Lc30ad system

An amount of 0.15 g of fresh germinating seeds from each treatment was homogenized in 1 ml of 80% methanol solution and stored at 4°C for 12 h. The sample was treated by ultrasound in an ice bath for 30 min and then centrifuged at 12,000 rpm at 4°C for 5 min. A total of 800 μl of the extract was used to purify the supernatants under reduced pressure at room temperature, then reconstituted by adding 80 μl of 80% methanol solution and loaded in the SPE column (Welchrom C18 SPE, 00559-11001). The sample was then washed twice with 160 μl of distilled water to enrich the plant hormones and then washed twice with 160 μl of 5% methanol aqueous solution. After discarding the washing liquid, the residues were washed four times with 160 μl of methanol solution, and the methanol washing liquid was collected. Further drying was performed under reduced pressure, and 100 μl of methanol was added, then the generated extracts were used to measure the hormone contents by injecting 10 μl of sample into an AB SCIEX-Triple-TOF^®-5600 + LC/MS/MS system (HPLC, Shimadzu LC30AD System, MS, AB Sciex TripleTOF 5600+).