Research in Plant Metabolites

Research in Plant Metabolites

Potential of Plant Extracts as Biostimulants to Enhance Growth, Sugar Metabolism, and Rebaudioside A Accumulation in Stevia rebaudiana Bertoni under In Vitro Conditions

Document Type : Original Article

Authors
Nastaran Kazemi 1
Abdollah Hatamzadeh 1
Masoumeh Khanahmadi 2
Omid Sohrabi 3
1 Plant Production Department, University of Guilan, Rasht, Iran
2 Department of Chemistry, Academic Center for Education, Culture & Research (ACECR), Kermanshah, Iran
3 Department of Horticultural Science and Engineering, Razi University, Kermanshah, Iran
10.22034/jrpsm.2026.597016.1133
Abstract
Background and Objective: Stevia rebaudiana is one of the most important medicinal plants and natural sweeteners, valued for producing glycosides such as stevioside and Rebaudioside A. Rebaudioside A is particularly important in the food industry and natural sweetener production because of its greater sweetness and more desirable taste. On the other hand, the extensive use of chemical growth regulators in micropropagation and in vitro culture, in addition to increasing production costs, may affect plant growth, metabolism, and secondary metabolite production. In recent years, the use of naturally derived biostimulants has received increasing attention as an approach more compatible with sustainable agriculture. Accordingly, this study aimed to investigate the potential of natural extracts of Bon-sorkh (Allium jesdianum) and dandelion (Taraxacum officinale) as biostimulants for improving growth and increasing Rebaudioside A content in Stevia rebaudiana.
Materials and Methods: In this study, explants were cultured on basal MS medium supplemented with different concentrations of hydroalcoholic extracts of Bon-sorkh and dandelion and compared with treatments containing BAP and NAA and a growth regulator-free control. The extracts were applied at 100, 200, and 400 mg/L, sterilized by cold filtration, and added to the sterile culture medium. Morphological traits, including leaf number, number of lateral branches, fresh weight, and dry weight, as well as chlorophyll a, chlorophyll b, and soluble sugar content, were evaluated. Finally, Rebaudioside A content was determined and compared by high-performance liquid chromatography (HPLC) to assess the effects of the treatments on Stevia-specific metabolite production.
Results: The results showed that the response of Stevia rebaudiana to the natural extracts depended on both extract type and concentration. Among the extract treatments, application of 400 mg L⁻¹ Bon-sorkh extract had the greatest effect on leaf number, resulting in 12.00 leaves, which represented an approximately 38.56% increase compared with the control. This treatment did not differ significantly in leaf number from the 2 BAP + 1 NAA treatment, which had a mean of 12.66 leaves. Fresh weight of plantlets treated with 400 mg L⁻¹ Bon-sorkh extract increased by 240.7% compared with the control, whereas the corresponding increase in the 2 BAP + 1 NAA treatment was 529.3%. Regarding dry weight, the 400 mg L⁻¹ Bon-sorkh extract treatment had a mean value of 0.60 g, representing a 361.5% increase compared with the control, whereas the corresponding PGR treatment resulted in a 515.4% increase in dry weight relative to the control. In addition to growth-related traits, the treatments significantly affected photosynthetic pigments and soluble sugars. The highest soluble sugar contents were recorded in the 400 mg L⁻¹ Bon-sorkh and dandelion extract treatments, with values of 4.73 and 4.44 mg g⁻¹ fresh weight, respectively, representing increases of 57.67% and 48% compared with the control (3 mg g⁻¹ fresh weight). HPLC analysis of four selected treatments showed that some of the selected treatments were associated with increased rebaudioside A content. Among the four selected treatments, the highest value was recorded in the 100 mg L⁻¹ dandelion extract treatment, with a mean of 8.64%, which was approximately three times that of the control. This was followed by the 2 BAP + 1 NAA and 400 mg L⁻¹ Bon-sorkh extract treatments, with mean rebaudioside A contents of 6.66% and 5.55%, respectively.
Conclusion: Based on the results, Bon-sorkh and dandelion extracts may be considered natural plant extracts with potential biostimulant activity for modulating growth and certain metabolic responses of Stevia rebaudiana under in vitro conditions. Bon-sorkh extract at 400 mg L⁻¹, particularly in terms of leaf number, showed a response comparable to that of the 2 BAP + 1 NAA treatment; however, its fresh and dry weights were lower than those obtained with the PGR treatment. Therefore, the findings of this study do not demonstrate the direct or complete replacement of BAP and NAA with natural extracts. However, because HPLC analysis was not performed for the other treatments, this finding cannot be generalized to all experimental treatments. Overall, the findings indicate the potential of natural extracts to modulate certain growth and metabolic traits of Stevia rebaudiana. Further characterization of the bioactive compounds present in these extracts and investigation of their mechanisms of action may provide a more comprehensive understanding of these responses in future studies.
Keywords
Bon-sorkh
HPLC
Glycoside
Biostimulant
Tissue culture
Abdalla, M. M. (2013). The potential of Moringa oleifera extract as a biostimulant in enhancing the growth, biochemical and hormonal contents in rocket (Eruca vesicaria subsp. sativa) plants. Int. J. Plant Physiol. Biochem, 5(3), 42-49. https://doi.org/10.5897/IJPPB2012.026    
Afzal, S., Chaudhary, N., & Singh, N. K. (2021). Role of soluble sugars in metabolism and sensing under abiotic stress. In Plant growth regulators: signalling under stress conditions (pp. 305-334). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-030-61153-8_14
Ahlawat, Y. K., Singh, M., Manorama, K., Lakra, N., Zaid, A., & Zulfiqar, F. (2024). Plant phenolics: neglected secondary metabolites in plant stress tolerance. Brazilian Journal of Botany, 47(3): 703-721. https://doi.org/10.1007/s40415-023-00949-x
Alhalawi, Z., & Ervita, L. (2025). The Effect of Stevia Consumption on Glycemic Control in Elderly Patients with Diabetes: A Literature Review. Journal of Community Development and Disaster Management, 7(2): 551-562. https://doi.org/10.37680/jcd.v7i2.7630
Kusvuran, S. (2021). Microalgae (Chlorella vulgaris Beijerinck) alleviates drought stress of broccoli plants by improving nutrient uptake, secondary metabolites, and antioxidative defense system. Horticultural Plant Journal, 7(3), 221-231. https://doi.org/10.1016/j.hpj.2021.03.007
Alum, E. U. (2024). The role of indigenous knowledge in advancing the therapeutic use of medicinal plants: challenges and opportunities. Plant signaling & behavior, 19(1): 2439255. https://doi.org/10.1080/15592324.2024.2439255
Ashrafi, K., Iqrar, S., Saifi, M., Khan, S., Qamar, F., Quadri, S. N., ... & Abdin, M. Z. (2022). Influence of plant growth regulators on glandular trichome density and steviol glycosides accumulation in Stevia rebaudiana. ACS omega, 7(35): 30967-30977. https://doi.org/10.1021/acsomega.2c02957
Askari, Y. (2022). Composition of essential oil of Dorema aucheri Boiss. and Allium jesdianum Boiss. medicinal plants. Int. J. Adv. Biol. Biomed. Res, 10(1): 72-83. DOI: 10.22034/IJABBR.2022.545885.1374
Ansar, Z., Baradaran Firouzabadi, M., Galeshi, S., Gholami, A., Parsaeian, M. (2019). Effects of marjoram and mountain thyme extracts on yield, yield components, and antioxidant enzymes of sesame under drought stress conditions. Journal Crop Improvement, 21:149–166. https://doi.org/10.22069/ejcp.2020.15979.2204 (in persian)
Blum, A. (2017). Osmotic adjustment is a prime drought stress adaptive engine in support of plant production. Plant, cell & environment, 40(1): 4-10. https://doi.org/10.1111/pce.12800
Dang Xuan, T., Xuan Chien, N., Dang Khanh, T., Duc Viet, T., & Ngoc Minh, T. T. (2025). Advancements and challenges in allelopathy: a global perspective on agricultural practices. Journal of Crop Health, 77(5): 151. https://doi.org/10.1007/s10343-025-01217-6
De Saeger, J., Van Praet, S., Vereecke, D., Park, J., Jacques, S., Han, T., & Depuydt, S. (2020). Toward the molecular understanding of the action mechanism of Ascophyllum nodosum extracts on plants. Journal of Applied Phycology, 32(1): 573-597. https://doi.org/10.1007/s10811-019-01903-9
DuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical chemistry, 28(3): 350-356. https://doi.org/10.1021/ac60111a017
Ehiagbonare, J. E., Onyibe, H. I., & Ehiagbonare, P. O. (2008). Conservation studies on four medicinal taxa of Southern Nigeria. Scientific Research and Essays, 3(2): 40-45.
Foidl, N., Makkar, H. P. S., & Becker, K. (2001). The potential of Moringa oleifera for agricultural and industrial uses. What development potential for Moringa products, 20. https://moringatrees.org/moringa-doc/the_potential_of_moringa_oleifera_for_agricultural_and_industrial_uses.pdf
Ghaderi, A., Moghaddam, M., & Rezvan, S. (2024). Assessment of the relationship between rebaudioside-A and stevioside content and growth, yield, and root characteristics of stevia (Stevia rebaudiana Bertoni) under drought Stress. Iranian Journal of Medicinal and Aromatic Plants Research, 40(3): 596-616. https://doi.org/10.22092/ijmapr.2024.365043.3428
Grauso, L., Emrick, S., de Falco, B., Lanzotti, V., & Bonanomi, G. (2019). Common dandelion: A review of its botanical, phytochemical and pharmacological profiles. Phytochemistry Reviews, 18(4): 1115-1132. https://doi.org/10.1007/s11101-019-09622-2
Han BongHee, H. B., Yu HeeJu, Y. H., Yae ByeoungWoo, Y. B., & Peak KeeYeoup, P. K. (2004). In vitro micropropagation of Lilium longiflorum'Georgia'by shoot formation as influenced by addition of liquid medium. https://doi.org/10.1016/j.scienta.2004.04.020
Hao, F., Deng, X., Yu, X., Wang, W., Yan, W., Zhao, X., & Han, L. (2024). Taraxacum: A review of ethnopharmacology, phytochemistry and pharmacological activity. The American Journal of Chinese Medicine, 52(01): 183-215. https://doi.org/10.1142/S0192415X24500083
Hazrati Yadkori, S., & Tahmasbi Sarvestani, Z. A. (2011). Effects of different levels of nitrogen fertilizer and foliar application of benzyl adenine (BA) hormone on growth and sucker production of aloe vera (Aloe vera L.). Iranian Journal of Medicinal and Aromatic Plants Research, 28(2): 210–223. (In Persian). https://doi.org/10.22092/ijmapr.2012.3023 
Heydargholinejad, F., Hamidoghli, Y., Ghasemi Omran, V. A., & Biparva, P. (2024). Effect of auxin and cytokinin on in vitro culture of water pennywort (Centella asiatica L.). Iranian Journal of Horticultural Science and Technology, 25(1): 119–128. (In Persian). http://journal-irshs.ir/article-1-635-fa.html
Javaheri, N., & Kaviani, B. (2022). Effect of auxin and cytokinin hormonal combination on micropropagation of Oriental lily (Lilium oriental hybrid "Casablanca") using bulb scale explants. Journal of Horticultural Science, 36(1): 57–69. (In Persian).
Joshi, J. R. (2025). Extraction of stevioside and rebaudioside A from Stevia rebaudiana Bertoni leaves. Unpublished thesis). Gujarat Technological University.
Kamanga, B. M., Cartmill, D. L., McGill, C., & Clavijo McCormick, A. (2025). Allelopathic effects of Moringa oleifera Lam. on cultivated and non-cultivated plants: Implications for crop productivity and sustainable agriculture. Agronomy, 15(8):1766. https://doi.org/10.3390/agronomy15081766
Kang, G., Yun, Q., Bao, G., Bao, L., Min, S. and Bai, L. (2024). Exploration of the Application of Allelopathy in Medicinal Plants in Agriculture and Medicine. International Journal of Frontiers in Medicine, 6(7).
Kumar, V., Zadokar, A., Kumar, P., Sharma, R., Sharma, R., Siddiqui, M. W., ... & Chandora, R. (2025). Advancing medicinal plant agriculture: integrating technology and precision agriculture for sustainability. PeerJ, 13: e19058. https://doi.org/10.7717/peerj.19058
Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. In Methods in enzymology (Vol. 148, pp. 350-382). Academic Press.
Mahedi, M., Shaili, S. J., Nurnobi, M., Sakil, A. R., & Rahman, A. (2025). Adoption of organic farming practices: A comprehensive review of trends, determinants, and challenges. Journal of Agricultural and Rural Research, 8(1): 26-45.
Masciulli, F., Ambroselli, D., Libero, M. L., Acquaviva, A., Di Simone, S. C., Chiavaroli, A., ... & Ingallina, C. (2025). Phytochemical composition and bioactivity of edible Taraxacum officinale: potential as an ingredient in brain health-oriented functional foods. Food & Function, 16(18): 7217-7229. https://doi.org/10.1039/d5fo02646f
Nehzomi, Z. S., & Shirani, K. (2024). A comprehensive review of the pharmacological properties and medicinal uses of Allium jesdianum Boiss. & Buhse. Journal of Medicinal Plants, 23(90): 1-16. http://dx.doi.org/10.61186/jmp.23.90.1
Nematillaevna, K. M. (2024). STUDING MEDICINAL PLANTS (PHYTONYMS) IN THE SECTION OF PHARMACEUTICAL TERMINOLOGY. Central Asian Journal of Multidisciplinary Research and Management Studies, 1(2): 4-7. https://doi.org/10.5281/zenodo.10673840
Ram, S., Yadav, S. K., Rathore, D., & Nema, R. K. (2025). Review on Stevia Rebaudiana: A Comprehensive Analysis of its Properties and Applications. Int. J. Adv. Multidiscip. Res. Stud., 5: 347-351.
Ramak, P., & Karimian, V. (2023). Effects of planting times on the chemical compositions, antioxidant activity and nutritional properties of Allium jesdianum Boiss. & Buhse leaves in Lorestan province. Ecopersia, 11(3): 175-185. https://doi.org/10.22034/ecopersia.11.3.175
Sadak, M. S., Dawood, M. G., Bakry, B. A., & El-Karamany, M. F. (2013). Synergistic effect of indole acetic acid and kinetin on performance, some biochemical constituents and yield of faba bean plant grown under newly reclaimed sandy soil. World Journal of Agricultural Sciences, 9 (4): 335-344. http://www.idosi.org/wjas/wjas9(4)13/5.pdf
Saleem, M., Nadeem, M. S. A., & Naz, S. (2025). Medicinal crops as a pathway to sustainable agriculture, food security, farmers' income, and sustainable development in Pakistan. Journal of Climate and Community Development, 4(1): 157-167. https://joccd.com/index.php/joccd/article/view/74/65
Saremi, R., Omidi, H., & Bostani, A. (2016). Effects of auxin and cytokinin on some morphophysiological characteristics of seedling and seed germination of stevia (Stevia rebaudiana Bertoni). Iranian Journal of Seed Research, 3(2): 57–70.  (In Persian). https://sid.ir/paper/263518/fa#:~:text=https%3A//doi.org/10.29252/yujs.3.2.57
Shirani, M., Aghazadeh, E., Yousefsani, B. S., & Shirani, K. (2024). Anticancer activity of Allium jesdianum extract: A potential therapeutic approach for Melanoma. https://doi.org/10.5812/jjnpp-139022
Sichanova, M., Geneva, M., Petrova, M., Miladinova-Georgieva, K., Kirova, E., Nedev, T., ... & Trendafilova, A. (2022). Improvement of Stevia rebaudiana Bertoni in vitro propagation and steviol glycoside content using aminoacid silver nanofibers. Plants, 11(19): 2468. https://doi.org/10.3390/plants11192468
Siddiqui, H., Sami, F., Faizan, M., Faraz, A., & Hayat, S. (2019). Brassinosteroid mediated regulation of photosynthesis in plants. In Brassinosteroids: Plant Growth and Development (pp. 185-217). Singapore: Springer Singapore. https://doi.org/10.1007/978-981-13-6058-9_7
Singh, P., Nayak, S. L., Sharkar, R., Sarkar, A., Dutta, S., & Paul, D. (2025). Application of modern techniques in cultivation of medicinal plants: A review. Plant Arch, 25(1): 1947-1962.
Sohrabi, O., Hatamzadeh, A., Ghasemnezhad, A., Samizadeh, H., & Erfani-Moghadam, V. (2025). A Preliminary Experimental Protocol for Enhanced Tomato Callus Formation and Growth via Several Medicinal Plant Extracts. International Journal of Horticultural Science and Technology, 12(1): 83-100. https://doi.org/10.22059/ijhst.2024.363254.677
Sohrabi, O., Hatamzadeh, A., Ghasemnezhad, A., Samizadeh, H., & Erfani-Moghadam, V. (2024). Exploring the effects of Medicinal Plant extracts on Tomato (Solanum lycopersicum L.) morphology, Biochemistry, and Plant Growth regulators under greenhouse conditions. International Journal of Horticultural Science and Technology, 11(3): 285-298. https://doi.org/10.22059/ijhst.2023.360638.649
Sohrabi, O., Hatemzadeh, A., Ghasemnezhad, A., Samizadeh Lahiji, H., & Erfani Moghadam, V. (2021). Study of the effects of some plant extracts on growth changes of tomato (Solanum lycopersicum L.) under in vitro and ex vitro conditions. Journal of Vegetable Science, 5(10): 181–197. (In Persian). https://doi.org/10.22034/iuvs.2022.543372.1184
Sosnowski, J., Truba, M., & Vasileva, V. (2023). The impact of auxin and cytokinin on the growth and development of selected crops. Agriculture, 13(3): 724. https://doi.org/10.3390/agriculture13030724
Taiz, L., Zeiger, E., Møller, I. M., & Murphy, A. (2015). Plant physiology and development (No. Ed. 6, pp. 761-pp).
Tarakhovskaya, E. R., Kang, E. J., Kim, K. Y., & Garbary, D. J. (2013). Influence of phytohormones on morphology and chlorophyll a fluorescence parameters in embryos of Fucus vesiculosus L.(Phaeophyceae). Russian journal of plant physiology, 60(2): 176-183. https://doi.org/10.1134/S1021443713020192
Tuyuc Amezquita, I. V. (2025). Micropropagation of Stevia rebaudiana: Optimizing Shoot Multiplication and Ex vitro Rooting Establishment.
ul Islam, S. N., Arshad, M., Ahmad, S., & Asgher, M. (2024). Role of sulfur and its crosstalk with phytohormones under abiotic stress in plants. Improving stress resilience in plants, 225-247. https://doi.org/10.1016/B978-0-443-18927-2.00010-8
Wahyuni, R., Solin, N. W. N. M., Hanum, C., Mawarni, L., & Septirosya, T. (2025). Environmental Stress, Elicitors and Steviol Glycoside Production in Stevia rebaudiana: A Systematic Literature Review. https://doi.org/10.21203/rs.3.rs-8073139/v1
Wang, Y., Chen, L., Li, Y., Li, Y., Yan, M., Chen, K., ... & Xu, L. (2016). Efficient enzymatic production of rebaudioside A from stevioside. Bioscience, Biotechnology, and Biochemistry, 80(1): 67-73. https://doi.org/10.1080/09168451.2015.1072457
Wu, Y., Sun, M., Zhang, J., Zhang, L., Ren, Z., Min, R., ... & Xia, Y. (2019). Differential effects of paclobutrazol on the bulblet growth of oriental lily cultured in vitro: growth behavior, carbohydrate metabolism, and antioxidant capacity. Journal of Plant Growth Regulation, 38(2): 359-372. https://doi.org/10.1007/s00344-018-9844-5
Youssef, N. M., Shaaban, S. A., Ghareeb, Z. F., & Taha, L. S. (2019). In vitro bulb formation of direct and indirect regeneration of Lilium orientalis cv.“Starfighter” plants. Bulletin of the National Research Centre, 43(1): 211. https://doi.org/10.1186/s42269-019-0246-z