Phenotypic characterization of pre-harvest sprouting resistance mutants generated by the CRISPR/Cas9-geminiviral...

Pre-harvest sprouting is a critical phenomenon involving germination of seeds in the mother plant before harvest under relative humid conditions and reduced dormancy. In this paper, we generated HDR mutant lines with one region SNP (C/T) and an insertion of 6 bp (GGT/GGTGGCGGC) in OsERF1 genes for pre-harvest sprouting (PHS) resistance using CRISPR/Cas9 and a geminiviral replicon system. The incidence of HDR was 2.6% in transformed calli. T1 seeds were harvested from 12 HDR-induced calli and named ERF1-hdr line. Molecular stability, key agronomic properties, physiological properties, and biochemical properties of target genes in the ERF1-hdr line were investigated for three years. The ERF1-hdr line showed significantly enhanced seed dormancy and pre-harvest sprouting resistance. qRT-PCR analysis suggested that enhanced ABA signaling resulted in a stronger phenotype of PHS resistance. These results indicate that efficient HDR can be achieved through SNP/InDel replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits. [BMB Reports 2024; 57(2): 79-85].