*3.6. Association of Grain Yield and Other Parameters under Heat Stress*

Knowledge of association among the yield, yield-related, and other agronomic parameters is very important because it provide the basic information regarding the selection of certain parameters, which can be utilized as marker of grain yield improvement. In control conditions (Figure 6A), PH showed positive highly significant association with TPP (*r* = 0.62 \*\*\*) and a negative but significant association with GY (*r* = −0.40 \*) and HI (*r* = −0.65 \*\*\*). TPP negatively associated with HI (*r* = −0.50 \*) and NDVI (*r* = −0.43 \*). GY showed highly positive association (*r* = 0.65 \*\*\*) with HI. SY showed a negative but significant association with NDVI (*r* = −0.42 \*) and HI (*r* = −0.49 \*). There was a highly significant association between GL and GW (*r* = 0.65 \*\*\*).

Similarly, under heat stress, PH showed a positive association with TPP (*r* = 0.63 \*\*\*), SY (*r* = 0.51 \*\*), and GL (*r* = 0.40 \*). PH also showed a negative but significant association with HI (*r* = −0.39 \*) and NDVI (*r* = −0.44 \*, Figure 6B). GY showed a highly significant association with HI (*r* = 0.89 \*\*\*) and a negative but highly significant association with HSI (*r* = −0.88 \*\*\*). HI and SY also showed a negative but significant association (*r* = −0.74 \*\*\*). Similarly, HI showed a negative association with HSI (*r* = −0.72 \*\*\*).

**Figure 6.** *Cont*.


**Figure 6.** Association studies between agronomic parameters under control (**A**) and heat stress (**B**). PH = Plant height (cm), TPP = Tillers per plant, GY = Grain yield per plant (g), SY = Straw yield per plant (g), HI = Harvest index, GW = 1000-Grain weight (g), GL = Grain length (mm), NDVI = Normalized difference vegetative index, PFP = Pollen fertility percentage, his = Heat susceptibility index, and CMS = Cell membrane stability. \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001.

### **4. Discussion**

Rice is a very important cereal crop for majority of the world's population [51]. Previously, it had been reported that the origin of rice varieties was not related to the degree of heat tolerance [52]. In general, the different growth stages of rice behave differently towards heat stress, but the flowering stage is a particularly sensitive stage [53]. Previous studies showed that rice production was optimum at 32–36 ◦C and a reduction in yield was observed at higher temperatures beyond that level [32,53,54]. The global climate is changing rapidly, and during the 21st century the expected increase in the earth's temperature will be 2 to 4.5 ◦C [55]. Climate is intrinsically connected with agriculture and an increase in temperature will significantly reduce crop production [11,56]. It is reported that with every 1 ◦C increase in temperature, rice production will decrease by 2.6% [57]. The population of the world is also increasing day by day and is expected to reach 9 billion by 2050. Keeping in mind the increasing temperature and population and its demand, green super rice (GSR) was developed through the utilization of the world's best germplasm material (Figure 7). GSR has the potential to cope different environmental stresses and maintain an overall grain yield [44]. Furthermore, to our knowledge, GSR lines have never been evaluated for heat stress.

**Figure 7.** Salient features of GSR and basmati lines. (**A**) Comparison of GSR and basmati lines under control conditions. (**B**) Panicle comparison of basmati and GSR lines. (**C**,**D**) Basmati lines under control and heat stress conditions. (**E**) Seed comparison and (**F**) the effect of disease on GSR lines. Bars (**A**,**C**,**F** = 10 cm; **B**,**D** = 1 cm; **E** = 1 mm).

In this study, twenty-two GSR lines, along with four local Pakistani varieties (controls), were studied under normal and heat stress conditions for grain yield and morphophysiological parameters. Several morpho-physiological parameters collectively contribute to the grain yield [5,24,58]. In this study, we observed a significant reduction in TPP, SY, HI, CMS, and, ultimately, the GY, for most of the genotypes under heat stress. However, certain traits, including PH, GW, GL, PFP, and NDVI, were less affected under heat stress and probably contributed towards overall heat tolerance. In general, the GSR lines were less affected as compared to local varieties for several traits, showing their potential for breeding heat-tolerant rice cultivars. This was possibly due to GSR having more photosynthates than the control varieties because they might absorb more resources or nutrients in a short period of time [59,60].

Heat stress at the flowering or anthesis stage caused pollen sterility, which may lead to the failure of fertilization and ultimately reduction in yield [11]. Pollen sterility, in most cases, is a major reason for reduced grain production in rice under heat stress [24]. Surprisingly, we did not observe a considerable loss of PFP under heat stress, showing that reduced pollen fertility is not a reason for reduced GY (Figure 4). Compatible with this finding, we observed an increase in SY under heat stress in most of the genotypes, suggesting that plants increased their vegetative growth and slowed down their reproductive growth, which is an important avoidance mechanism for heat tolerance. Heat stress causes cell injury, which leads to the leakage of ions in the susceptible genotypes [61]. The genotypes which show better cell membrane stability under heat stress are generally considered to

be heat tolerant [24,62]. However, we did not observe a very straightforward trend of CMS with GY, suggesting that it may not be very reliable to screen tolerant genotypes only based on CMS. In addition, prolonged multi-generational heat stress at the flowering stage may cause the accumulation of mutations that enable the plants to become acclimated under heat stress conditions [63]. In the current study, we found NGSR-16 to be the most heat-tolerant GSR line as it showed a minimum or no reduction in the GY, TPP, HI, TGW, and CMS compared to rest of the GSR lines. Among the local variants and overall, Kashmir Basmati outperformed in all the traits with the least reduction in GY under heat stress compared to the control, suggesting a better source of breeding heat-tolerant cultivars. Kashmir Basmati has previously been identified as a heat-tolerant cultivar in a separate study in Pakistan [23]. It is interesting to note that Kashmir Basmati was originally bred in Pakistan as a cold-tolerant rice cultivar for the Kashmir region, where it tolerates cold stress by producing heat shock proteins [64]. This suggests that there could be a common mechanism for cold- and heat-tolerance, possibly involving heat shock proteins, which needs to be further studied to understand the biochemical mechanism of heat tolerance in GSR.

Stress breeding programs depend on the reliable selection indices to screen good germplasm. Thus, it is important to evaluate the association between the final yield and other agronomic indices. We used correlation and PCA analyses to study the association of GY with other traits. Results showed that the GY had a significant positive association with HI but a negative correlation with GW and GL, suggesting that increasing the GW and GL may decrease the final GY probably via decreasing the total number of grains. Thus, an optimum GW and GL would be required for an optimum GY, which involves a sophisticated balance of source–sink translocation.
