Large-Scale Isolation of Ti Plasmid DNA

Use of the Agrobacterium tumefaciens Ti plasmids to introduce foreign genes into many plant species is now firmly established (1 ). Ti plasmids, which are of tern single copy molecules, typically of 180–220 kilobases (kb), are divided into three main groups: nopaline, octopine, and agropine types. This classification (2 ) is based on the synthesis of these unusual amino acids in plant cells transformed by these plasmids. Their synthesis is governed by the expression of T-DNA encoded genes that become integrated into the host plant DNA in the course of infection. A feature thought to be important in this transfer and integration is the 25-base pair (bp) imperfect direct repeat sequences (3 ) separated by the 22 kb of nopaline T-DNA, or the TL-(13 kb) and TR-(8 kb) of the bipartite octopine type T-DNA (4.5; see Fig.1 ). Other important features of the Ti plasmids include; the large virulence region, essential for transfer of the T-DNA; the ori region, responsible for the Ti plasmid replication functions; the opine catabolism region, which is associated with transfer of Ti plasmids from one bacterium to another (6 ).