STUDIES ON THE REPRODUCTIVE CAPACITY OF AESCULUS PARVIFLORA AND AESCULUS PAVIA: OPPORTUNITIES FOR THEIR IMPROVEMENT THROUGH INTERSPECIFIC HYBRIDIZATION DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ann Marie Chanon, M.S. ***** The Ohio State University 2005 Dissertation Committee: Approved by Dr. Pablo Jourdan Dr. Joseph C. Scheerens Adviser Dr. Daniel K. Struve Graduate Program in Horticulture and Crop Science Dr. Davis Sydnor Copyright by Ann Marie Chanon 2005 ABSTRACT The genus Aesculus, of the family Hippocastanaceae, is comprised of thirteen species, numerous botanic varieties, cultivars, and natural hybrids. All members of this genus can be easily identified by their palmately compound leaves, ornamental flowers and characteristic seeds from which the common name is thought to be derived. Most species of Aesculus are propagated from seed and the cultivars by budding or grafting. Aesculus are susceptible to two important foliar problems. The most important disease is leaf blotch caused by Guignardia aesculi, and the other is physiological leaf scorch. Both of these problems have limited the use of Aesculus in the landscape. Sufficient diversity exists within the genus to consider the development of superior horticultural types through controlled hybridization within and/or among species. To this end, this project focused on the floral, pollen, seed, and reproductive biology of Aesculus parviflora and Aesculus pavia as the foundation for the development of an Aesculus improvement project. Aesculus parviflora is a large, rounded, shrub which produces long panicles of white flowers in summer, with foliage resistant to blotch and scorch and good yellow fall color. Aesculus pavia is valued for its red flowers and its ability to hybridize with other species. Both species exhibited andromonecy and most flowers were functionally staminate. The sex ratio for both species was approximately 5.5%. Aesculus pavia ii panicles contained fewer flowers, 73 on average, with the complete flowers located predominately in the basal portion of the inflorescence. The average A. parviflora panicle contained 284 flowers with the complete flowers located predominately in the upper most apical quarter of the panicle. Anthesis for both species progressed base to tip. Complete flowers are present in A. pavia from the beginning of anthesis but do not appear in A. parviflora until the fifth day of anthesis. Staminate flowers are present throughout anthesis in both species. There appeared to be some plasticity in floral sex expression since mechanical modification increased the number of complete flowers per panicle. Pollen viability was assessed using five species of Aesculus and the interspecific hybrid A. × carnea through an in vitro pollen germination method. While fresh pollen germinated at acceptable levels across a broad range of sucrose concentrations and temperatures. The optimal test conditions for maximum germination were determined to be a combination of 20% sucrose and 15°C. Fresh pollen under these conditions had germination rates between 82-93% and all would make suitable male parents. The effect of storage temperature and time were assessed using these optimized conditions. The differences in pollen germination response from pollen stored at –20°C and –80°C was not significant but the duration of the storage time was highly significant. Short periods of storage resulted in only modest declines in pollen germination. However, extended storage (12 months) reduced pollen germination, as measure by these in vitro test conditions, to below the recommended threshold for fruit set. Seed propagation of Aesculus has been limited by the physiological conditions of recalcitrance and dormancy. The high moisture content and metabolism rate of the iii seeds make them susceptible to damage during initial seed handling and stratification. While the ability to produce radical growth without the benefit of stratification has been demonstrated for both A. parviflora and A. pavia, overall the performance was enhanced by a 60 day stratification period at 4°C. The 60 day stratification period improved the uniformity and increased the rate of the germination and emergence while minimizing the losses due to mold and pregermination. Inadequate or extended periods of stratification resulted in deterioration produced by the high rate of metabolism and subsequent mold infection. This chilling period, however, was not able to overcome the expression of epicotyl dormancy observed in most A. parviflora seedling, and they require a second period of chilling prior to the resumption of growth. The largest number and highest quality seedlings resulted form the 60 day stratification treatment. Studies on the floral biology, pollen viability, and seed management provided much of the essential information necessary to initiate a program for the genetic improvement of A. parviflora and A. pavia. However, successful hybridization programs rely on the accurate selection of superior parental stock. The selection of good maternal parents is based in part upon their ability to initiate and support seed development. One way to evaluate these traits is through the use of a provenance model which measures both the tree’s potential for seed set, seedling efficiency and its realized performance. Realized performance is measured by fruit and seed production. Fruit and seed production in this study was found to be influence significantly by the weather. Most panicles produced one fruit with the maximum number of fruit observed being nine. Most fruit were single seeded and multiple seeds per fruit negatively impacted seed fresh weight. Based in part on the provenance model three A. parviflora iv and two A. pavia were selected as maternal parents. For both A. parviflora and A. pavia, the frequency of seed maturation from both self pollinations and intraspecific pollinations was equal to or somewhat better than the fruit maturation for naturally occurring open pollinations. Maternal trees differed in their ability to develop fruit. In all cases, the success rate for interspecific pollinations was quite low. It was noted that there were two periods of fruit drop, one in the initial days after pollination and the other toward the end of the reproductive cycle. The provenance model despite it limitations proved to predict which trees would be superior in their fruit development potential and offers a mathematical way to assess breeding potential. Wide interspecific crosses have many challenges but great rewards. The limitation in fruit development will require to utilization of techniques like embryo rescue in conjunction with traditional breeding methodologies. v This work is dedicated in loving and lasting memory to my mother Mary Kathryn Chanon vi ACKNOWLEDGEMENT John Donne the seventeenth century English poet and clergy man wrote "no man is an island" and the same can be said of this doctoral project. It is no exaggeration to say that I would not have been able to undertake and complete this dissertation without the assistance of many people who generously gave of there time and talent, and who deserve special recognition. I want to begin by expressing my appreciation to my advisor, Dr. Pablo Jourdan, for allowing me the opportunity to experience true academic freedom in the pursuit of this degree, first by allowing me to explore a range of projects and then by entrusting me to design and conduct this research. I want to express my graduated to him for granting me the time to honor all my responsibilities both personal and professional and for having the confidence in me that I would complete the last portion of this project despite difficult circumstances. In addition, I would like to express my graduated to Dr. Joseph Scheerens, who really assumed the role of co-advisor during the completion of this manuscript. His mentoring, wit and wisdom made what seemed at times to be an overwhelming process, manageable. His professionalism, academic acumen and dedication were inspirational and have modeled for me the role of professor. He has set a standard of excellence that I hope to emulate with me own students. I would like to thank Dr. Dan Struve for encouraging me throughout my graduate school process and then especially for helping me with the harvesting of the Aesculus seed and providing me with the means to germinate and grow them. I want to recognize other members of my committee members, Dr. Davis Sydnor, and Dr, Glenn Howe for their assistance and helpful suggestions throughout the course of my studies. I give special thanks, to the staff at MCIC for their assistance and expertise with the scanning electron microscope. Thank you also to the staffs at The Holden vii Arboretum and Dawes Arboretum for allowing me the use of their laboratory facilities and access to their extensive Aesculus germplasm. I would like to publicly acknowledge and thank in a very heartfelt way the members of the greenhouse staff especially Jim Vent and Lee Duncan for their assistance in caring for my seedlings, and the members of the support staff especially Regina Vann-Hickok and Laura Raubenalt who my their tireless efforts help all of us with our work. I am grateful for the support and friendship that I received from my fellow graduate students, Audry Darrigues and Michelle Stanton who took time from their demanding schedules to make me feel welcome and then to helped me during the preparation of this document. I wish to thank my uncle, Rev. Alfred H. Winters, who has been very supportive of me through this most difficult year. I want to recognize the contributions of Mrs. Joan Wiegel, Mr. Edward Winters, and Mr. Richard Poruban who assisted me by critiquing portions of this manuscript. I offer my sincerest thanks to a host of friends and family for all the large and small things that you have done for me throughout this past year. Finally, I wish to acknowledge the contribution of my mother Mrs. Mary K.