A plasmid rescue technique for the recovery of plant DNA disrupted by T-DNA insertion

Arabidopsis mutants generated by insertion of the T-DNA from Ti plasmid 3850∶1003 serve as a starting point for the isolation of novel genes. The disrupted plant DNA can be recovered using a plasmid rescue technique utilizing high efficiency electroporation. Rescued plasmids are resistant to ampicillin and contain an origin of replication from pBR322. Plasmids generated from either the left or right border of the T-DNA that carry flanking DNA sequences can be identified by analyzing the products of restriction enzyme digests on agarose gels. The plasmids with flanking sequences can then serve as a starting point for cloning plant sequences that share homology to the DNA at the point of T-DNA insertion.