Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development

Summary Multilocular silique is a desirable agricultural trait with great potential for the development of high‐yield varieties of Brassica . To date, no spontaneous or induced multilocular mutants have been reported in Brassica napus , which likely reflects its allotetraploid nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed homologues of CLAVATA 3 ( CLV 3 ) for a secreted peptide and its related receptors CLV 1 and CLV 2 in the CLV signalling pathway using the CRISPR /Cas9 system and achieved stable transmission of the mutations across three generations. Each Bn CLV gene has two copies located in two subgenomes. The multilocular phenotype can be recovered only in knockout mutations of both copies of each Bn CLV gene, illustrating that the simultaneous alteration of multiple gene copies by CRISPR /Cas9 mutagenesis has great potential in generating agronomically important mutations in rapeseed. The mutagenesis efficiency varied widely from 0% to 48.65% in T 0 with different single‐guide RNA s (sg RNA s), indicating that the appropriate selection of the sg RNA is important for effectively generating indels in rapeseed. The double mutation of Bn CLV 3 produced more leaves and multilocular siliques with a significantly higher number of seeds per silique and a higher seed weight than the wild‐type and single mutant plants, potentially contributing to increased seed production. We also assessed the efficiency of the horizontal transfer of Cas9/ gRNA cassettes by pollination. Our findings reveal the potential for plant breeding strategies to improve yield traits in currently cultivated rapeseed varieties.