Fate of selectable marker DNA integrated into the genome of Nicotiana tabacum

Abstract

To compare the effects of different transformation methods on the integration behavior and structural stability of integrated foreign genes in plant cells, tobacco protoplasts were transformed with Escherichia coli plasmid pLGV2103neo DNA using the Ca phosphate DNA coprecipitation technique. Parallel transformations were done by cocultivation with Agrobacterium tumefaciens harboring the Ti plasmid derivatives pGV3850::2103neo or pGV3850::1103neo. A comparison of the fine structure of the integrated donor DNA obtained by direct gene transfer and by cocultivation indicates that the donor DNA in cells transformed by the former technique undergoes structural changes and concatemerizations, while the DNA integrated by the latter procedure is often unaltered. The cotransformed nopaline synthase gene, which is present in the donor Ti plasmid DNA, was inactivated in two out of nine cases. Once integrated, the arrays of selectable marker DNA appear to be structurally stable under different cell culture and selection conditions, as well as after genetic transmission.