ISSN 2169-3048
International Journal of Biochemistry and Biotechnology ISSN 2169-3048 Vol. 9 (3), pp. 001-010, March, 2020. © International Scholars Journals
Full Length Research Paper
Novel pGreen/pSoup dual-binary vector system in multiple T-DNA co-cultivation as a method of producing marker-free (clean gene) transgenic rice (Oriza sativa L) plant
A.S. Afolabi1, 2,* B. Worland1, J. Snape1 and P. Vain1
1Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, U.K.
2Department of Biological Sciences, University of East Anglia, Norwich, U.K.
Accepted 14 September, 2019
Abstract
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 b - 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.
Key words: pGreen/pSoup, dual-binary vectors, T-DNA co-cultivation, marker-free, clean gene, transgenic, rice, hygromycin, PPT.