Efficient Mesophyll Protoplast Isolation and Development of a Transient Expression System for Castor-Oil Plant (Ricinus communis L.)

Liu, Ying and Xue, Yingbin and Tang, Jianian and Chen, Jianping and Chen, Miao (2019) Efficient Mesophyll Protoplast Isolation and Development of a Transient Expression System for Castor-Oil Plant (Ricinus communis L.). BIOLOGIA FUTURA, 70 (1). pp. 8-15. ISSN 2676-8615 (print); 2676-8607 (online)

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Abstract

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 × 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).

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