Improved Agrobacterium-mediated co-transformation and selectable marker elimination in transgenic rice by using a high copy number pBin19-derived binary vector.

A high copy number, selectable marker gene (SMG)-free Agrobacterium binary vector pBin19ΔnptII was constructed by deleting the nptII gene from pBin19. The binary vectors with the RK2 and pVS replication origins exist in 12 and 3 copies, respectively, in Agrobacterium. The tobacco osmotin gene (ap24) was cloned in pBin19ΔnptII and the resultant plasmid pBin19ΔnptII-ap24 was mobilized into the Agrobacterium tumefaciens strain C58C1 Rif(r) harbouring the single-copy cointegrate vector pGV2260::pSSJ1. The T-DNA of the cointegrate vector harboured the hph (SMG) and gus genes. Transformation of Oryza sativa L. var. Pusa Basmati1 with Agrobacterium tumefaciens (pGV2260::pSSJ1, pBin19ΔnptII-ap24) yielded 14 independent hyg+/GUS+ transgenic plants. Southern blot analysis with hph and ap24 probes revealed that 12 out of the 14 transgenic plants were co-transformed and harboured hph, gus and ap24 genes. The new multi-copy binary vector yielded 86% co-transformation efficiency. SMG elimination by genetic separation of the cointegrate T-DNA with the hph/gus genes and binary vector T-DNA with the ap24 gene was accomplished in four out of ten primary co-transformants that were forwarded to the T₁ generation.