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University of Florida Thesis Or Dissertation Formatting THE PRODUCTION OF RED-FLESHED GRAPEFRUIT/PUMMELO TETRAPLOID PARENTS TO AID THE GRAPEFRUIT IMPROVEMENT PROGRAMS By DIVYA KAINTH 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 2010 1 © 2010 Divya Kainth 2 To my late father Dr. Satvinder Singh, my mother Mrs. Surinder Kaur and my beloved sister Kavya 3 ACKNOWLEDGMENTS I would like to express my sincere gratitude to my advisor Dr. Jude W. Grosser, the chairperson of my graduate committee for giving me an opportunity to work with him on such an interesting project and for giving me encouragement, moral and financial support throughout my degree program. I had a great learning experience under his constant scientific guidance. I would like to extend my gratitude to Dr. Frederick G. Gmitter, Dr. Jim H. Graham and Dr. Jose Chaparro for serving on my committee and for their help, academic support, and valuable advice and constructive criticism that helped me throughout my research experience. Deepest thanks to United States Department of Agriculture/ Cooperative State Research, Education, and Extension Service, Florida Citrus Production Research Advisory Council, New Varieties Development and Management Corporation/ Florida Department of Citrus and Barney and Harriett Greene for providing me financial support through my entire program. I would like to acknowledge my lab mates including postdoctoral research associates, graduate students, biological scientists and OPS employees. Many thanks to Dr. Manjul Dutt for his expert advice, Gary Barthe for providing me with all the equipments and chemicals I needed for my research. Special thanks to Milica Calovic who gave me training in somatic hybridization technique as well as helped me finding solutions to the research problems I faced, from her expertise and experience. I am also very thankful to my friends in my lab Monica Vasconcellos, Julie Gmitter, Jamuna, Elaine, Pamela for their support and friendship. I am thankful to Chuck and Ralph Story for taking care of my plants in the greenhouse. I want to thank Katherine Snyder and Paul Weikel for helping me with poster preparations and other technical things. Great thanks to the entire Citrus Research and Education Center (CREC) community for 4 making me feel at home away from home. I am very grateful to my friends Harsimran gill, Gaurav Goyal, Sunehali Sharma, Amit Kohli, Omar, Tejdeep, Ekta Pathak, Ashish and Jagroop Gill for their support through all thick and thin. Finally, I would like to thank my family and friends in India who supported my decision to come here and learn and their unconditional love. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 ABSTRACT ................................................................................................................... 10 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW ..................................................... 12 History of Grapefruit ................................................................................................ 12 Grapefruit Nutrition ................................................................................................. 14 Importance of Seedless Cultivars ........................................................................... 14 Problems in the Grapefruit Market .......................................................................... 17 Introduction of Pummelos in Breeding Programs .................................................... 18 History of Tetraploids in Citrus ................................................................................ 20 Somatic Hybridization ....................................................................................... 20 Spontaneous Altered Ploidy ............................................................................. 23 Colchicine Induced Polyploidy .......................................................................... 26 Objectives ............................................................................................................... 30 2 INDUCTION OF AUTOTETRAPLOIDS IN PUMMELO (CITRUS GRANDIS) THROUGH COLCHICINE TREATMENT OF MERISTEMATICALLY ACTIVE SEEDS IN VITRO ................................................................................................... 32 Introduction ............................................................................................................. 32 Materials and Methods............................................................................................ 34 Plant Materials .................................................................................................. 34 Colchicine Treatments ...................................................................................... 35 Ploidy Analysis ................................................................................................. 36 Results and Discussion........................................................................................... 37 Conclusion .............................................................................................................. 42 3 SELECTION OF ALTERED PLOIDY LEVELS FROM NATURAL POPULATIONS OF MONOEMBRYONIC PUMMELOS (CITRUS GRANDIS) ....... 43 Introduction ............................................................................................................. 43 Materials and Methods............................................................................................ 45 Plant Materials .................................................................................................. 45 Methodology ..................................................................................................... 45 Ploidy Analysis ................................................................................................. 47 Results and Discussion........................................................................................... 47 6 Conclusion .............................................................................................................. 49 4 PRODUCTION OF COLCHICINE INDUCED AUTOTETRAPLOIDS IN PUMMELO (CITRUS GRANDIS) VIA INDIRECT ORGANOGENESIS .................. 50 Introduction ............................................................................................................. 50 Materials and Methods............................................................................................ 52 Plant Materials .................................................................................................. 52 Colchicine Treatments ...................................................................................... 53 Ploidy Analysis ................................................................................................. 55 Results and Discussion........................................................................................... 55 Conclusion .............................................................................................................. 62 5 COLCHICINE INDUCED POLYPLOIDY IN VIVO IN PUMMELO (CITRUS GRANDIS L. OSBECK) .......................................................................................... 63 Introduction ............................................................................................................. 63 Materials and Methods............................................................................................ 64 Plant Materials .................................................................................................. 64 Materials and Methods ..................................................................................... 64 Colchicine Treatments ...................................................................................... 65 Ploidy Analysis ................................................................................................. 67 Results and Discussion........................................................................................... 67 Conclusion .............................................................................................................. 71 6 SOMATIC HYBRIDIZATION OF GRAPEFRUIT + PUMMELO TO PRODUCE GRAPEFRUIT/PUMMELO TYPE ALLOTETRAPLOIDS ........................................ 72 Introduction ............................................................................................................. 72 Materials and Methods............................................................................................ 73 Protoplast Isolation, Fusion, and Culture .......................................................... 73 Callus Recovery and Attempted Induction of Somatic Embryogenesis ............ 74 Modification of Protoplast Culture Conditions ................................................... 75 Use of in vitro plants .................................................................................. 75 Nurse culture .............................................................................................. 75 Treatments of ‘Ruby Red’ callus prior to suspension culture initiation ....... 75 Results and Discussion..........................................................................................
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