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In Vitro Tissue Culture of Wild Arachis

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In Vitro Tissue Culture of Wild Arachis IN VIRTO AND IN VIVO EVALUATION OF Arachis paraguariensis AND A. glabrata GERMPLASM By OLUBUNMI OLUFUNBI AINA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1 © 2011 Olubunmi O. Aina 2 To my late father Isaac O. Ajani 3 ACKNOWLEDGMENTS It is a great privilege to be mentored and advised by such a knowledgeable and experienced professor like Dr. Kenneth H. Quesenberry. I sincerely thank him for the opportunity to join his team, as well as for his patience and support. His desire to see his student succeed in every aspect of life is worthy of acknowledgment and emulation. I would like to express my gratitude to all the members of my supervisory committee, Drs. Mike Kane, Barry Tillman, Maria Gallo, and Yoana Newman for giving me so much support, reassurance, and inspiration throughout this project. I am also thankful to Dr. Fredy Altpeter whose assistance always exceeded expectation. The staff at the University of Florida College of Medicine Electron Microscopy Core Facility is acknowledged for their assistance with the histological studies. I am thankful to my past and present lab members, as well as Judy Dampier, Gearry Durden, Justin McKinney and Jim Boyer for their help with the field evaluation aspect of this study. I am thankful to April, Bensa, and their entire family, as well as the families of other soccer moms who have invited my son to their home for sleepovers when I needed to work overnight on this dissertation. Kathy, Rebecca, Iris, Christiana, Christine, Lena, Dorothy, Cheryl, and their wonderful families are greatly acknowledged for all their help and support in various ways. I thank my mother and my brothers, Femi and Bode, for supporting all my academic efforts, especially for their financial inputs. Lastly, I am thankful to my precious little son, Peace, for good behavior and cooperation throughout my studies. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 12 CHAPTER 1 INTRODUCTION .................................................................................................... 14 Hypotheses ............................................................................................................. 17 Objectives ............................................................................................................... 17 Rationale ................................................................................................................. 18 2 LITERATURE REVIEW .......................................................................................... 19 The Genus Arachis ................................................................................................. 19 Origin and Distribution ...................................................................................... 19 Reproductive Biology ....................................................................................... 21 Wild Arachis in Forage Systems ............................................................................. 22 The Uniqueness of Rhizoma Perennial Peanut ................................................ 22 Limitations to Genetic Improvement of Rhizoma Perennial Peanut .................. 23 Wild Arachis in Gene Introgression ......................................................................... 23 Potential Uses of Arachis paraguariensis ......................................................... 24 Hybridization Barriers ....................................................................................... 25 Introgression Pathways .................................................................................... 25 In Vitro Tissue Culture of Wild Arachis.................................................................... 27 Morphogenic Pathways .................................................................................... 27 Signal Transduction .......................................................................................... 30 The Challenges of In Vitro Tissue Culture of Wild Arachis ............................... 34 In Vitro Ploidy Manipulation .............................................................................. 36 Ploidy Detection ............................................................................................... 37 3 OPTIMIZING IN VITRO REGENERATION CONDITIONS FOR Arachis paraguariensis ........................................................................................................ 39 Materials and Methods............................................................................................ 41 Explant Source and Sterilization ....................................................................... 41 Experimental Design and Data Collection ........................................................ 42 Statistical Analysis ............................................................................................ 43 5 Histological Analysis ......................................................................................... 44 Tissue Culture Regeneration ............................................................................ 44 Experiment I ............................................................................................... 45 Experiment II .............................................................................................. 45 In Vitro Rooting ................................................................................................. 45 In Vitro Flowering ............................................................................................. 46 In Vivo Evaluation ............................................................................................. 47 Results .................................................................................................................... 48 Experiment I - The Role of 2,4-D in Induction of Embryogenesis ..................... 48 Experiment II - The Role of TDZ in Induction of Embryogenesis and Organogenesis .............................................................................................. 49 Rooting and Post-Acclimatization Survival of In Vitro-derived Plantlets ........... 50 The Effect of Photoperiod on In Vitro Flowering ............................................... 52 Performance of Plantlets in the Greenhouse and in the Field .......................... 53 Discussion .............................................................................................................. 54 4 IN VITRO INDUCTION OF TETRAPLOIDY IN Arachis paraguariensis .................. 69 Materials and Methods............................................................................................ 70 Plant Materials .................................................................................................. 70 Tissue Culture Initiation and Establishment ...................................................... 71 Culture Condition .............................................................................................. 72 Ploidy Determination by Flow Cytometry .......................................................... 72 Morphological and Fertility Observations.......................................................... 73 Experimental Design, Data Collection and Analysis ......................................... 73 Results .................................................................................................................... 74 Tetraploid Induction by Colchicine Application to Quartered-Seeds and Shoot-Tips ..................................................................................................... 74 Regeneration of Mixoploids from Colchicine-Treated Callus ............................ 75 Morphology, Fertility and Survival of Induced Tetraploid and Mixoploid ........... 76 Discussion .............................................................................................................. 77 5 SEED PRODUCTION IN A. glabrata AND TISSUE CULTURE REGENERATION FROM THE SEED-DERIVED EXPLANTS ................................ 84 Materials and Methods............................................................................................ 86 Seed Production ............................................................................................... 86 Seed Quality Evaluation ................................................................................... 86 Tissue Culture Regeneration ............................................................................ 87 Statistical Analysis ............................................................................................ 89 Results .................................................................................................................... 89 Seed Production ............................................................................................... 89 Seed Quality
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