BREEDING “CLEAN” TRANSGENIC ORNAMENTAL PLANTS

The risk that selectable marker genes may cause damages, either to human health or to environment, is raised in public opinion, but is not documented by scientific evidences.
The generation of “marker-free” plants would certainly contribute to the public acceptance of transgenic crops.
An additional step towards “clean” transgenic plant production is to avoid the insertion of ancillary DNA sequences.
We reported here our preliminary results to assemble Agrobacterium vectors suitable for the production of either “marker free” or “clean” transgenic plants.
The plasmid pGreen rolC does not contain antibiotic resistance genes on the T-DNA and then is suitable for “marker-free” transgenic plant production.
In a successive experiment the pGreen T-DNA was replaced with a 44 bp sequence containing exclusively multiple cloning sites (pGreen II adapter). The pGreen II adapter can be considered a “clean” vector, suitable for further cloning.
The pGreen II rolC and the pGreen II rolABC are the first derivative.
Plenty of shoots were regenerated in absence of selective antibiotic in the medium, following co-cultivation of leaf explants of O. ecklonis with A. tumefaciens AGL1 carrying pGreen rolC, pGreen II rolC or pGreen II rolABC. Six plant clones (18%), obtained following co-cultivation with A. tumefaciens pGreen rolC are expected to be of transgenic nature following PCR amplification.
Two of them, showed the characteristic phenotype of plants expressing rolC. RT-PCR confirmed the rolC expression in a plant clone.
The use of vectors carrying useful gene only, is proposed for the recovery of “clean” transgenic plants in absence of in vitro selection pressure.