Plant Transformation Using Agrobacterium tumefaciens

Agrobacterium tumefaciens is a widespread naturally occurring soil bacterium that causes crown gall, and has the ability to introduce new genetic material into the plant cell (Gelvin, 2003). The genetic material that is introduced is called T DNA (transferred DNA) which is located on a Ti plasmid. A Ti plasmid is a circular piece of DNA found in almost all bacteria.

This natural ability to alter the plant’s genetic makeup was the foundation of plant transformation using Agrobacterium. Currently, Agrobacterium-mediated transformation is the most commonly used method for plant genetic engineering because of relatively high efficiency. Initially it was believed that this Agrobacterium only infects dicotyledonous plants, but it was later established that it can also be used for transformation of monocotyledonous plants such as rice.

During transformation, several components of the Ti plasmid enable effective transfer of the genes of interest into the plant cells. These include:

  • T-DNA border sequences, which demarcate the DNA segment (T-DNA) to be transferred into the plant genome
  • vir genes (virulence genes), which are required for transferring the T-DNA region to the plant but are not themselves transferred, and
  • modified T-DNA region where the genes that cause crown gall formation are removed and replaced with the genes of interest.

Agrobacterium-mediated Plant Transformation Process

The Agrobacterium-mediated transformation process involves a number of steps: (a) isolation of the genes of interest from the source organism; (b) development of a functional transgenic construct including the gene of interest; promoters to drive expression; codon modification, if needed to increase successful protein production; and marker genes to facilitate tracking of the introduced genes in the host plant; (c) insertion of the transgene into the Ti-plasmid; (d) introduction of the T-DNA-containing-plasmid into Agrobacterium; (e) mixture of the transformed Agrobacterium with plant cells to allow transfer of T-DNA into plant chromosome; (f) regeneration of the transformed cells into genetically modified (GM) plants; and (g) testing for trait performance or transgene expression at lab, greenhouse and field level. Figure 1 illustrates Agrobacterium-mediated plant transformation. .

Figure 1: Agrobacterium-mediated Plant Transformation Process

Source: http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF

The overall advantages of using Agrobacterium-mediated transformation over other transformation methods are: reduction in transgene copy number, and intact and stable integration of the transgene (newly introduced gene) into the plant genome (Jones et al., 2005).

Further Reading

  • Gelvin B. S. (2003) Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool. Microbiology and Molecular Biology Reviews 67(1): 16–37

Literature Cited

  • Jones D.H., Doherty A, and Wu H. (2005) Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 1: 5
  • Kumar K.K. , Maruthasalam S., Loganathan M., Sudhakar D. and Balasubramanian P. (2005) An Improved Agrobacterium-Mediated Transformation Protocol for Recalcitrant Elite Indica Rice Cultivars. Plant Molecular Biology Reporter 23: 67–73
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