Obtaining marker-free plants with high efficiency will benefit the environmental release of transgenic crops.To achieve this point,a binary vector with three T-DNAs was constructed using several mediate plasmids,in which one copy of BAR gene expression cassette and two copies of VIP1 gene expression cassette were included.EHA101 Agrobacterium strain harboring the final construct was applied to transform soybean cotyledon nodes.Through 2-3 months regeneration and selection with 3-5 mg·L-1 glufosinate,transgenic soybean plants were obtained at 0.83%-3.16%,and the co-transformation efficiency of both genes in the same individual reached up to 86.4%,based on the southe blot test.Using PCR analysis,southe blot and northe blot tests,as well as leaf painting of herbicide in T1 progenies,41 plants were eliminated of BAR gene with the frequency of 7.6%.Among the T1 populations tested,the loss of the alien genes happened in 22.7% lines,the silence of the BAR gene took place in 27.3% lines,and VIP1 gene silence existed in 37.1% marker-free plants.The results also suggested that the plasmid with three T-DNAs might be an ideal vector to generate marker-free genetically modified organisms.