Recombination of selectable marker DNA in Nicotiana tabacum

A chimeric neomycin phosphotransferase II (NPT II) gene, which normally provides kanamycin resistance to transformed plant cells, was inactivated by in vitro deletions. Repair plasmids not containing plant-specific transcription signals but containing only the NPT II coding region (or parts of it) were used in co-transformation experiments involving direct DNA uptake into protoplasts isolated from Nicotiana tabacum. Recombination, or gene conversion mediated by homologous sequences produced active NPT II genes in about 1% of transformants, rendering these cells resistant to kanamycin. Analysis of the size of the active enzyme indicated that recombination had occurred producing an NPT II gene indistinguishable from the wild-type gene. Southern blot analysis revealed that the bulk of co-transformed donor plasmid DNA had suffered structural modifications; however, kanamycin resistance was inherited in a Mendelian fashion, indicating that at least one functional and structurally intact copy of the regenerated NPT II gene is integrated into the host genome.