Efficient mesophyll protoplast isolation and development of a transient expression system for castor-oil plant (Ricinus communis L.)

Introduction: We investigated the main factors affecting the efficacy of protoplast isolation, including leaf-obtaining period, cutting shapes of leaf material, enzyme concentration, enzymolysis time, and centrifugal speed. Methods: Protoplast isolation was optimal on the condition of 20 days of leaf materials, 2-mm filament of leaves, 1.6% RS and 0.8% R-10, 80 min of enzymolysis, and 700 rpm of centrifugation, resulting in the best yield (1.19 x 106 protoplasts/g FW) and vitality (80.34%) of mesophyll protoplasts. The transient expression vector pGFP1 with green fluorescent protein was transfected into the obtained protoplasts from castor by polyethylene glycol-mediated method with a transformation efficiency of 12.37%. Results: Moreover, the applicability of the system for studying the subcellular localization of RcFATA (an acyl-ACP thioesterase) was validated via the protoplast isolation and transient expression protocol in this study. Discussion: Collectively, the efficient mesophyll protoplast isolation and protoplast transient expression system facilitate to analyze the function of specific gene in castor (Ricinus communis L).