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Comparison of Mesophyll Protoplast Isolation and Transformation Between Panicum Virgatum and Panicum Hallii

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Comparison of Mesophyll Protoplast Isolation and Transformation Between Panicum Virgatum and Panicum Hallii University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Masters Theses Graduate School 12-2016 Comparison of mesophyll protoplast isolation and transformation between Panicum virgatum and Panicum hallii Andrea Grace Collins University of Tennessee, Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes Part of the Plant Biology Commons Recommended Citation Collins, Andrea Grace, "Comparison of mesophyll protoplast isolation and transformation between Panicum virgatum and Panicum hallii. " Master's Thesis, University of Tennessee, 2016. https://trace.tennessee.edu/utk_gradthes/4262 This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Andrea Grace Collins entitled "Comparison of mesophyll protoplast isolation and transformation between Panicum virgatum and Panicum hallii." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Master of Science, with a major in Plant Sciences. C. Neal Stewart, Major Professor We have read this thesis and recommend its acceptance: Scott C. Lenaghan, Tarek Hewezi Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Comparison of mesophyll protoplast isolation and transformation between Panicum virgatum and Panicum hallii A Thesis Presented for the Master of Science Degree The University of Tennessee, Knoxville Andrea Grace Collins December 2016 ii Copyright © 2016 by Andrea Grace Collins. All rights reserved. iii Acknowledgements I would like to thank all of the Neal Stewart lab members, present and past, including Dr. Kellie P. Burris, Dr. Jason N. Burris, Dr. Elizabeth M. Dlugosz, Joshua N. Grant, Dr. Scott C. Lenaghan, and Dr. C. Neal Stewart Jr. I would also like to thank Dr. Tarek Hewezi for serving on my committee. This research was supported by Advanced Research Projects Agency—Energy (ARPA-E) Award No. DE-AR0000313. Also big thanks to Dr. Tom Juenger from University of Texas at Austin for providing seeds from inbred populations of Panicum hallii var. filipes. iv Abstract Protoplasts are appropriate targets for genome editing, DNA functional screens and transgenesis. This project focuses on the comparison of inexpensive mesophyll protoplast isolation via the use of food-grade enzymes and transformation between diploid Panicum hallii Vasey (PAH) and polyploid cellulosic feedstock Panicum virgatum L (switchgrass), a relative of PAH. PAH has great potential as a C4 model species for crop and bioenergy research. Here an inexpensive switchgrass and PAH mesophyll protoplast isolation and transformation system was developed; the first protoplast system for PAH. Using low-cost commercial food-grade enzymes, a cost reduction of ~1000-fold was achieved compared to traditional protoplast isolating enzymes with a cost of $0.003 (USD) per reaction for switchgrass mesophyll protoplasts and $0.0018 (USD) per reaction for switchgrass cell-suspension culture-derived protoplasts. Additionally, PEG-mediated switchgrass mesophyll protoplast transformation was improved to a maximum 30.4 % over the previous transformation efficiency of 9.1 %, achieving higher transformation efficiency with a reduction in DNA quantity. In the first protoplast isolation system for PAH, an average fivefold increase in protoplast yield from PAH leaf tissue over the optimum switchgrass tissue protoplast isolation was shown. PAH yielded an average 7340 ± 1816 viable protoplasts per mg mesophyll tissue and switchgrass yielded 1468 ± 431 viable protoplasts per mg mesophyll tissue with both species having greater than 95 % viable protoplasts. With additional food-grade enzyme concentration optimization, an additional cost decrease to v $0.001 (USD) per reaction was shown. PAH mesophyll protoplasts have a diameter from 3.9- 28.1 µm [micrometer], with a mean of 13.5 µm, which are significantly smaller than switchgrass mesophyll protoplasts which range from 6.5- 39.4 µm with a mean of 17.4 µm. Polyethylene glycol (PEG)-mediated transformation of PAH protoplasts revealed an optimum transformation efficiency of 46.7 ± 5.5 % with switchgrass protoplast transformation efficiency of 9.3 ± 1.9 %. The methods in this project provide an essential step toward using P. hallii as a C4 panicoid model species. vi Table of Contents Chapter 1 Introduction ........................................................................................................ 1 Background ..................................................................................................................... 2 Potential model plants ..................................................................................................... 3 Transformation using Agrobacterium ............................................................................. 5 Protoplast isolation.......................................................................................................... 6 Cost of protoplast isolation ......................................................................................... 7 Protoplast transformation ................................................................................................ 8 PEG-mediated transformation .................................................................................... 8 Electroporation ............................................................................................................ 9 Chapter 2 Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.) .................................................................................. 10 Abstract ......................................................................................................................... 11 Introduction ................................................................................................................... 12 Materials and methods .................................................................................................. 14 Plant material ............................................................................................................ 14 Protoplast isolation.................................................................................................... 15 Plasmid ...................................................................................................................... 17 PEG-mediated transfection ....................................................................................... 18 Statistical analysis ..................................................................................................... 18 Results ........................................................................................................................... 19 Optimization of protoplast isolation using food-grade enzymes .............................. 19 Analysis of a renewable source for switchgrass leaf tissue ...................................... 21 Optimization of switchgrass protoplast transformation ............................................ 22 Isolation and transformation of cell culture-derived protoplasts .............................. 23 Discussion ..................................................................................................................... 24 Chapter 3 Efficient mesophyll-derived protoplast isolation and transformation of Panicum hallii Vasey ........................................................................................................ 34 Abstract ......................................................................................................................... 35 Introduction ................................................................................................................... 36 Materials and methods .................................................................................................. 38 Plant material ............................................................................................................ 38 Protoplast isolation.................................................................................................... 38 Size distribution ........................................................................................................ 39 PEG transformation .................................................................................................. 39 Statistical analysis ..................................................................................................... 40 vii Results ........................................................................................................................... 41 Discussion ..................................................................................................................... 42 Chapter 4 Conclusions and Recommendations ................................................................. 46 List of References ............................................................................................................. 50 Appendix ..........................................................................................................................
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