| Cultivated alfalfa, Medicago sativa L., is one of the most important leguminous forage, with the area of about 350 million hectares in the world and 13 million hectares in China. Compared with other forage species, cultivated alfalfa has higher protein content (17%-20%) and suitable feeding value. However, in order to match the greater demand of forage yield in stockbreeding, it is necessary that a number of salt-tolerant alfalfa varieties should be cultivated, especially with the extension of saline soil in grassland, the devastation of environmental balance in grassland, and the vegetation degeneration in the plain. In the process of breeding salt-tolerant alfalfa varieties, trans-gene technology is a more effective method than the traditional protocol, which has the defection of long period and slow effect. In present study, MsNHX1, cloned from salt-stress alfalfa, was inserted into the expression vector PBI121, and constituted the plant expression vector named PBINHX, and then PBINHX was introduced into Agrobacterium tumefaciens LBA4404. To identify the function and the expression level of MsNHX1, alfalfa, tobacco, and Arabidopsis were transferred by LBA4404. In the process of transferring alfalfa by leaf disks, we improved the tissue culture of alfalfa successfully, and achieved the high-frequency regeneration system with the frequencies of callus, differentiation, and seedling beyond 80%, which implied a stable basis for alfalfa transformation. After screened by antibiotic, the regeneration plants were obtained and some of them were positive lines by PCR analysis. To understand the physiological function of MsNHX1 under salt stress, we constructed an over-expression vector of MsNHX1 and gained the over-expression plants of tobacco and Arabidopsis. By preliminary analysis of phenotype, compared with wild plants, transgenic plants showed better performance in seed germination and seedling survival under NaCl stress. In 50mM NaCl, the seeds of wild type were more intensively inhibited than those of transgenic tobacco, with 66% decline of seed germination rate. In 200 mM NaCl, the damage degree of wild plants was greater than transgenic tobacco. By preliminary analysis of phenotype, the seedlings of transgenic Arabidopsis showed greater tolerance under NaCl stress. |
