*2.2. Data Collection*

Heat tolerance measurements were taken during the growing season when 50% of the crop was flowering. Each plant was divided into two equal parts by measuring plant height in such a way that the bottom portion was under the shade of the plant canopy while the upper portion was exposed to direct sunlight. Flowers in the top part of the plant were exposed to direct sunlight while flowers on the bottom part of the plant were under the leaf canopy and received indirect sunlight. Heat tolerance-related parameters including pollen germination (PG), pollen viability (PV), and cell membrane thermostability (CMT) were assessed under in vitro conditions while boll retention and boll weight were measured in vivo.

Flowers that showed dehiscence of anthers were collected from the field and immediately transported to the laboratory where pollen grains were deposited on pollen germination media. The media was prepared following the method explained by Burke et al. [9] with little modifications. The solid germination medium consisted of 2% (*w*/*v*) agarose (Product no. A4718, Sigma Aldrich, Merck, Darmstadt Germany), 25% (*w*/*v*) sucrose (Product no. S0389, Sigma Aldrich, Merck, Germany), 0.52 mM KNO3 (Product no. P8291, Sigma Aldrich, Merck, Darmstadt Germany), 3.06 mM MnSO4 (Product no. M7899, Sigma Aldrich, Merck, Darmstadt Germany), 1.66 mM H3BO3 (Product no. B6768, Sigma Aldrich, Merck, Darmstadt Germany), 0.42 mM MgSO4·7H2O (Product no. M2643, Sigma Aldrich, Merck, Darmstadt Germany) and 1.0 μM A3 gibberellic acid (Product no. G7645, Sigma Aldrich, Merck, Darmstadt Germany). The pH of the germination medium was brought to 7.6 before adding sucrose and agarose. The medium was autoclaved and poured into Petri plates (100 × 15 mm, Product no. P5856, Sigma Aldrich, Merck, Darmstadt Germany) under a laminar flow hood to avoid contamination. Plates were wrapped with cling

film tape then placed in a refrigerator until used. Pollen grains with pollen tube lengths greater than the diameter of the pollen grains themselves were considered to be germinated (Figure 1). Percent pollen germination was estimated using the following equation:

Pollen germination (%) <sup>=</sup> Number of germinated pollen grains Total number of pollen grains <sup>×</sup> <sup>100</sup>

**Figure 1.** An example of pollen tube growth. A pollen grain that has germinated its pollen tube is labeled as 'a' while non-germinating pollen grains are labeled as 'b'.

The triphenyl-tetrazolium chloride (TTC) test was used to test the viability of pollen grains [26]. Flowers that showed dehiscence of anthers were taken into the laboratory to test pollen viability. Fresh pollen grains were sprinkled on a glass slide (76 × 26 mm) by gently tapping the flower. Two to three drops of 0.5% 2,3,5-triphenyl tetrazolium chloride (Product no. 17779, Millipore, Merck, Germany) were added in a 15% sucrose solution (Product no. S0389, Sigma Aldrich, Merck, Germany). The slide was covered with a coverslip (20 × 20 mm) to prevent desiccation and then placed under sunlight for 60 min at 30–37 ◦C. After this exposure, slides were observed under a light microscope (Model XSZ 107BN, Manufacturer: Zenith Lab Inc., Zhejiang China). The pollen grains that changed to red color after exposure to the TTC solution were considered viable while non-viable pollen remained yellowish in color (Figure 2). Pollen viability percentage was estimated using the following equation:

$$\text{Polllen viability } (\%) = \frac{\text{Number of viable problem grains}}{\text{Total number of golden grains}} \times 100\%$$

Two leaves from the top of the plant and two leaves from the bottom of the plant were selected for measuring CMT following the protocol of Sullivan [27] and using the following equation:

$$\text{Cell membrane thermosstability } \left( \% \right) = \left[ \frac{1 - \text{T}\_1 / \text{T}\_2}{1 - \text{C}\_1 / \text{C}\_2} \right] \times 100$$

where, the subscripts 1 and 2 refer to the 1st and 2nd electrical conductivity (EC) readings, respectively, and T and C refer to the EC of heat-treated (T) and control (C) sets of test tubes. The EC value was measured by a portable EC meter (FieldScout EC 110 Meter).

Boll weight, boll retention percentage, chlorophyll content, and canopy temperature were measured at harvest. For boll weight, all bolls from plants within the plot were harvested and weighed using an analytical balance (least count = 0.01 g). Total boll weight was divided by the total number of selected bolls to get the average weight of an individual boll. Boll retention percentage was estimated as the number of fruiting positions on the plant that had bolls divided by the total number of fruiting positions. To measure boll retention, all fruiting squares were labeled 60 days after sowing (DAS). One hundred days after sowing, the number of labeled bolls was counted. Boll retention was calculated as follows:

> Boll retention <sup>=</sup> Number of labeled bolls 100 DAS Number of labeled fruiting squares 60 DAS <sup>×</sup> <sup>100</sup>

The leaf chlorophyll content was measured using a "SPAD 502 Plus" (Konica Minolta, Japan) chlorophyll meter which works on the principle of red and blue light absorption (therefore, the SPAD measurement has no units). Top and bottom canopy temperatures of upland cotton genotypes was measured using an infrared crop temperature meter (Model: 2956, Spectrum technologies, Inc., Plainfield, NJ, USA) at crop maturity (Table 2).

**Figure 2.** An example of results from the triphenyl-tetrazolium chloride (TTC) test. Viable pollen has changed to a red color (labeled as 'a') while non-viable pollen does not change color (labeled as 'b').


**Table 2.** Top and bottom canopy temperature in 13 cotton genotypes grown under field conditions in 2019 in Faisalabad, Pakistan.
