Efficient generation of targeted point mutations in the Brassica oleracea var. botrytis genome via a modified CRISPR/Cas9 system

Abstract

In this study, we used the modified CRISPR/Cas9 system to produce targeted point mutations in cauliflower. Acetolactate synthase (ALS) and Centromere-specific histone H3 variant (CENH3) genes were selected as the base-editing targets and hypocotyls of cauliflower were used as explants. For ALS gene, a C-to-T conversion in the Pro182 codon (CCT) can alter the encoded amino acid, likely resulting in herbicide resistance, and a C-to-T mutation in the Leu133 codon (CTT) in the CENH3 gene may produce a haploid inducer. Results indicated that the transformation efficiency was 1.8%–4.5% and the mutation efficiencies for the ALS and CENH3 genes were approximately 22% and 87%, respectively. The ALS mutant cauliflower showed strong herbicide resistance, with possible immediate implications for broadleaf weed control in cauliflower fields.

Keywords

• Cauliflower
• Targeted point mutations
• Base-editing
• CRISPR/Cas9
• ALS
• CENH3