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

Ying Liu; Yingbin Xue; Jianian Tang; Jianping Chen; Miao Chen

doi:10.1556/019.70.2019.02

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

Biol Futur. 2019 Jan;70(1):8-15. doi: 10.1556/019.70.2019.02. Epub 2019 Nov 16.

Authors

Ying Liu¹, Yingbin Xue¹, Jianian Tang^{1

2}, Jianping Chen³, Miao Chen^{4

5}

Affiliations

¹ Department of Biotechnology, Faculty of Agricultural Science, Guangdong Ocean University, Zhanjiang, Guangdong, P. R. China.
² State Key Laboratory for Conservation and Utilization of Subtropical Afro-Bioresources, South China Agricultural University, Guangzhou, Guangdong, P. R. China.
³ Department of Food Science and Engineering, College of Food Science and Technology, Guangdong Ocean University, Zhanjiang, Guangdong, P. R. China. cjp516555989@126.com.
⁴ Department of Biotechnology, Faculty of Agricultural Science, Guangdong Ocean University, Zhanjiang, Guangdong, P. R. China. czchenmiao@126.com.
⁵ State Key Laboratory for Conservation and Utilization of Subtropical Afro-Bioresources, South China Agricultural University, Guangzhou, Guangdong, P. R. China. czchenmiao@126.com.

PMID: 34554435
DOI: 10.1556/019.70.2019.02

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 X 106 protoplasts/g FW) and vitality (80.34%) of mesophyll protoplasts. The transient expression vector pGFPl 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 Re FATA (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).

Keywords: Ricinus communis; protoplast; subcellular localization; transient expression.