Avni Mann, Juhi Kumari, Roshan Kumar, Pawan Kumar, Akshay K. Pradhan, Deepak Pental, Naveen C. Bisht

Plant Biotechnology Journal; First published: 04 August 2023; https://doi.org/10.1111/pbi.14121

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.

See https://onlinelibrary.wiley.com/doi/10.1111/pbi.14121?fbclid=IwAR0PbSL6y0PpnWJ8Tod-RfI82HVgZQnSkOU4KxwiTYjHnSt8Po1X7aOp1Ro

Fig. 1: Genomic location of gRNA target sites and generation of GTR1::GTR2(GEd) construct. (a) Genomic localization of target sites of three gRNA viz., gRNA1 (targeting BjuGTR1-A1, BjuGTR1-B1, BjuGTR1-A2, and BjuGTR1-B2), gRNA2 (targeting BjuGTR2-A1, BjuGTR2-B1, and BjuGTR2-B2), and gRNA3 (targeting BjuGTR2-A2, BjuGTR2-A3, and BjuGTR2-B3) were designed from the second exon of the target BjuGTR genes. (b) T-DNA map showing SpCas9-based BjuGTR1::GTR2(GEd) transformation construct used for targeting the BjuGTRs in B. juncea cv. Varuna. The three sgRNA fragments containing gRNA1, gNRA2, and gRNA3 were cloned sequentially into the multiple cloning sites of pZP200debar:SpCas9 vector, containing the bar gene as the plant selection marker.