ABSTRACT
Because of their large size (100 kb), Ti plasmids themselves are not readily manipulated. However, two characteristics of the DNA mobilization machinery have made it possible to harness Agrobacterium as a means of gene delivery, so that today the bacterium provides the preferredmethod for transforming cells of both dicotyledonous andmonocotyledonous plants. The first feature is that the vir genes and the T-DNA need not reside on the same plasmid [11,12]. Ti plasmids that are disarmed by removal of the T-DNA segment propagate in A. tumefaciens but are incapable of producing crown gall tumors. Virulence is restored in an A. tumefaciens cell carrying a disarmed Ti plasmid when the Ti plasmid’s T-DNA is introduced into the cell as part of a separate replicon [11]. The second significant characteristic concerns the nature of the T-DNA itself. The T-DNA region of a Ti plasmid encodes genes for the synthesis of both plant growth hormones and unusual amino acid derivatives known as opines. Examples of opines include octopine, histopine, nopaline, mannopine, and agrocinopine [9]. These derivatives serve as nutrients for the A. tumefaciens cells and constitute the benefit the bacteria derive from the expression of the opine genes within the plant. Although the hormone and opine genes play an integral part in crown gall tumor formation, they are not necessary for mobilization of the T-DNA. Any DNA element that is placed between the RB and LB can be mobilized by the vir gene products. These two properties have permitted the adaptation of the Ti plasmid into a binary plasmid system for DNA delivery (Fig. 1). A donor vector able to replicate in both A. tumefaciens and Escherichia coli carries the RB and LB sequences between which one or more genes of interest may be cloned. Following introduction of the donor plasmid into an Agrobacterium strain that carries a resident disarmed Ti plasmid, the genes that are flanked by the border sequences are subject to mobilization upon activation of the vir genes. Most often activation is achieved by the exogenous addition of acetosyringone.
