Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression an
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Plant Methods Open Access
METHODOLOGY
Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays Snigdha Poddar1,2, Jaclyn Tanaka1, Jamie H. D. Cate1,2,3, Brian Staskawicz1,4 and Myeong‑Je Cho1*
Abstract Background: An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results: Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experi‑ ments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleopro‑ teins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion: The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice. Keywords: Protoplast isolation, Calli, Pause point, Transfection, Genome editing assay, Rice
*Correspondence: [email protected] 1 Innovative Genomics Institute, University of California, Berkeley, CA 94720, USA Full list of author information is available at the end of the article
Background Rice (Oryza sativa L.) is a vital crop that provides staple calories for approximately half of the global population and is a model organism for basic research of monocotyledon plant biology [1, 2]. Amidst rapid population growth, climate change, and threats posed by pests and pathogens, the need to address food security via improved agricultural outp
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