Salt-tolerant transgenic perennial ryegrass (Lolium perenne L.) obtained by Agrobacterium tumefaciens-mediated transformation of the vacuolar Na+/H+ antiporter gene

The objective of this study was to obtain a salt-tolerant perennial ryegrass (Lolium perenne L.) by transforming it with a rice vacuolar membrane Na+/H+ antiporter gene via the Agrobacterium-mediated method. To optimize the transformation conditions, two Agrobacterium tumefaciens strains, LBA4404 and EHA105, carrying plasmid pCAMBIA3301, were used to transform embryogenic calli of perennial ryegrass; two factors affecting transformation efficiency, acetosyringone and mannitol, were then determined. Addition of acetosyringone during the incubation of bacteria and the co-cultivation process increased the transformation rate. Transformation efficiency could be further increased by osmotic treatment with 0.4 mol/l mannitol before infection. Using this optimized transformation system, the rice vacuolar Na+/H+ antiporter gene, OsNHX1, was transferred into perennial ryegrass by Agrobacterium-mediated method. The transformants were confirmed by both PCR and Southern blot analysis. The resultant transgenic ryegrass had better salt-tolerance. After stress treatment for 10 weeks with 350 mmol/l NaCl, transgenic plants survived, while wild-type plants did not. The leaves of transgenic plants accumulated higher concentrations of Na+, K+ and proline than those of the control plants. These results demonstrate the OsNHX1 gene was successfully transferred into perennial ryegrass by Agrobacterium-mediated method, and the salt-tolerance of perennial ryegrass was improved by overexpression of the OsNHX1 gene.