Location: Children's Nutrition Research CenterTitle: Development of a rapid and efficient protoplast isolation and transfection method for chickpea (Cicer Arietinum)
|CHENG, NINGHUI - Children'S Nutrition Research Center (CNRC)|
Submitted to: MethodsX
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/5/2020
Publication Date: 8/8/2020
Citation: Cheng, N., Nakata, P.A. 2020. Development of a rapid and efficient protoplast isolation and transfection method for chickpea (Cicer Arietinum). MethodsX. 7:101025. https://doi.org/10.1016/j.mex.2020.101025.
Interpretive Summary: Chickpea is the second most widely grown legume crop worldwide and is a source of food for millions of people especially in developing countries. Chickpea yields are below their theoretical potential due in part to their susceptibility to certain environmental factors such as drought and pathogens. Recent chickpea genome sequencing efforts identified many of the genes of the plant. Determining which of these genes are responsible for traits such as drought or pathogen susceptibility remain largely unknown because there is no suitable system to express and test the function of the genes identified by the genome sequencing effort. Here we report the development of a simple and efficient transient gene expression system. Using leaves from chickpea seedlings, we have established a procedure that enables the isolation of individual chickpea cells. In addition, we have optimized a method to efficiently deliver and express selected chickpea genes into these cells. This protocol provides a platform that will allow rapid screening and characterization of gene expression and function. Knowledge gained through such studies will benefit current efforts to improve our ability to grow and produce chickpeas.
Technical Abstract: Chickpea (Cicer arietinum L.) 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. 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.