Selection of the Appropriate Melatonin Concentration

Green mustard seeds were provided by Danong Korea Ltd., Gyeonggi-do, South Korea. The seeds were surface-sterilized in 2.5% sodium hypochlorite and tested for viability, according to a method previously described by Ke et al. (2018). The germinated seeds were sown in plastic pot trays (28 cm × 54 cm) filled with horticultural soil (Shinsung Mineral Co., Ltd., South Korea). Then, two to three seeds were planted in each pot. The mustard seedlings were pre-cultivated under natural light in a greenhouse at Kyungpook National University, Daegu, South Korea (35.53′°N, 128.36′°E), with 46.7% relative humidity (RH) and 25°C/19°C (day/night) temperature, and the seedlings were watered daily. Furthermore, 3-week-old seedlings with uniform sizes were transferred to pots (10 cm × 10 cm) and subjected to different treatments. Pre-cultivated seedlings were kept for 2 days to allow them to acclimatize. Then, all mustard seedlings were randomly divided into two groups: (i) normal control, grown with only distilled water (100 mL/plant); and (ii) salinity treatment, irrigated with 150 mM NaCl (100 mL/plant). Each treatment contained a triplicate of 15 plants each. Each group was treated for 7 days. Afterward, the salinity-stressed seedlings were treated with 0, 0.1, 1, 5, and 10 μM melatonin (100 mL/plant, once a day) on their roots for 16 days. The melatonin (ChemScene LLC., Monmouth Junction, NJ, United States) solution (10 μM, stock) was prepared by dissolving the solute in distilled water (2.32 mg × 1,000 mL), followed by dilution with distilled water to prepare different concentrations. Plants of each concentration were measured for several plant growth characteristics. Plant growth parameters, such as plant height, leaf length, and leaf width were measured after day 4 (4DAT), day 8 (8DAT), day 12 (12DAT), and day 16 (16DAT) of treatment. Moreover, the 1 μM melatonin concentration was found to be the most optimal concentration for further studies.