DEVELOPING A MODEL OF ORCHID SEED GERMINATION: IN VITRO STUDIES OF THE THREATENED FLORIDA SPECIES BLETIA PURPUREA By TIMOTHY R. JOHNSON 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 Timothy R. Johnson 2 To my wife, Danielle, and my son, Finley… we did it! 3 ACKNOWLEDGMENTS First, I thank my colleagues and lab mates—Dr. Phil Kauth, Dr. Scott Stewart, Nancy Philman, Daniela Dutra, Jonathan Jasinski, James J. Sadler and Nguyen Hoang—for years of help designing and carrying out experiments in the laboratory and in the field. I also thank Larry Richardson, Wade Gurly and the staff of the Florida Panther National Wildlife Refuge for assistance with field work, for protecting experiments from wildfires and for making the refuge feel like home. Plant identification would not have been possible without the help of Lucas Majure from the Laboratory of Molecular Systematics and Evolutionary Genetics at the Florida Museum of Natural History (University of Florida). I also wish to recognize Dr. Mike Kane, my “Virgil”, major advisor and mentor, who has guided me through this process and taught me how to be a teacher, researcher and mentor. Additionally, Dr. Wil Taylor gave me the keys to his electron microscopy lab when I was an undergraduate and told me to go look at stuff. I cannot seem to stop. My committee also deserves recognition for making this challenging process fun and exciting: thank you Drs. Dennis Gray, Deborah Miller, Hector Pérez and Carrie Reinhardt Adams. I thank my friends and family for humoring my desire to stay in school for so long and supporting me all along the way. My chief-emotional-supporter has been my wife, Danielle, who has gone so far as to help me collect data in June in south Florida simply because I asked for her help; I love you more for it. Much of this work was made possible by grants from the U.S. Fish and Wildlife Service, as well as from the University of Florida Alumni Fellowship program. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 11 ABSTRACT ................................................................................................................... 14 CHAPTER 1 REVIEW OF LITERATURE .................................................................................... 17 Plant Conservation ................................................................................................. 17 The Orchidaceae .................................................................................................... 19 Flower Morphology and Pollination Biology ............................................................ 20 Orchid Seed Physiology .......................................................................................... 22 In Situ Germination ........................................................................................... 24 Orchid Seed Dormancy .................................................................................... 25 The Orchid-Fungi Symbiosis ............................................................................ 28 Orchid fungi ................................................................................................ 28 Anatomy of orchid mycorrhizae .................................................................. 29 Fungal specificity ....................................................................................... 30 Physiology and ecology of mycotrophy ...................................................... 31 Restoration of Orchid Populations .......................................................................... 32 Conservation Genetics............................................................................................ 34 Species of Study ..................................................................................................... 37 Project Description .................................................................................................. 39 Project Objectives ............................................................................................ 39 Rationale and Significance ............................................................................... 40 2 THE EFFECTS OF TEMPERATURE AND ILLUMINATION ON GERMINATION AND EARLY SEEDLING DEVELOPMENT ............................................................ 45 Background ............................................................................................................. 45 Methods .................................................................................................................. 46 Seed Collection and Storage ............................................................................ 46 Seed Sowing and Culture Conditions ............................................................... 47 Data Collection and Statistical Analysis ........................................................... 48 Results .................................................................................................................... 49 Discussion .............................................................................................................. 50 3 GERMINATION AND SEEDLING DEVELOPMENT IS ENHANCED BY ILLUMINATION, MINERAL SALT NUTRIENTS AND NON-ALCOHOL SUGARS .. 58 5 Background ............................................................................................................. 58 Materials and Methods............................................................................................ 60 Seed Collection ................................................................................................ 60 Estimate of Seed Viability ................................................................................. 60 Germination and Early Seedling Development in the Presence of Sucrose ..... 61 Germination and Early Seedling Development in the Presence of Sucrose and Mineral Salts .......................................................................................... 62 Germination and Early Seedling Development in the Presence of Various Carbohydrates ............................................................................................... 63 Statistical Analysis ............................................................................................ 63 Results .................................................................................................................... 65 Effects of Sucrose and Illumination on Germination and Early Seedling Development on Water Agar ......................................................................... 65 Effects of Sucrose and Illumination on Germination and Early Seedling Development on Mineral Nutrient Agar ......................................................... 67 Germination and Development of Seeds Cultured in the Presence of Various Carbohydrates ................................................................................. 69 Discussion .............................................................................................................. 70 Carbohydrate Utilization by Germinating Seeds and Developing Seedlings .... 71 Role of Nutrients in Regulating Germination and Seedling Development ........ 73 Effect of Light on Germination and Seedling Development .............................. 74 Germination in the Absence of Nutrients and Carbohydrates ........................... 76 4 EFFECTS OF SUGAR ALCOHOLS ON GERMINATION AND SEEDLING DEVELOPMENT IN THE PRESENCE OF THE GERMINATION-PROMOTING CARBOHYDRATES FRUCTOSE AND SUCROSE ................................................ 91 Background ............................................................................................................. 91 Materials and Methods............................................................................................ 93 Seed Collection, Sterilization and Viability........................................................ 93 Asymbiotic Culture and Experimental Treatments ............................................ 94 Data Collection and Statistical Analysis ........................................................... 94 Results .................................................................................................................... 96 Interactions Between Sucrose and Sorbitol ...................................................... 96 Interactions Between Sucrose and Mannitol .................................................... 96 Interactions Between Fructose and Sorbitol ..................................................... 98 Interactions Between Fructose and Mannitol .................................................... 99 Discussion .............................................................................................................. 99 5 EFFECTS OF GIBBERELLIC ACID AND ABSCISIC ACID ON GERMINATION AND SEEDLING DEVELOPMENT ....................................................................... 110 Background ........................................................................................................... 110 Materials and Methods.........................................................................................