Compound Analysis of Transplanted Leaves

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).