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%.