Methods of Genetic Transformation: Agrobacterium tumefaciens

Abstract

The soil phytopathogen Agrobacterium tumefaciens induces tumors, known as crown galls, mainly on dicotyledonous plants. Such tumors are generated by a complex, multi-step transformation process. Agrobacterium has been routinely utilized for the transfer of genes to dicotyledonous plants, and monocotyledonous plants, including important cereals, were thought until recently to be outside the range of this technology since they are generally not considered within the host range of crown gall. Various attempts to infect monocotyledons with Agrobacterium were made in the 1970’s and 1980’s, but conclusive evidence of integrative transformation was not obtained until recently. This delay occurred in part because many methods for the transformation of dicotyledons depend heavily on the cell divisions that are induced by wounding. Similar approaches in monocotyledons failed, because they do not exhibit active responses to wounding. Wounding is necessary for formation of a crown gall and the main roles of wounding are to produce compounds that activate the virulence genes of Agrobacterium and to induce DNA synthesis in host cells. Efficient protocols for Agrobacterium-mediated transformation have recently been developed for rice, maize, barley and wheat. A key point in these protocols is the use of tissues that consist of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, which are co-cultivated with Agrobacterium in the presence of acetosyringone, which is a potent inducer of the virulence genes. The advantages of Agrobacterium-mediated transformation include the transfer of pieces of DNA with defined ends with minimal rearrangement, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and the high quality and fertility of both monocotyledonous and dicotyledonous transgenic plants. Stable inheritance and expression of transgenes in the progeny has also been demonstrated. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues that contain ‘competent’ cells are infected. The success with cereals highlights the critical importance of numerous experimental factors, which include the genotype of the plants, the type and age of the tissue that is inoculated, the vector, the strain of Agrobacterium, the selectable marker genes and selection agents, and the various conditions of tissue culture.