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.