Efficient and versatile rapeseed transformation for new breeding technologies.

Many gene functions are widely studied and understood in Arabidopsis; however, the lack of efficient transformation systems often limits the application and verification of this knowledge in crop plants. Brassica napus L., a member of the Brassicaceae family, is usually transformed by Agrobacterium-mediated hypocotyl transformation, but not all growth types are equally amenable to transformation. In particular, winter rapeseed, which requires vernalization to initiate flowering, is recalcitrant to in vitro regeneration and transformation. The analysis of gene functions in rapeseed is further complicated by the allotetraploid nature of its genome and the genome triplication within the Brassica genus, which has led to the presence of a large number of gene homologs for each Arabidopsis ortholog. We have established a transformation method that facilitates the regeneration of winter rapeseed by using the WUSCHEL gene from Beta vulgaris. This allowed us to efficiently transform a winter and spring rapeseed genotype in small-scale experiments. As proof of principle, we targeted BnCLV3 and BnSPL9/15 with CRISPR/Cas9 and showed that entire gene families are effectively edited using this transformation protocol. This allowed us to simultaneously study many redundantly acting homologous genes in rapeseed. We observed mutant phenotypes for BnCLV3 and BnSPL9/15 in primary transformants, indicating that biallelic knockouts were obtained for up to eight genes. This allowed an initial phenotypic characterization to be performed already a few months after starting the experiment.