GC FID (Gas Chromatography with Flame Ionization Detector) - Measurement * Roughly 80 mg of seed material is weighed into Precellys grinding tubes (ceramic beads, 2 ml capacity). The large seed amount is to reduce inter-seed variation. * Seeds are ground at 5500 rpm for 25 seconds three times, with a wait time of 30 seconds between each grinding step. * 1 ml of hexane containing the internal standards cholesteryl-heptadecanocate and cholestanol at a concentration of 0.00002M (20 µM) are added to samples. * Samples are vortexed, and then incubated at 30°C in a rotary incubator at 200 rpm for 1 hour. * Samples were then centrifuged at 14000 rpm for 1 minute to sediment seed material. The hexane supernatant was removed using Pasteur pipettes and transferred to 3 ml, glass trident vials. * A further two hexane extractions were performed (with no internal standards added), with 1 ml of pure hexane added to each sample, incubation at 30°C for 1 hour at 200 rpm, with a prior sample vortex step, centrifugation at 14000 rpm and finally, separation and pooling of the hexane fractions. * The 3 ml of seed lipid extract was dried down using a Genevac EZ-2 at ? 40°C for 40 minutes. * 1.5 ml of 1.5 M potassium hydroxide in 95% ethanol was added to the lipid sample (roughly 10 mg), and incubated at 80°C for 1 hour (from 80 mg total seed, 1ml total suspension, 250 ul used). * The samples are cooled, and 1 ml of a 1% (w/v) NaCl solution is added, followed by 1 ml of hexane. * Samples are vortexed, centrifuged and the upper hexane phase is removed. * A further hexane extraction is carried out with 1 ml of hexane, which is pooled with the first. * Samples are dried in a rotary evaporator or under a stream of nitrogen. * Free sterols are then silated using 100 µl of BSTFA + TMCS (99:1) for 1 hour at 85°C. (100 ul heptane added, 1 ul injection into GC-FID). *GC program: * Initial temp: 200°C, to 300°C at 16°C/min, then to 325°C at 4°C min and held for 4.5 minutes. * Inlet temp: 325°C, split-less injection. * Column: HP-1MS UI, 30 m x 0.25 mm x 0.25 µm, flow = 1 ml/ min. * FID temp: 325°C attribute entity variable_of scale_of acronym method_of