Stacking of Hordeum vulgare vacuolar sodium/proton antiporter and a bar gene in wheat for salt and herbicide tolerance

Abstract

Abiotic stresses and emerging climate change patterns are forecasted to be the biggest challenge to food security. Salt and drought are the critical abiotic stresses responsible for the wheat yield gap with irrigated and fertile lands. In this study, the Hordeum vulgare NHX1 gene, which encodes for vacuolar Na+/H+ antiporter, was transformed in two wheat varieties, FSD-2008 and Galaxy. The HvNHX1 gene expression cassette was developed under a constitutive viral promoter (2X CaMV35S). The construct was assembled in pSB219, a monocot transformation vector containing the herbicide tolerance gene (bar). The transgenic plants were initially screened by two rounds of BASTA selection (2 mg/L and 3 mg/L). PCR later confirmed the putative transgenics. The transformation efficiency was estimated to be 0.4% for Galaxy and 0.2% for FSD-2008, respectively. Expression analysis of the NHX1 gene in T2 transgenics and non-transgenic controls through qRT-PCR revealed a 12 fold higher expression of the transgene in Galaxy and onefold higher expression in FSD-2008. Under salt stress, the transgenic lines displayed increased chlorophyll content, reduced electrolyte leakage, and higher relative water content in their leaves than in the control plants. Moreover, under stress conditions (200 mM NaCl), the transgenic lines yielded higher biomass and seed weight than non-transgenic controls. The results demonstrated that the constitutive expression of the HvNHX1 gene in wheat resulted in better grain yield than parent lines. Additionally, the bar gene co-transformed with the HvNHX1 confers herbicide (BASTA) resistance in salt-tolerant wheat transgenics.