Establishment Of An Agrobacterium-mediated Transformation System And Obtaining Of Salt-, Freezing-Tolerant Transgenic Perennial Ryegrass (Lolium Perenne L.)

Perennial ryegrass {Lolium perenne L.) is one of the most important turfgrass species, Currently, worldwide water supply is waning and the salinity of soil is increasing. To grow on these dry and salty lands, grasses such as turfgrass need to have salt-tolerance. Turfgrass grow in dry area can reduce the vaporization of water and decrease the accumulation of soluble salt on soil surface, thus maintain or even improve the soil condition. Furthermore, salt-tolerant turfgrass can be irrigated with secondly cleansed water containing high salinity. At the same time, each year most of the turfgrass are faced with different kinds of winter injury in transition and northern climate region, which had great effects on turf qulity. The purpose of the paper is to obtain salt-tolerant and freezing- or drought-tolerant perennial ryegrass by over-expression of OsNHX1 and AtDREBlA gene.The protocol of efficient regeneration system of perennial ryegrass was studied at first. Callus formation and regeneration via embryogenic calli were observed in all the six used cultivars seeds. Growth regulators and their concentration supplemented had great effect on callus induction and regeneration of perennial ryegrass. The optimized tissue culture conditions for seeds of perennial ryegrass were supplementation of 9mg/l 2, 4-D in callus induction medium, 3mg/l 2, 4-D in embryogenic calli medium, 0.1mg/l 2, 4-D and 0.1mg/l 6-BA in callus regeneration medium. No growth regulators were used in root induction medium. Under the most optimized conditions, significant difference were found among the cultivars with 'TopGun' having the highest callus induction and regeneration rates which were 92.6% and 48.9%, respectively. Using the optimized regeneration conditions, two factors affecting transformation efficiency, acetosyringone and mannitol, were determined. Addition of 20μM acetosyringone during the incubation of bacteria and the co-cultivation process and osmotic treatment with 0.4mol/l mannitol for 12h before infection increased transformation efficiency to 19.39%.Using the optimized transformation system, the rice vacuolar Na+/H+ antiporter gene, OsNHXl and Arabidopsis AtDREBlA gene were transferred into perennial ryegrass by Agrobacterium-mediated method.Four resistant lines were obtained by Agrobacerium-mediated transformation of a vacuolar Na+/H+ antiporter OsNHXl gene in rice. PCR and hybridization indicated that the OsNHXl cDNA have been inserted into perennial ryegrass genome. The obtained transgenic ryegrass showed better salt-tolerance. The leaves of transgenic plants accumulated higher contents of Na+, K+ and Pro than those of the control plants.Transgenic plants over-expressing AtDREBlA exhibited dwarf phenotype compared with the wild-type plants. The leaf length and width of transgenic plants were considerably shorter than the wild-type plants. The leaves of transgenic plants showed dark-green in conrast to the wild-type plants. Compared with the wild-type plants, the content of chlorophyll of transgenic plants increased. Transgenic plants over-expressing AtDREBlA displayed high level of tolerance to freezing. The content of MDA in transgenic plants was lower than that of wild-type whichindicated that the damage to the membrane of transgenic plants was lighter than that of wild-type plants.In the paper, OsNHX1and AtDREB1A genes were successfully transferred into perennial ryegrass by Agrobacterium-mediated method, the transgenic plants showed salt- and freezing- tolerance, which can lay good foundations for turfgrass improvement, quicken the breeding process thus provide significant economic and social values.