Lipid Profiling of Plant Tissue

Leaf discs (2 cm diameter) were cut from 6 different plants (two plants per replicate). Total lipids were extracted from freeze dried tissue as previously reported (Petrie et al., 2012 (link)), run on a TLC plate (20 cm², Silica gel 60, Merck) and developed using a two-step solvent system. Samples were first run to 12 cm in chloroform:methanol:acetic acid:water (68:22:6:4, v:v:v:v), followed by a second separation in hexane:diethyl ether:acetic acid (70:30:1, v:v:v). TAG, DAG and MAG bands were visualized using iodine vapor and isolated from the silica gel. Fatty acid methyl esters were prepared from each isolated lipid fraction and analyzed by GC-FID (7890A GC, Agilent Technologies, Palo Alto, CA, USA) equipped with a 30 m BPX70 column (SGE, Austin, TX, USA) as described previously (Petrie et al., 2012 (link)). Peaks were integrated with Agilent Technologies ChemStation software (Revion B.04.03).