CONSERVATION OF BEGONIA GERMPLASM THROUGH SEEDS: CHARACTERIZATION OF GERMINATION AND VIGOR IN DIFFERENT SPECIES THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By: Steven Robert Haba, B.S. Graduate Program in Horticulture and Crop Science The Ohio State University 2015 Thesis Committee: Dr. Pablo Jourdan, Advisor Dr. Mark Bennett Dr. Claudio Pasian Dr. Mark Tebbitt Copyrighted by Steven Robert Haba 2015 ABSTRACT Begonia is one of the most speciose genera of angiosperms, with over 1500 species distributed throughout tropical and subtropical regions; it is also a very important ornamental group of plants displaying a high degree of morphological diversity. This genus is a priority for conservation and germplasm development at the Ornamental Plant Germplasm Center located at The Ohio State University, which currently holds approximately 200 accessions, maintained primarily as clonal plants. In an effort to expand germplasm work in seed storage of Begonia, and in response to a scarcity of published information about begonia seed biology we initiated a project to develop baseline information about germination, dormancy, and stress tolerance of begonia seeds. Because of the extremely small size of begonia seeds (ca. 200 µm) I adapted germination and viability testing protocols typical of Arabidopsis research, to develop relatively efficient quantitative protocols for seed studies. Using this methodology seeds can be routinely germinated on 1% agar plates at 25°C and 16 hours light. To examine the variation in seed characteristics among Begonia accessions in the collection, I selected six species from diverse environments and from different sections of the genus for which we had abundant seed and compared their germination patterns in response to temperature and light, tolerance to high humidity/high temperature stress, and dormancy. I have determined that begonia seeds are desiccation-tolerant (orthodox), require light for germination (photoblastic), germinate under a wide range of temperatures, and mostly ii appear to lack any strong dormancy — depending on species, and are tolerant of various level(s) of stress. I found that Begonia ulmifolia, B. fischeri, and B. dregei are tolerant to high levels of stress (120 hours at 41°C) whereas B. boliviensis, B. grandis subsp. evansiana and B. subvillosa are less so. In addition, B. dregei appears to have some dormancy, which was observed through extended dark treatments (2-52 weeks) and exposure to low temperatures (12 and 15°C). Furthermore, B. grandis subsp. evansiana appears to show secondary dormancy as a response to extended dark treatments (2-52 weeks). Overall, I was able to establish a quantitative germination protocol that allowed for examination of different species to various environments. Tetrazolium testing was also possible after treatment of seeds with sodium hypochlorite. This work has established baseline information about seeds of Begonia that can be applicable to other physiological and conservation studies and useful for maintaining and characterizing a seed collection at the OPGC. iii ACKNOWLEDGEMENTS In the beginning of the master’s thesis, it seems as though you’ve taken on a task that is tangible, but it doesn’t take long before the reality sets in. Both the research and the writing have been a tremendous learning experience for my personal growth as well as in how to collaborate and incorporate other viewpoints into a project. I would like to thank my immediate advisor, Dr. Pablo Jourdan, for his incredible energy and ability to consistently provide “suggestive” guidance, which allows individuals, such as myself, to pursue goals and interests of my own. Pablo’s positivity and fruitful ideas were essential to the success of this project and to the everyday excitement at the Ornamental Plant Germplasm Center. I feel very privileged to have worked under someone who is passionate and intelligent about horticulture, as well as individuality. I equally would like to thank Dr. Mark Tebbitt, as our mutual interests in plants and horticulture has created a plethora of new information on Begonia seeds, as well as a glance at the overwhelming diversity of Begonia. I was extremely lucky to have a Begonia systematist and a knowledgeable, friendly botanist to talk to about Begonia only several hours away. Much gratitude is needed for Dr. Mark Bennett and Dr. Claudio Pasian, both professors were crucial steps of the ladder to get my writing and research skills to where they are today. I greatly appreciate Claudio’s rigorous organization and I thank Mark iv Bennett for his critical comments and ideas about experimental design and protocol development. In addition, I would like to thank J.C. Jang, as we would not been able to develop this project if it were not for the adoption of the germination tests used in his lab. His suggestions gave us a platform for which we were able to develop something that works time and time again. Many thanks are warranted for the staff, students, and interns at the Ornamental Plant Germplasm Center, as their ability to work with me on various projects, to go above and beyond their immediate responsibilities, have been much appreciated. I would like to specifically thank Fernanda Brunetta Godinho, as her hard work was not only tedious and extreme, but crucial to the rigorous data generated for this project. None of this would have been possible without the continued love and support of my parents, Pamela and Jim Haba, and willingness to allow me to pursue my interests, as they have since childhood. Last but not least, I would like to thank my girlfriend, Leith Fava, for her appreciation, reliability, and perpetual love. v VITA March 31, 1987………………………………………………….….Born, Cleveland, Ohio 2007………………………………..A.S. Horticulture Science, The Ohio State University 2009………………………………………..B.S. Crop Science, The Ohio State University 2009-2010………………………………………...Section Grower, Green Circle Growers, Oberlin, Ohio 2010 to present..................Research Assistant/Greenhouse Coordinator, Ornamental Plant Germplasm Center, Columbus, Ohio PUBLICATIONS Haba, Steven R. and P. Jourdan. Begonias at the Ornamental Plant Germplasm Center. The Begonian. March/April 2012. FIELDS OF STUDY Major Field of Study: Horticulture and Crop Science vi TABLE OF CONTENTS Page ABSTRACT ........................................................................................................................ ii ACKNOWLEDGEMENTS ............................................................................................... iv VITA .................................................................................................................................. vi LIST OF FIGURES ........................................................................................................... ix LIST OF TABLES ........................................................................................................... xiv 1. The genus Begonia .......................................................................................................... 1 Introduction ................................................................................................................. 1 Begonias at the Ornamental Plant Germplasm Center ................................................ 4 Begonia seed ................................................................................................................ 6 Seed anatomy and morphology ................................................................................... 8 Desiccation tolerance .................................................................................................. 9 Germination ................................................................................................................. 9 Study objectives ........................................................................................................ 13 2. A protocol for Begonia seed germination ..................................................................... 20 Introduction ............................................................................................................... 20 Material and methods ................................................................................................ 25 Results ....................................................................................................................... 28 Discussion ................................................................................................................. 31 3. Comparison of seed characteristics among six Begonia species .................................. 46 Introduction ............................................................................................................... 46 Seed testing ................................................................................................................ 48 Material and methods ................................................................................................ 55 Results ....................................................................................................................... 63 Discussion ................................................................................................................. 69 vii Conclusions ............................................................................................................... 80 Appendices: Appendix A Begonia Seed Cleaning ......................................................................