1200 mass spectrometer

Stover samples (∼0.2 mg) from WT and bmr lines were pyrolyzed in a Varian 1079 programmable temperature vaporization (PTV) injector mounted on a Varian 3800 gas chromatograph connected to a Varian 1200 mass spectrometer. The PTV injector was operated at a split ratio of 1:100 at 450°. The pyrolysate was led on a capillary column (25 m, 0.32 mm i.d., fused silica coated with SGE BPX5). Helium (1.2 ml/min) was used as the carrier gas. The GC program started with a hold at 70° for 4 min, followed by a temperature increase to 150° at a rate of 2°/min. The temperature was subsequently increased to 250° at a rate of 4°/min, then to 320° at a rate of 30°/min. The mass spectrometer was operated at 1.0 kV. The mass range included m/z 45 to 350 and was scanned every 0.2 sec. The identification of compounds was based on Ralph and Hatfield (1991) . To compare the proportion of the hydroxycinnamic acids p-coumaric acid and ferulic acid in the biomass, stover was subjected to pyrolysis-TMAH-GC-MS as described by Saballos et al. (2012) (link). Samples of approximately 0.2 mg stover from BTx623 and the novel bmr mutants were trans-methylated with 2.5% (v/v) tetramethyl ammonium hydroxide (TMAH; Sigma-Aldrich, St. Louis, MO) in methanol (Mulder et al. 1992) and pyrolyzed in a Varian 1079 PTV injector mounted on a Varian 3800/1200 GC-MS as described above.