Phosphomannose isomerase: A versatile selectable marker forArabidopsis thaliana germ-line transformation
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A new selection system using mannose has been evaluated for germ-line transformation ofArabidopsis thaliana. Although mannose itself has no adverse effects on plant cells, it leads to an accumulation of mannose-6-phosphate, which depletes intracellular stores of inorganic phosphate. This results in an inhibition of plant cell growth. The selection system uses theEscherichia coli pmi gene that encodes phosphomannose isomerase (PMI). Transgenic plants carrying thepmi gene can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the PMI activity. Germ-line transformation ofA. thaliana followed by sterile selection on 2–5 mM of mannose resulted in the isolation of mannose-6-phosphate-resistant progeny in about 2.5% of the treated seed, consistent with transformation rates using other selection schemes. Integrative transformation was confirmed by Southern hybridization. Analysis of PMI enzyme activity demonstrated a 5-fold range of activity levels, although these differences had little effect on the ability to select transformed plants or on the growth of transformed plants on mannose. Finally, mannose selection using thepmi gene could be accomplished in sterile plates and in soil, making this an extremely versatile tool forA. thaliana transformation.
Key wordsgerm-line transformation Arabidopsis thaliana mannose mannose-6-phosphate resistance phosphomannose isomerase
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- Altmann T, Damm B, Halfter U, Willmitzer L, and Morric P-C (1992) Protoplast transformation and methods to create specific mutants inArabidopsis thaliana. In: Koncz C, Chua N-H, and Schell J (eds), Methods in Arabidopsis Research, pp 310–330, World Scientific Publishing Co., River Edge NJ.Google Scholar
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, and Struhl K (1995) Current Protocols in Molecular Biology, 3rd edition. John Wiley & Sons, New York, NY.Google Scholar
- Baskin T, Remillong E, and Wilson J (2001) The impact of mannose and other carbon sources on the elongation and diameter of the primary root ofArabidopsis thaliana. Austral J Plant Phys 28: 481–488.Google Scholar
- Ferguson J, Street H, and David SD (1958) The carbohydrate nutrition of tomato roots IV. The inhibition of excised root growth by galactose and mannose and its reversal by dextrose and xylose. Ann Bot 22: 525–538.Google Scholar
- Malca I, Endo R, and Long M (1967) Mechanism of glucose counteraction of inhibition of root elongation by galactose, mannose and glucosamine. Phytopathology 57: 272–278.Google Scholar
- Sigma-Aldrich Fine Chemicals (2001) Biochemicals and Reagents for Life Science Research. Sigma-Aldrich, St Louis MO.Google Scholar