A CRISPR/Cas9 Induced Male-Sterile Line Facilitating Easy Hybrid Production in Polyploid Rapeseed (Brassica napus)

Rapeseed is a globally significant oilseed crop cultivated to meet the increasing demand for vegetable oil. In order to enhance yield and sustainability, breeders have adopted the development of rapeseed hybrids as a common strategy. However, current hybrid production systems in rapeseed have various limitations, necessitating the development of a simpler and more efficient approach. In this study, we propose a novel method involving the targeted disruption of Defective in Anther Dehiscence1 of Brassica napus (BnDAD1), an essential gene in the jasmonic acid biosynthesis pathway, using CRISPR/Cas9 technology, to create male-sterile (m-s) lines. BnDAD1 was found to be dominantly expressed in the stamen of rapeseed flower buds. Disrupting BnDAD1 led to decreased levels of α-linolenic acid and jasmonate in the double mutants (DMs), resulting in the defect of anther dehiscence and pollen maturation. By crossing the DM m-s lines with male-fertile lines, a two-line system was demonstrated, enabling the production of F1 seeds. The m-s trait of the bndad1 DM lines was maintainable by applying exogenous methyl jasmonate and subsequently self-pollinating the flowers. This breakthrough holds promising potential for harnessing heterosis in rapeseed and offers a simpler and more efficient method for producing hybrid seeds.