Screening of Elite Coarse Rice Lines for Drought Stress Simulated by Polyethylene Glycol (PEG) at Seedling Stage

| Among various abiotic stresses causing serious impact on crop growth, drought poses a significant reducing stress towards crop yield and its stability over the time and locality. In terms of sensitivity to this environmental stress, rice, which is a drought-sensitive crop, exhibits notable varietal variability. for the estimation of elite coarse rice lines to withstand against the drought stress especially during early plant developmental phases and germination and seedling stage, an experiment was carried out from June to July 2021 at the


Introduction
A biotic stresses, which are increasing throughout the world, have a negative impact on agriculture's productivity by affecting the growth of plants, which lead to the failure of crop and pose a serious threat to crop production (Wu et al., 2011;Musclo et al., 2014).Due to drastic change in climate and increased in the population of world, it is becoming difficult to meet the human requirements (food, shelter, and other necessities) (HongBo et al., 2005;Akram, 2007).Rice plant is sensitive to drought conditions, as it exhibit negative impact when subjected to the moisture stress condition at critical stages of development (Cha-Um et al., 2010) and approximately 50% of the world's rice crop production is impacted by water shortage conditions (Mostajean and Rahimi-Eichi, 2009).The impact of water stress was greater during the germination and seedling stages, which shows the crop sensitivity to moisture stress at these stages, effect the overall success of a crop (Tsago et al., 2014;Khayatnezhad et al., 2010).Water is a limiting abiotic stress that lowers plant yield, drought stress cause reduction in the development process of rice plant (Purbajanti et al., 2017).The water scarcity cause drought condition which triggers the numerous plant responses include cell division, cell growth, stomata conductress, photosynthesis rate, transpiration rate and eventually yield of plant (Chaves and Oliveira, 2004;Cattivelli et al., 2008;Tuna et al., 2010).Plants respond differently under water deficit conditions, some shows tolerance and some shows avoidance mechanism (Nazar et al., 2015).
Rice crop is significantly affected by the drought stress which limits its production per unit area in Pakistan.Several factors can contribute to drought stress in rice crops, the major factor is climate change that results in increased temperatures and changing rainfall pattern, which can lead to drought stress in rice crop (Faroq et al., 2015).Second one is the water scarcity and limited access to irrigation water at the time of transplanting or even lateral crop stages specially at flowering time dry weather and heat stress conditions also cause evapotranspiration losses of water both in the file and affect crop physiology.Soil conditions including high salinity and poor crop management practices for irrigation such as less irrigation due to fear of increase cost of production due to power expenditures for ground water irrigation also led to drought stress in rice crop.On an average almost 1500 liters of irrigation water is utilized to gain 1 Kg of rice, major outflow goes in the form of evapotranspiration, percolation, and seepage.Under the present scenario of climate change and increased population pressure water availability to raised crops is challenging.Screening of rice genotypes resilient to climatic stresses specially drought stress is prerequisite for drought tolerant variety development.
Plants are screened for moisture stress using a substance called polyethylene glycol (PEG).The rice crop's stunted development is caused by a lack of water, and this condition also affected the rate of photosynthesis and the length of the charging period, which in turn reduced the grain yield of rice (Sayar et al., 2008).Breeders should give careful consideration to morphological analyses of the traits linked to tolerance and yield improvement when selecting genetic components for the development of new variety.In breeding projects, choosing, and using features related to yield and tolerance could result in the development of sustainable varieties in stressprone areas (Masud et al., 2022).
Water scarcity shows strong associated connection to limit the rice crop productivity and quality, and its effects are expected to become more severe with increasing climate variability and change.Despite extensive research efforts, we could not reveal a concise and open information related to drought tolerance mechanism of rice.
There is a need to unveil the impact of drought on rice crop which may be achieved by artificially inducing drought stress by the application of polyethylene glycol (PEG) as it produces almost similar level of hindering effects on plant.PEG treatment induces osmotic stress in plants, simulating drought stress, and which ultimately helps to figure out the genotypes with tolerance potential to withstand drought levels.
PEG-induced drought stress has been widely adopted method as it is reliable and repetition of similar stress is possible for the confirmation of genetic potential of genotypes and to test the tolerance efficacy as well.reproducible method for screening rice germplasm for drought tolerance.PEG treatment has also been used to identify key characters which may pose a significant role towards the drought tolerance by improving water use efficiency and normalizing the osmotic adjustment.
The basic objective of this experiment was to reveal the potential of rice germplasm for their genetic capability to withstand the severity of drought stress being induced at various levels by polyethylene glycol (PEG) during early growth stages.The study aims to identify the most suitable parent plants with higher tolerance to PEG, an indicative of drought tolerance, which further may be employed in our breeding program in different crossing schemes.The use of PEG as a stress inducer in this experiment provides an opportunity to assess the ability of rice plants to survive under limited water availability conditions, which is an essential trait for rice cultivation in regions with limited water resources.By identifying the genotypes with higher PEG tolerance, the study can provide insights into the genetic mechanisms lying behind the ability to tolerate drought stress.Exploitation of the potential parent genotypes having higher degree of tolerance may lead towards the development of improved drought tolerant rice verities.

Experimental site
Experiment was carried out at Rice Molecular Breeding Laboratory of Rice Research Program, Crop Sciences Institute, National Agriculture Research Centre, Islamabad between June to July 2022.

Material
In this experiment 10 coarse rice GSR lines obtained from PGRI, NARC were used against various levels of drought stress (Table 2) at germination and seedling stage.

Method
The rice lines were tested against 4 concentrations of Polyethylene glycol 6000 (PEG-6000) at 0, 10, 15 and 20% induced drought stress.For the execution of experiment, completely randomized design was adopted with 2 factors and two replications.First factor was 10 GSR lines while second factor was four (0, 10, 15 and 20%) level of drought stress.The 5% sodium hypochlorite solution was used to immerse the seeds for 5 min followed by washing thrice with distilled water.Petri plates containing filter paper were arranging in CRD design, 5 seeds of each entry (rice line) were transferred into petri plates and treated with respective treatments.Distilled water was used in treatment 1 whereas for other treatments first two days 5ml of PEG solution was used twice a day and then on later days 5 mL PEG solution was used once a day.Each entry treated with respective treatment was allowed to germinate at 25 o C. The data on germination was noted on daily basis until full germination.In tenth day germinated seeds were taken out for data collection.

Parameters studied
The parameters examined in the study were as follows: Seedling fresh and dry weight, stress tolerance index, germination rate and percentage with stress tolerance index (GSTI) and vigor index.
Germination percentage: After 10 days of seed sowing germination percentage was calculated by using following formula (Yari et al., 2010).
Germination (%) = (germinated seed/ total number of seeds) * 100 Germination stress tolerance index: The germination stress tolerance index (GSTI) was determined as a percentage and for the calculation of GSTI promptness index was required.Promptness index was calculated by using the following formula (Ashraf et al., 2008).whereas seedling fresh weight was measured in (g) by using digital weighing balance and seedlings were dried 24 hours at 70ºC then by using digital weighing balance seedling dry weight was measured.

Seedling fresh and dry weight stress tolerance index:
The following formula was used to compute the stress tolerance index for seedling fresh weight and dry weight.
Vigor index: According to Sagar (2017), the vigor index (VI) was determined using the method below: Where SL= length of the seedling and GE= germination rate (germination percentage).

Statistical analysis
Data collected was analyzed statistically for analysis of variance (ANOVA) by using R studio agricolae package) and (least significant differences (LSD) was applied at 0.05% for comparisons of means among treatments.Correlation and principal component analysis (PCA) were done by using R Studio 4.2.1 (Facto extra package).

Results and Discussion
To evaluate the importance of specific traits (variables), a two-step process was followed.First, an analysis of variance was conducted, and then a correlation matrix was generated for all the traits.The results, shown in Table 4, revealed that several variables exhibited significant positive correlations.These correlations could be explored further using principal component analysis (PCA).The Eigen values in Table 5 explain the partitioning of total variance that account into each component (principal component) and explained the variance percentage and cumulative proportion for all components (Raza et al., 2017).For the number of principal components in Table 5, eigenvalues greater than 1 were taken into consideration Figure 9 scree plot, in this regard PC1 and PC2 were retained Table 6.Principal component analysis was used to evaluate the relationship between the attributes of 10 elite lines of rice.PCA classified 6 traits into two components, PC1 and PC2 contributed 84.5% of total variance, PC 1 with highest variance of 65.7% and PC2 with 18.8% as observed in Figure 10.The plant life cycle's most crucial stage is germination.
To ascertain the tolerance level of rice germplasm under moisture stress (water deficit) circumstances, the effects of increased moisture stress generated by PEG on seed germination and the response of elite coarse GSR lines by increasing the PEG concentrations were examined.The mean square analysis of variance (Table 3) showed that for germination emergence (GE) percentage and for germination rate was highly significant among treatments.The results obtained from the study showed that maximum percentage germination was found in control (T0) whereas zero percent germination (seeds were not germinated) was observed at 20% PEG concentration.In Figure 1 graph showed that increased in concentration of PEG result in decreased germination percentage.Under control (T0) condition all genotypes showed 100% germination whereas in treatment two at 10% PEG maximum germination (100%) was observed in NRPC 9 and NRPC 10 followed by NRPC 1, 5, 6 and NRPC 7 with 80% germination whereas minimum GE (60%) was found in NRPC 2, 3, 4 and NRPC 8.In treatment three at 15% PEG concentration NRPC 9 showed maximum GE (80%) and minimum GE (20%) was observed in NRPC 3, 4 and 8. Table 3 shows vigor index among treatments was highly significant.In Figure 4 graph showed the vigor index of all rice lines highest index was found in control (T0) condition ( 336) in NRPC 1 and the lowest vigor index was observed at 15% PEG induced moisture stress (1) in NRPC 3.Under treatment two 10% PEG induced water stress highest index was found in NRPC 3 (64).At 15% PEG imposed moisture stress highest vigor index was observed in NRPC 9 (27).
The GSTI (germination stress tolerance index) showed similar pattern in Figure 3, declining as PEG concentration increased.Figure 3 graph demonstrated that NRPC 6 at 10% PEG-induced stress had the greatest GSTI (100), while NRPC 3 and NRPC 4 had the lowest (50).The highest GSTI was found in NRPC 7 (68) and the lowest (10) in NRPC 3 and NRPC 8 at 15% PEG concentration.The water potential in the osmotic solution decreases as PEG concentrations rise because there is less oxygen available for the seed to germinate (Purbajanti et al., 2019;Ashaduzzaman et al., 2020).ANOVA showed (Table 3) significant result among treatments for seedling fresh weight, within treatment significant and between genotypes non-significant.Figure 6 revealed that maximum fresh weight was observed in control (T0) condition (0.1573 g) in NRPC 1 followed by T1 (10% PEG) with fresh weight of (0.1 g).At 15 % PEG (T2) highest seedling fresh weight was observed in (0.014g) in NRPC 6.In all the green super rice lines seedling fresh weight decreased by increasing PEG concentrations.For seedling dry weight, the analysis of variance (Table 3) revealed significant results within treatments and nonsignificant results within genotypes and for genotype x treatment interaction was not significant.The results of the Seedling Fresh and Dry Weight Stress Tolerance Index (SFWSTI and SDWSTI) showed that as the level of drought rose, seedling fresh weight stress tolerance decreased.The SWFSTI of rice seedlings subjected to higher PEG concentrations showed a discernible decline (Figure 8).In NRPC 7, the greatest SWFSTI value (96.0) was found at 10% PEG concentration, and the smallest value (21.7) was found at 15% PEG concentration.Similar outcomes were seen in Figure 7, where SDWSTI decreased as the level of drought stress increased.Several studies revealed consistent trend of decreased seedling fresh and dry weight under more severe drought stress conditions ( Ji et al., 2012;Mostajeran and Rahimi-Eichi, 2009).The low availability of water to plants is due to osmotic stress which causes a reduction in cell elongation and division process therefore by lowering cell growth and the turgor pressure as a result, there is a corresponding decrease in dry mass and biomass.(Farooq et al., 2015;Sagar, 2017;Roy et al., 2018;Ashaduzzaman et al., 2020;Islam et al., 2018).The ANOVA showed (Table 3) significant variance among treatments whereas non-significant among genotypes for seedling height.The interaction among genotype and treatment showed significant difference at 1% alpha.The graph in Figure 2 showed maximum seedling height was observed in NRPC1 (3.2800 cm) in control condition (T0).Seedling height decreased with increasing PEG concentrations at 10% PEG maximum seedling height was found in NRPC 3 (1.0050cm) and at 15%PEG concentration maximum height was found at NRPC 1 (0.3250 cm).
The water stress condition inhibits the emergence of radicle is due to decreased water potential gradient between seed and the external environment, therefore impairs height of seedling (Murillo-Amador et al., 2002;Sokoto and Muhammad, 2014).This could be a result of the negative impacts of water stress on the growth of root cells, which would impede nutrient uptake and negatively impact photosynthesis, which is necessary for biomass buildup and, consequently, shoot and root elongation.Therefore, it appears that under water stress, plants' ability to absorb and use water is reduced to the point where their droughttolerance mechanisms cannot support continued normal development (Ashaduzzaman et al., 2020;Islam et al., 2018;Magar et al., 2019).

Conclusions and Recommendations
The results of this experiment led to the conclusion that the drought has a distinct impact on the seed germination and seedling growth, as PEG concentration increases the percentage of seeds that germinate decrease.Selection may be based on these traits at an early growth stage for the purpose of screening large populations against drought stress.
Screening the germplasm at an early growing stage would be more productive, cost effective and labor intensive.The study also showed that variance among genotypes for germination stress tolerance index (GSTI) was proven to be a valid indicator of drought tolerance in rice.Germination was not seen at 20% PEG concentration, all lines survived at 10% PEG, but at 15% PEG under conditions of drought stress, resistant lines could be exploited further in breeding initiatives to create drought-tolerant cultivars.

Figure 2 :
Figure 2: Effect of drought stress on seedling height (cm).Based on treatment PC1 and PC2 divide into 3 groups in Figure10, the traits SDW and SFW depicted positive and close association with each

Figure 4 :
Figure 4: Effect of drought stress on the vigor index (VI).

Figure 5 :
Figure 5: Seedling dry weight under different level of drought stress (SDW).

Figure 6 :
Figure 6: Seedling fresh weight under different level of drought stress (SFW).

Figure 9 :
Figure 9: Scree plot showing variance of each principal component.

Table 1 :
Germplasm used in the present study.

Table 2 :
Different level of drought stress (PEG induced).

Table 3 :
Mean square of the analysis of variance of measured traits in rice genotypes under drought stress.

Table 4 :
Correlation matrix of the variables.

Table 5 :
Eigen analysis of the variables.

Table 6 :
Structure of first four principal components.