Genetic Transformation Of Alfalfa With GmDREB1 Gene And Salt Tolerance Of Three Transgenic Alfalfa Variety Seeds

High salinity is one of the most important environmental limiting factors of forage growth and productivity. There is 100 million ha of arable land in China, of which 6.67 million ha is saline land, and another 0.3 million hectares of saline-alkali wasteland. Grassland in the western of Jilin Province is an important forage producing area, but also one of the most serious saline-alkali lands in China. In recent decades, with the impact of climate change and increasement of intensive human activities, the area of saline-alkali soil and the degree of saline-alkali have grown constantly. Relevant data shows that leguminous grass has almost completely disappeared in some of this region, while the halophytes have been developing rapidly.Alfalfa (Medicago sativa L.), an important leguminous forage plant, has a certain degree of salt tolerance. It is a highly significant way to improve vegetation of Western Jilin, develop and utilize saline-alkali land resources rationally by using plant genetic engineering technique to make specific genetic improvement of alfalfa and to adapt saline-alkali conditions.This study mainly consists of two parts as follows:Part one: The first true leaves of Alfalfa as recipient were infected by Agrobacterium tumefaciens strain LBA4404 which was transformed by pBI121 with GmDREB1 gene, Callus inducted from transgenic leaves were used for plant tissue culture operation with antibiotics (Kan) resistance including differentiation, rooting, and seedlings. Then PCR were used to analyze transgenic alfalfa plants, and the results showed that 119 individual plants were positive. Three kinds of transgenic plants harboring SsNHX1 gene, SsNHX1-PDH45 double gene and GmDREB1 gene were transplanted on Stud-farm, Changling Country, Jilin Province on May 1st, 2009. Among them, the transgenic plants harboring SsNHX1 gene were propagated by cutting and layering methods. In conclusion, 116 transformants with SsNHX1 gene, 112 transformants with SsNHX1-PDH45 double gene and 75 transformants with GmDREB1 gene were obtained, and the seeds were harvested.Part two: Seeds from three transgenic alfalfa transformants with SsNHX1 gene, SsNHX1-PDH45 double gene and transformants with GmDREB1 gene, and Gongnong-1 alfalfa seeds were used to investigate the effects of germination and growth under different gradient of NaCl conditions (0mM, 100mM, 200mM, 300mM and 400mM). By statistical analysis, results showed that the germinating potentiality, germinating rate, germination index, vigor index, root natural weight and nature fresh weight in three transgenic alfalfa seeds were significantly higher than these in Gongnong-1 seeds(p<0.05) with the concentration at 200-400 mM. The final conclusion was that: The integration and expression of stress-resistant genes gave salt resistance of transgenic alfalfa.This research transferred GmDREB1 gene into alfalfa tested by PCR and transgenic plants were obtained. Three transgenic alfalfa were transplanted in field and propagated by cutting and layering. Mass of T1 seeds from transformants were obtained. Salt tolerance assays of three transgenic alfalfa T1 seeds and Gongnong-1 seeds demonstrated that the salt tolerance of transgenic alfalfa T1 seed had an obvious progress compared with the control. It provides the theoretical basis for further research on feasibility of planting in large areas of saline-alkaline land.