Abstract: In order to establish a high frequency and rapid regeneration system for the genetic transformation of alfalfa. Six cultivars of alfalfa were applied to investigate some factors which affect alfalfa genetic transformation system, such as ultrasonication, the concentration and time of agrobacterium tumefaciens infection and so on, by sonication assisted agrobacterium-mediated. Experimental results showed that, the best combination for alfalfa cotyledon transformation and resistant callus information were as follows: the concentration of agrobacterium tumefaciens infection OD600 was 0.4-0.5, and the appropriate time of infection was 10-15 min, with 10 minutes’ of ultrasonication in the meantime. PCR amplification of the DNA of kanamycin resistance callus revealed that, transformant and plasmid DNA amplified about 546 bp distinct stripe with the length respectively, which means salt tolerance gene SeNHX1 have integrated into alfalfa callus.