Quantifying ABA in Soybean Seeds

For analysis of ABA content in soybean seeds, the previously protocol we used^{14 (link)} were employed in this study. Firstly, the seeds were ground to powder in liquid nitrogen, and 300 mg of seeds powder was homogenized and extracted for 24 hours in methanol containing D6-ABA (OIChemIm Co. Ltd.) as an internal standard. Purification was performed with an Oasis Max solid phase extract cartridge (Waters) and eluted with 5% formic acid in methanol. Subsequently, the elution was dried and reconstituted, and it was then injected into a liquid chromatography–tandem mass spectrometry system consisting of an Acquity ultra performance liquid chromatograph (Acquity UPLC; Waters) and a triple quadruple tandem mass spectrometer (Quattro Premier XE; Waters). Three biological replications were performed.