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| Techniques Used for Generating Transgenic Plants |
As with bacteria, the ability to genetically modify plants depends on obtaining genetically identical populations and readily manipulating DNA. How do you "clone" a plant? Many plant species naturally undergo asexual reproduction by fragmentation, where segments from a parent plant regenerate a new plant. It is also possible to grow plants in culture from small explants. Another method is to culture plants from totipotent cells found in plant meristems. These plant cells can divide and differentiate into the various types of specialized cells. In a test tube, plant cells will divide and form an undifferentiated callus. When hormones in the culture medium are adjusted, the callus will sprout shoots and roots and eventually develop into a plantlet that can be transplanted to soil. To clone a plant - perhaps a plant with new genes - the growing callus is simply subdivided. Thousands of genetically identical plants can be generated in this way.
How do you get a plant to take up a gene? Researchers working with rice often use the soil bacterium Agrobacterium tumefaciens. This bacterium, the cause of crown gall disease in many fruit plants, is well known for its ability to infect plants with a tumor-inducing (Ti) plasmid. A section of the Ti plasmid, called T-DNA, integrates into chromosomes of the plant. Recombinant DNA can be added to the T-DNA, the gall-inducing genes removed, and infection by the bacteria - containing the recombinant plasmid - will provide for transfer of novel genes to plant embryos.
Although Agrobacterium tumefaciens works for introducing plasmids into rice, not all plants are equally susceptible to this bacterium. Researchers interested in modifying crops such as wheat and corn have turned to other methods for delivering genes to plant cells. One approach is to use a "gene gun," (Fig. 3) which fires plastic bullets filled with DNA-coated metallic pellets. An explosive blast or burst of gas propels the bullet toward a stop plate. The DNA-coated pellets are directed through an aperture in the stop plate, and then penetrate the walls and membranes of their cellular targets. Some projectiles penetrate the nuclei of cells, where occasionally the introduced DNA integrates into the DNA of the plant genome. Transformed cells can then be cloned in culture.
Marker genes are often included in DNA constructs so that plants that have acquired the novel DNA can be selected. In plants, marker genes include those for herbicide resistance. Plants that grow in the presence of the herbicide are assumed to possess the transgene of interest. The transgenic plant embryos are cultivated in tissue culture. Once mature plants are obtained they are evaluated for the activity of the introduced gene, any unintended effect on plant growth, and product yield and quality. The ability of the gene to be expressed in subsequent plant generations is also evaluated.
Not all genes are expressed in every tissue of a plant. When golden rice was developed it was necessary to ensure that the novel genes were expressed in the endosperm of the seed. The endosperm of a seed is the starchy component that provides energy and nutrients for the developing plant embryo. Regulatory DNA sequences upstream from the specified genes were introduced into the recombinant Ti plasmids. Such regulatory regions influence where and when a gene will be expressed. (See the Genomics unit.) The regulatory regions chosen for golden rice provide an uninhibited transcription of the genes in endosperm.