Cell Press Acta Medica Okayama 22111247 28 5 2019 Genome-Edited Triple-Recessive Mutation AltersSeed Dormancy in Wheat 1362 1369 EN Fumitaka Abe Division of Wheat and Barley Research, Institute of Crop Science, NARO Emdadul Haque Division of Wheat and Barley Research, Institute of Crop Science, NARO Hiroshi Hisano Institute of Plant Science and Resources, Okayama University Tsuyoshi Tanaka Division of Basic Research, Institute of Crop Science, NARO Yoko Kamiya Kihara Institute for Biological Research, Yokohama City University Masafumi Mikami Graduate School of Nanobioscience, Yokohama City University Kanako Kawaura Kihara Institute for Biological Research, Yokohama City University Masaki Endo Division of Applied Genetics, Institute of Agrobiological Sciences, NARO Kazumitsu Onishi Department of Agro-Environmental Science, Obihiro University of Agriculture and Veterinary Medicine Takeshi Hayashi Division of Basic Research, Institute of Crop Science, NARO Kazuhiro Sato Institute of Plant Science and Resources, Okayama University 1Common wheat has three sets of sub-genomes, making mutations difficult to observe, especially for traits controlled by recessive genes. Here, we produced hexaploid wheat lines with loss of function of homeoalleles of Qsd1, which controls seed dormancy in barley, by Agrobacterium-mediated CRISPR/Cas9. Of the eight transformed wheat events produced, three independent events carrying multiple mutations in wheat Qsd1 homeoalleles were obtained. Notably, one line had mutations in every homeoallele. We crossed this plant with wild-type cultivar Fielder to generate a transgene-free triple-recessive mutant, as revealed by Mendelian segregation. The mutant showed a significantly longer seed dormancy period than wild-type, which may result in reduced pre-harvest sprouting of grains on spikes. PCR, southern blotting, and whole-genome shotgun sequencing revealed that this segregant lacked transgenes in its genomic sequence. This technique serves as a model for trait improvement in wheat, particularly for genetically recessive traits, based on locus information from diploid barley. No potential conflict of interest relevant to this article was reported.