Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool

Abstract

Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this "natural genetic engineer" for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.

FIG. 1.

Schematic representation of a typical octopine-type Ti plasmid (A) and the T-DNA region of a typical octopine-type Ti plasmid (B). (A) The T-DNA is divided into three regions. T_L (T-DNA left), T_C (T-DNA center), and T_R (T-DNA right). The black circles indicate T-DNA border repeat sequences. oriV, the vegetative origin of replication of the Ti plasmid, is indicated by a white circle. (B) The various T-DNA-encoded transcripts, and their direction of transcription, are indicated by arrows. Genes encoding functions involved in auxin synthesis (auxin), cytokinin synthesis (cyt), and the synthesis of the opines octopine (ocs), mannopine (mas), and agropine (ags) are indicated.

FIG. 2.

Schematic representation of the steps involved in gene replacement by double homologous recombination (homogenotization [107, 277]). The green lines represent regions targeted for disruption. (A) An antibiotic resistance gene (in this case, encoding a β-lactamase that confers resistance to carbenicillin) has been inserted into the targeted gene that has been cloned into an IncPα plasmid (containing a kanamycin resistance gene [kan] in its backbone) and introduced into Agrobacterium. Double homologous recombination is allowed to take place. (B) Following double homologous recombination, the disrupted gene is exchanged onto the Ti plasmid (pTi). (C) A plasmid of the same incompatibility group as the first plasmid is introduced into Agrobacterium. An example is the IncPα plasmid pPH1JI, containing a gentamicin resistance gene (gent). (D) Because plasmids of the same incompatibility group (in this case IncPα) cannot replicate independently in the cell at the same time, selection for gentamicin resistance results in eviction of the other IncPα plasmid, onto which has been exchanged the wild-type gene.