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The Development of an Efficient Method of Agrobacterium-Mediated Transient Expression in Soybean (Glycine Max)

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The Development of an Efficient Method of Agrobacterium-Mediated Transient Expression in Soybean (Glycine Max) The Development of an Efficient Method of Agrobacterium-mediated Transient Expression in Soybean (Glycine max) THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Jessica Lynn King Graduate Program in Horticulture and Crop Science The Ohio State University 2013 Master's Examination Committee: Leah McHale, Advisor John Finer Christopher Taylor Copyrighted by Jessica Lynn King 2013 Abstract Transient expression refers to the rapid gene expression which occurs directly following transgene introduction. Transient expression using agroinfiltration permits efficient delivery of DNA to a large number of cells and can be applied to the rapid analysis of gene function. Although agroinfiltration systems have been developed for many plant species ranging from model plants to ornamental, vegetable, and cereal crops, one has yet to be developed for soybean. In the present study, we have optimized conditions including the addition of reducing agents and surfactants to the infiltration buffer and the application and timing of sonication and vacuum treatments to develop an agroinfiltration transient expression system for whole soybean seedlings. Each of these factors, as well as the strain of Agrobacterium tumefaciens and the soybean cultivar, had a significant effect on transient expression. The addition of dithiothreitol (DTT) to the infiltration buffer in combination with 30s sonication and three 5-minute periods of negative pressure provided the optimal conditions for transient GUS expression in soybean. Agrobacterium strain J2 yielded the highest expression levels and the greatest expression levels were observed in soybean cultivar Peking. ii Acknowledgments First and foremost, I would like to sincerely thank my husband John for all of your support and patience throughout my college career. Thank you for all of your visits, phone calls, gifts, cards, and much-needed vacations to help me get through! I am also extremely grateful and blessed to have such a loving and supportive family! Thank you to my sisters Stephanie, Kristen, Elizabeth, and Gracie and my brother Aaron as well as my parents for being my foundation and support system. A special thanks to my advisor Dr. Leah McHale for all of your guidance and patience while working with me, and for introducing me to the vast world of horticulture and crop science. Thank you especially for all of your time spent discussing my work and the hours spent editing my writing. It has been a pleasure to learn from you while working on my project. Our lab has been a lot of fun to work in and I’ve enjoyed working with such great people. I would also like to thank my committee members Dr. John Finer and Dr. Chris Taylor for challenging me as a researcher as well as their valuable suggestions on my research project. I have learned a lot from our meetings and discussions not only about my iii research topic but also how to think like a scientist and ask the right questions when designing an experiment. Thanks to Angie Parker, Elizabeth Baskin, Christine Dubler, and our lab manager/”miracle worker” Amanda Gutek for all of your help with my project. Dr. Veena Ganeshan and Mao Huang for your advice with my project as well as Kamila Rezende Dazio de Souza, Rhiannon Schneider, Brad Snyder, and Qianli Shen for sharing the radio, engaging in discussions during lab meeting as well as all of the “insightful” conversations we’ve had. Also, thank you to the United Soybean Board and the OARDC SEEDS Grant for funding support. iv Vita May 2006.................................................... ...Maplewood Jr/Sr High School 2010…............................................................B.S. Agriculture & Biological Engineering, Pennsylvania State University 2011 to Present.................................... ...........Graduate Research Associate, Horticulture & Crop Science, The Ohio State University Fields of Study Major Field: Horticulture and Crop Science v Table of Contents Abstract ............................................................................................................................... ii Acknowledgments ............................................................................................................. iii Vita ...................................................................................................................................... v Table of Contents ............................................................................................................... vi List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Chapter 1: Literature Review ............................................................................................. 1 Introduction..............................................................................................................1 Overview of Gene Introduction.........................................................................1 Transient Expression..........................................................................................6 Factors Affecting Transient Expression.............................................................7 Research Applications and Objectives.............................................................10 Chapter 2: Method optimization for Agrobacterium-mediated transient expression in soybean..............................................................................................................................14 Introduction............................................................................................................14 Materials and Methods...........................................................................................18 Results....................................................................................................................25 vi Discussion..............................................................................................................29 References ......................................................................................................................... 45 vii List of Tables Table 2.1 Previous studies for the development of Agroinfiltration in which sonication and vacuum infiltration were used.....................................................................................32 Table 2.2 A. tumefaciens strains used in this study............................................................33 Table 2.3 Effects of infiltration buffers and sonication time on transient GUS expression in soybean...........................................................................................................................................34 Table 2.4 Effects of Agrobacterium strain on transient expression of GUS in soybean...35 Table 2.5 Effects of soybean genotype on transient GUS expression in soybean.............36 viii List of Figures Figure 2.1 Effects of vacuum time period on transient GUS expression of strain J2 in Williams 82........................................................................................................................37 Figure 2.2 Examples of transient GUS expression observed in leaves for each treatment............................................................................................................................38 Figure 2.3 Mean GUS activity in monofoliate leaves of vacuum infiltrated seedlings with treatments of varied sonication times and supplements to the infiltration buffer..............39 Figure 2.4 Leaf images representative of each of the five A. tumefaciens strains used to infiltrate Williams 82 under optimal conditions................................................................41 Figure 2.5 Effects of different A. tumefaciens strains on transient GUS expression in infiltrated seedlings as measured by image analysis of histochemically stained monofoliate leaves.............................................................................................................42 Figure 2.6 Leaf images representative of each of the five soybean cultivars and one isoline infiltrated with A. tumefaciens strain J2 under the optimal conditions..................43 Figure 2.7 Transient GUS expression of strain J2 in six soybean cultivars......................44 ix Chapter 1: Literature Review Overview of Gene Introduction Transformation, involving the introduction of foreign DNA to the target cell, is a common method used to study the function and expression of a gene of interest, as well as incorporate a desired gene into a host genome. Transformation can be either stable or transient. Stable transformation of soybean starts with the introduction of DNA into a single cell and ends with the generation of whole plants from the single altered cell using the process of tissue culture. Transformation provides an additional tool, which can be used to complement conventional breeding. The foreign DNA is composed of a promoter, coding sequence, and terminator, which are all necessary for the expression of a gene of interest. DNAs are introduced into the nucleus of the target cell and become integrated into a chromosome (Matsumoto et al. 1990). As the DNAs are integrated, stable transformation results in a heritable change to the organism. In contrast to stable transformation, transient
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