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Selectable Marker Genes in Transgenic Plants

Summary of
"Selectable marker genes in transgenic plants: applications, alternatives and biosafety"
by Brian Miki and Sylvia McHugh

From the Journal of Biotechnology, Volume 107, Issue 3 , 5 February 2004, Pages 193-232, with permission from Elsevier. The complete paper is available on the Journal of Biotechnology Web site.

The Journal of Biotechnology is not subject to the Official Languages Act, and therefore the paper at that link is not available in French.

Marker genes are central to the research and development of transgenic plants. As scientists work to create plants that exhibit a desired new trait—for example, improved disease resistance—they insert a marker gene into the plant, along with the gene for the new trait. The marker gene clearly flags for researchers the cells that have the new trait (the transformed cells) so that they can quickly find, from among hundreds of young plants, those that have the desired characteristic.

The paper "Selectable marker genes in transgenic plants: applications, alternatives and biosafety" by Brian Miki and Sylvia McHugh, examines:

• the scientific literature on 50 marker genes
• their adoption or level of readiness for use in crop plants
• their biosafety

It also reviews the literature on marker-free transgenic plants.

The literature shows that while many marker genes exist, only a few are widely used. However, a variety of selection systems is essential because no single marker gene works well in all situations. This is particularly the case as scientists continually develop new transgenic plants and new uses for them. Much research is underway on alternative genes and strategies, and promising developments are at hand or on the horizon.

How Selectable Marker Genes Work

Selectable marker genes work by "selecting" certain cells—either the natural or transformed cells, depending on the system—for either destruction or proliferation, again depending on the system. One such gene, for example, is the gene that is resistant to the antibiotic kanamycin. If researchers wanted to create a cotton plant that can fight bollworms, they might inject the plant tissue with a gene that makes the plant toxic to the insect, along with the gene that resists kanamycin. They would then apply kanamycin to all of the cells. Only the transformed cells would be able to resist the kanamycin and survive. These would be nurtured into laboratory plantlets.

Antibiotics and Herbicides

The marker genes most commonly used are those that are resistant to antibiotics and herbicides. These genes work by killing non-transformed plant cells and allowing the transformed cells to survive. The literature examination revealed that three of these selection systems were used in more than 90 percent of the cases. These were the genes resistant to the antibiotics kanamycin or hygromycin and the herbicide phosphinothricin.

Targeting the Plant's Metabolism

While antibiotic and herbicide marker genes usually act on a specific process in the plant (that is, they code for a specific enzyme that degrades the antibiotic or herbicide), a new approach involves manipulating the plant's metabolic or biosynthetic pathways. This is done using either agents such as antibiotics, herbicides and drugs or, in an important new development, non-toxic elements such as phytohormones (growth substances) and carbon supply which are natural to the plant.

An example is the gene coding for (manA). Because the non-toxic approach appears to yield greater transformation frequencies and is effective for many species, it is of major interest for studying crop plants. It also addresses public concerns that the use of antibiotic-resistance genes could increase human and animal tolerance to antibiotics, and that herbicide-resistance genes could cause environmental problems.

Altering the Plant's Development

The genes that control a plant's ability to grow from a single leaf or shoot segment provide opportunities to develop new selectable marker genes without the need for selective agents such as antibiotics or genes that confer resistance to or detoxification of such agents. The ipt gene, for example, enhances shoot development by modifying the plant's hormone levels. Scientists have shown that some of these genes (those that control the formation of organs during embryonic development) may function as selectable marker genes.

However, the paper points out that this strategy is still very new and complicated to implement. It also notes that both this approach and the ones that alter the plant's metabolism may require more testing as such changes can also create unexpected alterations, as was reported in some cases.

"Reporter" Genes

Advances are also being made in non-selectable marker genes, or "reporter" genes, which allow scientists to physically see the transformed cells. Reporter genes are important partners to marker genes in terms of improving transformation systems and recovering transgenic plants. The gene coding for the enzyme β-glucuronidase (GUS) is the most widely used. However, others are becoming more popular, such as the gene coding for green fluorescent protein (GFP) from jellyfish. This is due to practical advantages they offer, such as the test being non-destructive (unlike GUS) and easy to apply.

GFP has also become valuable in field trials for monitoring gene expression (detecting the plants in which the new gene is working) and for following pollen flow. New ways of using reporter genes—for instance, as visible markers for observing transgenic escapes (the movement of genes from modified plants into other plants)—can extend their importance. Reporter genes that can be detected by taste or smell may also be considered.

Marker-free Transgenic Plants

One research area still in its infancy but growing rapidly is the removal of the marker genes from the plants once the genes have done their job. The scientific literature describes several such strategies and, although all are more difficult to implement or less efficient than systems that leave the markers in the plants, a couple show promise.

Marker-free transgenic plants offer several advantages. These include:

• addressing public questions about biosafety
• simplifying the regulatory process
• allowing the use of more experimental marker genes that have not been extensively tested for biosafety

However, this research area is still at the experimental stage and the paper reports that it is too soon to rate its commercial significance.

Environmental Questions

Key environmental questions are about gene flow (the potential spread of genetically modified traits to other crops or plant relatives) and horizontal gene transfer (DNA from genetically modified plants to other organisms). While the literature review shows that neither aspect poses notable risk, scientists are nonetheless addressing them.

Several approaches are being developed to restrict gene flow, although most are still in early development and have limitations. With regard to horizontal gene transfer, some publications recommend that antibiotics widely used for clinical or veterinary purposes not be used as selectable markers, and that antibiotic resistance genes be removed as new technologies become available. While the marker genes used so far have proven safe, it may be more difficult to predict the impact of individual markers that alter plant metabolism should they transfer to wild species.

Conclusion

While many of the advances described in the paper are still in early development, they show significant promise. The development of new marker technologies will continue to be important in the creation of transgenic plants.