TISSUE CULTURE OF Trifolium polymorphum, T. carolinianum, Adesmia latifolia, A. bicolor AND Lotononis bainesii By MARIA LAURA VIDOZ 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 2006 Copyright 2006 by Maria Laura Vidoz To my mother and to God, for their unconditional love. ACKNOWLEDGMENTS I would like to thank God for revealing His presence throughout my life, for His guidance during hard times and for His many blessings. I am thankful to my mother for her unconditional support and encouragement, for devoting so much time to my education, for giving me a deep love of nature, making it difficult to put into words how much she means to me. I would like to thank my supervisory committee chair (Dr. Kenneth Quesenberry) for his advice and knowledge, and for introducing me to his wonderful family, all of which helped me professionally and personally. I am also grateful to the other members of my committee (Dr. Mimi Williams, Dr. Maria Gallo and Dr. David Wofford) for their critics and suggestions. I thank Judy Dampier for her technical support, friendship and advice, making my stay in Gainesville a positive experience. I thank Loan Ngo for her technical assistance and sincere friendship. I also appreciate the technical support of Lindsay and Kailey Place. I am thankful to Luis Mroginski and Dr. Hebe Rey, who introduced me to the tissue culture world and constantly encouraged me to grow in my career. I would like to thank Lorena, Carlos, Gaby, Raquel, Jorge and Sonali for their company during these two years, and to all my friends in Argentina for their support. iv TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES............................................................................................................ vii LIST OF FIGURES .............................................................................................................x ABSTRACT...................................................................................................................... xii CHAPTER 1 INTRODUCTION ........................................................................................................1 2 LITERATURE REVIEW .............................................................................................5 Tissue Culture Concepts as Applied to the Fabaceae Family.......................................5 Applications of Tissue Culture for the Fabaceae Family .............................................9 Production of Plants Free from Certain Specific Pathogens ...............................10 Micropropagation ................................................................................................11 Production of Dihaploid, Homozygous Plants ....................................................12 Generation of Interspecific Hybrids: Embryo Rescue and Protoplast Fusion.....12 Plant Regeneration after Transformation Protocols ............................................14 Medium and Long-term Germplasm Conservation and Plant Material Exchange..........................................................................................................15 3 MATERIALS AND METHODS ...............................................................................17 Plant Material..............................................................................................................17 Culture Conditions......................................................................................................17 Evaluation and Experimental Design .........................................................................19 4 TISSUE CULTURE OF Trifolium polymorphum AND T. carolinianum .................21 Introduction.................................................................................................................21 Materials and methods................................................................................................22 Trifolium polymorphum.......................................................................................22 Trifolium carolinianum .......................................................................................23 Results and Discussion ...............................................................................................23 Trifolium polymorphum.......................................................................................23 v Trifolium carolinianum .......................................................................................25 Conclusions.................................................................................................................27 5 PLANT REGENERATION OF Adesmia latifolia AND A. bicolor ..........................31 Introduction.................................................................................................................31 Materials and Methods ...............................................................................................32 Plant Material ......................................................................................................32 Basal Media Experiment .....................................................................................33 Factorial Experiments with TDZ and BAP .........................................................34 Induction Time Experiments ...............................................................................35 Type of Explant Experiment ...............................................................................35 Results and Discussion ...............................................................................................36 Effect of Basal Medium on Shoot Organogenesis ..............................................36 Influence of TDZ on Organogenesis ...................................................................38 Influence of BAP on Organogenesis ...................................................................40 Induction Time for Adventitious Bud Formation................................................42 Effect of Explant Type on Shoot Organogenesis ................................................44 Conclusions.................................................................................................................45 6 PLANT REGENERATION OF Lotononis bainesii...................................................61 Introduction.................................................................................................................61 Materials and Methods ...............................................................................................62 Cotyledon Culture ...............................................................................................62 Leaflet Culture.....................................................................................................62 Type of Explants..................................................................................................63 Results and Discussion ...............................................................................................63 Cotyledon Culture ...............................................................................................63 Leaflet Culture.....................................................................................................65 Type of Explant ...................................................................................................67 Conclusions.................................................................................................................68 7 SUMMARY AND CONCLUSIONS.........................................................................74 APPENDIX ADDITIONAL TABLES FOR TWO GENOTYPES OF Adesmia latifolia.....................77 LIST OF REFERENCES...................................................................................................86 BIOGRAPHICAL SKETCH .............................................................................................95 vi LIST OF TABLES Table page 3-1 Plant material used as a source of explants in the experiments. ..............................20 4-1 Mean percentage of explants producing callus from petiole pieces of T. polymorphum in three basal media after 30 days of culture. ...................................29 4-2 Number of buds per explant in the responsive genotypes of T. carolinianum cultured on medium supplemented with 10 µM TDZ after 30 and 60 days of culture.......................................................................................................................29 5-1 Effect of three basal media on percentage of adventitious bud formation, mean number of buds and regeneration index in A. bicolor and A. latifolia after 60 days of culture. .........................................................................................................47 5-2 Effect of TDZ concentration on percentage of adventitious bud formation, mean number of buds per explant, and regeneration index in A. latifolia after 30 days of culture. .................................................................................................................48 5-3 Effect of TDZ concentration on percentage of adventitious bud formation, mean number of buds per explant, regeneration index, number of shoots, shoot length and percentage of ex vitro survival in A. latifolia. ...................................................48