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University of Florida Thesis Or Dissertation Formatting Template GREENHOUSE ACCLIMATIZATION AND FIELD ESTABLISHMENT OF THE ENDANGERED GHOST ORCHID, DENDROPHYLAX LINDENII By JAMESON COOPMAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017 © 2017 Jameson Coopman To my family, who has been a solid foundation for my ambition To my advisor, Michael Kane, for mentoring me in many ways To Owen Haugen, whose friendship and support kept me going ACKNOWLEDGMENTS I would like to acknowledge all of my committee members, Dr. Rebecca Darnell, Dr. Hector Perez, Dr. Larry Zettler, and my advisor Dr. Michael Kane for their assistance and input throughout this adventure. I would like to thank Dr. Larry Zettler for the use of his fungal mycobionts for experimentation. I thank Larry Richardson for his help with the out-planting experiments and for providing some amazing photos. I would also like to express gratitude to Mark Danaher, the Florida Panther National Wildlife Refuge, and all those who work there for allowing me to conduct my field research there. Components of this research were originally supported by funding from the U.S. Fish & Wildlife Service under contract F12AC01245. I would like to acknowledge my peers, Ben Hughes, Paulina Quijia, Candice Prince, and Hoang Nguyen for being sounding boards for ideas and questions. Additionally, I thank Pandora, YouTube, Netflix, and Spotify for keeping me productive when I needed to be. I especially thank Calvin Harris and Jessie Reyez for “Hard to Love”, which I listened to on repeat for a week straight writing this thesis. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 13 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 15 Orchidaceae ........................................................................................................... 15 Orchid Mycorrhizae ................................................................................................. 18 Orchid Conservation ............................................................................................... 21 Plant of Study: Dendrophylax lindenii ..................................................................... 24 Project Objectives ................................................................................................... 32 2 GREENHOUSE ACCLIMATIZATION PROTOCOLS FOR FIELD ESTABLISHMENT OF IN VITRO-DERIVED DENDROPHYLAX LINDENII PLANTS .................................................................................................................. 33 Introduction ............................................................................................................. 33 Materials and Methods............................................................................................ 36 Plant Material Source and Maintenance ........................................................... 36 Greenhouse Acclimatization ............................................................................. 37 Statistical Analysis ............................................................................................ 39 Results and Discussion........................................................................................... 40 3 EFFECT OF ROOT PRUNING AND GENERATION OF NEW ACTIVE ROOTS ON GREENHOUSE ACCLIMATIZATION AND ROOT ATTACHMENT OF D. LINDENII ................................................................................................................. 53 Introduction ............................................................................................................. 53 Materials and Methods............................................................................................ 56 Plant Material Source and Culture Maintenance .............................................. 56 Root Pruning .................................................................................................... 57 Effects of Pruning on Root Attachment ............................................................ 58 Statistical Analysis ............................................................................................ 59 Results .................................................................................................................... 59 Discussion .............................................................................................................. 60 5 4 IN VITRO DESICCATION TOLERANCE OF THE EPIPHYTIC ORCHID DENDROPHYLAX LINDENII .................................................................................. 71 Introduction ............................................................................................................. 71 Materials and Methods............................................................................................ 74 Plant Material Preparation ................................................................................ 74 Desiccation Study ............................................................................................. 75 Relative Water Content .................................................................................... 77 Statistical Analysis ............................................................................................ 78 Results .................................................................................................................... 78 Plant Survival ................................................................................................... 78 Plant Fresh Mass ............................................................................................. 79 Tissue Water Status ......................................................................................... 79 Root Number .................................................................................................... 80 Active Root Tip Number ................................................................................... 80 Total Plant Root Length .................................................................................... 81 Discussion .............................................................................................................. 81 5 THE EFFECT OF GREENHOUSE ACCLIMATIZATION AND ATTACHMENT MATERIAL ON THE OUT-PLANTING PERFORMANCE OF DENDROPHYLAX LINDENII ................................................................................................................. 97 Introduction ............................................................................................................. 97 Materials and Methods.......................................................................................... 100 Plant Material ................................................................................................. 100 Preparation Methods for Direct Out-planting vs Greenhouse Acclimatization 101 Comparative Performance of Out-planting Methods ...................................... 102 Statistical Analysis .......................................................................................... 103 Results .................................................................................................................. 103 Survival........................................................................................................... 103 Root Number and Attachment ........................................................................ 104 Active Root Tip Number ................................................................................. 105 Total Plant Root Length .................................................................................. 105 Discussion ............................................................................................................ 106 6 EFFECT OF MYCOBIONT INOCULATION ON THE GREENHOUSE ACCLIMATIZATION AND FIELD ESTABLISHMENT OF DENDROPHYLAX LINDENII ............................................................................................................... 126 Introduction ........................................................................................................... 126 Materials and Methods.......................................................................................... 128 Plant Material ................................................................................................. 128 Mycobiont Isolates .......................................................................................... 129 In Vitro Mycobiont/Plant Co-culture for Symbiotic Greenhouse Acclimatization ............................................................................................ 130 Symbiotic Greenhouse Acclimatization .......................................................... 130 Peloton Verification ........................................................................................ 131 6 In Vitro Mycobiont/Plant Co-culture for
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