Selecting the future of plant transformation
The PMI system from Syngenta is a novel, safe and environmentally friendly selectable marker for plant transformation. It offers an efficient alternative to using antibiotic resistance and herbicide tolerance markers in genetically engineered crops. PMI has been developed in response to public concern about the use of existing marker genes.

Marker genes
A selectable marker gene is required in the early stages of plant transformation. When inserting these new genes into plant cells, not all of them are transformed. The marker gene is inserted into the plant cell at the same time as the new gene of interest, thereby enabling selection of the transformed cells. Commonly used marker genes enable transformed cells to survive treatment with antibiotics or herbicides while eliminating non-transformed cells. No health or environmental risks have been identified for these types of marker genes, but there is a public concern about the use of antibiotic resistance marker systems in crops.

The technology
The PMI technology makes use of the inability of most plants, for example maize, most small grains, potato and sugar beet, to metabolize the simple sugar mannose. Transgenic plants expressing the enzyme phosphomannose isomerase (PMI) encoded by the manA gene from E. coli (Miles H et al, 1984 Gene 32 pp41-48) are able to convert mannose-6-phosphate to fructose-6-phosphate, which can then be utilized. When placed on a medium containing either predominantly mannose or even mannose as the sole sugar source, non-transformed tissue remains dormant and becomes outgrown by the transformed tissue. Mannose itself has no adverse effect on plant cells. The selection is believed to occur as a result of its phosphorylation to mannose-6-phosphate by hexokinase. In tissue that doesn't contain the PMI enzyme, mannose-6-phosphate accumulates and the cells stop growing.
The PMI system offers an effective alternative to antibiotic resistance or herbicide tolerance marker genes in plant species that do not already possess the manA gene. It has been shown to be effective in a range of commercially important crop plants and plant model species. It has been successfully used for transformation of Arabidopsis and commercially important crops such as maize, rice, cassava, wheat, barley, sugar beet, watermelon, tomato, squash, cabbage, sunflower and oilseed rape. We have observed no differences in agronomic performance (such as yield) between maize plants transformed with the manA gene compared to non-transformed maize plants. PMI can be used with a variety of standard plant transformation protocols, including:

• Agrobacterium
• Biolistics™
• Floral dip
• Protoplast transformation

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Diagram of the PMI System

The advantages

• Non toxic - By using the manA gene as a selectable marker, the end product is a harmless and physiologically important sugar for plant metabolism. Instead of using a toxic compound, such as an antibiotic or herbicide to kill non-transformed cells, they are simply deprived of a source of carbohydrate.
• Rapid and efficient - For many applications, the PMI system has proven to be superior in performance both in the transformation frequency and the time required for the selection-regeneration process.
• Versatile - The system is extremely versatile both with respect to range of plant species it has been successfully applied to and with respect to transformation methods it can be used with
• Trait Stacking - The PMI system can be used as an additional marker for stacking traits.
• Addresses public concern - The use of antibiotic resistance in plant transformation is currently a major public concern, despite the fact that the safety of these existing markers has been comprehensively tested and proven over several years. The PMI system doesn't use antibiotics for the selection process and therefore addresses these public concerns. While Syngenta stands by the safety of other marker genes, PMI provides an alternative.

The data

		Results for transformation using PMI show that, if anything, it is more efficient than traditional methods. For example, as shown by the above bar charts, the transformation frequency and selection efficiency of sugar beet is reportedly enhanced by more than 5-fold. In addition, rooting problems seen, for example, with kanamycin-based selection methods, do not occur with the PMI system. The PMI selection system has been optimized for several genotypes of various crops with almost no escapes. Co-transformation frequencies can be as high as 94% depending on the gene of interest and are no lower than 60%. PMI ELISA studies have shown that more than 70% of PCR positive events express the protein.
		Molecular and expression analysis have demonstrated that the manA gene is transmitted to progeny in a Mendelian fashion. Once the PMI system is optimized for a particular genotype or transformation method, the frequency of escapes is low while the co-transformation and self-fertility of T0 plants are high. Results in maize have also shown transformation frequencies to be increased by up to three times with PMI when compared to BASTA selection after biolistic transformation, (see Wright et al, submitted).

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Safety aspects
Safety of the PMI marker gene: PMI has undergone rigorous safety checks, including its toxicity, allergenic potential and effects on biochemical composition of plant tissue. It was found that:

• The manA gene is naturally present and expressed in mammals, so the products of the PMI system are already widely present.
• PMI was readily digested in simulated mammalian gastric and intestinal fluids, indicating a low allergenic potential.
• Transgenic plant events expressing PMI do not have altered glycogen profiles.
• PMI transgenic maize events were indistinguishable from their non-transgenic counterparts with respect to the compositional analysis of grain when measuring moisture, ash, fiber, fat, protein, b-carotene, xanthophylls and vitamin C.
• Agronomic characterization, including yield, showed no differences between PMI transgenic maize events and their non-transgenic counterparts.
• No adverse effects of PMI were found during an acute oral mouse toxicity study.

Relevant publication:

" Phosphomannose Isomerase, A Novel Selectable Plant Selection System: Mode of Action and Safety Assessment " (80KB, PDF)
The above article was published in: Proceedings of the 6th International Symposium on The Biosafety of Genetically Modified Organisms, Saskatoon, Canada, (eds. C. Fairbairn, G. Scoles & A. McHughen) University Extension Press, Univ. Saskatchewan, pp. 171 - 178.

Safety of existing marker genes: The safety of the existing marker genes has been comprehensively tested and proven over years of use. For example, the results of extensive studies concludes that the antibiotic-resistance marker gene used in Syngenta Bt-176 maize poses no health risk or threat to the effectiveness of antibiotics used in humans or animals. This is supported by the opinions of some of the world's leading experts1 on antibiotics and micro-organisms, who have concluded that the possibility of DNA transfer from Bt-176 maize to bacteria living in the gut of animals is virtually zero. Bt maize has been extensively tested and over 30 independent scientific committees around the world have concluded that it is as safe as conventional maize.

¹Antibiotic Resistance via the Food Chain: Fiction or Reality Case Study: Ampicillin; Sept. 23-24, 1996.

Bibliography
Evans R, Wang A, Hanten J, Altendorf P & Irvin Mettler, 1996. A positive selection system for maize transformation. In Vitro 32, 3, Part II:72A abstract.

Haldrup A, Petersen SG & Okkels FT, 1998. The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent. Plant Molecular Biology 37 pp287-296.

Haldrup A, Petersen SG & Okkels FT, 1998. Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry. Plant Cell Reports 18 pp76-81.

Joersbo M, 1999. Advances in the selection of transgenic plants. Research Advances in Phytochemistry.

Joersbo M & Okkels FT, 1996. A novel principle for selection of transgenic plant cells: positive selection. Plant Cell Reports 16 pp219-221.

Joersbo M, Donaldson I, Kreiberg J, Petersen SG, Brundstedt J & Okkels FT, 1998. Analysis of mannose selection used for transformation of sugar beet. Molecular Breeding 4 pp111-117.

Melanson D, Roussy I & Hansen G, 1999. The use of phosphomannose isomerase as a selectable marker to recover transgenic Arabidopsis plants. Crown Gall Conference, Houston, Texas.

Negrotto D, Jolley M, Beer S, Wenck AR & Hansen G, 2000. The use of phosphomannose isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Reports ~ in press.

Privalle LS, Wright M, Reed J, Hansen G, Dawson J, Dunder EM, Chang Y-F, Powell ML & Meghji M, 2000. Phosphomannose isomerase, a novel selectable plant selection system: mode of action and safety assessment. Proceedings of the 6th International Symposium on the Biosafety of GMOs.

Reed JN, Chang Y-F, McNamara DD, Beer S & Miles PJ, 1999. High frequency transformation of wheat with the selectable marker mannose-6-phosphate isomerase (PMI). In Vitro 35:57-A abstract P-1079.

Stein JC & Hansen G, 1999 Mannose induces an endonuclease responsible for DNA laddering in plant cells. Plant Physiol. 121:71-79.

Wang AS, Evans RA, Altendorf PR, Hanten JA, Doyle MC & Rosichan JL, 2000. A mannose selection system for production of fertile transgenic maize plants from protoplasts. Plant Cell Reports 19, 7 ~ in press.

Wright M, Dawson J, Dunder E, Suttie J, Reed J, Kramer C, Chang Y & Wang H, 2000. Efficient Biolistic® transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphate mannose isomerase, pmi gene as the selectable marker. Submitted for publication.