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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]

GTR1 and GTR2 transporters are components of the source to sink translocation network of glucosinolates, which are major defence metabolites in the Brassicaceae. These transporters can be genetically manipulated for reduction of seed‐glucosinolates without inhibiting glucosinolate biosynthesis, thereby maintaining the inherent defence potential of plants. However, the different roles of GTRs in influencing tissue‐specific distribution of glucosinolates in agriculturally important Brassica crops are yet unknown. Here, we report functional characterization of two groups of glucosinolate transporters (GTR1 and GTR2) from Brassica juncea based on gene expression data, biochemical analysis, gene‐complementation studies in GTR‐deficient mutants and RNAi‐based knockdown followed by insect feeding experiments. Although both GTRs showed ubiquitous expression patterns and broad substrate specificity, the single‐gene knockdown lines displayed different phenotypes. The GTR2‐knockdown plants showed a significant reduction of glucosinolates in seeds and a higher accumulation in leaves and pods, while the GTR1‐knockdown plants displayed a smaller reduction of glucosinolates in seeds and significantly lower glucosinolate levels in leaves. Consequently, knockdown of GTR2 resulted in higher resistance towards the generalist pest, Spodoptera litura. Overall, our study highlights the distinctive roles of B. juncea GTRs in tissue‐specific accumulation of glucosinolates and the potential for manipulating GTR2 for enhanced nutrition and plant defence. The glucosinolate transporters GTR1 and GTR2 exhibit differential potential for tissue‐specific manipulation of glucosinolate content for enhanced nutrition and defence traits in the oilseed, Brassica juncea. [ABSTRACT FROM AUTHOR]