*3.1. Physiological Traits*

The viability and germination of pollen grains were higher in the bottom part of the plant as compared to pollen that developed in flowers on the top part of the plant (Figure 3). The maximum viability of pollen grains in the bottom position was seen in CYTO-608 (47%) followed by MNH-1026 (43%), and the lowest value was observed in MNH-1016 (28.67%). The top flowers of CYTO-608 shown in Figure 4 and N-1048 showed the highest pollen viability measures of 40.33% and 36%, respectively, while MNH-1016 and CIM-602 had the lowest pollen viability of 21% for both genotypes (Table 4). Tukey's mean comparison test for pollen viability revealed non-significant differences for the genotypes CYTO-608, N-1048, and AA-933. On average, these genotypes performed well in both top and bottom positions (Table 5). Mean values of pollen viability across all genotypes to compare top and bottom positions of plants revealed a significant difference between top and bottom positions. Pollen viability at the bottom position exhibited more value compared to the top position (Figure 3).

**Figure 3.** Means averaged across all genotypes to compare top and bottom positions of plants for different traits at maturity. The error bars are standard errors. The letters show Tukey's mean comparison where different letters show significant differences between top and bottom positions for each trait. (**a**) PV (Pollen viability), PG (Pollen germination), CMT (Cell membrane thermostability) and boll ret (boll retention), (**b**) Chl. Content (Chlorophyll content), (**c**) Boll wt (Boll weight).

The highest pollen germination from flowers at the bottom position was observed in AA-933 (31.33%) while in the top position, both AA-933 and CRS-2 showed 24% pollen germination. The lowest value for this parameter was observed in N-1048 with 12.67% and 9% germination in the bottom and top positions, respectively (Table 4). Overall, pollen from flowers that bloomed on top parts of the plant showed less germination when compared to pollen from bottom flowers (Figure 3). Pollen germination estimates were also lower than pollen viability estimates. Tukey's mean test revealed a non-significant difference between CRS-2, Cyto-608, and AA-933. It was observed that these genotypes performed well in both viability and germination tests, except CRS-2 which showed good pollen germination (Table 5). This indicates that most of the viable pollen of CRS-2 did germinate. Variation in pollen tube length was also observed as shown in Figure 5.

**Figure 4.** Results from staining pollen with triphenyl-tetrazolium chloride (TTC). (**a**) Pollen from flowers from the bottom positions of CYTO-608 shows the highest pollen viability. (**b**) Pollen from flowers from the top positions of NIBGE-2 shows the lowest pollen viability.

**Table 5.** Mean values for six measures of heat tolerance in 13 cotton genotypes grown in 2019 in Faisalabad, Pakistan.


PV = Pollen viability; PG = Pollen germination; Chl. Cont. = Chlorophyll content; CMT = Cell membrane thermostability; Boll Wt. = Boll weight; Boll Ret. = Boll retention. Means with the same letters in each column are not significantly different according to Tukey's test.

**Figure 5.** The observed variation in pollen tube germination where (**a**) Bottom flowers from AA-933 showed maximum pollen germination (**b**) Top flowers from N-1048 showed lowest pollen germination.

Cell membrane thermostability (CMT) and chlorophyll content were also significantly different for genotypes and plant positions (Table 3). The CMT values for top leaves (67.96%) were lower than values for bottom leaves (78.17%) (Figure 3). At the bottom of the plant, the maximum value for CMT was recorded for the genotype AA-933 (92.87%) followed by MNH-1016, CYTO-608, and CIM-602 which presented 92.50%, 92.35%, and 92.31% CMT, respectively (Table 4). Leaves of the bottom branches had more chlorophyll content as compared to leaves from the top branches (Figure 3). The genotypes CIM-602, CYTO-608, and AA-933 showed the highest chlorophyll contents in bottom branches (103.8, 87.13, and 78.83, respectively) while CIM-602 also had the highest chlorophyll content in leaves of top branches (96.9) (Table 4).
