Rapid Low-Cost Method for Protoplast Isolation from Suspension Cultures of Switchgrass

Protoplast extraction from plant cell culture is often a very expensive and laborious process that utilizes highly purified enzymes, with varying activity at a range of temperatures and pH, which are mixed into solutions in-house directly before use. Protoplast extraction and isolation has many advantages in the design of plant systems for analyzing synthetic biology constructs, including ease of non-agro transformation, and uniformity of culture. Considering the importance of switchgrass (Panicum virgatum) as a bioenergy feedstock, and the need to generate plants with lower recalcitrance to reduce processing costs, a low-cost protoplast screening system is highly desirable. Despite these advantages, previous research on protoplast isolation from switchgrass involved a relatively high percentage of multiple enzymes for extraction and was cell-type dependent, making protoplast isolation cost-prohibitive. In this study, a low-cost method for protoplast isolation was developed using food-grade enzymes in combination with liquid suspension cultures of switchgrass. These enzymes were much cheaper than typical enzymes used in vitro, and were amenable to mixed use due to similar temperature and pH optima. In addition, it was possible to directly transform the protoplasts using non-agro transformation protocols, further validating this system for analysis of synthetic promoters and novel transgenes. The establishment of a cost-effective, highly reproducible protocol for protoplast extraction and transformation from switchgrass cells represents a significant achievement for the future of genetically modified switchgrass.