Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2004 Factors influencing regeneration of plantlets from leaf strips of strawberry (Fragaria x ananassa Duch.) Kristi Lee Whitley Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Recommended Citation Whitley, Kristi Lee, "Factors influencing regeneration of plantlets from leaf strips of strawberry (Fragaria x ananassa Duch.)" (2004). LSU Master's Theses. 3657. https://digitalcommons.lsu.edu/gradschool_theses/3657 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. FACTORS INFLUENCING REGENERATION OF PLANTLETS FROM LEAF STRIPS OF STRAWBERRY (FRAGARIA X ANANASSA DUCH.) A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in The Department of Horticulture Kristi Lee Whitley B.S. University of Southern Mississippi, 1983 December 2004 ACKNOWLEDGEMENTS I wish to express my sincere appreciation to my husband Rodney for allowing me the opportunity to pursue this degree. Many thanks to Dr. Charles E. Johnson for his support throughout this endeavor. His tireless effort and total dedication to teaching and research along with his seemingly infinite intelligence have been my driving force and inspiration. Thanks to my dear sister in Christ, Sherry Zorzi, for using her God-given intelligence to proofread this paper. Also, I would like to thank Amy Blanchard, Gloria McClure, Victoria Hurst, Donna Elisar, Dr. Leon Standifer, Dr. Ed O’Rourke, Dr. Ed Bush, and the other staff and fellow graduate students of the horticulture department for their assistance and encouragement. Dr. Jeff Keuhny and Dr. Don LaBonte are also to be thanked for their service on my committee. The past three years have been the dirtiest, sweatiest, stinkiest, most enjoyable and rewarding years of my life. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS............................................................................................................ ii ABSTRACT.................................................................................................................................... v CHAPTER 1. INTRODUCTION ................................................................................................... 1 1.1 History ............................................................................................................................... 1 1.2 Taxonomy and Biology ..................................................................................................... 2 1.3 Economic Importance........................................................................................................ 5 1.4 Economic Impact of Diseases Related to Strawberry........................................................ 7 1.5 Regeneration...................................................................................................................... 8 1.6 Transformation .................................................................................................................. 9 1.7 Agrobacterium Gene Constructs...................................................................................... 11 1.8 Kanamycin....................................................................................................................... 12 1.9 Leaf Tissue....................................................................................................................... 13 1.10 Phytohormones .............................................................................................................. 14 1.11 Darkness Period............................................................................................................. 16 1.12 ‘Chandler’...................................................................................................................... 16 1.13 Rubus ............................................................................................................................. 18 1.14 Problems with Strawberry Regeneration....................................................................... 18 1.15 Objectives ...................................................................................................................... 20 1.16 Literature Cited.............................................................................................................. 20 CHAPTER 2. INFLUENCE OF LENGTH OF INITIAL DARK PERIOD AND NUMBER OF MEDIA SUBCULTURES ON PLANTLET REGENERATION RATE OF LEAF STRIPS OF STRAWBERRY, RASPBERRY AND BLACKBERRY ............................................................ 25 2.1 Introduction...................................................................................................................... 25 2.2 Materials and Methods .................................................................................................... 26 2.3 Results.............................................................................................................................. 28 2.3.1 ‘Chandler’ ................................................................................................................ 28 2.3.2 ‘Cardinal’ ................................................................................................................. 29 2.3.3 Blackberry and Raspberry........................................................................................ 33 2.4 Discussion........................................................................................................................ 33 2.5 References........................................................................................................................ 34 CHAPTER 3. AGROBACTERIUM-MEDIATED TRANSFORMATION OF ‘CHANDLER’ AND ‘CARDINAL’ STRAWBERRY ......................................................................................... 36 3.1 Introduction...................................................................................................................... 36 3.2 Materials and Methods .................................................................................................... 37 3.3 Results.............................................................................................................................. 46 3.4 Discussion........................................................................................................................ 49 3.5 References........................................................................................................................ 49 CHAPTER 4.INFLUENCE OF PHYSIOLOGICAL AGE OF LEAVES OF ‘CHANDLER’ STRAWBERRY GROWN IN VITRO ON PLANTLET REGENERATION............................. 51 4.1 Introduction...................................................................................................................... 51 4.2 Materials and Methods .................................................................................................... 51 4.3 Results.............................................................................................................................. 53 iii 4.4 Discussion........................................................................................................................ 55 4.5 References........................................................................................................................ 56 CHAPTER 5. CONCLUSIONS AND SUMMARY.................................................................... 57 5.1 Conclusions...................................................................................................................... 57 5.2 Summary.......................................................................................................................... 58 5.3 References........................................................................................................................ 59 APPENDIX: STATISTICAL ANALYSIS AND PREVIOUS WORK ON REGENERATION OF STRAWBERRY ..................................................................................................................... 60 VITA............................................................................................................................................. 81 iv ABSTRACT Influence of media age, initial dark period, inoculation with Agrobacterium, and leaf maturity on the regeneration and genetic transformation of two strawberry cultivars was investigated. Four cultivars of blackberry and one raspberry were included to test their response to media age and dark period. Leaf strips of ‘Chandler’ and ‘Cardinal’ strawberry, blackberry, and raspberry were exposed to a four-week dark treatment on modified MS media and subcultured to fresh media. One group of leaf strips was left on the original media for the entire eight weeks. Another group of explants, along with the media treatments was given an additional four-week dark period. ‘Chandler’ strawberry leaf strips exposed to four weeks dark and subcultured onto fresh media produced 19% more plantlets than ‘Chandler’ exposed to eight weeks dark. ‘Chandler’ given a four-week dark period and subcultured onto fresh media produced 58% more plantlets