Box 2.5: Gene Transfer Technologies

Two primary methods currently exist for introducing new transgenic genetic material into plant genomes in a functional manner. For plants known as dicots (broad-leaved plants such as soybean, tomato, and cotton), transformation is usually brought about by use of the bacterium, Agrobacterium tumefaciens.  Agrobacterium naturally infects a wide range of plants by inserting some of its own DNA directly into the DNA of the plant. By taking out the undesirable traits associated with Agrobacterium infection and inserting a gene of interest into the Agrobacterium DNA that will ultimately be incorporated into the plant’s DNA, any desired gene can be transferred into a dicot’s DNA following bacterial infection. The cells containing the new gene subsequently can be identified (with the aid of selectable markers) and grown (using plant cell culture techniques) into a whole plant that now contains the new transgene incorporated into its DNA. 

Plants known as monocots (including cereals such as maize, wheat, and rice) are not readily infected by Agrobacterium. The external DNA that is to be transferred into the plant’s genome is coated on the surface of small tungsten balls and the balls are physically shot into plant cells (the biolistic method, using a gene gun). Some of the DNA comes off the balls and is incorporated into the DNA of the recipient plant. These transformed cells can similarly be identified and grown via cell culture into a whole plant that contains the foreign DNA.

In the case of Agrobacterium-mediated gene transfer, its success varies with the plant species, and even between strains within the one species. There are continuing efforts to increase the efficiency of transformation techniques, for example by the selection of particular strains of Agrobacterium.

Improving gene control

In the early transformation efforts, genes were inserted at random into the genome. The location at which the gene was placed randomly on the plant chromosome has been shown to affect the level of gene expression. (because chromosomal proteins modulated gene expression). For various reasons, an introduced gene may not express itself properly, for example if multiple copies of the gene are inserted into the plant (a feature termed gene silencing).

Much research effort is going into improving gene control and regulation, so as to increase the precision of the transformation process. Ideally, a single copy of a gene will be able to be inserted into a particular part of the genome. Such improvements in gene control will allow precise gene placement and possibly also gene replacement.

Multiple gene control

Where multiple genes are required to produce a desired plant type (phenotype), an important prerequisite is to be able to switch on all the required genes simultaneously and to a similar degree. Yet coordinating the expression of more than one gene has proved difficult. Two types of strategies are employed when introducing multiple genetic modifications. These are simultaneous or sequential transformation. In simultaneous transformation, several genes are either collected into one transforming molecule and introduced into the target plant, or they may be simultaneously inserted while on different transforming molecules. In sequential transformation, a transgenic plant is re-transformed with a second gene, or two plant lines receive one transgene each and their progeny are crossed and the resulting double transformant selected from amongst the next generation progeny.

(Sources: Van Montague and Bursenns 2002; Elborough & Hanley, 2002).

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