Abstract: The GM-gene deletor which was induced by pAB5 promoter was transformed into hybrid poplar clone 353 by Agrobacterium tumefaciens mediated transformation method. Using the orthogonal design method with the frequency of explants obtained resistant shoots as measure index, several key factors of transformation were optimized. The result showed that the optimized system bacteria cell density (OD₆₀₀) 0.8, 8 min infection incubation, 200 μmol/L acetosyringone in co-culture medium, 35 mg/L kanamycin, 350 mg/L cefotaxime in differentiation medium, and the selecting methods in rooting phase were applied under the selection pressure of 25 mg/L kanamycin. By adopting the optimized system of genetic transformation, the frequency of explants obtained resistant shoot of clone 353 was increased to 80.00%. PCR detection showed that the GM-gene deletor expressed in the genome of Populus. This optimized transformation system provides technical support for the detection of effectiveness of GM-gene deletor system in Populus.