Quantification of Phytohormones in Seeds

The content of Gibberellin (GA3), indoleacetic acid (IAA) and Abscisic Acid (ABA) in seeds of different colors were determined by High-Performance Liquid Chromatography (HPLC)(Agilent-1290, Agilent Technologies, USA) coupled to a mass spectrometer (MS/MS)(AB Sciex-6500 Qtrap, Allen-Bradley, USA). Each sample was replicated four times (3 colors × 4 replicates). The brief process was that 1.5g seed material was ground in liquid nitrogen, followed by adding mixed solution of isopropyl alcohol, water and hydrochloric acid. Then the specimen was shaken for 30 minutes respectively after adding standard solution and methylene chloride. Next, the specimen was centrifuged (13000 r/min) for 5 minutes to gain organic substance. Next, the organic substance was dried using Termovap Sample Concentrator (NDK200-2N, Hangzhou Miu Instruments Co., Ltd, China) and redissolved by methyl alcohol. After that, the solution was centrifuged again (13000 r/min) for 10 minutes and then filtrated. The whole solvent extraction was operated at 4 °C. Finally, the hormones were measured by HPLC-MS/MS.