IN VITRO PHYSIOLOGY OF RECALCITRANT TISSUE CULTURED PLANTS IN THE NYMPHAEACEAE, ALISMATACEAE, AND ORCHIDACEAE By HOANG NGUYEN 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 2016 © 2016 Hoang H. Nguyen To Huong, Mom, Dad, Huy, Dale and Dr. Kane ACKNOWLEDGMENTS “I once dreamed that I could walk through the trees that are full of blooming ghost orchids” said Larry Richardson of the Florida Panther National Wildlife Refuge. Dear Larry, your dream is coming true. And so is mine. My dream, reflected through this study and this dissertation, could not be completed without the continuing support from friends, colleagues, and instructors. I deeply thank my major advisor, Dr. Michael Kane, who has guided me from the first days in the US. He is not only my teacher, my mentor, but he is also my cheerleader. Thank you for letting me do what I love, and giving me room to grow. I would like to thank my committee members, Drs. Charles Guy, Dennis Gray, Gregory Macdonald, and Hector Perez for their guidance. I especially appreciate Drs. Sandra Wilson, Hector Perez, Stuart McDaniel, and Thomas Colquhoun for allowing me to use their research equipment and spending their time to discuss my research with me. I thank Drs. Thomas Sheehan, Harold Koopowitz, Carlos Fighetti, and Mr. Frank Smith of American Orchid Society, who inspired me tremendously with their humility, knowledge, and true love of orchids. This study is proof of the strong collaboration between the University of Florida, the Florida Panther National Wildlife Refuge (Larry Richardson, Mark Danaher, Ben Nottingham, Kevin Godsea), and Illinois College (Dr. Lawrence Zettler). The orchid research was financially supported by funding from U.S. Fish & Wildlife Service under contract F12AC01245. The mycorrhizal fungus strains in this study were isolated by Ellen Radcliffe, Dr. Zettler’s student. 4 I am grateful for the friendship and support of of Tanh Nguyen, Nguyet Doan, Addison Nguyen, Oren Ehrlich, Cong Dang, Trang Nguyen, Yen Nguyen, Kien Nguyen,Tu Nguyen, Manh Tran, Du Huynh, Lien Dieu, Phu Nguyen, Dung Phan, Long Kap, Brian Owens, John Ingram, Marian Sheehan, John Bourret, JJ Sadler, Nancy Philman, Tim Johnson, Jonathan Jasinski, Phil Kauth, Rinnie Rodenius, Ben Hughes, Paulina Quijia, Jameson Coopman, Wendy Vidor, Adrienne Smith, Candace Prince, Nick Genna, Tia Tyler, Leah Cobb, Ryan Dickson, Gabriel Campbell, and Kim Backer- Kelley. I thank you all for your friendship and for the years we have spent together. I am blessed to know Stacey and Anthony Barber, Dale and Karen, Haskell, Rose Steeves, Fe Almira, Cuong Nguyen, Tuan Nguyen, Anh Lac, Qui Lac, Lien Duong, Ly Lam, Meredith Course, Atossa Shaltouki, Sally Kim, Bart Schutzman, Edritz Javelosa, Sandy Dang, Anh Lan, Moo Prasert, and their family members. Their hearts are among the biggest and their minds are the most beautiful. With the unconditional love of my wife, mom, dad, and my little brother, I am the richest man in the world. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES .......................................................................................................... 10 LIST OF FIGURES ........................................................................................................ 11 LIST OF ABBREVIATIONS ........................................................................................... 14 ABSTRACT ................................................................................................................... 15 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 17 Plant Propagation ................................................................................................... 17 Plant Micropropagation ........................................................................................... 18 Aquatic Plant Micropropagation ........................................................................ 20 Orchid Micropropagation .................................................................................. 23 Orchid Propagation from Seeds ....................................................................... 26 Plant Tissue Culture Applications in Conservation ........................................... 28 Wetland loss .............................................................................................. 29 Orchid conservation ................................................................................... 30 Micropropagation Industry ...................................................................................... 32 Tissue-culture Recalcitrant Plants .......................................................................... 34 Recalcitrance during Culture Initiation .................................................................... 35 In vitro Culture Initiation .................................................................................... 35 Aquatic Plant Culture Initiation ......................................................................... 36 Recalcitrance during Shoot Multiplication ............................................................... 38 Carry-over Effects ............................................................................................ 38 Carry-over Effects and the Reliability of Plant Tissue Culture Research .......... 40 Recalcitrance during Ex vitro Acclimatization ......................................................... 41 Species of Study ..................................................................................................... 44 Nymphaeaceae Waterlilies. .............................................................................. 44 Traditional waterlily propagation ................................................................ 46 Waterlily in vitro propagation ...................................................................... 47 Sagittaria latifolia .............................................................................................. 48 Ghost Orchid (Dendrophylax lindenii) ............................................................... 50 Project Description .................................................................................................. 54 Project Objectives ............................................................................................ 54 Rationale and Significance ............................................................................... 55 6 2 IN VITRO CULTURE ESTABLISHMENT AND GERMINATION OF NYMPHAEACEAE WATERLILIES ......................................................................... 57 Introduction ............................................................................................................. 57 Materials and Methods............................................................................................ 60 Explant Sources ............................................................................................... 60 Ovule Development in Nymphaea × ‘Madame Ganna Walska’ ........................ 61 Experiment 1: Effects of sterilization method on sterility of N. × ‘Madame Ganna Walska’ ovule tissue .................................................... 61 Experiment 2: Effects of surface sterilization and seed density on in vitro seed germination of Nymphaea × ‘Madame Ganna Walska’ .......... 62 Experiment 3: Effects of light and temperature on germination of Nymphaea × ‘Madame Ganna Walska’ .................................................. 63 Experiment 4: Effects of temperature and seed coat removal on germination of Nymphaea ‘Mayla’ ........................................................... 64 Experiment 5: Effects of seed coat inhibitors on Nymphaea ‘Mayla’ excised embryo development ................................................................. 64 Experiment 6: Effects of GA3 on seed germination of Nymphaea ‘Mayla’ .. 65 Experiment 7: Effect of sterilization methods on sterility and germination of Victoria embryos ................................................................................. 66 Statistical analysis ...................................................................................... 67 Results and Discussion........................................................................................... 67 Ovule development in Nymphaea × ‘Madame Ganna Walska’ ........................ 68 Effects of Sterilization Methods on Sterility of Nymphaea × ‘Madame Ganna Walska’ Ovary ............................................................................................... 69 Effects of Seed Surface Sterilization and Density on Germination ................... 70 Effects of Light and Temperature on Germination of Nymphaea × ‘Madame Ganna Walska’ .............................................................................................. 71 Effects of Temperature on N. ‘Mayla’ Seed and Excised Embryo in vitro Germination ................................................................................................... 72 Effects of Seed Coat Inhibitors on Excised Embryo Development ................... 73 Effects of GA3 on Seed Germination of Nymphaea ‘Mayla’ .............................. 74 Effect of Surface Sterilization Methods on Sterility of Victoria Seeds and Embryos .......................................................................................................