Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays
Tóm tắt
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. 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 experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. 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.
Tài liệu tham khảo
Birla DS, Malik K, Sainger M, Chaudhary D, Jaiwal R, Jaiwal PK. Progress and challenges in improving the nutritional quality of rice (Oryza sativa L.). Crit Rev Food Sci Nutr. 2017;57:2455–81.
Jackson SA. Rice: the first crop genome. Rice. 2016;21:1–3.
Romero FM, Gatica-Arias A. CRISPR/Cas9: development and application in rice breeding. Rice Sci. 2019;26:265–81.
Sheen J. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol. 2001;127:1466–75.
Su P-H, Yu S-M, Chen C-S. Spatial and temporal expression of a rice prolamin gene RP5 promoter in transgenic tobacco and rice. J Plant Physiol. 2001;158:247–54.
Ni J, Yu Z, Du G, Zhang Y, Taylor JL, Shen C, et al. Heterologous expression and functional analysis of rice glutamate receptor-like family indicates its role in glutamate triggered calcium flux in rice roots. Rice. 2016;9:1–14.
Hiei Y, Komari T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc. 2008;3:824–34.
Andrieu A, Breitler J, Siré C, Meynard D, Gantet P, Guiderdoni E. An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves. Rice. 2012;5:23.
Christou P. Genetic transformation of crop plants using microprojectile bombardment. Plant J. 1992;2:275–81.
Kirienko DR, Luo A, Sylvester AW. Reliable transient transformation of intact maize leaf cells for functional genomics and experimental study. Plant Physiol. 2012;159:1309–18.
Prelich G. Gene overexpression: uses, mechanisms, and interpretation. Genetics. 2012;190:841–54.
Jaganathan D, Ramasamy K, Sellamuthu G, Jayabalan S, Venkataraman G. CRISPR for crop improvement: an update review. Front Plant Sci. 2018;9:985.
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21.
Wu F-H, Shen S-C, Lee L-Y, Lee S-H, Chan M-T, Lin C-S. Tape-Arabidopsis sandwich—a simpler Arabidopsis protoplast isolation method. Plant Methods. 2009;5:16.
Yao X, Zhao W, Yang R, Wang J, Zhao F, Wang S. Preparation and applications of guard cell protoplasts from the leaf epidermis of Solanum lycopersicum. Plant Methods. 2018;14:26.
Gao L, Shen G, Zhang L, Qi J, Zhang C, Ma C, et al. An efficient system composed of maize protoplast transfection and HPLC–MS for studying the biosynthesis and regulation of maize benzoxazinoids. Plant Methods. 2019;15:144.
Shan X, Li Y, Zhou L, Tong L, Wei C, Qiu L, et al. Efficient isolation of protoplasts from freesia callus and its application in transient expression assays. Plant Cell Tissue Organ Cult. 2019;138:529–41.
Priyadarshani SVGN, Hu B, Li W, Ali H, Jia H, Zhao L, et al. Simple protoplast isolation system for gene expression and protein interaction studies in pineapple (Ananas comosus L.). Plant Methods. 2018;14:95.
Li Z, Murai N. Efficient plant regeneration from rice protoplasts in general medium. Plant Cell Rep. 1990;4:216–20.
Utomo HS, Croughan SS, Croughan TP. Suspension and protoplast culture of US rice cultivars. Plant Cell Rep. 1995;15:34–7.
He F, Chen S, Ning Y, Wang G-L. Rice (Oryza sativa) protoplast isolation and its application for transient expression analysis. Curr Protoc Plant Biol. 2016;1:373–83.
Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc. 2014;10:2395–410.
Page MT, Parry MAJ, Carmo SE. A high-throughput transient expression system for rice. Plant Cell Environ. 2019;42:2057–64.
Toriyama K, Hinata K. Cell suspension and protoplast culture in rice. Plant Sci. 1985;41:179–83.
Thompson JA, Abdullah R, Cocking EC. Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose. Plant Sci. 1986;47:123–33.
Mathur J, Koncz C. PEG-mediated protoplast transformation with naked DNA. In: Martinez-Zapater JM, Salinas J, editors. Methods in molecular biology, Vol 82: Arabidopsis protocols. Totowa: Humana Press; 1998. p. 267–76.
Cho M-J, Ha CD, Lemaux PG. Production of transgenic tall fescue and red fescue plants by particle bombardment of mature seed-derived highly regenerative tissues. Plant Cell Rep. 2000;11:1084–9.
Mickael M, Roberto V, Min-Hee J, Ok-Jae K, Seokjoong K, Jin-Soo K, Riccardo V, Chidananda NK. DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front Plant Sci. 2016;7:1904.
Woo J, Kim J, Kwon S, Corvalan C, Cho SW, Kim H, Kim S-G, Kim S-T, Choe S, Kim J-S. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol. 2015;33:1162–4.
Nanjareddy K, Arthikala M-K, Blanco L, Arellano ES, Lara M. Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis. BMC Biotechnol. 2016;16:53.
Xie K, Yinong Y. RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant. 2013;6(6):1975–83.
Sandhya D, Jogam P, Allini VR, Abbagani S, Alok A. The present and potential future methods for delivering CRISPR/Cas9 components in plants. J Genet Eng Biotechnol. 2020;18:25.