Efficient mesophyll protoplast isolation and development of a transient expression system for castor-oil plant (Ricinus communis L.)
2019
Liu, Ying | Xue, Yingbin | Tang, Jianian | Chen, Jianping | Chen, Miao
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. 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 × 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%. 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. Collectively, the efficient mesophyll protoplast isolation and protoplast transient expression system facilitate to analyze the function of specific gene in castor (Ricinus communis L).
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