Synergistic and Antagonistic Effects of pH Interactions with Sodium Chloride on The In Vitro Growth of (Medicago Sativa L.)

Volume 17, Issue 2
Spring 2026
Page 39-46

Document Type : Research Paper

Authors

1 Department of Biology, College of Science, University of Duhok. Kurdistan Region, IRAQ

2 Department of Environmental Science /College of Science / University of Zakho

3 Scientific Research Center, College of Science, Duhok University, Kurdistan Region, IRAQ

Abstract
Salinity and medium pH are critical abiotic factors influencing plant growth, yet their interactive effects remain poorly understood. This study aimed to evaluate the primary and interacting effects of medium pH and sodium chloride (NaCl) on the in vitro development of Medicago sativa L. (cv. CUF 101). A 3 × 4 factorial design was employed to investigate pH levels of 4.5, 6.5, and 8.5 in conjunction with NaCl concentrations of 0, 60, 120, and 180 mM in a completely randomized design, with growth traits (root and shoot length, biomass, and leaf number) measured after 21 days of germination. This approach allowed us to capture responses across varying gradients of saline-pH stress. The effects of pH, salinity, and their interaction were all found to be highly significant (p < 0.01). Near-neutral pH (6.5) consistently maximized biomass and exhibited antagonistic effects by alleviating the growth inhibition caused by NaCl. Conversely, acidic conditions (pH 4.5) combined with NaCl produced synergistic effects, resulting in disproportionate shoot elongation without an accompanying increase in their biomass. Under alkaline conditions (pH 8.5) with NaCl, the interaction also acted synergistically, severely inhibiting root elongation and indicating an early failure of root establishment. Salinity imposed a range of penalties on all traits, exhibiting significant reductions beyond approximately 120 mM and approaching a critical collapse at 180 mM. The correlations among traits indicated a trade-off in stress allocation: root length was positively correlated with total biomass and negatively correlated with shoot elongation and leaf number. These findings demonstrate that precise pH management is essential for optimizing salt tolerance in alfalfa with implications for cultivar screening under saline-alkaline stress.

Keywords

Subjects
[1].   Safarnejad, A., Collin, H.A., Bruce, K.D., McNeilly, T. (1997). Characterization of alfalfa (Medicago sativa L.) following in vitro selection for salt tolerance. In: Tigerstedt, P.M.A. (eds) Adaptation in Plant Breeding. Developments in Plant Breeding, vol 4. Springer, Dordrecht. https://doi.org/10.1007/978-94-015-8806-5_9
[2].   Dave, K., Kumar, A., Dave, N., Jain, M., Dhanda, P. S., Yadav, A., & Kaushik, P. (2024). Climate Change Impacts on Legume Physiology and Ecosystem Dynamics: A Multifaceted Perspective. Sustainability16(14), 6026. https://doi.org/10.3390/su16146026
[3].   Hassani, A., Azapagic, A., & Shokri, N. (2021). Global predictions of primary soil salinization under a changing climate in the 21st century. Nature Communications, 12(1), 6663. https://doi.org/10.1038/s41467-021-26907-3
[4].   Balasubramaniam, T., Shen, G., Esmaeili, N., & Zhang, H. (2023). Plants’ Response Mechanisms to Salinity Stress. Plants12(12), 2253. https://doi.org/10.3390/plants12122253
[5].   Munns, R., & Tester, M. (2008). Mechanisms of salinity tolerance. Annual Review of Plant Biology, 59(1), 651–681. https://doi.org/10.1146/annurev.arplant.59.032607.092911
[6].   Li, Y., Guo, S., Chen, X., & Zhou, Y. (2022). Physiological and transcriptomic analysis reveals the regulatory role of melatonin in salt tolerance of alfalfa (Medicago sativa L.). Frontiers in Plant Science, 13, 919177. https://doi.org/10.3389/fpls.2022.919177
[7].   Niu, J., Hao, W., Zhang, Y., & Zhou, Y. (2022). Exogenous melatonin promotes the growth of alfalfa (Medicago sativa L.) under NaCl stress through multiple pathways. Ecotoxicology and Environmental Safety, 233, 113938. https://doi.org/10.1016/j.ecoenv.2022.113938
[8].   Yousef, A. N., Danial, G. H., Ibrahim, D. A., & Barakat, S. E. (2024). Propagation and callus regeneration of potato (Solanum tuberosum L.) cultivar ‘Desiree’ under salt stress conditions. Science Journal of University of Zakho, 12(3), 294-298. https://doi.org/10.25271/sjuoz.2024.12.3.1236.
[9].   Hasanuzzaman, M., Raihan, M. R. H., Masud, A. A. C., Rahman, K., Nowroz, F., Rahman, M., Nahar, K., & Fujita, M. (2021). Regulation of Reactive Oxygen Species and Antioxidant Defense in Plants under Salinity. International Journal of Molecular Sciences22(17), 9326. https://doi.org/10.3390/ijms22179326
[10].    Guo, S., Wang, S., Zhang, J., & Li, Y. (2023). Exogenous melatonin improves alfalfa growth and alleviates salinity stress by enhancing antioxidant capacity and photosynthetic performance. Agronomy, 13(7), 1727. https://doi.org/10.3390/agronomy13071727
[11].    Panchal, P., Miller, A.J., Giri, J., (2021). Organic acids: versatile stress-response roles in plants. J. Exp. Bot. 72, 4038–4052.  Doi: 10.1093/jxb/erab019
[12].    Wang, X., Liu, L., Wang, B., Dingxuan, Q., & Zhou, G. (2021). Variations in ion transportation and accumulation in alfalfa plants under NaCl and na2hco3 stresses with different pH levels. Grassland Science, 67(3), 258-266. https://doi.org/10.1111/grs.12313  
[13].    Stefanov, M. A., Rashkov, G. D., Yotsova, E. K., Dobrikova, A. G., & Apostolova, E. L. (2023). Impact of Salinity on the Energy Transfer between Pigment–Protein Complexes in Photosynthetic Apparatus, Functions of the Oxygen-Evolving Complex and Photochemical Activities of Photosystem II and Photosystem I in Two Paulownia Lines. International Journal of Molecular Sciences24(4), 3108. https://doi.org/10.3390/ijms24043108
[14].    Guo, R., Zhou, J., Hao, W., Chu, L., & Feng, W. (2020). Comparative responses of photosystem II and photosystem I to salinity–alkalinity stress in alfalfa (Medicago sativa L.). Plant Signaling & Behavior, 15(10), 1832373. https://doi.org/10.1080/15592324.2020.1832373
[15].    Mulet, J., Campos, F., & Yenush, L. (2020). Editorial: ion homeostasis in plant stress and development. Frontiers in Plant Science, 11. https://doi.org/10.3389/fpls.2020.618273
[16].    Basu S, Gangopadhyay G, Mukherjee BB. 2002. Salt toler-ance in rice in vitro: Implication of accumulation of Na+, K+and proline. Plant Cell Tiss. Org. Cult. 69: 55-64 https://doi.org/10.1023/A:1015028919620
[17].    Bai, G., He, F., Shan, G., Wang, Y., Tong, Z., Cao, Y., & Yuan, Q. (2024). Effect of Saline Irrigation Water on Alfalfa Growth and Development in Saline–Alkali Soils. Agronomy14(12), 2790. https://doi.org/10.3390/agronomy14122790
[18].    Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
[19].    Habash, A. N. Y. (2013) Overexpression of Pyruvate, Orthophosphate Dikinase Facilitates Phosphate Uptake and Allows Better Growth of Tobacco in Alkaline Soil http://dx.doi.org/10.17169/refubium-16775
[20].    Yousef, A.N. (2025). Synergistic and Antagonistic Impacts of pH Interaction with NaCl Salt on the In Vitro Growth of Asparagus officinalis L.Zanin Journal of Science and Engineering. DOI: https://doi.org/10.64362/zjse.154
[21].    Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics, 11(1), 1–42. https://doi.org/10.2307/3001478
[22].    Wang, Y., Wang, J., Guo, D., Zhang, H., Che, Y., Li, Y., Tian, B., Wang, Z., Sun, G., & Zhang, H. (2021). Physiological and comparative transcriptome analysis of leaf response and physiological adaptation to saline–alkali stress across pH values in alfalfa (Medicago sativa). Plant Physiology and Biochemistry, 167, 953–964. https://doi.org/10.1016/j.plaphy.2021.07.040
[23].    Guo, R., Zhou, Z., Cai, R., Liu, L., Wang, R., Sun, Y., Wang, D., Yan, Z., & Guo, C. (2024). Metabolomic and physiological analysis of alfalfa (Medicago sativa L.) in response to saline and alkaline stress. Plant Physiology and Biochemistry, 207, 108338. https://doi.org/10.1016/j.plaphy.2024.108338 
[24].    Oldroyd, G. E. D., & Leyser, O. (2020). A plant's diet, surviving in a variable nutrient environment. Science (New York, N.Y.)368(6486), eaba0196. https://doi.org/10.1126/science.aba0196
[25].    Wegner, L. H., & Zimmermann, U. (2004). Bicarbonate-induced alkalinization of the xylem sap in intact maize seedlings as measured in situ with a novel xylem pH probe. Plant Physiology, 136(3), 3469–3477. https://doi.org/10.1104/pp.104.043844
[26].    Zhao, Y., Chen, Y., Liu, S., Li, F., Sun, M., Liang, Z., Sun, Z., Yu, F., Rengel, Z., & Li, H. (2023). Bicarbonate rather than high pH in growth medium induced Fe-deficiency chlorosis in dwarfing rootstock quince A (Cydonia oblonga Mill.) but did not impair Fe nutrition of vigorous rootstock Pyrus betulifoliaFrontiers in plant science14, 1237327. https://doi.org/10.3389/fpls.2023.1237327
[27].    Hager, A. (2003). Role of the plasma membrane H-ATPase in auxin-induced elongation growth: Historical and new aspects. Journal of Plant Research, 116(6), 483–505. https://doi.org/10.1007/s10265-003-0110-x  
[28].    Takahashi, K., Hayashi, K., & Kinoshita, T. (2012). Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis. Plant physiology159(2), 632–641. https://doi.org/10.1104/pp.112.196428
[29].    Li, X., Liu, H., He, F., Li, M., Zi, Y., Long, R., Zhao, G., Zhu, L., Hong, L., Wang, S., Kang, J., Yang, Q., & Lin, C. (2024). Multi-omics integrative analysis provides new insights into alkaline stress in alfalfa. Plant Physiology and Biochemistry, 215, 109048. https://doi.org/10.1016/j.plaphy.2024.109048 
[30].    Shavrukov, Y. (2013). Salt stress or salt shock: Which genes are we studying? Journal of Experimental Botany, 64(1), 119–127. https://doi.org/10.1093/jxb/ers316 
[31].    Guo (Kaiwen), G., Zhao, Y., Li, C., & Zhang, C. (2020). Effects of salt concentration, pH, and their interaction on photosynthesis of alfalfa (Medicago sativa L.). Plant Signaling & Behavior, 15(10), 1832373. https://doi.org/10.1080/15592324.2020.1832373 
[32].    Guo, S., Ma, X., Cai, W., Wang, Y., Gao, X., Fu, B., & Li, S. (2022). Exogenous proline improves salt tolerance of alfalfa through modulation of antioxidant capacity, ion homeostasis, and proline metabolism. Plants, 11(21), 2994. https://doi.org/10.3390/plants11212994 
[33].    Guo, W., Chen, J., Liu, L., Ren, Y., Guo, R., Ding, Y., Li, Y., Chai, J., Sun, Y., & Guo, C. (2024). MsMIOX2, encoding a MsbZIP53-activated myo-inositol oxygenase, enhances saline–alkali stress tolerance by regulating cell wall pectin and hemicellulose biosynthesis in alfalfa (Medicago sativa). The Plant Journal, 120(3), 998–1013. https://doi.org/10.1111/tpj.17032 
[34].    [34]. Taiz, L., Møller, I. M., Murphy, A., & Zeiger, E. (2022). Plant physiology and development (7th ed.). Oxford University Press. https://global.oup.com/ushe/product/plant-physiology-and-development-9780197577240
[35].    Liu, L., Si, L., Zhang, L., Guo, R., Wang, R., Dong, H., & Guo, C. (2024). Metabolomics and transcriptomics analysis reveals the response mechanism of alfalfa to combined cold and saline–alkali stress. The Plant Journal, 119(4), 1900–1919. https://doi.org/10.1111/tpj.16896 
[36].    Xu, W., Liu, Q., Wang, B., Zhang, N., Qiu, R., Yuan, Y., Yang, M., Wang, F., Mei, L., & Cui, G. (2024). Arbuscular mycorrhizal fungi communities and promoting the growth of alfalfa in saline ecosystems of northern China. Frontiers in Plant Science, 15, 1438771. https://doi.org/10.3389/fpls.2024.1438771 
[37].    Wang, T., Yang, J., Cao, J., Zhang, Q., Liu, H., Li, P., Huang, Y., Qian, W., Bi, X., Wang, H., & Zhang, Y. (2025). MsbZIP55 regulates salinity tolerance by modulating melatonin biosynthesis in alfalfa. Plant Biotechnology Journal, 23(6), 2125–2139. https://doi.org/10.1111/pbi.70035