Volatile Organic Compound (VOC) Collection and Analysis
VOCs were collected from each of the plants within the various salt treatments. Individual tomato plants were placed in a 9-liter glass chamber, and VOCs were collected for 12 hours using a push-pull system at a push flow rate of 1 L/min and a pull flow rate of 0.8 L/min, following which plant fresh and dry shoot weights (FW and DW) were recorded. Ten plants were sampled per treatment. Volatiles were collected using adsorbent filters containing 45 mg HayeSep Q (Hutchison Hayes Separation Inc., USA) and were eluted by passing 150 μl dichloromethane through the traps into 2.0 ml glass vials equipped with a 250 μl glass insert. 5 µl of internal standards octane (40 ng µl-1) and nonyl acetate (40 ng µl-1) were added to the samples (Helms et al. , 2019).
VOCs were analyzed by GC-MS using an Agilent 7890A gas chromatograph equipped with an Agilent HP-5MS UI (30m x 0.25 mm x 0.25 µm) column coupled to an Agilent 5975C mass spectrometer configured with standard EI tune settings. Splitless injections were performed at an inlet temperature of 250°C using helium carrier gas with a constant flow rate of 0.7 mL/min. After 1 µl sample injection, the column was maintained at 40°C for 2 minutes. The oven temperature was increased by 10°C per minute and held at 300°C for 4 minutes. Target compounds were identified by comparing mass spectra and retention indices published in NIST17, Adams, and the University of Göteborg libraries (Helms et al. , 2017; Lin, Chen, et al. , 2021). Compounds with quality scores >85 were selected for further analysis. The abundance of each compound per gram of fresh plant tissue was calculated using the internal standard of nonyl acetate. A principal component analysis (PCA) was performed on the analyzed compounds for visual representation.