Cell Press Acta Medica Okayama 2666-1667 1 2 2020 Protocol for Genome Editing to Produce Multiple Mutants in Wheat 100053 EN Fumitaka Abe Division of Basic Research, Institute of Crop Science, NARO Yuji Ishida Plant Innovation Center, Japan Tobacco Inc. Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Masaki Endo Division of Applied Genetics, Institute of Agrobiological Sciences, NARO Toshihiko Komari Plant Innovation Center, Japan Tobacco Inc. Seiichi Toki Division of Applied Genetics, Institute of Agrobiological Sciences, NARO Kazuhiro Sato Institute of Plant Science and Resources, Okayama University Here, we describe a protocol for producing multiple recessive mutants via genome editing in hexaploid wheat (Triticum aestivum) cv. Fielder. Using Agrobacterium-delivered CRISPR/Cas9 and three sub-genome-specific primer sets, all possible combinations of single, double, and triple transgene-free mutants can be generated. The technique for acceleration of generation advancement with embryo culture reduces time for mutant production. The mutants produced by this protocol can be used for the analysis of gene function and crop improvement. For complete details on the use and execution of this protocol, please refer to Abe et al. (2019). No potential conflict of interest relevant to this article was reported.