2.3 Pollen development in Exacum Styer Group genotype 01-37-37 by acetocarmine stain. (A) Prophase I, anther length 0.4 cm; (B) Telophase I, anther length 0.4 cm; (C) Metaphase II and Telophase II, anther length 0.4 cm; (D) Tetrads, anther length 0.5 cm; (E) Microspore released from tetrads, anther length 0.5 cm; (F) Mature but non-viable pollen, at anthesis, anther length 1.1 cm. Scale bar = 10 urn 36 Fig. 2.4 Microsporogenesis in Exacum Styer Group genotype 01-09-01 stained with DAPI. (A) Uninucleate, anther length 1 cm; (B) Prophase during mitotic division, anther length 1 cm; (C) Metaphase during mitotic division, anther length 1 cm ; (D) Telophase during mitotic division; anther length 1 cm; (E) Early binucleate, anther length 1.1-1.2 cm; (F) Late binucleate at anthesis, anther length 1.2 cm.