Genetic transformation of Sr22 gene in a high yielding susceptible cultivar of commercial wheat (Triticum aestivum L.).

Abstract

In this study, the Sr22 gene was isolated and prepared for transformation in disease-susceptible commercial high-yielding wheat (Triticum aestivum L.) cultivar Lasani-2008. The Sr22 fragment was initially inserted in plasmid pUC57 for sequence confirmation before performing further experiments. After confirmation, Sr22 was subcloned in pGreen0029 which helped in further cloning and ligation. pUC57-Sr22 was restricted with Nru1 and BamH1, while pGreen0029 was restricted with EcoRV and BamH1 and ligated. From pGreen0029, Sr22 was eluted and ligated in pJIT163 to insert the 2 × 35S promoter and CaMV terminator using Xho1 and BamH1 and Sal1. At this stage, the expression cassette was completed. The 2 × 35Sp-Sr22-CaMVt was then ligated in pGreen0029 and transferred to Agrobacterium along with pSOUP. pSOUP helped pGreen0029 to insert 2X35Sp-Sr22-CaMVt in the callus of Lasani-2008, along with kanamycin-resistant gene. Transgenic callus was used for regeneration of the whole plant by tissue culture. Transgenic plants were further tested by PCR, qPCR and SDS-PAGE. The transgenic Lasani-2008 showed substantial resistance against stem rust in both seedling and adult plant stages. The results also showed that transgenic Lasani-2008 has increased average yield of grains (i.e., 4893 ± 148kg/ha) as compared to non-transgenic Lasani-2008 (i.e., with average yield of gains 4762 ± 103kg/ha). Sr22 containing lines and the transgenic developed in this study can be used in breeding systems. Transgenic seeds developed will be shared with breeding institutes and breeders should use this information to develop new varieties.