Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium -mediated co-transformation with the same selectable marker gene

Rice chitinase ( chi 11) and tobacco osmotin ( ap 24) genes, which cause disruption of fungal cell wall and cell membrane, respectively, were stacked in transgenic rice to develop resistance against the sheath blight disease. The homozygous marker-free transgenic rice line CoT23 which harboured the rice chi 11 transgene was sequentially re-transformed with a second transgene ap 24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260∷pSSJ1 and a multi-copy binary vector pBin19∆ npt II- ap 24 in the same cell. pGV2260∷pSSJ1 T-DNA carried the hygromycin phosphotransferase ( hph ) and β-glucuronidase ( gus ) genes. pBin19∆ npt II- ap 24 T-DNA harboured the tobacco osmotin ( ap 24) gene. Co-transformation of the gene of interest ( ap 24) with the selectable marker gene (SMG, hph ) occurred in 12 out of 18 T 0 plants (67%). Segregation of hph from ap 24 was accomplished in the T 1 generation in one (line 11) of the four analysed co-transformed plants. The presence of ap 24 and chi 11 transgenes and the absence of the hph gene in the SMG-eliminated T 1 plants of the line 11 were confirmed by DNA blot analyses. The SMG-free transgenic plants of the line 11 harboured a single copy of the ap 24 gene. Homozygous, SMG-free T 2 plants of the transgenic line 11 harboured stacked transgenes, chi 11 and ap 24. Northern blot analysis of the SMG-free plants revealed constitutive expression of chi 11 and ap 24. The transgenic plants with stacked transgenes displayed high levels of resistance against Rhizoctonia solani . Thus, we demonstrate the development of transgene-stacked and marker-free transgenic rice by sequential Agrobacterium -mediated co-transformation with the same SMG.