*2.2. Measurement of Relative Electrolyte Leakage and Proline Content*

Relative electrolyte leakage, which measures cellular membrane integrity, is frequently used to evaluate plant stress tolerance [36]. The upper third of the fully expanded leaves from treated and non-treated plants of 17-03 and H1023 were excised and used to generate leaf discs (9 mm in diameter). Three replicates were used for each line, with 20 leaf discs per replicate. The leaf discs were placed into 50 mL centrifuge tubes containing 25 mL of distilled deionized water and shaken at 60 rpm for 12 h in the dark at 25 ◦C. The electrolyte leakage (R1) of the solution was measured using a portable magnetic conductivity meter (DDB-303A, Shanghai, China) at 25 ◦C. The solutions were boiled for 30 min and then cooled to room temperature. The electrolyte leakage in the boiled solution (R2) was then determined using the same method. The relative electrolyte leakage (%) was calculated as (R1/R2) × 100.

The proline content was measured as described by Ben et al. [37]. Briefly, 200 mg of ground leaf sample was extracted with 2 mL of 3% sulfosalicylic acid in boiling water for 10 min. The samples were cooled and centrifuged at 4000 rpm for 15 min. Then, 1 mL of the supernatant was transferred to a new 15 mL test tube. Then, 1 mL of glacial acetic acid and 1 mL of acid-ninhydrin reagent were added. The mixture was boiled for

30 min and cooled in an ice bath to terminate the reaction. Next, the reaction mixture was partitioned by adding toluene (3 mL). After static delamination, the upper liquid was centrifuged at 4000 rpm for 15 min. The absorbance of the organic phase was measured in a spectrophotometer (Schimadzu, Japan) at a 520 nm wavelength. The proline content of the leaf samples was calculated using a standard curve constructed with known amounts of proline.
