Development of genetically modified rust resistant wheat: A breakthrough by dinted introgression of novel DREB2C and HSFA2 genes under stress induced expression

• Addressing stripe rust, a major wheat disease caused by Puccinia striiformis f. sp. tritici (Pst), prevalent in regions such as Pakistan. • Employing transgenic technology to enhance resistance against stripe rust and abiotic stresses in wheat. • Pyramiding HSFA2 and DREB2C genes under dual expression is an effective strategy for controlling stripe rust and providing heat resistance. • Transgenic wheat plants exhibited significant tolerance to stress, with minor spotting under constitutive expression conditions. Wheat is a major staple food worldwide yet numerous yield limiting agents affect its productivity. Stripe rust is a major culprit in this context and efforts have been made to culminate this pathogen using conventional as well as advanced innovative techniques. Transgenic technology is of significant importance in this context and numerous success stories are evident to prove its worth. In the current study, two novel genes HSFA2 and DREB2C were expressed in an elite wheat genotype Akbar, Fakhre-e-Bhakhar under constitutive CAMV35S promoter and stress inducible rd29 Promoters. The shoot cut method was used for the Agrobacterium-mediated transformation and putative transformants were selected on kanamycin 50 mg/L. The resultant transformants were tested through PCR for transgene integration whereas expression analysis was carried out through realtime qPCR. Expression of both of the aforementioned genes was found to be higher under rd29 promoter as compared with transgene(s) expression under CAMV35S promoter. In the bioassay, transgenic wheat plants demonstrated significant tolerance to stress, exhibiting only minor spotting under constitutive expression conditions. Upon exposure to stress, these plants showed exceptional resistance to stripe rust, producing large, bold grains compared to individual trait expressions and negative controls. Subsequently, the DREB2C gene was knocked out to determine if stripe rust control was specifically attributed to this gene. Following the knockout, the onset of stripe rust was comparable to that of the negative control. This led to the conclusion that pyramiding the DREB2C gene with HSFA2 through dual expression represents a novel and highly effective strategy for controlling the widespread stripe rust in wheat. This approach also offers resistance to high temperatures (above 32°C) from the pollination stage through to maturity.