Screening of genotypes under water deficit condition

Introduction

Rice (Oryza sativa L.) is one of the most important cereal crops and serves as a major source of food and nutritional energy for a large proportion of the world's population, particularly in Asia. Rice production is highly dependent on adequate water availability, and water scarcity has become an increasingly important constraint to sustainable rice production. Among the various abiotic stresses, drought is considered one of the major factors limiting rice growth, productivity and yield, particularly in rainfed and poorly irrigated ecosystems. Approximately 45% of the world's rice-growing area is under rainfed conditions, where intermittent and unpredictable water shortages frequently occur during the crop growth period (Verulkar et al., 2010) [1] . Drought stress occurs when the availability of water in the soil becomes insufficient to meet the water requirements of plants. Rice is particularly vulnerable to water deficit because its productivity is strongly influenced by water availability throughout the crop growth cycle. Drought can occur at different developmental stages, including germination, seedling establishment, vegetative growth, flowering and grain filling, with the magnitude of damage depending on the severity and duration of stress and the developmental stage of the crop. Water deficit can result in reduced cell expansion and division, stomatal closure, impaired photosynthesis, reduced biomass accumulation and premature leaf senescence. Consequently, prolonged or severe drought stress can cause substantial reductions in grain yield and yield components (Nahar et al., 2018; Panda et al., 2021) [2,3] . At the seedling stage, several morphological and physiological traits can be effectively used for preliminary screening of rice genotypes. Germination percentage, germination index, root length, shoot length, fresh weight, dry weight, relative water content (RWC), and membrane injury are among the commonly evaluated traits. Nahar et al. (2018) [2] demonstrated that these morpho-physiological parameters could effectively differentiate drought resistant and drought-sensitive rice cultivars under PEG-induced water stress. Relative water content (RWC) is an important indicator of the plant's water status and provides an estimate of the ability of a genotype to maintain tissue hydration under drought conditions. Genotypes capable of maintaining comparatively higher RWC under water deficit are generally better able to sustain cellular functions and physiological activity. Similarly, membrane injury or membrane stability can provide an indication of drought-induced cellular damage (Kumar et al., 2015) [4] . Screening at the early seedling stage provides a relatively rapid and economical approach for identifying contrasting genotypes before undertaking detailed physiological, biochemical, molecular and field-level investigations. Controlled screening methods, includ ing PEG-induced osmotic stress allow genotypes to be exposed to relatively uniform stress conditions. Therefore, screening of rice genotypes under drought stress is an essential step for identifying droughttolerant genetic resources and understanding the physiological basis of drought adaptation. Evaluation of germination, seedling growth, relative water content, membrane stability and other stress-responsive traits can help distinguish tolerant and susceptible genotypes. The identification of contrasting genotypes can subsequently facilitate their utilization in physiological and biochemical studies as well as in breeding programmes aimed at developing high-yielding rice cultivars adapted to drought-prone environment.

Materials and Methods

Twenty rice genotypes were used for the initial screening of drought resistance. Seeds were soaked in distilled water for 12 h and subsequently surface-dried under room temperature for 2 h. The seeds were then placed in petri plates for germination. Three days after germination, drought stress was imposed by applying polyethylene glycol (PEG-6000) solution with an osmotic potential of -10 bar to simulate water deficit conditions. The seedlings were maintained under these stress conditions until 10 DAG. At 10 DAG various seedling growth parameters were recorded.

The germination percentage was calculated as described in the Association of Official Seed Analysts (Firoozeh et al., 2012) [5] by following formula.

𝐺𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑖𝑜𝑛 % = 𝑁𝑜.𝑜𝑓 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑 𝑇𝑜𝑡𝑎𝑙 𝑁𝑜.𝑜𝑓 𝑠𝑒𝑒𝑑 × 100

Germination index

The germination index (GI) was calculated as described in the Association of Official Seed Analysts (Firoozeh et al., 2012) [5] by following formula. GI =

𝑁𝑂. 𝑜𝑓 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑 𝐷𝑎𝑦𝑠 𝑜𝑓 𝑓𝑖𝑟𝑠𝑡 𝑐𝑜𝑢𝑛𝑡

𝑁𝑜. 𝑜𝑓 𝑔𝑒𝑟𝑚𝑖𝑛𝑎𝑡𝑒𝑑 𝑠𝑒𝑒𝑑 𝐷𝑎𝑦𝑠 𝑜𝑓 𝑓𝑖𝑟𝑠𝑡 𝑐𝑜𝑢𝑛𝑡

Five randomly selected seedlings were taken from each growing treatment to calculate the length of shoot. They were measured with a measuring scale and expressed in centimeters.

Five randomly selected seedlings were taken from each growing treatment to calculate the weight of shoot. Weight of shoots were measured by balance and expressed in miligram.

Five randomly selected seedlings were taken from each growing treatment to calculate the length of root. They were measured with a measuring scale and expressed in centimeters.

Five randomly selected seedlings were taken from each growing treatment to calculate the weight of root. Weight of roots were measured by balance and expressed in milligram.

The method described by Weatherley (1950) [6] was used for the determination of relative water content of rice genotypes under control as well as water deficit stress conditions. Pre-weighed leaf samples of rice were transferred in a petri dish filled with at least 100 mL distilled water so that leaves remain submerged for six hours. Then the leaves were taken out, dried by blotting paper and measured the turgid weight. After that, turgid leaf samples were kept in oven at 80 °C overnight and weighed until constant weight was obtained. RWC was calculated using the following formula:

𝑅𝑊𝐶 % = 𝐹𝑟𝑒𝑠ℎ 𝑤𝑒𝑖𝑔ℎ𝑡 -𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑇𝑢𝑟𝑔𝑖𝑑 𝑤𝑒𝑖𝑔ℎ𝑡 -𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 × 100

Membrane injury (MI) was determined by using the method of Suvillan (1971) [7] with some modification. Fresh leaf tissues i.e. 0.1 g were taken and placed in beaker containing 100 ml of distilled water. They were kept in hot water bath at 40 °C for 30 minutes. After 30 minutes samples were cooled to room temperature and electrical conductivity (EC) was recorded for both control (C 1 ) and treatment (T 1 ). Then the tubes were kept in boiling water bath at 100 °C for 15 minutes. After cooling, again electrical conductivity was measured in control i.e. C 2 and in treatment i.e. T 2 . Membrane injury was calculated by using the following formula:

Results and Discussion

Total 20 genotypes were assessed for drought resistance using germination percent, germination index, relative water content (RWC), membrane injury (MI), shoot length, root length, shoot weight and root weight.

Germination percentage is a key parameter in stress physiology, especially when studying drought tolerance. Germination percentage is an important indicator used to screen and identify drought-tolerant genotypes at the early growth stage, helping select crops that can establish successfully under limited water conditions. The genotypes exhibited significant variability in germination percentage are measured and presented in (2010) [8] , Kim et al. (2001) [9] , Radhouane (2007) [10] and Xu et al. (2015) [11] . They have suggested that PEG-induced drought stress reduced germination percentage. Sabesan et al. (2016) [12] studied an in vitro study utilizing polyethylene glycol to screen Indica rice genotypes for drought resistance. They observed germination percentage varied from 54.50 % to 100 %. They have also reported that PEG-induced osmotic stress can cause hydrolysis of storage compounds that further lower the internal osmotic potentials of the seed. Similar finding was observed by Vibhuti et al. (2015) [13] and Akte et al. (2016) [14] . Our findings suggest the genotype R 2307-73-1-38-1 possess better germination quality, while genotype R-RF-212 having poor germination quality, consistent with the findings of Bewley et al. (2012) [15] who emphasized that rapid and uniform germination is linked to improved seed vigor and early seedling establishment.

Germination index (GI)

Germination Index (GI) is a measure that evaluates both the speed and the uniformity of seed germination. It gives more weight to seeds that germinate earlier, so it reflects seed vigor and germination performance under stress conditions. In most drought stress experimental studies, the germination index is important character due to measure of germination speed and extent of seed germination, which may help to researchers to identify genotypes that can germinate quickly and survive under drought stress conditions.

The germination index of 20 genotypes of rice was analyzed at vegetative stage and the data are depicted in Table 4.1. The germination index was found significantly differed among the rice genotypes and it was varied between 6.13 to 14.23. However, significantly maximum germination index was found in genotype R 2307-73-1-38-1, which was at par with R 2272-515-2-287-1 and AAUDR-1. Significantly minimum germination index was found in genotype R-RF-212.

Shoot length is an important, rapid and reliable screening parameter in vitro drought study of rice. It helps identify genotypes that can sustain growth under water deficit. Shoot length was found significantly differed among the rice genotypes (Table 4.1). However, the shoot length of all genotypes of rice was recorded between 1.33 to 7.42 cm. Significantly the highest and the lowest shoot length was found in genotype R 2307-73-1-38-1 (7.42 cm) and genotype R-RF-212 (1.33 cm), respectively. [16] studied identification of in-vitro PEG mediated drought tolerance genotype in rice (Oryza sativa L.). They have recorded that the shoot lengths were reduced under PEG stress (4.05-10.65 cm) as compared to control condition (5.60-13.65 cm).

Under drought stress condition the shoot weight of rice is a key physiological and screening character in vitro study. The shoot weight of rice genotypes was measured at vegetative stage and data presented in Table 4.1. Significantly the highest and the lowest shoot length was found in genotype R 2307-73-1-38-1 (23.1 mg) and genotype R-RF-212 (3.9 mg), respectively. Our results are accordance with various scientists, Saddique et al. (2020) [17] , Liu et al. (2017) [18] , Pirdashti et al. (2003), Garg et al. (2017) [19] and Xu et al. (2020) [20] . They have indicated that generally drought stress damages rice plants which can reflects on shoot weight.

Root length is a most important character in screening of rice genotypes under drought stress condition. Roots are directly responsible for water uptake and plant survival under water-deficit conditions. Longer root systems are often associated with greater drought tolerance, as they enable plants to extract water from deeper soil layers.

The data presented in Table 4.1 indicated that root length was significantly differed among the rice genotypes. Maximum and minimum root length observed in genotype R 2307-73-1-38-1 (10.14 cm) and in genotype R-RF-212 (3.30 cm), respectively. Our finding is accordance with Shovon et al. (2020). They have found that the maximum value of root length was found in BRRI Dhan-28 (6.80 cm) followed by Dular and the lowest was recorded in Begunbichi (2.28 cm). For drought effect root length for most of the plants were decreased compared to the control conditions which is a common adverse effect of drought. Thus, the reduction of root length may occur due to decreased cell division under the stress condition.

Relative water content provides a direct, quantitative measure of plant water status which helping identify rice genotypes that can maintain hydration, sustain physiological activity and tolerate drought stress under in vitro conditions. [21] .

In specifically drought condition the membrane injury character serves as a reliable and rapid screening criterion for identifying drought tolerant/susceptible genotypes. Drought-induced oxidative stress causes damage to cellular membranes, resulting in increased electrolyte leakage. Genotypes exhibiting lower membrane injury maintain better membrane integrity, cellular function and water status under stress conditions. The membrane injury of rice genotypes was analyzed and the data are depicted in Table 4.1. The membrane injury was found significantly varied between 1.40 % to 77.57 %. Significantly higher and lower membrane injury was found in genotype R 2307-73-1-38-1 (77.57 %) and genotype R-RF-212 (17.90 %), respectively. Our results are agreement with Mehta et al. (2023) [22] . They have indicated that the membrane injury enhanced during water stress condition.

Conclusion

The screening study demonstrated significant variation among the rice genotypes in their response to water-deficit conditions. Genotype R 2307-73-1-38-1 exhibited superior performance, as evidenced by higher germination percentage, better seedling growth, and greater relative water content, coupled with lower membrane injury. In contrast, genotype R-RF-212 showed comparatively poor germination and seedling growth, lower relative water content, and higher membrane injury under water-deficit conditions. Based on the combined physiological and morphological responses, R 2307-73-1-38-1 was identified as the drought-resistant genotype, whereas R-RF-212 was identified as the drought-susceptible genotype. The results indicate that early-stage traits such as germination, seedling growth, relative water content, and membrane injury can serve as useful and reliable indicators for the preliminary screening and identification of contrasting rice genotypes for drought tolerance.

Acknowledgement

Root weight (mg)Root weight is an important parameter for in vitro screening of rice genotypes under drought stress. Roots are the first organs responsible for sensing and responding to water deficit. Root weight was observed significantly differed among the rice genotypes (Table 4.1). However, the root weight of all rice genotypes was recorded between 4.8 to 22.4 mg. Significantly the maximum and minimum root weight was found in genotype R 2307-73-1-38-1(22.37 mg) and genotype R-RF-212 (4.8 mg), respectively. Our results are accordance with various scientists, Rajput et al. (2022), Sokoto and Muhammad (2014), Akte et al. (2016) [14] and Islam et al. (2018). They have suggested that PEG-induced drought stress reduced root weight.International Journal of Agriculture and Food Science https://www.agriculturaljournals.com

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