*2.3. Plant Materials and Treatments*

Marie Galante-85 and CRI-12 semi-wild accessions of *G. hirsutum* were used, as they are tolerant to drought and salt stresses. Seeds of the two accessions were provided by the Institute of Cotton Research, Chinese Academy of Agricultural Sciences, where the entire experiment was performed with a Complete Random design. In the preliminary steps, the cotton seeds were soaked in dd H2O overnight to allow the seed coat to soften. The soaked seeds were then grown in folded absorbent papers vertically placed in mini rectangular plastic buckets, which had been filled with distilled water halfway and left for a period of four days [31]. Upon germination (the sixth day), the healthy seedlings were transplanted to a Hoagland nutrient solution medium in the greenhouse. In the greenhouse, transplanted seedlings were treated by a 16 h light-8 h dark photoperiod with specified temperatures of 28 ◦C during the day and 25 ◦C at night. The relative humidity in the experimental room was maintained at 60–70%, as previously described [32]. The entire Hoagland nutrient solution medium was replenished when the seedlings reached the three-true-leaf stage, and freshly prepared solutions of 17% of glycol PEG-6000 and 250 mM of sodium chloride compounds were immediately added to simulate drought and salt stresses, respectively [33]. The healthy tissues of the root and leaf were collected from nine plants of each category for RNA extractions after stress exposure at the following time

intervals: 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h. Three biological replications were considered in each case. Untreated plants were considered as the control. The harvested tissues were directly frozen in liquid nitrogen and transferred to the fridge at −80 ◦C for storage up to the total RNA extraction.
