Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco
The transfer DNA (T-DNA) mobilized into plant cells by Agrobacterium tumefaciens seems to integrate rather randomly into the plant genome. We analyzed a target site in the genome of Nicotiana tabacum before and after integration of a T-DNA. Clones presenting right and left T-DNA/plant DNA junctions were used as probes to identify and isolate a unique 1.8-kilobase EcoRI fragment corresponding to the plant DNA target site for a T-DNA insertion event. Comparison of the nucleotide sequences of the plant DNA portions of the T-DNA junction clones with the original plant DNA target revealed that several types of rearrangements resulted from insertion of the T-DNA. The most dramatic alteration was a 158-base-pair direct repeat of target plant sequences at the left and right T-DNA junctions. In addition, there were deletion and insertion events at the ends of the right and left copies of the 158-base-pair repeat. The variety of target-site rearrangements suggests that T-DNA insertion is a multistep process of recombination accompanied by local replicative and repair activities mediated by host-cell enzymes.