“Clean Gene” transformation technology for rice
Philippe Vain – John Innes Centre
Plant transgenic approaches are still limited by uncontrolled factors affecting the integration and behaviour of transgenes. Strategies to control transgene integration and to limit unwanted or multiple integrated DNA sequences are central to the developmentof advanced plant transformation technologies. Among these, the production of marker-free transgenic plants is particularly important. Selectable marker genes required for the transformation process are generally unwanted, especially in sequential transformation experiments or in transgenic crops.
A new “clean gene” technology has been developed to generate transgenic rice plants free of undesirable selectable marker genes (such as antibiotic resistance genes) and containing simple transgenic locus. This technology involves the use of a natural vector for plant transformation (Agrobacterium tumefaciens) containing multiple binary plasmids (pGreen/pSoup system). pGreen is a small binary plasmid unable to replicate in Agrobacterium without the presence of another binary plasmid, pSoup, in the same strain. Both pGreen and pSoup can carry a T-DNA with different transgenes. When co-transferred into the rice genome, the transgenes carried by pGreen (in blue Fig1) and pSoup (in red Fig 1) can integrate at unlinked loci allowing the recovery of rice plant progeny containing only the gene of interest (on pGreen T-DNA) but without any selectable marker gene (on pSoup T-DNA).
Transgenic locus composition and T-DNA linkage configuration were determined in a large population of transgenic rice plants transformed with pRT binary vectors (based on the pGreen/pSoup system) in order to assess the performance of the “clean gene” strategy (Afolabi et al., 2004). Around one third of the transgenic loci generated by this system contained only the pGreen-based pRT T-DNA and 2% of the progeny plants recovered were free of selectable marker gene (plant containing only the gene of interest carried by the pGreen-based pRT T-DNA). The pRT vectors have been used to produce rice plants containing only cystatin gene for nematode resistance.
