Ethanol → (+)-gamma-Tocopherol

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GMT expression vectors pMON36503 (FIG. 9), pMON36505 (FIG. 11) and pMON36506 (FIG. 12) are transformed into the soybean line A3244 using Agrobacterium mediated transformation. See, for example the methods described by Fraley et al., Bio/Technology 3:629-635 (1985) and Rogers et al., Methods Enzymol. 153: 253-277 (1987). Ten bulked seeds from the R1 generation are ground and the resulting soy meal is used for tocopherol analysis. Twenty five to forty mg of the soy meal is weighed into a 2 mL micro tube, and 500 μl 1% pyrogallol (Sigma Chemicals, St. Louis, Mo.) in ethanol containing 5 μg/mL tocol, is added to the tube. The sample is shaken twice for 45 seconds in a FASTPREP (Bio101/Savant) using speed 6.5. The extract is then filtered (Gelman PTFE acrodisc 0.2 μm, 13 mm syringe filters, Pall Gelman Laboratory Inc, Ann Arbor, Mich.) into an autosampler tube. HPLC is performed on a ZORBAX silica HPLC column, 4.6 mm×250 mm (5 μm) with a fluorescent detection using a Hewlett Packard HPLC (Agilent Technologies). Sample excitation is performed at 290 nm, and emission is monitored at 336 nm. Tocopherols are separated with a hexane methyl-t-butyl ether gradient using an injection volume of 20 μl, a flow rate of 1.5 ml/min, and a run time of 12 min (40° C.). Tocopherol concentration and composition is calculated based on standard curves for α, β, γ and δ-tocopherol using Chemstation software (Agilent Technologies, Palo Alto, Calif.). As shown in FIGS. 14-16, several lines from each construct completely or substantially converted δ and γ-tocopherol, leaving α and β-tocopherol as the only detectable tocopherol isomers.

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