title: Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene.
abstract: Rice chitinase (chi11) and tobacco osmotin (ap24) 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 chi11 transgene was sequentially re-transformed with a second transgene ap24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260::pSSJ1 and a multi-copy binary vector pBin19∆nptII-ap24 in the same cell. pGV2260::pSSJ1 T-DNA carried the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes. pBin19∆nptII-ap24 T-DNA harboured the tobacco osmotin (ap24) gene. Co-transformation of the gene of interest (ap24) with the selectable marker gene (SMG, hph) occurred in 12 out of 18 T(0) plants (67%). Segregation of hph from ap24 was accomplished in the T(1) generation in one (line 11) of the four analysed co-transformed plants. The presence of ap24 and chi11 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 ap24 gene. Homozygous, SMG-free T(2) plants of the transgenic line 11 harboured stacked transgenes, chi11 and ap24. Northern blot analysis of the SMG-free plants revealed constitutive expression of chi11 and ap24. 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.
authors.affiliation: Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India.
authors.forename: Mangu Venkata
authors.initials: MV
authors.lastname: Ramana Rao
authors.forename: Chidambaram
authors.initials: C
authors.lastname: Parameswari
authors.forename: Rajasekaran
authors.initials: R
authors.lastname: Sripriya
authors.forename: Karuppannan
authors.initials: K
authors.lastname: Veluthambi
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D018628:Gene Dosage; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005819:Genetic Markers; D005822:Genetic Vectors; D006720:Homozygote; D007113:Immunity, Innate; D012275:Oryza / Q000235:genetics* / Q000276:immunology / Q000382:microbiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010935:Plant Diseases / Q000276:immunology / Q000382:microbiology; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000276:immunology / Q000382:microbiology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012232:Rhizoctonia / Q000472:pathogenicity; D014170:Transformation, Genetic; D019076:Transgenes
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; D002688:Chitinases