5975c inert xl ms detector

At transplant and three weeks after transplant (harvest), height from the substrate surface to the meristem, leaf area of two (seedling) or four (harvest) most recently fully expanded leaves (measured with LI-300; LI-COR Biosciences), stem diameter (harvest reps 2 and 3) at the base, and shoot fresh mass were recorded. Additionally, the number of branches >2.5 cm and node number (rep 2 and 3) were recorded at harvest. Tissue was placed in a forced-air oven maintained at 75°C for at least 3 d, weighed, and dry mass was recorded. Three weeks after transplant, the two most recent, fully mature leaves of five plants from each treatment were detached, frozen, and stored at -20°C until gas chromatography mass spectrometry (GCMS) analysis as reported in Walters et al. [25 (link)] from a method derived from [26 (link)] Schilmiller et al. Tissue was ground in liquid nitrogen, and compounds were extracted with methyl tert-butyl ether (MTBE) with a tetradecane internal standard. Samples were analyzed using an Agilent 7890A GC and single quadrupole MS with 5975C inert XL MS detector (Agilent, Santa Clara, CA). Compound concentrations were normalized to the sample internal standard and leaf dry weight, then quantified using the standard calibration curves of 1,8 cineole, eugenol, linalool, and methyl chavicol with a tetradecane internal standard (Millipore Sigma; St. Louis, MO).

Two weeks after sowing the two most recent, fully mature leaves of five plants per treatment, per replication (10 total plants), were detached, frozen, and stored at -20°C until GCMS analysis. In a method derived from Schilmiller et al. (2010) (link), tissue was ground in liquid nitrogen, and an aliquot was placed in a 1.5 mL microcentrifuge tube containing 500 μL of methyl tert-butyl ether (MTBE) with 10 ng ⋅ μL^–1 of tetradecane internal standard and gently rocked for 3 min. Samples were centrifuged to pelletize the tissue and 150 μL of supernatant was transferred to GS auto sampler vials. Samples were analyzed using an Agilent 7890A GC and single quadrupole MS with 5975C inert XL MS detector (Agilent, Santa Clara, CA). Standards were utilized to identify metabolites in addition to m/z values in the ChemStation database. Peak areas were integrated using MassLynx V4.1 QuanLynx software (Waters Corporation, Milford, MA). The compound concentrations were normalized to the sample tetradecane internal standard and leaf dry weight, then quantified using the standard calibration curves of 1,8 cineole, eugenol, linalool, and methyl chavicol with a tetradecane internal standard (Millipore Sigma; St. Louis, MO).