Effect of increasing salinity levels on growth of rice seedlings in hydroponics

Introduction

Rice (Oryza sativa L.) serves as a primary food source for more than three billion people worldwide,a major cereal crop and a primary source of nutrition. The overall number of people on world has reached about 8 billion, and current estimates indicate that 10 billion by 2050. Given that 3.5 billion people worldwide suffer from deficiencies in at least one important nutrient and that over 800 million people are estimated to be hungry, our current ways of producing food are unable to keep up with this growing population [1] . Overcoming the uncertainty of abrupt atmospheric extremes, however, is currently difficult. Growing abnormal climate changes and global warming present a variety of biotic and abiotic difficulties for crops [2] . One of the most important environmental factors endangering rice production is salinity. Salinity is the second crucial abiotic stress that drastically decreases agricultural yield [3] . Due to the high salt content, millions of hectares of suitable rice fields in Asia and Africa are not being used (IRRI, 2020). Salinity poses an existential threat to sustainable agriculture because, according to studies, one-third of all cultivable land globally is impacted by salt, and 50% of all arable land could be salinised by 2050 [4] . Salinity affected 2.1% of India's cultivated area, with 2.96 million hectares of saline soil and 3.77 million hectares of sodic soil. White alkali or saline soil contains EC>4, pH<8.5, and ESP<15; sodic soil has EC<4 dsm-1, ESP>15, and pH = 8.5 to 10 [5] . The adverse effects of salinity stress emphasise how urgent it is to produce rice genotypes that can resist salt to maintain food security, especially in areas where soil salinisation is prominent [6] . Rice plants are more susceptible to salt stress during the seedling and reproductive stages, which can have several damaging effects that significantly reduce production and quality [4] . A trusted strategy for determining how genotypes respond to salt stress is hydroponic cultivation.

Several crops, including rice, have been utilised in hydroponic culture experiments to evaluate how various plant species respond to salinity stress [7] . Yoshida solution served as a nutrient solution for all of these experiments. The macronutrients and micronutrients necessary for healthy crop development are provided in Yoshida solution, which is frequently used to cultivate rice plants in hydroponic culture [8] . By disrupting the physiological mechanism of the seed, salt stress causes an adverse effect on seed germination. It diminishes the growing seeds water potential, food reserve, and protein content [9] . Rice responds and adapts to salt stress in an extremely complex manner. Root growth inhibition, leaf rolling, lowered plant height and tiller number, and spikelet sterility are all consequences of salt stress that eventually result in a lower yield [10] . Raising salinity reduces fresh weight, dry weight, shoot length, root length, and seed germination, according to multiple research [11] . Hence this study aim to understand how the root length, shoot length and morphological parameters reduce in different levels of salinity.

Materials and Methods:

Rice genotypes included 100 varieties from different states of India, including Pokkali (a landrace) as a tolerant check and IR 20 as a sensitive check variety. This research study was conducted in a laboratory at Karunya Institute of Technology and Sciences in 2025. Two distinct treatments and one control were used in the trial. The different concentrations are 0, 4 and 8 EC's. The standard protocol of salinity screening by Yoshida followed the experiment [12] and IRRI score. We used the trays that have the capacity of holding water up to 5 litres. The Styrofoam sheets were cut according to the depending upon the of the trays. The trays were filled with Yoshida nutrient medium and distilled water. A Styrofoam (polystyrene) sheet was prepared with 100 evenly spaced holes and stitched with mesh below the holes to hold the seeds. The nutrient solution was prepared as a stock as the solution should be changed once in 5 days and the pH and EC's should be maintained perfectly. The PH was adjusted to 5.1 by pH meter using 1N HCL and 1N NaOH when necessary. The standard Yoshida medium. Ammonium nitrate (91.4 grams per litre), potassium sulphate (97.8 grams per litre), potassium dihydrogen phosphate (29 grams per litre), dipotassium phosphate (8 grams per litre), calcium chloride hexahydrate (175 grams per litre), and magnesium sulphate heptahydrate (324 grams per litre) were among the macronutrients. The micronutrients included 1.5 grams of manganese chloride tetrahydrate, 0.074 grams of ammonium molybdate tetrahydrate, 0.93 grams of boric acid, 0.035 grams of zinc sulphate heptahydrate, 0.03 grams of copper sulphate pentahydrate, 10.5 grams of ferric sodium EDTA, and 2.5 grams of ferrous sulphate [13] . The exact amount of chemicals was prepared as a stock. The observed parameters are root length, shoot length, Root and shoot fresh weight, Root and shoot dry weight, Germination percentage, Seedling vigour index, and salinity tolerant index,

Traits observed

Germination percentage: For each replication and for each treatment, 3 seeds were sown, and so far germination percentage has been taken.

Germination percentage =

Number of seeds germinated

Total number of seeds sown × 100 Shoot length: Shoot length was measured by centimeter scale, which was used for all the replications and the control also. Root length: Root length was measured using a cm scale, which was used in all the replications and the control also.

Fresh weight:

The fresh weight of the seedling was measured using a weighing balance Dry weight: Dry weight of a seedling was measured by a weighing balance; the seedlings were placed in a hot air oven for 24 hours at 60 degree Celsius. Stress tolerance index used to check the tolerance level of rice seedlings: STI = Yp Ys/Yp2 Yp = Average seedling dry weight of a non-stressed condition. Ys = Average seedling dry weight under stressed conditions. Yp = Average seedling dry weight of all genotypes at non stressed condition [14] .

Statistical analysis

The experiment was carried out in factorial completely randomised block design (FCRD) with the genotypes of 100 and two levels, the experiment was replicated thrice. The data on morphological traits were observed with SES score. The statistical analysis for ANOVA and cluster was done in STAR software, and the PCA and correlation was done in R software. Based on how similar the genotypes were under 4 and 8 EC's circumstances, the dendrogram divided them into multiple clusters. While genotypes in separate clusters performed differently, genotypes within the same cluster performed similarly. While smaller clusters reflected distinct types, larger clusters indicated shared responses across diverse genotypes. The genotypes with varied genetic backgrounds are highlighted by this grouping. Principal component analysis demonstrated that the first component (PC1) contributed significantly to the interpretation of the observed variability, explaining 61.81% of the overall variation among the genotypes. 16.59% of the variation was explained by the second principal component (PC2), followed by PC3, PC4, PC5, and PC6, which explained 8.96%, 6.05%, 3.95%, and 1.12%, respectively. The first two components combined contribution was 78.40%, indicating that a significant amount of the overall variability is successfully contained within these dimensions. The first two principal components are sufficient to represent the diversity among the genotypes, but the following components contribute minimally to the overall variation, as evidenced by the significant decrease in the percentage of variance from PC1 to the subsequent components. Genotypes appeared to be distinct according to salt levels in the PCA score plot. The majority of genotypes under t1 (control) were located on the negative side of PC1, whereas t2 occupied an intermediate position and t3 (high salinity) mostly appeared on the positive side.

Variable tolerance among genotypes is highlighted by the broader distribution of genotypes under t2 and t3, which indicates more diversity in response to salinity. While genotypes on the negative side show sensitivity, a few genotypes located further on the positive side under t3 regarded as salt-tolerant.All things considered, the PCA verifies that salinity has a major impact on trait variation and helps in the identification of genotypes that are resistant. Adequate expression of genotypic variation at the seedling stage proved attainable by maintaining uniform salinity conditions throughout the experiment. Under saline conditions, significant differences were observed among genotypes in different traits. The continuous variations in response demonstrate that the screening conditions were successful in distinguishing genotypes according to their salinity tolerance.

Results and Discussion

Under hydroponic salinity conditions, rice genotypes performed in a way that clearly distinguished between moderate and severe stress levels. As salinity increased from 4 dS m⁻¹ to 8 dS m⁻¹, there was a noticeable decrease in growth and vigour. Based on the genotypes' overall performance, a threshold level of 4 dS m⁻¹ was identified, at which most genotypes indicated a significant decrease in growth. This suggests that only a few genotypes survive higher stress levels, whereas several genotypes can withstand low salinity. The only two genotypes that remained to grow stably even at 8 dS m⁻¹ were Pokkali and Ezhome-4. Strong tolerance mechanisms that enable them to maintain normal growth without significantly lowering seedling vigour are suggested by their uniform performance under high salinity. A comparatively larger number of genotypes indicated increased adaptation at the threshold level of 4 dS m⁻¹. Genotypes like Ezhome-1, Ezhome-2, Ezhome-3, Kuzhiyadichan, Kullakar, and TRY-3 showed good growth and endured higher vigor, along with Pokkali and Ezhome-4. Genotypes listed above are limited to moderate salinity, however, as demonstrated by their inability to maintain equivalent performance at 8 dS m⁻¹. As a result, these genotypes can be categorized as moderately tolerant. The significance to identify a threshold level in salinity screening is shown by the evident decrease in the number of performing genotypes above 4 dS m⁻¹. It helps in distinguishing genotypes that show actual tolerance from those that only survive in less stressful conditions. The threshold level successfully differentiated stable performers from genotypes that were susceptible and moderately tolerant in this study.IR 20, TPS-3, and TPS-5, on the other hand, performed weakly even at 4 dS m⁻¹ and were negatively impacted at greater salt levels. These genotypes showed a significant decrease in seedling vigour and growth, indicating a high exposure to salinity stress. The ability of genotypes demonstrating tolerance to maintain root and shoot growth under stressful conditions was another important finding. Better water uptake and internal stability are likely promoted by this balanced growth, which makes it easier for the plant to withstand salinity stress. This classification pattern was also confirmed by the genotypes performance-based grouping. Significant difference based on salt response was demonstrated by the grouping of tolerant genotypes together and the development of separate clusters for susceptible genotypes. Overall, salt tolerance at the seedling stage was successfully determined through the hydroponic screening system. The determination of 4 dS m⁻¹ as a threshold level provides a useful standard for future screening studies. According to the findings, Ezhome-1 to Ezhome-3, Kuzhiyadichan, Kullakar, and TRY-3 were found to be moderately resistant under the threshold level, while Pokkali and Ezhome-4 were found to be highly tolerant under severe salinity. The susceptible response from TPS-3, TPS-5, and IR 20 confirms the screening method's predictability.

Conclusion

The research demonstrates that assessing rice genotypes in well-maintained hydroponic settings can give an accurate representation of how various genotypes react to rising salinity stress. Salinity tolerance is a complex trait that cannot be assessed using one example or parameter, as demonstrated by the variation in response among genotypes.

Identifying an important stress level improves the precision of choosing potential genotypes and aids in the improvement of screening techniques. The results of this study indicate that rather than depending on a single trait and a single level of tolerance, future breeding efforts should focus on combining various adaptive traits. The observed genotype diversity provides an opportunity to develop superior cultivars that are more suited to saline environments. Overall, the methodology used in this study can be a useful structure for early screening and selection in breeding programmes.

Acknowledgement

AcknowledgmentThe authors sincerely express their heartfelt gratitude to the farmers who generously provided the seed materials for this International Journal of Advanced Biochemistry Research https://www.biochemjournal.com study. Their cooperation and willingness to share valuable resources made this research possible. We deeply appreciate their support and contribution to advancing agricultural research.

References

1. Díaz-Rodríguez AM, Salcedo Gastelum LA, Félix Pablos CM, Parra-Cota FI, Santoyo G, Puente ML, et al. The current and future role of microbial culture collections in food security worldwide. Front Sustain Food Syst. 2021;4:614739.
2. Sahu M, Maurya S, Jha Z. in vitro selection for drought and salt stress tolerance in rice: an overview. Plant Physiol Rep. 2023;28(1):8-33.
3. Beulah P, Manasa Y, Karthika G, Nagaraju P, Jaldhani V, Gobinath R, et al. Physiological changes and evaluation of Saltol1 introgressed backcrossed inbred lines (BILs) for early seedling stage salinity tolerance in rice (Oryza sativa L.). Russ J Plant Physiol. 2024;71(1):10.
4. Changappa V, Tamilselvan A, Ramadoss BR, Jothiganapathy K, Krishnaswamy P, Lakshminarayanan A, et al. Identification of novel allelic combination for salt tolerance through targeted association mapping in rice. Genet Resour Crop Evol. 2024;71(1):129-143.
5. Kumar P, Sharma PK. Soil salinity and food security in India. Front Sustain Food Syst. 2020;4:533781.
6. Villalobos-López MA, Arroyo-Becerra A, QuinteroJiménez A, Iturriaga G. Biotechnological advances to improve abiotic stress tolerance in crops. Int J Mol Sci. 2022;23(19):12053.
7. Hidayah A, Nisak RR, Susanto FA, Nuringtyas TR, Yamaguchi N, Purwestri YA. Seed halopriming improves salinity tolerance of some rice cultivars during seedling stage. Bot Stud. 2022;63(1):24.
8. Yoshida S, Forno DA, Cock JH. Laboratory manual for physiological studies of rice. Manila: International Rice Research Institute; 1971.
9. Wu L, Huo W, Yao D, Li M. Effects of solid matrix priming (SMP) and salt stress on broccoli and cauliflower seed germination and early seedling growth. Sci Hortic. 2019;255:161-168.
10. Razzaq A, Ali A, Safdar LB, Zafar MM, Rui Y, Shakeel A, et al. Salt stress induces physicochemical alterations in rice grain composition and quality. J Food Sci. 2020;85(1):14-20.
11. Shafeeqa T, Ravichandran V, Senthil A, Arul L, Radhamani S. Varietal difference in seed germination under different levels of salt stress in rice. Agric Sci Dig. 2025;45(3).
12. Gregorio GB, Senadhira D, Mendoza RD. Screening rice for salinity tolerance. Manila: International Rice Research Institute; 1997.
13. Singh R, Flowers T. The physiology and molecular biology of the effects of salinity on rice. In: CRC Press; 2010. p. 899-939.
14. Fernandez GCJ. Effective selection criteria for assessing plant stress tolerance. In: Adaptation of food crops to temperature and water stress. 1992.