Platanthera chapmanii: culture, population augmentation, and mycorrhizal associations By Kirsten Poff, B.S. A Thesis In Plant and Soil Science Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Approved Dr. Jyotsna Sharma Chair of Committee Dr. Scott Longing Dr. John Zak Dr. Mark Sheridan Dean of the Graduate School August, 2016 © 2016, Kirsten Poff Texas Tech University, Kirsten Poff, August 2016 ACKNOWLEDGEMENTS First I would like to thank my mentor and advisor, Dr. Jyotsna Sharma for all of her help and support. She has challenged and encouraged me throughout my program and the duration of this project. Thanks to her, I am light-years ahead of where I was two years ago. Texas Parks and Wildlife is also gratefully acknowledged for funding portions of this study. I also wish to express my gratitude to Dr. John Zak for his enthusiasm and for encouraging my love of microbes. I also gratefully thank Dr. Scott Longing for his advice, and constructive comments. I sincerely thank all three committee members for all the time and energy they have spent on me throughout the duration of my project. I gratefully acknowledge Dr. Jason Woodward for his encouragement and recommendations as well. I also acknowledge Dr. Cynthia McKenney and Mr. Russel Plowman for their support; I now have a passion for teaching, and a much better understanding of what it is like to teach college level courses. I want to also thank Mr. Robby Carlson for his time and technological assistance. I wish to extend a heartfelt thank you to my lab mates for their time and help. I extend my gratitude to Niraj Rayamajhi and Bianca Walker for teaching me a multitude of lab techniques. I want to also thank Jaspreet Kaur and Dr. Eeva Terhonen, specifically for all there help with data analyses. I thank Pablo Tovar for teaching me how to troubleshoot, none of my sequences would exist without him. I thank all of them for their friendship. I gratefully acknowledge the efforts of my professors at Texas Tech ii Texas Tech University, Kirsten Poff, August 2016 University, I also thank the Plant and Soil Science Department administrative staff and chair Dr. Eric Hequet, all your advice and wisdom was well received. To my parents, Leslie and Kevin Poff, and my siblings Kevin Scott Poff, Crista Poff and Caroline Poff, thank you for all of your support and love. Last, I want to thank Daniel Smith for his inspiration, love and encouragement, and for coming with me on this journey. This would not have been possible without all of these people, I am very lucky and thankful to have them. iii Texas Tech University, Kirsten Poff, August 2016 TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................... ii LIST OF TABLES ................................................................................................ vii LIST OF FIGURES ............................................................................................... ix I. INTRODUCTION AND BACKGROUND ........................................................ 1 Introduction ....................................................................................................... 1 Orchid classification ..................................................................................... 1 Orchid biology .............................................................................................. 2 Orchid Conservation ..................................................................................... 6 Genus Platanthera ......................................................................................... 8 Platanthera chapmanii .................................................................................. 9 Background ..................................................................................................... 10 In vitro seed germination ...................................................................................... 10 Greenhouse culture of plants ................................................................................ 14 Population augmentation ...................................................................................... 16 Mycorrhizal associations ...................................................................................... 19 Techniques for mycorrhizal identity ..................................................................... 22 Summary of Research Gaps .................................................................................. 24 Objectives of the study.................................................................................... 26 Significance of the study ................................................................................. 26 Literature Cited ............................................................................................... 27 II. COLD-MOIST STRATIFICATION IMPROVES GERMINATION IN A TEMPERATE TERRESTRIAL NORTH AMERICAN ORCHID ...................... 38 Abstract ........................................................................................................... 38 iv Texas Tech University, Kirsten Poff, August 2016 Introduction ..................................................................................................... 39 Materials and methods .................................................................................... 43 Seed stratification ....................................................................................... 43 Seed plating and germination assessment .................................................. 44 Data analysis ........................................................................................................ 45 Results ............................................................................................................. 46 Seed germination .................................................................................................. 46 Seedling development............................................................................................ 47 Discussion ....................................................................................................... 48 Literature Cited ..................................................................................................... 52 III. PLATANTHERA CHAPMANII: NUTRIENT SUPPLEMENTATION AND POPULATION AUGMENTATION .................................................................... 63 Abstract ........................................................................................................... 63 Introduction ..................................................................................................... 64 Materials and Methods .................................................................................... 69 Nutrient supplementation ........................................................................... 69 Population augmentation ............................................................................ 70 Results ............................................................................................................. 72 Nutrient supplementation ...................................................................................... 72 Population augmentation ............................................................................ 73 Discussion ....................................................................................................... 73 Literature Cited ..................................................................................................... 77 IV. DIVERSITY OF MYCORRHIZAE FORMING TULASNELLACEAE IN A TEMPERATE TERRESTRIAL ORCHID IN EX SITU AND IN SITU ENVIRONMENTS ............................................................................................... 83 v Texas Tech University, Kirsten Poff, August 2016 Abstract ................................................................................................................. 83 Introduction ........................................................................................................... 85 Materials and methods .......................................................................................... 90 Literature cited .................................................................................................... 105 V. CONCLUSIONS ............................................................................................ 127 Literature Cited ............................................................................................. 130 vi Texas Tech University, Kirsten Poff, August 2016 LIST OF TABLES 2.1 Effect of cold-moist stratification (0, 8, or 12 weeks) on seed germination was experimentally tested in Platanthera chapmanii. An Analysis of Variance . (ANOVA) was conducted (a); Germination of seeds was categorized as Stage 0 (no further development), Stage 1 (germination; rhizoid development), or Stage 2 (leaf primordium development). Mean number of seeds that were plated in an experimental unit, mean number of viable seeds, and mean percent viability are presented. Mean germination percentages were calculated by using total number of seeds and number of viable seeds separately. Means followed by the same letter in each column were statistically similar based on Fisher’s Least Significant Difference (LSD) test…................................................55 2.2 Effect of cold-moist stratification (0, 8, and 12 weeks) on seedling