Hormone quantification
The IAA level was determined by Zoonbio Biotechnology Co., Ltd. 0.5 g
samples were ground in a pre-cooled mortar that contained 5 mL
extraction buffer composed of isopropanol/hydrochloric acid. The extract
was shaken at 4°C for 30 min. Then, 10 mL dichloromethane was added, and
the sample was shaken at 4°C for 30 min and centrifuged at 13,000 rpm
for 5 min at the same temperature. The organic phase was dried under
N2 gas and the pellet dissolved in 150 μL methanol
(0.1% methane acid) and filtered with a 0.22-μm filter membrane. The
purified product was then subjected to high-performance liquid
chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. HPLC
analysis was performed using a ZORBAX SB-C18 (Agilent Technologies)
column (2.1 mm × 150 mm; 3.5 mm). The mobile phase A solvents consisted
of methanol/0.1% methanoic acid, and the mobile phase B solvents
consisted of ultrapure water/0.1% methanoic acid. The injection volume
was 2 μL. MS conditions were as follows: the spray voltage was 4500 V;
the pressure of the air curtain, nebulizer, and aux gas were 15, 65, and
70 psi, respectively; and the atomizing temperature was 400°C.