Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.)

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T-0 plants were edited very efficiently (56.30%-67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi-allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T-1 and T-2 progeny. We also obtained transgene-free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off-target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi-dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi-dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.