title: Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.).
abstract: A switchgrass protoplast system was developed, achieving a cost reduction of ~1000-fold, a threefold increase in transformation efficiency, and a fourfold reduction in required DNA quantity compared to previous methods. In recent years, there has been a resurgence in the use of protoplast systems for rapid screening of gene silencing and genome-editing targets for siRNA, miRNA, and CRISPR technologies. In the case of switchgrass (Panicum virgatum L.), to achieve economic feasibility for biofuel production, it is necessary to develop plants with decreased cell wall recalcitrance to reduce processing costs. To achieve this goal, transgenic plants have been generated with altered cell wall chemistry; however, with limited success owing to the complexity of cell walls. Because of the considerable cost, time, and effort required to screen transgenic plants, a protoplast system that can provide data at an early stage has potential to eliminate low performing candidate genes/targets prior to the creation of transgenic plants. Despite the advantages of protoplast systems, protoplast isolation in switchgrass has proven costly, requiring expensive lab-grade enzymes and high DNA quantities. In this paper, we describe a low-cost protoplast isolation system using a mesophyll culture approach and a cell suspension culture. Results from this work show a cost reduction of ~1000-fold compared to previous methods of protoplast isolation in switchgrass, with a cost of $0.003 (USD) per reaction for mesophyll protoplasts and $0.018 for axenic cell culture-derived protoplasts. Further, the efficiency of protoplast transformation was optimized threefold over previous methods, despite a fourfold reduction in DNA quantity. The methods developed in this work remove the cost barrier previously limiting high-throughput screening of genome-editing and gene silencing targets in switchgrass, paving the way for more efficient development of transgenic plants.
authors.affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, TN, 37996, USA.
authors.forename: Kellie P
authors.initials: KP
authors.lastname: Burris
authors.affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, TN, 37996, USA.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Dlugosz
authors.affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, TN, 37996, USA.
authors.forename: A Grace
authors.initials: AG
authors.lastname: Collins
authors.affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, TN, 37996, USA.
authors.forename: C Neal
authors.initials: CN
authors.lastname: Stewart
authors.affiliation: Center for Renewable Carbon, University of Tennessee, Knoxville, TN, 37996, USA. slenagha@utk.edu.|Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville, TN, 37996, USA. slenagha@utk.edu.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Lenaghan
mesh_terms: D056804:Biofuels; D018929:Cell Culture Techniques; D002473:Cell Wall / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003362:Cost-Benefit Analysis; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008897:Panicum / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011523:Protoplasts / Q000378:metabolism*; D015203:Reproducibility of Results; D014162:Transfection / Q000191:economics / Q000379:methods*
chemical_list: D056804:Biofuels; D008164:Luminescent Proteins; D010940:Plant Proteins
keywords: Enzymatic digestion; Protoplasts; Switchgrass; Transformation