HAU16008-02-9 - Agrobacterium-Mediated Plant Transformation...

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35 From: Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and Protocols Edited by: L. Peña © Humana Press Inc., Totowa, NJ 2 Plant Transformation Agrobacterium -Mediated Gene Transfer Abhaya M. Dandekar and Henry J. Fisk Summary Plant transformation is the process by which DNA is introduced into plant cells or tissues. The DNA can come from virtually any source. Gene transfer methodology has become part of an essential technology to manipulate plants for both scientific and com- mercial purposes. Transgenic plants, the products of this methodology, are useful for dissecting the mechanism(s) of plant gene regulation. This technology is also useful for identifying and evaluating agriculturally useful traits (genes) as well as for their intro- duction into commercially valuable crops. One of the most efficient methods for gene transfer employs Agrobacterium tumefaciens and takes advantage of the naturally evolved crown gall-inducing mechanisms of DNA transfer present in this common soil pathogen. Much has been learned about the mechanisms of this form of DNA movement and subsequent crown gall induction. This information has been applied to develop meth- ods that result in the formation of gall-free, genetically transformed plants. This chapter describes a detailed protocol for Agrobacterium -mediated transformation of tobacco cells and their subsequent selection and regeneration into transgenic plants. Key Words: Agrobacterium tumefaciens ; genetic selection; plant transformation; regeneration; transgenes, transgenic plants. 1. Introduction The production of transgenic plants involves the marriage of two critical yet distinct basic technologies. The first directs the introduction of new genetic material into plant cells (transformation); whereas the second uses methods based in tissue culture to regenerate the resulting transformed cells into transgenic plants. Of the various methods developed to introduce DNA into plant cells, most include a transformation step that is mediated by Agro-
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36 Dandekar and Fisk bacterium tumefaciens (1–4) . In nature, Agrobacterium tumefaciens is the causative agent of crown gall disease and was discovered at the turn of the last century. However, approx 75 yr passed before it was determined that this ubiq- uitous soil microorganism is capable of interkingdom DNA transfer (5) . The crown gall (tumor) represents a manifestation of the transfer and expression of bacterial DNA in plant cells. This highly evolved and elegant mechanism of transforming plant cells has been harnessed by plant biotechnologists for both knowledge and profit. For the purposes of this chapter, we highlight some of the salient features of Agrobacterium -mediated transformation of plant cells and their regeneration into transgenic plants as these features need to be under- stood from a basic perspective to carry out effectively the procedures presented.
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