EXPLOITATION OF PUMMELO (CITRUS MAXIMA (BURM.) MERRILL) THROUGH BREEDING, PLOIDY MANIPULATION, AND INTERSTOCKS FOR IMPROVEMENT OF CULTIVATED CITRUS By ETHAN RIRIE NIELSEN 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 Ethan Ririe Nielsen To my loving wife, Harmonie ACKNOWLEDGMENTS I would like to thank my supervisory committee chair Dr. Jude Grosser for his insights and guidance throughout this long journey towards the attainment of the highest degree in education. I admire his dedication to developing better Citrus and improving the livelihood of the growers that look to him for solutions, which he freely gives. I also want to thank my other committee members, Dr. Jose X. Chaparro who taught me many insights in perennial plant breeding, Dr. Fred Gmitter, Jr., Dr. Arnold Schumann, and Dr. Kevin Folta whose excitement for discovery through the scientific method is infectious. I want to thank my fellow Lab members: Dr. Manjul Dutt, Gary Barthe, Dr. Ahmad Omar, Dr. Miliça Ćalović, and Elaine Moreira. Thanks to my fellow graduate students Aditi Satpute and Flavia Zambon, and the other undergraduates and lab assistants who helped make this possible. Thanks to Dr. James Graham for his instructions on working with citrus canker and Kayla Gerberich who assisted with the canker inoculum preparation. Thanks to Dr. Larry Duncan and Dr. Fahiem EL-Borai Kora for the assistance with nematodes. Thanks to Dr. Timothy Ebert for assistance with statistical analyses. Thanks to my wife and children who let me pursue my dream of becoming a plant breeder. Thanks to my parents for instilling in me the values and work ethic that have helped me to succeed. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES ........................................................................................................ 10 LIST OF ABBREVIATIONS ........................................................................................... 12 ABSTRACT ................................................................................................................... 13 CHAPTER 1 INTRODUCTION .................................................................................................... 15 2 REVIEW OF LITERATURE .................................................................................... 19 History of Cultivation in Florida ............................................................................... 19 Taxonomy ............................................................................................................... 22 Genetic Variation in Citrus ...................................................................................... 23 Ancestral and Contemporary Cultivars ................................................................... 24 Pummelo (Citrus maxima (Burm.) Merrill) ........................................................ 24 Grapefruit (Citrus x paradisi Macfadyen)........................................................... 27 Oranges ............................................................................................................ 30 Citron, Lemon, and Lime .................................................................................. 32 Mandarin and Mandarin Hybrids ...................................................................... 32 Kumquats ......................................................................................................... 33 Trifoliate Orange (Poncirus trifoliata [L.] Raf.) .................................................. 34 Papedas ........................................................................................................... 35 Near Citrus Relatives ....................................................................................... 36 Rootstocks .............................................................................................................. 36 Interstocks ........................................................................................................ 39 Protein and RNA Signals .................................................................................. 40 Citrus Breeding ....................................................................................................... 40 Mutation Breeding ............................................................................................ 41 Polyploidy ......................................................................................................... 41 Protoplast Culture and Somatic Hybridization .................................................. 43 Seedless Citrus ................................................................................................ 44 Interploid Hybridization ..................................................................................... 44 Cybrids in Citrus ............................................................................................... 46 Diseases of Citrus ................................................................................................... 47 Citrus Canker ................................................................................................... 48 Citrus Canker Inoculation ................................................................................. 49 Huanglongbing ................................................................................................. 50 CLas Detection ................................................................................................. 52 Nematode Caused Diseases ............................................................................ 53 Objectives ............................................................................................................... 54 5 3 EVALUATION OF CITRUS CANKER RESISTANCE IN PUMMELO PARENT SELECTIONS ......................................................................................................... 56 Background and Objective ...................................................................................... 56 Materials and Methods ............................................................................................ 59 Material Background ........................................................................................ 59 Pummelo Candidate Selection ......................................................................... 60 Bacterial culture and inoculum preparation ...................................................... 61 Leaf Inoculation ................................................................................................ 62 Candidatus Liberibacter asiaticus Testing ........................................................ 63 Results and Discussion ........................................................................................... 64 4 INTERPLOID HYBRIDIZATION TO PRODUCE SEEDLESS TRIPLOID HYBRIDS OF PUMMELO AND GRAPEFRUIT ....................................................................... 85 Background and Objective ...................................................................................... 85 Materials and Methods ............................................................................................ 87 Easy Peel Grapefruit ........................................................................................ 90 Embryo Rescue for Interploid Crosses ............................................................. 91 Flow Cytometry for Ploidy Determination ......................................................... 91 Seedling Grafting .............................................................................................. 92 Citrus Canker Inoculations ............................................................................... 92 Results and Discussion ........................................................................................... 93 Citrus Canker Inoculation ................................................................................. 95 Preliminary Evaluation of Field trees ................................................................ 98 5 TESTING OF SELECTED PUMMELO AND HYBRID INTERSTOCKS TO MITIGATE HLB SYMPTOMS IN COMMERCIAL SWEET ORANGE/SWINGLE TREES ................................................................................................................. 119 Background and Objectives .................................................................................. 119 Materials and Methods .......................................................................................... 120 Stick Grafting .................................................................................................. 121 Control Trees .................................................................................................. 123 Rootstocks ...................................................................................................... 123 HLB Development .......................................................................................... 123 CLas Detection ............................................................................................... 124 Growth Data ..................................................................................................