Dt 102h

To optimize the condition, 50‐gram powder of M. piperita was dispersed in 250 ml of ethanol/water (0:100, 50:50, and 100:0) solvent. Then, the Erlenmeyer flask of the samples was placed in the ultrasonic bath (DT 102H, Bandelin). To find the optimum extraction condition with ultrasonic waves, different variables consisting of a ratio of ethanol to water (0:100, 50:50, and 100:0), the temperature (25, 45, and 65°C), and time (5, 27.5, and 50 min) were investigated. Then, the prepared solution was filtered via Whatman filter paper. The filtered solution exposed vacuum conditions until the complete evaporation of solvents. The prepared samples were kept at −18°C at the freezer until the experiments (Farahmandfar, Asnaashari, et al., 2019 (link); Hammi et al., 2015 (link)).

To preparation of elicitors, 0.138 g salicylic acid (Sigma-Aldrich, Münich, Germany) was first dissolved in 100 μL 1 N NaOH and made up to 1 mL with sterile distilled water, then passed through a 0.45-micron nitrocellulose filter and used in hairy root cultures at concentrations of 100, 200 and 300 μM. The stock of 1000 mg/L of water-soluble chitosan (Chitoplant; Sigma-Aldrich, Münich, Germany) was prepared and filtered through a 0.45-micron nitrocellulose filter before use. The filtered chitosan was applied to the hairy root cultures at concentrations of 100, 200, and 400 mg/L. Also for ultrasonic treatment, the hairy roots were placed in the ultrasonic bath (Bandelin, DT 102 H, 35 kHz, 320 W, Belgium) for 1, 2, and 4 min. Subsequently, the elicited hairy roots were placed on a 110 rpm shaker at room temperature in the dark. After 2 and 4 days, the hairy roots were harvested. After harvest, excess water was removed from the roots using Whatman filter paper and the roots were weighed with an analytical balance (AND Weighing FX-5000i Precision Balance 5200 × 0.01 g).

First, 50‐gram powder of P. khinjuk fruit was dispersed in 250 ml of ethanol/water (60:40) solvent. Then the Erlenmeyer flask of the samples was placed in the ultrasonic bath (DT 102H; BANDELIN) (35 kHz, for 50 min at 65°C) (Estakhr et al., 2020).