Phenotyping for the Seedling Stage of the Drought Stress Tolerance in Rice (

The ever-increasing demand for rice raises the need to increase productivity by developing drought-tolerant rice varieties. Drought tolerance is a complex polygenic trait that largely depends upon plant developmental stages and showed genotype specific variability. The experiment was conducted using drought tolerant (Binadhan-19, BRRI dhan83) and drought susceptible (BRRI dhan26, BRRI dhan48) rice genotypes at the glasshouse of Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh to characterize the seedling stage drought tolerance as well as disclose the variability of the genotypes for morphological and biochemical traits. A completely randomized design (CRD) with three replications and treatments (control and drought stress imposed by 20% PEG) was used for the experiment. Data on shoot length, root length, fresh root weight, fresh shoot weight, dry root weight, dry shoot weight, chlorophyll content (SPAD value), MDA and H 2 O 2 were recorded and analyzed through appropriate breeding tools. Significant variation (p<0.01) was observed for genotype, treatment and genotype (G) × treatment (T) interactions viz. , shoot length, root length, root fresh weight, shoot fresh weight, root dry weight, shoot dry weight, chlorophyll, H 2 O 2 and MDA. Drought stress leads to a significant decrease in root and shoot growth whereas the level of H 2 O 2 and MDA increased significantly. A greater decrease in root and shoots growth was observed in susceptible genotypes (BRRI dhan28, BRRI dhan48) compared to tolerant genotypes (BRRI dhan71, Binadhan-19). In contrast, a limited increase in H 2 O 2 and MDA was recorded in tolerant genotypes compared to susceptible genotypes. H 2 O 2 showed a significant positive correlation with root and shoot characteristics under control conditions, whereas H 2 O 2 showed a significant negative correlation with chlorophyll content under drought conditions. MDA showed a significant negative correlation with most of the studied traits under well-watered conditions. Considering all of the traits at the seedling stage, the genotype Binadhan-19 is considered a drought-tolerant genotype both under well-watered and drought conditions and this genotype was selected for further study under direct field conditions as well as for genetic improvement against drought stress.

a major threat to agriculture particularly in developing nations. Climate change globally, influences the regularity and level of hydrological fluctuations, and causes various abiotic stresses for plants (Turral et al., 2011). Amongst the abiotic factors, drought is the most imperative and a major limitation for rice production in rainfed ecosystems (Pandey & Shukla, 2015;Alam et al., 2022).
Rice is the principal food crop of Bangladesh as well as second important food grain in the world. About 34.7 million tons of rice is produced in Bangladesh per annum and 75% of the cultivable land is used for rice production (BBS, 2016). Rice sector contributes one-half of the agricultural gross domestic products and one-sixth of the national incomes. In Bangladesh, rice productivity and sustainability are continually threatened by abiotic stresses like drought, heat and cold stresses. The situation becomes worse due to climate change that may multiply the frequency and severity of such abiotic stresses. Drought associated with high temperature plays a key role for impacting negatively on crop yield. Yield loss of rice due to slight drought is from 10-30% and might be up to 70-90% under severe drought (Rahman, 2011). Importantly, climate change model predicts 33% rice yield decrease in 2100 in Bangladesh (Karim et al., 2012) whereas we need to increase rice yield to feed the ever-increasing population. Any decline in rice production through climate change would thus critically impair food security in the country. Hence, there is an urgent need to develop improved rice varieties or management techniques that are more resilient to drought stress to ensure food security and also for sustainable agricultural production. Considering the above facts, the present research work was therefore undertaken to fulfil the following objectives: to characterize rice genotypes for drought stress tolerance and to establish physiological and biochemical markers linked to drought stress tolerance.

MATERIALS AND METHODS: Experimental materials
Four rice genotypes including two drought tolerant genotypes (Binadhan-19 and BRRI fhan71) and two drought sensitive genotypes (BRRI dhan28 and BRRI dhan48) were used as plant materials. The seeds were collected from Bangladesh Rice research institute (BRRI) and Bangladesh Institute of Nuclear agriculture (BINA).

Design and treatments of the experiment
The experiment was conducted in completely randomized design (CRD) with three replications and treatments used for these experiments were as follows: control (C) and drought stress imposed by 20% PEG.

Time and experimental site
The experiment was conducted in the glasshouse of Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh-2202, and executed during the period of January to March 2022. Seedlings establishment and growth in hydroponic system the rice seeds were surfaced sterilized soaked in distilled water for 24 h and germinated on wet filter paper embedded in petri dishes. Sprouted seeds were sown in a line on Styrofoam sheet floating in trays containing normal water under glasshouse condition. The seedlings of each line were arranged in two sets of floats (one for stress treatment and the other for control). The seedlings in this setup were kept in water at 28 ºC for 3 days. On the 4th day, the seedlings were transferred to grow in Peter's nutrient medium solution and grown under open glasshouse conditions by adjusting pH 5.1 to 5.2 NaOH and/or HCl whenever necessary.

Hydroponic drought treatment
Under hydroponic condition, 14 days old seedlings were subjected to drought stress grown primarily in Peter's nutrient solution, containing 20% PEG-6000 with osmotic potential (OP) of -4.05 to -4.95 MPa for 5 days at (28±2) ºC. However, the non-stressed plants were grown in a normal strength of the hydroponic Peter's nutrient solution.

Data on morphological and biochemical traits at the seedling stage
The following data on shoot length (cm), root length (cm), fresh root weight (mg), and fresh shoot weight (mg) were calculated from five seedlings per genotype for each replication. Data on dry root weight (mg) and dry shoot weight (mg) were estimated after oven drying of the samples at 60 o C for 3 days. Data on biochemical parameters viz., chlorophyll content (SPAD value), MDA and H 2 O 2 were measured from flag leaf tissues after 7 days of drought treatment.

Determination of Malondialdehyde (MDA)
MDA was measured from leaf tissue following the standard method as described by Heath and packer (1968) and the MDA content was determined using 155 mM -1 cm -1 extinction coefficient and expressed as nmolg -1 FW.

Determination of Chlorophyll Content (SPAD value)
Chlorophyll content of flag leaf, 1st, 2nd and 3rd leaves was measured at the end of the drought stress using SPAD meter (Model: SPAD 502).

Statistical analysis
Data recorded for different parameters were compiled and tabulated in proper form for statistical analysis which was carried out in Minitab 17 statistical software package (Minitab Inc. State College, Pennsylvania) and software R, version 3.3.2. Various statistical tests such as two-factor ANOVA test, mean performance, combined effects of genotype treatment, percent reduction and correlation coefficient were performed to assess the varietal performance of the studied genotypes.

Calculation of percent yield reduction
For comparing stressed plants with control plants, the percent reduction was calculated by the following formula: value of control plants ×100

Morphological traits
The result of analysis of variance (ANOVA) for all the characters (viz., shoot length, root length, root fresh weight, shoot fresh weight, root dry weight, shoot dry weight, chlorophyll, H 2 O 2 , MDA) showed highly significant (P≤.001) variation among the genotypes studied. The present study found a considerable variation in shoot length. On average, shoot length was the highest in BRRI dhan48 (31.84 cm) and the lowest in BRRI dhan71 (19.65 cm) under control condition ( Table 1). Drought stress resulted in a remarkable decrease in shoot length and the highest decrease was observed in BRRI dhan48 (36.99 %) followed by BRRI dhan28, Binadhan-19 and BRRI dhan71 (25.08, 21.06, and 15.12%, respectively) compared to control (Fig. 1). Drought stress lead to a significant decrease in root length among all the genotypes studied. The highest reduction was observed in drought susceptible BRRI dhan28 (25.58 %) whereas the lowest reduction was found in BRRI dhan71 (9.53 %) (Fig. 1). The result found that the highest fresh shoot weight was noted in BRRI dhan48 (1066.78 mg) and the lowest was recorded in BRRI dhan71 (343.00 mg) ( Table 1). Drought stress resulted in significant reduction in fresh shoot weight among all of the genotypes; the highest reduction was found in BRRI dhan48 (77.18 %) whereas the lowest reduction was found in BRRI dhan71 (44.73 %) as compared to control (Fig. 1).
For shoot dry weight, the highest reduction was observed in drought susceptible BRRI dhan48 (66.55 %) whereas the lowest reduction was found in BRRI dhan71 (17.22 %) compare to their control (Fig. 1). Drought stress resulted in a sharp decrease in root fresh weight among all genotypes; the highest reduction was found in BRRI dhan48 (72.21 %) and the lowest reduction was found in BRRI dhan71 (53.30 %) related to their control (Fig. 1). Under drought stress condition, maximum decrease was recorded in BRRI dhan48 (64.26 %) and the lowest in BRRI dhan71 (22.69 %) as compared to control (Fig. 1).

Biochemical traits
In the study, the treatment effects showed a significant variation in MDA content among the genotypes. MDA was noted maximum in BRRI dhan48 (25.98 nmolg -1 ) and minimum in Binadhan-19 (22.17 nmolg -1 ) under control condition ( Table 1).
Drought stress lead to a significant increase in MDA content in all of the genotypes; the highest induction was found in BRRI dhan28 (185.76 %) and the lowest increase in Binadhan-19 (104.23 %) as compared to control (Fig. 1) 1). Imposition of drought stress resulted in a significant decrease in chlorophyll content (SPAD) among all of the genotypes compared to control. Due to drought stress, the highest decrease in SPAD was found for BRRI dhan28 (56.49 %) and the lowest in Binadhan-19 (39.51%) (Fig. 1).

Estimation of phenotypic correlation co-efficient among nine characters of rice genotypes under control and drought stress conditions at the seedling stage
Correlation co-efficient is a measure of intensity or degree of linear relationship between two variables. In the present study, relationship among morphological and biochemical traits were studied through analysis of correlation between them. Phenotypic correlation co-efficient among 9 traits of 4 rice geno-types are presented in Table 2. In the present study out of 72 associations, 27 associations were significant at phenotypic level in control condition and 12 associations were significant at phenotypic level in drought stress condition ( Table 2). The positive and non-significant associations referred information of inherent relation among the pairs of combination. The negative and non-significant association referred a complex linear linkage among the pair of combinations.

DISCUSSION:
The ultimate goal of plant breeding is to develop superior crop varieties with potential to give maximum yield under optimum growth condition as well as able to produce a satisfactory yield under any stressful conditions. The negative effects of drought stress are well studied but the variability of drought stress response at various plant developmental stages by using tolerant and sensitive genotypes has not been well documented. In the present study, an attempt was made to investigate the impact of drought stress on morphological and biochemical traits by using drought sensitive and drought tolerant rice genotypes. A detail discussion of the results found is presented below.
Root length was severely affected by the induction of drought. As drought decreases the growth of plants, the decrease in root length might be due to the negative consequence of drought stress. The reduction in root length due to drought was also found by other researchers (Lanna et  In a nutshell, the decrease in growth and development of drought stressed seedlings might be due to the negative effects of high osmotic potential of the nutrient solution that lowered absorption of water and nutrient (Shrivastava et al., 2015).

Phenotypic correlations of morphological and biochemical traits at the seedling stage
In the present experiment, the relationship among morphological and biochemical traits were studied through phenotypic correlation (

CONCLUSION:
Drought in the field of agriculture is a major obstacle which detrimentally affects rice in terms of growth and productivity leading to low yield potential and food insecurity. Drought tolerance is a complex polygenic trait that is genotype specific and largely depends upon plant developmental stage. The study was attempted to identify the potential determinates of drought tolerance at seedling stage in rice through the assessment of morphological and biochemical traits. The experiment was conducted following a CRD design with three replications and two treatments (control and drought) to fulfill the proposed objectives. The results of this experiment have been summarized as follows: One group of seedlings was subjected to drought stress (20% PGE) for 72 hrs. After the completion of drought stress treatment, data on nine morphological and biochemical parameters were recorded and analyzed through appropriate breeding tools. The results of analysis of variance showed significant variation due to genotypes, treatments and genotype × treatment interaction for most of the studied traits.
In response to drought stress, a significant decrease in root and shoot growth characteristics were recorded whereas the level of H 2 O 2 and MDA increased significantly. A greater decrease in root and shoot characteristics was observed in the sensitive genotypes (BRRI dhan28, BRRI dhan48) as compared to tolerant genotypes (BRRI dhan71, Binadhan-19). In contrast, a limited increase in H 2 O 2 and MDA content were recorded in the tolerant genotype as compared to tolerant genotype. H 2 O 2 showed significant positive correlation with root and shoot characteristics whereas it showed non-significant negative correlation with chlorophyll content under control conditions. Under drought stress conditions, H 2 O 2 showed significant negative correlation with chlorophyll content. MDA showed nonsignificant correlation for most of the studied traits under controlled conditions whereas it showed significant negative correlation with most of the studied traits. Considering all of the traits at the seedling stage, the genotype Binadhan-19 considered as drought tolerant genotype both under control and drought condition.

ACKNOWLEDGEMENT:
The author would like to thanks research cell, Noakhali Science and Technology University for financial support to conduct this research and special thanks to BINA and BRRI for providing the seeds and research facilities.

CONFLICTS OF INTEREST:
There is no conflict of interest