Abstract: Stems of Populus simonii×P. nigra ‘Baicheng’ in vitro plants were selected as explants and MS medium was used to establish tissue culture system by adjusting the hormone concentrations of 6-BA, NAA, TDZ and IBA. Based on the tissue culture system, the optimal concentration of kanamycin and infection time were confirmed to establish Agrobacterium tumefaciens-mediated genetic transformation system for P. simonii×P. nigra ‘Baicheng’. By using this system, the transgenic plants overexpressed a key tension wood formation regulator LBD39（Lateral Organ Boundaries Domain） were created successfully. The results showed that the tissue culture system consisted of three stages, including adventitious bud differentiation induction(MS+ 0.5 mg·L^-1 6-BA+0.1 mg·L^-1 NAA+0.001 mg·L^-1 TDZ, shoot differentiation rate=92.6%), stem induction(MS+0.2 mg·L^-1 6-BA+0.05 mg·L^-1 NAA, multiplication coefficient=6.5) and rooting induction(1/2 MS+0.4 mg·L^-1 IBA, rooting percentage=100%). The optimal kanamycin concentration for genetic transformation was 30 mg·L^-1 and the optimal infection time was 20 min, and the transgenic plants were obtained successfully after 30 d of adventitious bud differentiation induction, 15-30 d of stem induction and 25 d of rooting induction with 2% transformation efficiency respectively. By using this system, five overexpressed plants of LBD39 were obtained, and the transformation efficiency was 3.3%.