5.5 Phytohormones extraction and quantification
The extraction and purification of auxin (IAA), abscisic acid (ABA) and
jasmonic acid (JA) were carried out following previously described
method (Fu et al., 2012;
Pan et al., 2008) with slight
adjustments. The sample was pulverized in liquid nitrogen. 0.1g of
powder was homogenized in 1 ml of ethyl-acetate containing 25 µl of
internal standards of d2-IAA (Sigma-Aldrich), d5-JA (QCC) and d6-ABA
(OlchemIm Ltd, Czechoslovakia), agitated for 12 h at 140 rpm at 4°C,
centrifuged for 10 minutes at 12000 rpm at the same temperature and the
supernatant collected. The sample was re-extracted once with 1 ml
ethyl-acetate. The supernatants were pooled, dried using nitrogen gas,
precipitate re-suspended in 0.5 ml of 70% methanol, and centrifuged for
10 min at 12000 rpm at 4°C. Three aliquots of 0.2 ml of sample
supernatant were placed in separate snap-cap vials and analyzed using
HPLC-mass spectrometry as previously described
(Pan et al., 2019). The Agilent Zorbax
XDB C 18 column (150 x 2.1 mm, 3.5 µm) was employed for the HPLC
analysis as described earlier (Chen et al.,
2018).