CRISPR/Cas9-mediated mutagenesis of the RING-type E3 ubiquitin ligase, TaGW2, in the elite Canadian cultivar AAC Brandon increases grain size and weight but reduces total grain number per plant

Grain size is a key determinant of yield in bread wheat and remains an important target for genetic improvement in elite cultivars. The gene Grain Weight 2 (TaGW2), encoding a RING-type E3 ubiquitin ligase, is a negative regulator of grain development in cereals and represents a promising target for precision breeding. In this study, we used CRISPR/Cas9 to disrupt TaGW2 in the widely grown Canada Western Red Spring wheat cultivar AAC Brandon. A single guide RNA targeting exon 1 was designed to edit the A, B, and D homeologs. Following Agrobacterium-mediated transformation, heat-shock-enhanced editing, and segregation of the transgene, we obtained transgene-free lines carrying homozygous mutations in either the A subgenome or both A and B subgenomes, whereas no D subgenome mutants were recovered. Frameshift mutations in the A and B copies introduced premature stop codons and reduced transcript abundance in mature grains (Zadoks growth stage 90-99). Edited lines showed a 9% increase in grain area and ~14% higher thousand grain weight compared with wild type. However, grain number per plant declined by approximately 30%, in the double mutant, resulting in no significant change in total grain weight per plant. These results confirm TaGW2 as a negative regulator of grain size in elite Canadian wheat line.