J w da wax capillary column

Twelve seeds from different siliques were harvested at 27, 38 and 45 DAF for each biological repeat. In total, six biological repeats were used. FA extraction was performed as previously described (Woodfield et�al. 2017 (link), Woodfield et al. 2018 (link)). Seed samples were incubated in 1.2 ml of isopropanol at 70�C for 30 min to inactivate any endogenous (phospho-) lipases. Nonadecanoic acid (19:0; Sigma, St. Louis, Missouri, USA) was used as an internal standard. Fatty acid methyl esters (FAMEs) were analyzed by an Agilent GC-MS device (5,973 inert mass spectrometer combined with 6,890 N gas chromatograph) equipped with an Agilent J&W DA-WAX capillary column (30 m � 0.25 mm � 0.25 �m; Lung et�al. 2017 (link)). The oven temperature was set to 170�C for 3 min, increased to 220�C at 4�C�min⁻¹, and held at 220�C for 15 min (Woodfield et�al. 2017 (link)). FAMEs were routinely identified by comparing the retention time of peaks with the Supelco 37 Component FAME MIX standard (Sigma) but had been identified fully in previous work (Woodfield et�al. 2017 (link)).

Twelve seeds from different siliques were collected at 38 DAF for each biological repeat. In total, six biological repeats were used. Fatty acid extraction was conducted as previously described^{28 ,31 (link)}. Fatty acid methyl esters (FAMEs) were obtained by incubating seed samples in 1.2 ml of isopropanol at 70 °C for 30 min followed by incubation in 3 ml of 2.5% H₂SO₄ in methanol:toluene (2:1, v/v) at 70 °C for 2 h. Nonadecanoic acid (19:0) (Sigma) (20 µg) was added as an internal standard. 2 ml of 5% NaCl and 3 ml of hexane were mixed with the samples, and the upper phase was collected after brief centrifugation. Subsequently, another 3 ml of hexane was mixed with the samples, and the upper phase was collected. The combined hexane phase was dried by nitrogen gas. FAMEs were analysed by an Agilent GC–MS device (5973 inert mass spectrometer combined with 6890N gas chromatograph) equipped with an Agilent J&W DA-WAX capillary column (30 m × 0.25 mm × 0.25 µm). The oven temperature was 170 °C for 3 min, increased at 4 °C min⁻¹ to 220 °C, and held for 15 min at 220 °C^{31 (link)}. FAMEs were identified by comparing the retention time of peaks with the Supelco 37 Component FAME MIX standard (Sigma).