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标题：Development of a rapid and efficient protoplast isolation and transfection method for chickpea ( Cicer arietinum)
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作者：Ninghui Cheng ; Paul A. Nakata
期刊名称：MethodsX
印刷版ISSN：2215-0161
电子版ISSN：2215-0161
出版年度：2020
卷号：7
页码：1-10
DOI：10.1016/j.mex.2020.101025
语种：English
出版社：Elsevier
摘要：AbstractChickpea (Cicer arietinumL.) is the second most important grain legume worldwide. Recent advances in the sequencing of the chickpea genome has provided a new and valuable resource to aid efforts in gene discovery and crop trait improvement. Technical difficulties in stable chickpea transgenics and the lack of a transient expression system for rapid analysis of gene expression and function; however, has limited the usefulness of this genomic resource. As a step toward alleviating this limitation, we report here the development of a simple and efficient transient gene expression protocol. Using leaves from chickpea seedlings, we have established a procedure that enables the generation of large quantities of vital chickpea protoplasts within only a few hours. In addition, we have optimized a PEG-calcium-mediated transfection method to efficiently deliver exogenous DNA into the chickpea protoplast. The current study is the first to present a detailed step-by-step procedures for protoplast isolation, evaluation, transfection, and application in chickpea. In addition, we optimize the transfection efficiency which has not been previously reported. Our protoplast transfection approach provides a platform that will allow rapid high-throughput screening and systematic characterization of gene expression and function. Knowledge gained through such studies will benefit current efforts to improve chickpea production and quality.•Modified enzymatic digestion solution for higher yield and viability.•Optimize transfection of chickpea protoplasts.Graphical abstractDisplay Omitted
关键词：Protoplast;Transfection;Chickpea;Transient expression;Subcellular localization