Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz)

BMC Biotechnology

Methodology Article

Xiao-Shan Geng¹  Qin Liu¹  Jin-Ping Liu¹  Jun-Zheng Wu¹  Li-Juan Luo¹  Kai-Mian Li² 

[1] Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, College of Agriculture, Hainan University, 570228, Haikou, Hainan Province, China;The Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, 571101, Haikou, Hainan Province, China;

关键词: Cassava;    Manihot esculenta;    Protoplast isolation;    Transient expression system;    Subcellular localization;   

DOI  :  10.1186/s12896-017-0349-2

 received in 2016-12-01, accepted in 2017-03-07,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundCassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced.MethodsFully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization.ResultsIn this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 107 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H+/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system.ConclusionWe optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

【 授权许可】

CC BY   
© The Author(s). 2017