An Improved Leaf Protoplast System for Highly Efficient Transient Expression in Switchgrass (Panicum virgatum L.)

As a robust perennial C4-type monocot plant and a native species to North America, switchgrass (Panicum virgatum) has been evaluated and designated as a strong candidate bioenergy crop by the U.S. DOE. Although genetic modifications of switchgrass have been used to successfully reduce the recalcitrance of switchgrass biomass for biofuel production, the generation of transgenic switchgrass is still a slow and laborious process. A transient protoplast system can provide an excellent platform to accelerate the selection of genes-of-interest for tailoring switchgrass biomass. However, partially due to the lack of the complete genomic information, the attempts to optimize the transient protoplast system for switchgrass remain scarce. In this chapter, we provide an improved protocol for switchgrass protoplast isolation, increased transformation efficiency using CsCl gradient ultracentrifugation-derived plasmid DNA and extended application of the transient switchgrass protoplast system to analyze protein expression using western blot.