Key message Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T seeds. Abstract The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-medi- ated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T and T generations indicates 1 2 that putative mutation induced in the T plant is transmitted to the T generation. The inheritable mutation induced in the T 0 1 1 plant was also detected. This result indicates that continuous induction of mutations during T plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA
Plant Cell Reports – Springer Journals
Published: Jan 15, 2018
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