CRYOPRESERVATION AND IN VITRO TECHNIQUES FOR THE CONSERVATION OF SELECTED FLORIDA NATIVE ORCHIDS By BENJAMIN A. HUGHES 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 2017 © 2017 Benjamin A. Hughes To Allison and Ronin ACKNOWLEDGMENTS I thank Dr. Michael Kane for his guidance, patience, and generosity in helping me develop into a competent researcher and scientist. You saw something in me years ago that I certainly didn’t. I thank Dr. Rebecca Darnell, Dr. Greg MacDonald, Dr. Carrie Reinhardt- Adams, and Dr. Wagner Vendrame for serving on my supervisory committee and providing me necessary challenges to grow. I thank Dr. Héctor Pérez for acting as a sounding board since I was an undergraduate. There are too many other faculty and staff to name in the Environmental Horticulture Department that have helped me but thank you as well. I thank my peers that helped me throughout this process, beginning with my fellow graduate students: Paulina Quijia, Charles Stewart, Christian Christenson, Candice Prince, Gabriel Campbell, Jameson Coopman, Tia Tyler, and Amber Gardner. Sharing ideas, problem- solving, and commiserating with you was essential to my graduate experience. I also thank my friends not in academia: Dom, Cory, Chris, Sam, Ethan, Johnathan, Adam, Mike, and Matt, amongst others, for all the good times that distracted me from work and helped keep me sane. Finally, I thank my family, especially my parents, Neil and Jennie, supporting and encouraging me throughout this process even though they weren’t always sure what that entailed. I thank my Aunt Rose for the family dinners and always asking how she could help me. Thanks to Charlie for the laughs, debates, and beers. Most importantly, I thank Allison Bechtloff for her love, support, and understanding. She has kept me grounded throughout this process and I doubt I would have finished without her. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 LIST OF ABBREVIATIONS ........................................................................................................11 ABSTRACT ...................................................................................................................................12 CHAPTER 1 LITERATURE REVIEW .......................................................................................................14 Introduction .............................................................................................................................14 Orchid Pollinators ...................................................................................................................16 Embryology ............................................................................................................................17 Gametogenesis .................................................................................................................17 Fertilization and Embryo Development ..........................................................................19 Orchid Seeds and Seedling Development ...............................................................................21 Orchid Mycorrhizae ................................................................................................................23 Orchid Conservation ...............................................................................................................24 In Vitro Orchid Propagation ...................................................................................................25 Vegetative Propagation ...................................................................................................25 Seed Propagation .............................................................................................................27 Liquid Media ...................................................................................................................29 Bioreactors .......................................................................................................................30 Orchid Germplasm Banking ...................................................................................................34 Low-Temperature Storage ...............................................................................................34 Cryopreservation .............................................................................................................36 Controlled rate cooling .............................................................................................36 Vitrification without plant vitrification solution ......................................................37 Vitrification with plant vitrification solution ...........................................................37 Direct Orchid Seed Cryopreservation .............................................................................38 Orchid Seed Cryopreservation by Vitrification ...............................................................42 Orchid Seed Cryopreservation by Encapsulation-Dehydration ......................................45 Protocorm Cryopreservation ...........................................................................................47 Florida Native Orchid Conservation ...............................................................................47 Project Description .................................................................................................................49 Project Objectives ............................................................................................................49 Rationale ..........................................................................................................................49 2 DIRECT CRYOPRESERVATION OF SELECTED FLORIDA NATIVE ORCHID SEED ......................................................................................................................................51 5 Introduction .............................................................................................................................51 Materials and Methods ...........................................................................................................54 Plant Material ..................................................................................................................54 Protocol Optimization for Direct Cryopreservation ........................................................55 Direct Cryopreservation ..................................................................................................56 Statistical Analysis ..........................................................................................................56 Results.....................................................................................................................................57 Protocol Optimization .....................................................................................................57 Direct Cryopreservation ..................................................................................................58 Further Seedling Development ........................................................................................58 Scanning Electron Microscopy ........................................................................................59 Discussion ...............................................................................................................................59 3 EFFECTS OF WATER CONTENT AND LOW-TEMPERATURE STORAGE ON CRYOPRESERVATION OF TWO FLORIDA NATIVE ORCHIDS ..................................74 Introduction .............................................................................................................................74 Materials and Methods ...........................................................................................................76 Plant Material ..................................................................................................................76 Experiment 1: Effect of Seed Moisture Content on Direct Cryopreservation .................77 Experiment 2: Effect of Low-Temperature Storage on Orchid Seed Cryopreservation ..........................................................................................................78 Statistical Analysis ..........................................................................................................79 Results.....................................................................................................................................80 Moisture Content .............................................................................................................80 Low-Temperature Storage ...............................................................................................81 Discussion ...............................................................................................................................82 Moisture Content .............................................................................................................82 Low-Temperature