Abstract: In order to establish a highly efficient plant regeneration system suitable for gene transformation, the immature embryos of “Hua 30”, which is a improved barley variety derived from anther culture, was used as explants to study the effects of different medium, hormone combination and carbon sources on callus induction and plant regeneration. The results indicated that the combination medium of modified N6, MS and B5 + 2 mg/L 2, 4-D + 1 mg/L ABA + 30 g/L sucrose was optimal for callus induction. The addition of Cu2+ had effects on inhibiting immature embryos germination and improving callus quality on callus induction. The highest regeneration frequency could be obtained by adding 2 mg/L 6-BA in differentiation medium. Meanwhile, some factors that affect transient expression including bacterial concentrations, infection time and co-culture period were investigated by detecting transient expression of GUS gene to increase the frequency of transient expression of exogenes mediated by Agrobacterium tumefaciens and to optimize the transformation conditions in barley variety “Hua 30”. The results showed that the higher transient expression frequency of GUS gene could be resulted from Agrobacterium tumefaciens bacterial concentration of OD600=0.5, 15 min infection, 2 d co-cultivation.