Improved Salt Tolerance Of Alfalfa (Medicago Sativa. L) By Introducing GmDREB1 Gene

Salt stress adversely affects crop productivity and forage yield. With ingravescent fresh water scarcity, the situation of drought and soil salinity get even worse in the arid and semi-arid areas of China. Alfalfa (Medicago sativa.L) is a protein-riched legume forage, acting an important role in agriculture and animal husbandry and ecological construction in arid and semi-arid areas of western JiLin Province and Northeast of China. However, salt stress at 50-200 mM NaCl significantly limits the productivity of alfalfa. Traditional breeding strategies have generated very few crop varieties with improved stress tolerance. Contrary to the classical breeding approaches, direct introduction of genes by genetic engineering seems a more attractive and quick solution for improving stress tolerance. Dehydration-responsive element binding proteins (DREB), specifically interacting with DRE element, could up-regulation of a whole array of downstream genes in response to stress and are regarded as important genetic resources for improving plant stress tolerance.In the current study, a soybean DREB orthologue, GmDREB1, was amplified by using genetic method. Subsequently, we constructed the recombinant expression vectors Rd29A: GmDREB 1:GFP and transformed it into Agrobacterium tumefaciens LBA4404. With Agrobacterium tumefaciens mediated transformation method, GmDREB1 was introduced into alfalfa cultiva, Gongnong1, and thereafter 200 regenerated plants from 4 lines were gained. PCR and Southren blot analysis demonstrated that the exogenous gene had been integrated into the chromosome of all the four transgenic lines. Furthermore, plants from line 29A-2 and 29A-4 carried a single active T-DNA locus and were applied for further analysis. Northern blot analysis shown thant GmDREB1 transcripts were stably detected in salt-treated line 29A-2 and 29A-4 plants. Together with the facts that under normal conditions no morphological differences were found between transgenic plants and wild-type plants, Rd29A promoter was comfirmed to play its role as expected. Moreover, GmDREB1 transgenic plants displayed strong tolerance to moderated salt stress(200mM NaCl). The four physiological indices of transgenic plants were all better than those of wild control under moderated salt stress treatments (200mM NaCl). Northern bolt analysis furthern confirmed thant GmDREB1 increased P5CS transcript levels thus led to more accumulation of free proline in transgenic plants.Overall, we the first to intruduce a DREB gene into alfalfa and improve its salt tolerance. Also we make sure that incorporating a key regulator gene such as DREB gene under the control of Rd29A promoter is an efficient approach to minimize stress damage to crops. Futher study on screening the downstream genes of GmDREB1 and the concrete way of GmDREB1 to affect its downstream genes will be carried out soon in the future.