Abstract: The high frequency regeneration system of Populus pseudo-cathayana ×P. deltodides Barry CV.‘Shan Hai Guan’ was established with leaf explants in our study. Furthermore, the factors related to transformation including Agrobacterium strains, pre-culture time, Agrobacterium concentration, inoculation time and acetosyringone (AS) concentration were evaluated via GUS transient expression. The results indicated that, the optimal adventitious induction culture was MS medium supplemented with 0.5mg.L-1 6-BA and 0.1mg.L-1 NAA, on which the regeneration rate reached 95% and 10.07 average adventitious buds were obtained. The application of Agrobacterium GV3101, no pre-culture, bacterium concentration at OD600=0.4~0.6, 10 min inoculation and supplement of 200μmol/L AS in co-culture medium produced the highest transient GUS expression detected by fluorescence quantification method, which were demonstrated optimal for high frequency transformation of ‘Shan Hai Guan’.