Measuring acyl lipid content and fatty acid composition is important in plant lipid research, and direct transmethylation followed by analysis with gas chromatography‐flame ionization detection has been broadly used in various studies. However, many existing protocols are time‐consuming and labor‐intensive, and the long reaction time under high temperature may artificially change fatty acid compositions of the samples. In this study, we optimized the direct transmethylation method for seed and leaf samples with a wide range of seed and tissue sizes, oil contents, and fatty acid profiles, which can be completed in 2 h. We also demonstrated that commercial disposable pipet tips, as well as polypropylene tubes under certain conditions, could be reliably used to replace glass tubes, Pasteur pipettes, and rubber bulbs to improve efficiency. Taken together, this optimized and generalized method could be used as a reliable and less time‐ and labor‐intensive approach to analyze acyl lipid content and fatty acid profile of plant samples.
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