Growth Performance of Rice Genotypes at the Seedling Stage under Different Salinity Stresses

Rice (Oryza sativa L.) is a staple food in Asian countries, accounting for 90% of total food production. As an Asian country, rice is the third most important crop in Malaysia where the self-sufficiency is about 75% since the average yield is about 4.5 t ha^-1 season^-1 which needs to expand to around 7 t ha^-1 season^-1 to meet the increasing demand (Tan et al., 2015). So, further research and technological advancement are needed to expand rice production in Malaysia (Shultana et al., 2020).  However, the climate change effect like soil salinity is a major global barrier boosting rice production around the world (Gregorio et al., 1997). In Malaysia, salinity is reported to affect around 100,000 hectares of rice crop land by 2056 and the cultivation of salt-sensitive rice genotypes can reduce rice yield by 50% (Selamat and Ismail, 2008). A previous study suggested that rice is sensitive to salt stress, especially at the seedling and reproductive stages (Aref and Rad, 2012). Soil contaminated with high levels of sodium or chloride ions can interfere with the plant’s ion uptake system and interfere with the uptake of essential ions such as K⁺, Ca²⁺ and NO^3- by the root system (Ashraf and Foolad, 2007). Individually, high concentrations of Na⁺ in the soil can damage plant membrane systems, causing electrolyte leakage and oxidative damage (Mandhania et al., 2006). On the other hand, high concentrations of Cl^- in the soil can damage the plant chlorophyll production mechanism causing leaf chlorosis (Tavakkoli et al., 2010).
       
Screening of rice genotypes for salt tolerance at the early seedling stage relies on agronomical and biochemical characteristics. The seedling stage screening of rice genotypes allows faster screening, which is complicated at the vegetative and reproductive stages (Gregorio et al., 1997; Ali et al., 2014). Even though, several studies on rice genotype screening at the seedling stage have been reported, there is a lack of findings on Malaysian salt-tolerant rice genotypes. Therefore, this study was conducted to screen out potential salt-tolerant rice genotypes that could be used for future breeding materials to develop salt-tolerant varieties suitable to cultivate in the coastal saline region of Malaysia.

Plant materials
 
Twenty-one rice genotypes with different genetic backgrounds were used in this experiment (Table 1).
 

 
Experimental location and design
 
This study was conducted in a glasshouse at Field 10, Faculty of Agriculture, Universiti Putra Malaysia (UPM) at 2°59` N, 101°42` E and 45 m above sea level. The trial was set up in a randomized complete block design following three replications with 10 plants per trial.
 
Experiment setup
 
Twenty-one rice genotypes with different origins (Table 1) were tested against seven different salinity levels. The rice variety Pokkali and FL478 were used as a salt-tolerant check and Binadhan-7 was used as a salt susceptible check.  The rice genotypes were screened following a hydroponic system using the IRRI standard protocol (Gregorio et al., 1997). The seeds were surface sterilized, immersed in distilled water for 24 h and then incubated for 48 h at 30°C. Pre-germinated seeds were seeded on a floating Styrofoam (38 cm length and 33 cm width) having 100 holes bottom attached with a nylon net which is adjusted in a 12-L plastic tray (34 cm height and 27 cm width).
 
Application of nutrient solution and adjustment of salinity
 
Nutrient solution (ferrous sulphate heptahydrate and Peter Water Soluble Fertilizer 20:20:20) was added after seven days of planting. The nutrient solution must touch the bottom of the Styrofoam nylon mesh to ensure nutrient uptake by the roots. The nutrient solution was salinized using NaCl maintaining a salinity concentration of 0.3 (control), 2, 4, 6, 8, 10 and 12 dSm^-1. The EC of the cultures was monitored with an EC meter (Hanna HI 4321, Weilheim, Germany). The pH of the nutrient solution was adjusted to 5 using a pH meter (Hanna HI 98304).
 
Seedling rating for salinity tolerance
 
According to Gregorio et al., (1997), the modified standard evaluation score (SES) was used to evaluate salt stress symptoms. This assessment distinguishes susceptible genotypes from resistant and moderately resistant genotypes. Initial and final scores were recorded at 14 and 21 days after salinity treatment.
 
Data collection
 
The data on root length, shoot length, root fresh weight, shoot fresh weight, root dry weight and shoot dry weight were measured after 24 days of salt application. The shoots were separated from the roots, washed twice with tap water and twice with deionized water, bagged and oven-dried at 70Ú C for 3 days. Oven-dried shoot samples were ground using a mechanical grinder and sieved then the content of Na⁺, K⁺ and Ca²⁺ were measured. All elemental analyses were performed by acid digestion using the Micro-Kjeldahl method (Thomas et al., 1967). Determinations were quantified with an atomic absorption spectrophotometer (AAS). The Na⁺/K⁺ ratio was determined from the concentration of Na⁺ and K⁺ and the Na/Ca ratio was calculated from the concentration of Na and Ca.
 
Statistical analysis
 
The collected data were statistically analyzed following Analysis of Variance (ANOVA) using SAS 9.4 software. Means were compared using LSD test (P<0.05).

The salinity tolerance level of different rice genotypes
 
All the rice varieties tested showed uniform growth under non-salinized conditions. Whilst, under salinized conditions, the plants showed a wide phenotypic variation. Among 21 rice genotypes, Pokkali, FL478 and Binadhan-10 were found to tolerate 12 dSm^-1 of salinity. BRRI dhan73, BRRI dhan61, Binadhan-8, BRRI dhan67 and BRRI dhan47 were tolerant at 10 dSm^-1; Putra-1 were moderately tolerant at 8 dSm^-1 and MR263, MR284, MR211 and MRQ74 were tolerant at 4 dSm^-1 and rest of the genotypes were salt susceptible. This finding is per Kranto et al., (2016) who stated that Pokkali is more salt tolerant than other rice cultivars. Muti et al., (2021) also found that Pokkali, FL478 and Binadhan-10 are more salt-tolerant than the other rice genotypes.
 
The effect of salinity on the root length of tested rice varieties
 
The root length did not vary significantly under the non-salinized condition among the rice genotypes (Table 2). At 2 dSm^-1, significantly the highest root length was recorded for the rice genotype MR297 (28.25 cm). Further, at 4 dSm^-1, the highest root length was recorded for the rice genotypes Pokkali (26.49 cm) and FL478 (26.32 cm). Likewise, at 6 dSm^-1, FL478 (25.04 cm) and Pokkali (24.76 cm) were recorded for the highest root length. Similarly, at 8, 10 and 12 dSm^-1, Pokkali (23.57 cm, 21.54 cm and 18.83 cm respectively) was recorded for the highest root length. The lowest root length reduction was recorded by the rice genotypes FL478 (0.17%), BRRI dhan61 (2.03%), BRRI dhan47 (5.92%), Pokkali (13.15%), Pokkali (20.63%) and Pokkali (30.61%) respectively at 2, 4, 6, 8, 10 and 12 dSm^-1.
 

       
Salt stress had a considerable effect on root length, but this varied with rice genotypes. Earlier, it was reported that at high salt concentrations, roots were reduced rapidly (Hoque et al., 2015). Previously, root length reduction in response to salt stress was reported by Acosta-Motos et al., (2017) and Ali et al., (2014). The phenotypic screening showed that tolerant genotypes grow their roots and shoots more quickly than susceptible genotypes and are less vulnerable to salt-induced damage. This may be because of their inherent defence mechanisms against salinity damage.
 
The effect of salinity on the shoot length of 21 rice genotypes at the seedling stage
 
The rice genotype Pokkali consistently produced the highest shoot length regardless of salinity concentrations (Table 3). At 2 and 4 dSm^-1 of salinity, the shoot length of the tested genotypes did not vary significantly. However, at 6, 8, 10 and 12 dSm^-1, Pokkali and FL478 consistently produced the highest shoot length. Moreover, no significant difference in shoot length was recorded by the rice genotype Pokkali (38.71 cm), FL478 (34.44 cm) and Binadhan-10 (31.70 cm) at the highest level of salinity (12 dSm^-1). Regarding the shoot length reduction, Binadhan-10 showed the lowest reduction which is reduced by 2.19% and 9.33% at 2 and 4 dSm^-1 respectively. The rice genotypes Pokkali and FL478 were reduced by 24.19%, 38.85%, 56.09% and 24.71%, 41.09% and 57.73% at 8, 10 and 12 dSm^-1 respectively.
 

       
The rice genotypes Pokkali and FL478 consistently generated the longest shoots with the least amount of shoot length reduction. The lowest salt accumulation in the cell wall did not affect cell wall elasticity resulting in increased shoot length. These results support those of Bhowmik et al., (2009) who have screened 11 rice varieties for salinity tolerance and observed that resistant varieties have higher plant height and dry biomass. Besides, tolerant plants can impede the upward translocation of sodium ions by maintaining a higher shoot-to-root ratio for better survival under salinity stress (Vaishnav et al., 2019).
 
Effect of salinity on root and shoot dry weight
 
Root dry weight did not vary significantly among the rice varieties at 2 and 4 dSm^-1 of salinity while significant variation was observed at 6, 8, 10 and 12 dSm^-1 of salinity (Table 4 and 5). The highest root dry weight was recorded by the rice genotype FL478 (0.25g) at 6 dSm^-1 of salinity. Moreover, at the highest saline condition (12 dSm^-1), FL478 was recorded for the highest root dry weight (0.15 g). Shoot dry weight did not vary significantly among the rice varieties in non-saline conditions and also at 2 dSm^-1 of salinity. Shoot dry weight significantly varied at 4, 6, 8, 10 and 12 dSm^-1. At all salinity levels, the highest shoot dry weight was recorded by the rice genotype Pokkali (0.98 g).
 

 

       
A significant decrease in shoot and root dry weight with increasing salinity was recorded in 21 rice genotypes. Hakim et al., (2014) stated that the decrease in the dry weight of shoots and roots may be attributed to several factors such as (i) decreased photosynthesis per unit leaf area since salt stress leads to an insufficient supply of carbohydrates required for shoot growth, (ii) lower water potential because of reduced turgor pressure and (iii) direct growth retardation due to disruption of mineral supply. Moreover, salinity affected the final cell size and cell production rate, resulting in decreased shoot and root dry weight. Likewise, Khatun et al., (2013) and Al-Saady (2015) observed a decrease in the shoot and root length and biomass for all tested wheat cultivars with increasing NaCl concentration.
 
Effect of salinity on Na/K and Na/Ca ratio
 
The ratio of Na: K and Na: Ca of 21 rice genotypes varied significantly at 2, 4, 6, 8, 10 and 12 dSm^-1 of salinity (Table 6 and 7). The lower ratio of Na/K and Na/Ca was consistently measured from the rice genotype Pokkali and FL478 at different salinity levels. The accumulation of Na⁺ and impairment of K⁺ nutrition are the key features of salt-stressed plants. The influx of K⁺ and efflux of Na⁺ is a common strategy of plants to maintain the cytoplasmic Na⁺/K⁺ ratio under salt stress conditions. Therefore, several reports stated the beneficial effects of high K⁺/Na⁺ ratios on crop salinity tolerance. According to the findings of  Hoque et al., (2015),  the K⁺/Na⁺ ratio is a scientifically established and genetically accepted measure of plant salinity tolerance. Several scientists have concluded that as salinity increases, sodium levels in rice increase and potassium levels decrease (Solangi et al., 2016; Theerawitaya et al., 2015).
 

 

 
Based on the Na⁺/K⁺ ratio, the rice genotype Pokkali, FL478 and Binadhan-10 showed high tolerance to extreme salinity. Hasan et al., (2015) reported a larger accumulation of toxic ions (Na⁺) in the leaf of salt susceptible variety causing a sharp decline of K⁺ content and K⁺/Na⁺ ratios causing ion poisoning and physiological injuries. Ahmad et al., (2006) reported a significant increase in Na⁺ ions and a decrease in K⁺ ions in the shoots and roots of two barley cultivars with increasing salinity. The current investigation showed a lower accumulation of calcium ions with the increase in salinity. However, tolerant plants showed a higher accumulation of Ca thus reducing the ratio of Na/Ca. Calcium is essential for the maintenance of cell membrane integrity (Reshna and Beena, 2021). Salt-sensitive genotypes expressed more nutritional imbalance while the salt-tolerant varieties were able to maintain balance among the nutrients in the tissues (Hakim et al., 2014). Calcium reduced uptake of Na⁺ concomitant with higher tissue K⁺/Na⁺ in seedlings, comparatively more in salt-tolerant Nona Bokra than in salt-sensitive IR-64, together with a significant increase in root PM H⁺ATPase in the former, but not in the latter (Gupta and Shaw, 2021).

To select a better pool of varieties, analyzing the physicochemical properties of the rice varieties at 12 dSm^-1 would be the way forward and those genotypes would be used as donor parents in the MABC program. The promising rice varieties that are tolerant at 12 dSm^-1 would be taken into consideration for a hybridization program in Malaysia to breed salinity-tolerant genotypes. Hence, the rice varieties Pokkali, FL478 and Binadhan-10 could be considered for further research.