Identification and determination of metabolites in plant cell biotechnology by gas chromatography and gas chromatography/mass spectrometry : Application to Nonpolar Products of Chrysanthemum cinerariaefolium and Tagetes Species

Abstract

Gas-liquid chromatography (g.l.c.) and gas-liquid chromatography/mass spectrometry (g.c./m.s.) were used for profile analysis and for identification, confirmation and determination of traces of nonpolar metabolites synthesized in plant tissue, calli and in suspension-cultured cells of Chrysanthemum cinerariaefolium (pyrethrum) or Tagetes species (marigold). When dried leaf samples (1–20 mg) were tested, six pyrethrins were detected simultaneously at the picogram level with an electron capture detector. This method permits the early selection of high yielding tissues and calli for the development of plant cell cultures. α-Terthienyl was quantified at the nanogram level with the electron capture detector; calibration graphs were linear between 0.2 and 20 ng and the minimum detectable quantity was about 0.1 ng. The concentration of α-terthienyl in air-dried roots of T. erecta was 140 μg g −1. However, this procedure used alone is not reliable, because α-terthienyl and the pyrethrin Cinerin I have the same retention time. Mass spectrometry is needed for identification and confirmation. Profile analysis of a leaf extract of T. minuta by high-resolution g.c./m.s, and accurate mass measurements showed 13 nonpolar compounds, of which 9 were tentatively identified; they include 2 sesquiterpenoids, 3 thiophenes, 2 sterols with a precursor and ethyl linolenate. Analyses of T. minuta indicated that in calli and cell suspensions, plant sterols are synthesized but sesquiterpenoids or thiophenes are not produced.