Alfalfa varieties ‘Zhongmu No.2’ was acted as the wild-type material.Agrobacterium-mediated method was used to introduce dehydrin gene(AmDHN) into alfalfa.PCR and southern blot methods were used to detect the presence of AmDHN in transgenic alfalfa plants.The expressions of AmDHN and cold-related genes were detected in transgenic plants of T₀ generations through RT-PCR and qRT-PCR.The content of proline and MDA of two plants were detected.Cold resistance of the transgenic alfalfa plants was detected by molecular level and physiological characteristics,which will provide a basis for new transgenic alfalfa material with resistance to cold stress.The main results are summarized as follows:(1)Expression of AmDHN was determined at different levels in transgenic alfalfa plants.(2)After being treated with 4 ℃,expressions of cold-related genes(CBF2,CBF3,ProDH and CAS17) were dramatically higher than WT.Expressions of CBF2,CBF3 and CAS17 increased significantly after being treated 5 h,reached an extremely significant level(P＜0.05),as compared with WT plants.The level of ProDH transcripts reached the maximum at 7 d after treatment by 4 ℃,their maximum were 2.5,4,1.6 and 3 times than WT,respectively.(3)The contents of both proline and MDA were increasing with extending treatment time.The content of proline of transgenic plants was significantly higher than that of WT.The content of MDA was significantly lower than that of WT,both goes up to an extremely significant level at 14 d after the treatment.These results suggested that transgenic plants may be more resistance to cold stress compared with WT.These imply that AmDHN proteins may protect the enzyme activity and membrane systems from freezing injury.Simultaneously,AmDHN may regulate indirectly cold tolerance of the plants through regulating the expressions of cold-related genes.