Mini–Ti: A New Vector Strategy for Plant Genetic Engineering

Abstract

Agrobacterium tumefaciens has the natural ability to insert a specific part of its Ti (tumor–inducing) plasmid, called T–DNA, into the chromosomal DNA of host plant cells, causing tumorous growth. Foreign DNA artificially introduced into T–DNA is inserted into the plant genome by this natural vector system. Genes on the Ti plasmid required for T–DNA transfer, called VIR (virulence) genes, are situated outside T–DNA. We have separated T–DNA from the VIR functions onto a MINI–Ti plasmid that replicates in E. coli and contains full–length T–DNA (including the 25 basepair border signals) from pTi T37, a nopaline–type Ti plasmid. We present evidence that MINI–Ti, when transferred to Agrobacterium by cointegration with a shuttle vector, in the presence of helper plasmid pAL4404 containing VIR genes, can incite nopaline positive tumors, evidence of T–DNA transfer. It is not clear whether MINI–Ti functions in trans to the VIR genes, or whether spontaneous cointegration of pAL4404 with MINI–Ti/pRK (the shuttle form of MINI–Ti) accounts for the tumor–inducing ability of the bacteria. The MINI–Ti approach simplifies procedures for exploiting T–DNA as a gene vector for plants.