Selectable marker genes are the sine qua non for the development of genetically modified crops, and all such commercially released GM crops have these marker genes. Marker genes render resistance in plant cells against antibiotics or herbicides and thus enable scientists to effortlessly select a rare transformed plant cell after co-introducing the desired gene along with a marker. Antibiotic or herbicide added to the plant culture media kills the normal plant cells while the few transformed cells survive, grow, and develop into whole plants.

Beyond the laboratory, these markers have no role and thus their presence in crops and food has provoked much public concern. The perceived risk of marker genes to environmental safety and health has led scientists to explore alternative systems such as the removal of marker genes using the Cre-lox system or transposable elements, and the use of cytokinin glucuronides with the GUS gene. A new selection strategy reported by Danish scientists involves a novel selectable marker system using a gene for an enzyme that metabolizes mannose-6-phosphate, a plant growth-inhibiting phosphorylated sugar, and thus the strategy may prove to be a better alternative to the use of antibiotic marker genes (1).

The most widely used selectable marker is the nptII gene that codes for neomycin phosphotransferase II, which confers resistance to antibiotic kanamycin or geneticin. Considerable research on the nptII has shown that it is safe for both the environment and the consumer (2).

However, such information is not available for other marker genes including some herbicide resistance genes, which may carry a risk of potential gene flow to weedy relatives. There are also some concerns that antibiotic marker proteins could compromise the therapeutic efficiency of orally administered antibiotic.

The new selectable system relies on the use of phospho- mannose isomerase (PMI). PMI is fairly ubiquitous in nature; however, aside from leguminous plants, it is not found in most plants, especially cereals. Plant cells without this enzyme are unable to survive in a tissue culture medium containing mannose-6-phosphate as a sole carbon source. Incubation of plant cells in the presence of mannose-6-phosphate results in phosphate and ATP starvation thus depleting energy from critical functions such as cell division and elongation (3).

The mannose-6-phosphate toxicity in plant cells has also recently been shown to cause apoptosis or programmed cell death through induction of an endonuclease responsible for DNA laddering (4).

The manA gene encoding PMI was cloned from E. coli by researchers at Danisco Biotechnology. Plant cells transformed with this gene can convert mannose-6-phosphate to fructose-6-phosphate, which is easily metabolized. Morten Joersbo and colleagues (1) observed that use of mannose selection in sugar beet resulted in a ten-fold increase in transformation frequency when compared to traditional kanamycin selection. According to researchers, such increased efficiency may occur because transformed cells are actively encouraged to grow rather than just allowed to survive. In the traditional selection system using antibiotics or herbicides, the transgenic cells convert the selective agent to a detoxified compound that may still exert a negative influence on the plant cells. Further, the release of toxic metabolites by dying adjacent cells may also inhibit the growth of transformed cells. In contrast, mannose selection essentially provides a metabolic advantage to the transformed cells while the untransformed cells are starved and progressively lose their viability, according to Joersbo et al.

Novartis Agribusiness Biotechnology Research, Inc., which has licensed the PMI gene selection system, has found this marker to be very effective in the selection of wheat and maize transgenics with an astoundingly high frequency of transformation of 25% and 50%, respectively. Novartis scientists have found that PMI protein is very safe as is evident from many studies including mouse toxicity assays (3).

The protein was readily digested in simulated mammalian gastric and intestinal fluids indicating a low allergenic potential. Sugar beet and maize plants containing the manA gene had identical biochemical profiles, yield, and nutritional composition when compared to control plants. The gene encoding this activity has been cloned from several bacteria and yeast species and also from humans. PMI has also been purified and studied from yeast, bacteria, pigs, and humans.

The PMI-mannose appears to be an ideal selectable system for plant transformation as it obviates the need for antibiotic or herbicide markers and also provides improved recovery of transformed plants. Researchers interested in obtaining the manA gene and more information about this system may contact Dr. Andy Beadle at andrew.beadle@seeds.novartis.com.

Sources

1. Joersbo M, et al. 1998. Analysis of mannose selection used for transformation of sugar beet. Molecular Breeding 4:111-117.

2. Fuchs R, et al. 1993. Safety assessment of the neomycin phosphotransferase II (NPTII) protein. Bio/Technology 11:1543-1547.

3. Privalle LS, Meghji M, and Powell L. 1999. Safety assessment of a novel plant selectable marker: Phosphomannose isomerase. Abstract No. 395. Annual Meeting of the American Society of Plant Physiologists (July, 1999; Baltimore, MD).

4. Stein JC and Hansen G. 1999. Mannose induces an endonuclease responsible for DNA laddering in plant cells. Plant Physiology 121:1-9.

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