*2.2. Cell Membrane Thermostability*

In the last week of the heat stress, the flag leaf samples from control and stressed plants were collected in pre-labeled 20 mL glass tubes. The leaf segments were treated with distilled water used for conductivity measurements. Cell membrane thermostability was measured by following the method of [48] and following formula:

$$\text{CMS\%} = \frac{1 - \left(\frac{\text{T1}}{\text{T2}}\right)}{1 - \left(\frac{\text{C1}}{\text{C2}}\right)} \times 100$$

where T and C refer to stressed and control plants, respectively. T1 and T2 are electrode conductance measurements before and after autoclave, while C1 and C2 are electrode conductivity measurements before and after autoclave.

### *2.3. Pollen Fertility Test*

A pollen fertility test was performed for all 26 genotypes following the method of [4] using light microscope (Nikon digital sight DS-Fi2). Spikelets were collected from the heat-treated plants on the following day in the morning before anthesis and preserved in formaldehyde solution (FAA). FAA was prepared with 1:1:18 ratio of formaldehyde, acetic acid, and ethanol, respectively. Anthers were placed on slide and pollens were extracted by crushing the anthers with the help of forceps. Then 50 μL of 1% potassium iodide (I2-KI) solution was added to the slide and covered with cover slip for visualization under compound microscope. Pollens that stained black with a circular shape were counted as fertile, while the irregular red-orange pollens were considered sterile [31]. At the end, pollen fertility percentage (PFP) was calculated as followed:

$$\text{PFP} = \frac{\text{No. of fertile pollens}}{\text{Total No. of pollens}} \times 100$$
