2.7 FA measurement
The freeze-dried tobacco seeds or leaves were suspended with methanol
solution containing 1M HCl, and heated at 80°C for 2 h. 2 mL of 0.9%
(w/v) NaCl and 2 mL hexane were added to the supernatant to extract the
FA methyl esters, and the organic phase with methyl heptadecanoate as an
internal standard was used for the following analysis. Gas
chromatography was then performed on a GC–2014 gas chromatograph system
(Shimadzu,http://shimadzu.com/an/gc/gc2014.html)
as described previously (Chen, Wang, et
al., 2012; Siloto et al., 2006;
Z. Wang et al., 2014). Each FA species
was identified by the characteristic retention times, and their
concentrations were normalized against the internal control.