Quantitative Analysis of Plant Hormones

Samples of 0.2 g were ground in an ice-cold mortar with 8 mL of 80% (v/v) methanol extraction medium that contained 1 mM butylated hydroxytoluene as an antioxidant 22. The extracts were incubated at 4 °C for 4 h and then centrifuged at 3500 r.p.m. for 8 min at 4 °C. The supernatants were passed through Chromosep C18 columns (C18 Sep-Pak Cartridge; Waters Corporation, Millford, MA, USA), which were washed with 10 mL of 100% and 5 mL of 80% (v/v) methanol. The hormone sediments were dried under nitrogen gas and then dissolved in 2 mL of phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatine (pH 7.5).
Phytohormones were separately analysed using GA₁₊₃, GA₄₊₇, IAA and ABA ELISA Kits produced by the Phytohormones Research Institute, China Agricultural University, China. Yang’s method for quantitative phytohormone determination was used^{23 (link)}.