MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation of Meepa A. Lokuge Candidate for the Degree: Doctor of Philosophy _______________________________________ Dr. Kenneth G. Wilson, Director _______________________________________ Dr. David A. Francko, Co-director _______________________________________ Dr. Susan R. Barnum, Reader _______________________________________ Dr. Nancy Smith-Huerta, Reader _______________________________________ Dr. Richard E. Lee Graduate School Representative ABSTRACT TISSUE CULTURE, GENETIC TRANSFORMATION, AND COLD TOLERANCE MECHANISMS IN COLD-HARDY PALMS By Meepa A. Lokuge Palms are a familiar and characteristic feature of tropical landscapes. Some palm species survive temperatures below -6.70C (200 F) and few survive temperatures below -17.70C (00 F). Needle palm, cabbage palm and Chinese windmill palm are very resistant to cold under USDA Plant Hardiness Zone 6 conditions. The first part of this study was undertaken to develop a tissue culture system for the clonal propagation of cold-hardy palms with desired characters and with the ultimate goal of producing a system for genetic transformation. Windmill palm was regenerated from shoot apical meristem tissues via indirect organogenesis, giving rise to viable plants that fully acclimated to greenhouse conditions. With cabbage palm 1.5 µM dicamba was optimal for the induction of somatic embryogenesis from zygotic embryos. The second part of this study was aimed at developing a genetic transformation system for cold-hardy palms. Cabbage palm was selected because it’s widespread use throughout USDA Zone 8 and previous data suggest that with minor improvement in cold tolerance this palm could be grown in even colder areas. Cabbage palm zygotic embryos were successfully transformed with the marker genes gfp and gus using the two most common plant transformation methods, biolistic and Agrobacterium-mediated transformation. Results indicated that Agrobacterium -mediated transformation gave more promising results when compared with the biolistic method. Plants exhibit two strategies for surviving extremely cold weather: freeze avoidance and freeze tolerance. Both strategies involved supercooling mechanisms and other adaptations that have not been characterized in palms. The final part of this dissertation was aimed at studying these mechanisms using the most cold- hardy palm, the needle palm, as a model system. According to our results needle palm supercooling capacity is already pronounced even in warm-incubated foliage and does not change significantly after exposure to cold-acclimating conditions. To further investigate the molecular mechanisms underlying this cold tolerance, a proteomic approach was used to examine initial changes of the leaf proteome upon cold treatment. Protein identification was difficult due to non- availability of relevant genome sequences. Nevertheless, 2- dimensional gel electrophoresis suggested that significant changes in protein products occur in needle palm leaves when challenged with non-lethal cold. Keywords Cold-hardy palms; regeneration; somatic embryogenesis; genetic transformation; supercooling; proteomics; Chinese windmill palm (Trachycarpus fortunei); cabbage palm, (Sabal palmetto); needle palm (Rhapidophyllum hystrix) TISSUE CULTURE, GENETIC TRANSFORMATION, AND COLD TOLERANCE MECHANISMS IN COLD-HARDY PALMS A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Botany By Meepa A. Lokuge Miami University Oxford, Ohio 2006 Dissertation Director: Dr. Kenneth G. Wilson © Meepa A Lokuge 2006 Table of Contents Chapter 1: Introduction 1 Literature Cited 29 Chapter 2: Regeneration of Trachycarpus fortunei (Hook) H. Wendl. (Chinese windmill palm) plants via organogenesis 43 Abstract 43 Introduction 43 Materials and Methods 45 Results and Discussion 47 Literature Cited 54 Chapter 3: Induction of somatic embryogenesis in Sabal palmetto; Walter Schultes & Schultes F: Morphological observations and 2-D protein profile Comparison 62 Abstract 62 Introduction 62 Materialsand Methods 64 Results and Discussion 66 Literature Cited 70 Chapter 4: Genetic transformation of Sabal palmetto Walter Schultes & Schultes F. zygotic embryos using biolistic and Agrobacterium-mediated Methods 86 Abstract 86 Introduction 86 Materials and Method 87 Results and Discussion 93 Literature Cited 98 Chapter 5: Investigations on cold tolerance mechanism in needle palm (Rhapidophyllum hystrix) and identification of cold responsive Protein 106 Abstract 106 Introduction 107 Materials and Methods 109 Results and Discussion 113 Literature Cited 117 Chapter 6 Conclusions 134 Literature Cited 138 iiiv Tables Table 2-1 Callusing frequency of Trachycarpus fortunei zygotic embryos grown on media containing differential 2, 4-D concentrations 61 Table 3-1 Effect of different auxins and cytokinins on embryogenic callus induction in S. palmetto 81 Table 3-2 Effect of different dicamba concentrations on embryogenic callus induction in S. palmetto 82 Table 3-3 Total protein content of the different stages of embryogenesis in S. palmetto 83 Table 3-4a Total number of protein spots in gels from different stages of S. palmetto tissue culture 83 Table 3-4b Qualitative analysis of spots from S. palmetto tissue culture gels 84 Table 3-4c Quantitative analysis of spots from S. palmetto tissue culture gels 85 Table 4-1a GUS activity in the tissues placed on media with different osmotica from biolistic experiment 102 Table 4-1b ANOVA for transient GUS expression for optimization of media for biolistic method 102 Table 4-2a Percentage of tissues showing GUS activity when infected with EHA105:pCAMBIA 1301 103 Table 4-2b Percentage of tissues showing GUS activity when infected with EHA105:pCAMBIA 1305.1 104 Table 4-2c Percentage of tissues showing GUS activity when infected with EHA105:pCAMBIA 1305.2 105 Table 5-1 Supercooling points of needle palm leaves treated at 4 C for 14 days 129 Table 5-2 Relative water content of needle palm leaves treated at 4 C for 14 Days 130 Table 5-3 Spot number comparison between gels obtained from needle palm leaf protein extracts treated at 4 C for 14 days 131 Table 5-4 Spot identification by MALDI-TOF-MS analysis and database search for proteins spots that have been upregulated 132, 133 ivv Figures Figure 2-1 Trachycarpus fortunei callus derived from mature zygotic Embryos 58 Figure 2-2 Different stages of plant regeneration of T. fortunei via indirect Organogenesis 59 Figure 2-3 Scanning electron micrographs of leaf stomata form different stages of T. fortunei regeneration 60 Figure 3-1 Sabal palmetto seed morphology 73 Figure 3-2 Major steps of S. palmetto seedling development 74 Figure 3-3 Stereomicrographs of major stages in the development of somatic embryos from S. palmetto zygotic embryos 75 Figure 3-4 A Scanning electron micrograph of 4-week callus tissue of S. Palmetto 76 Figure 3-4B Scanning electron micrograph of 6-week old tissue culture 77 Figure 3- 4C Scanning electron micrograph of 7-week old tissue cultures 78 Figure 3-5 Two-dimensional gel electrophoresis proteome map of S. palmetto tissue cultures 79 Figure 3-6 Selected regions of Fig. 3-5 to highlight some of the differentially expressed proteins 80 Figure 4-1 pCAMBIA 1301 vector 90 Figure 4-2 pCAMBIA 1305.1 vector 91 Figure 4-3 pCAMBIA 1305.2 vector 92 Figure 4-4 Histochemical GUS assay on bombarded and Agrobacterium co-cultivated mature zygotic embryos of S. palmetto 101 Figure 5-1 Graph of average supercooling point in Rhapidophyllum hystrix leaves vs. days at 40 C 121 Figure 5-2 Supercooling points and relative water content of R. hystrix treated at 40 C for 2 weeks 122 Figure 5-3A 2DE gel image of protein extract from R. hystrix leaves of at 260C 123 Figure 5-3B 2DE gel image of protein extract from leaves R. hystrix leaves t Treated at 40 C for 2 days 123 Figure 5-3C 2D E gel image of protein extract from R. hystrix leaves treated at 40 C for 4 days 124 Figure 5-3D 2DE gel image of protein extract from R. hystrix leaves treated at 40 C for 8 days 124 Figure 5-3E 2DE gel image of protein extract from R. hystrix leaves treated at 40 C for 10 days 125 Figure 5-3F 2DE gel image of protein extract from R. hystrix leaves treated at 40 C for 14 days 125 Figure 5-4A Western blot for dehydrin detection from a day 0 (cold untreated) leaf sample from R. hystrix 126 Figure 5-4B Western blot for dehydrin detection from day 14 (cold treated) leaf sample from R. hystrix 126 v Figure 5-5A Identification of superoxide dismutases (SODs) in R. hystrix leaves treated at 40 C for 14 days 127 Figure 5-5B Identification of Cu/Zn isoform and Mn-SOD isoform in R. hystrix using potassium cyanide 127 Figure 5-5C Identification of Cu/Zn isoform and Mn-SOD isoform in R. hystrix using hydrogen peroxide 128 viv Dedication Dedicated in loving memory of my parents Sirisena Lokuge and Kusuma Lokuge. viiv Acknowledgements I would like to acknowledge many people who helped me through this study. First, I would like to thank my major advisor Dr. Kenneth G. Wilson and my co-advisor, Dr. David A. Francko whose intellectual guidance and enthusiasm made this degree possible and for that I am tremendously grateful and deeply honored. You have been wonderful mentors, always ready to listen to my problems, showed me different ways to approach problems and inspired me to be persistent to accomplish this dissertation. I will cherish the memory of working with both of you in the years to come. Besides my advisors, I would like to thank my committee members, Dr. Susan R. Barnum, Dr. Nancy Smith-Huerta, and Dr. Richard E. Lee. A special thank to my committee member Dr. Richard E. Lee for being encouraging and supportive during my times of hardship in research. I am grateful to Dr. John W. Hawes from the Department of Chemistry and Biochemistry, Miami University for his help with my proteomics work. I am also grateful to Dr.