Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low‐seed, high‐leaf glucosinolate oilseed mustard with uncompromised defence and yield.

Summary: Glucosinolate content in the two major oilseed Brassica crops—rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 μmoles/g of seed dry weight, DW), making the protein‐rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9‐based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR‐edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR‐edited lines displayed a defence response at par or better than that of the wild‐type line. The GTR‐edited lines were equivalent to the wild‐type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low‐seed, high‐leaf glucosinolate lines with an uncompromised defence and yield. [ABSTRACT FROM AUTHOR]