Novel pGreen/pSoup dual-binary vector system in multiple T-DNA co-cultivation as a method of producing marker-free (clean gene) transgenic rice (Oryza sativa L.) plants
The possibility of producing marker-free transgenic rice plants using a novel dual binary pGreen/pSoup vectors, in multiple T-DNA co-cultivation, was investigated and demonstrated to be feasible. The T-DNA in pSoup (pRT47) vector was engineered to contain the selection marker hygromycin phosphotransferase (aphIV) gene ( plus intron in 5 UTR), and the green fluorescent protein (gfp) as a reporter gene both driven by the CaMV35S promoter and the nopaline synthase terminator. T-DNA in the pGreen (pRT18) vector harboured the phosphinothricin acetyl transferase ( bar), as selection marker gene, and the beta- glucuronidase (gusA) plus intron as a reporter gene, both driven by the maize 5 ubiquitin region and the nopaline synthase terminator. Both the pGreen and pSoup plasmids were transformed into E. coli strain DH5 using the PEG-transformation technique and into Agrobacterium strains AGL1using a freeze-thaw method. AGL1 was then used to transform embryogenic nodular units (ENU), derived from mature seeds of the model rice genotype Nipponbare. Selection on herbicide (PPT) or antibiotic ( hygromycin) of co- cultured ENUs led to the production of numerous independently transformed callus clones containing both T-DNAs from the selected and unselected vector. While co-transformation frequencies were 71% and 80% for the hygromycin only and herbicide ( PPT) only selection, respectively, data showed that co- expression frequency is most useful for the production of marker free transgenic rice. About half (50%) of the independent transgenic plant lines contained at least one unlinked T-DNA integration. In this work, we showed for the first time, that the novel dual-binary pGreen/ pSoup can efficiently produce marker-free transgenic rice.
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