title: Improved Agrobacterium-mediated co-transformation and selectable marker elimination in transgenic rice by using a high copy number pBin19-derived binary vector.
abstract: 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.
authors.affiliation: Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Palkalai Nagar, Madurai 625021, Tamil Nadu, India.
authors.forename: Rajasekaran
authors.initials: R
authors.lastname: Sripriya
authors.forename: Manoharan
authors.initials: M
authors.lastname: Sangeetha
authors.forename: Chidambaram
authors.initials: C
authors.lastname: Parameswari
authors.forename: Balamani
authors.initials: B
authors.lastname: Veluthambi
authors.forename: Karuppannan
authors.initials: K
authors.lastname: Veluthambi
mesh_terms: D015139:Blotting, Southern; D020090:Chromosome Segregation / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics; D012275:Oryza / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012231:Rhizobium / Q000235:genetics*; D012641:Selection, Genetic; D014026:Nicotiana / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers; D010940:Plant Proteins; C036483:T-DNA; C073139:osmotin protein, Nicotiana tabacum; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D005966:Glucuronidase