Abstract:Genotype is a key restrictive factor for genetic transformation in alfalfa. A high frequency-regeneration potential alfalfa cultivar named Gongnong No.1 was screened from 7 cultivars by embryonic callus inducing test， and was successfully used in establishing a high efficiency genetic transformation system. Additionally, we analyzed several factors affecting the genetic transformation and optimized the suspension culture conditions, and established sonication-assisted Agrobacterium-mediated transformation system in alfalfa primarily. The suspension callus from alfalfa hypocotyls were taken as explants, as concentration was determined at 100 μmol·L-1, sonication treatment time was 8 s, kanamycin concentration was 30 mg·L-1 and co-culture time was 4 days. Conclusively, large numbers of transgenic plants were obtained by the transformation system. Molecular analysis confirmed that exogenous vacuolar Na+/H+ antiporter gene from Suaeda corniculata was integrated into the genome of alfalfa.