Date of Award
Master of Science
C. Neal Stewart
Scott C. Lenaghan, Tarek Hewezi
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 $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.
Collins, Andrea Grace, "Comparison of mesophyll protoplast isolation and transformation between Panicum virgatum and Panicum hallii. " Master's Thesis, University of Tennessee, 2016.