Multiple Shoot and Leaf Induction on Stevia rebaudiana through In-vitro Plant Tissue Culture Technique
DOI:
https://doi.org/10.65141/jeraff.v6i1.n1Keywords:
Stevia rebaudiana, plant tissue culture technique, micropropagation, cytokinin, gibberellic acidAbstract
Stevia rebaudiana, a natural sweetener 100 to 300 times sweeter than sucrose, is gaining recognition for its potential health benefits as a sugar substitute, particularly for individuals with conditions like diabetes and obesity. The high demand for Stevia-based products has led to challenges in supply, prompting the need for research to enhance the plant's growth characteristics and address affordability concerns, ultimately benefiting marginalized sectors and those seeking healthier sugar alternatives. The study employed plant tissue culture techniques, utilizing 60 explants distributed across five distinct media formulations with varying concentrations of 6-benzylaminopurine (BA) and gibberellic acid-3 (GA3) to assess the number of shoots, number of leaves, and shoot height at the termination stage. Results of One-Way Analysis of Variance (ANOVA) revealed that there is a significant difference in the number of shoots and leaves formed when compared according to different treatments (p<0.01<α=0.05). The results of Tukey post-hoc tests highlight a significant increase in the number of shoots and roots when using MS media supplemented with 1ppm GA3 + 2ppm BA, compared to most other media formulations. This underscores the crucial role of cytokinin supplementation in the media for enhancing economically valuable attributes, which, in turn, can contribute to increased supply in Stevia rebaudiana production.
References
[1] Amghar, I., Diria, G., Boumlik, I., Gaboun, F., Iraqi, D., Labhilili, M., Mentag, R., Meziani, R., Mazri, M., Ibriz, M., & Abdelwahd, R. (2021). An efficient regeneration pathway through adventitious organogenesis for the endangered Argania spinosa (L.) Skeels. Journal of Plant Research, 34(11), 355–367. https://doi.org/10.1007/s42535-021-00208-y
[2] Cablinan, A. L. (2024). Agrilime and effective microorganisms as growth and yield enhancer of soybean (Glycine max L.). Linker (The Journal of Emerging Research in Agriculture, Fisheries and Forestry), 4(1), 71–87. https://doi.org/10.65141/jeraff.v4i1.n6
[3] Chowdhury, A. I., Alam, R. A., Raian, M. M., Rahman, T., Islam, S., & Oumma, H. (2022). Effect of stevia leaves (Stevia rebaudiana Bertoni) on diabetes: A systematic review and meta-analysis of preclinical studies. Food Science & Nutrition, 10(9), 2868–2878. https://doi.org/10.1002/fsn3.2904
[4] Creswell, J. W., & Creswell, J. D. (2018). Research design: Qualitative, quantitative, and mixed methods approaches (5th ed.). Sage Publications. https://edge.sagepub.com/creswellrd5e
[5] Cui, W., Song, Q., Zuo, B., Han, Q., & Jia, Z. (2020). Effects of gibberellin (GA4+7) in grain filling, hormonal behavior, and antioxidants in high-density maize (Zea mays L.). Plants, 9(8). https://doi.org/10.3390/plants9080978
[6] Domagalska, M. A., & Leyser, O. (2011). Signal integration in the control of shoot branching. Nature Reviews Molecular Cell Biology, 12(3), 211–221. https://doi.org/10.1038/nrm3088
[7] El-Showk, S., Ruonala, R., & Helariutta, Y. (2013). Crossing paths: Cytokinin signalling and crosstalk. Development, 140(7), 1373–1383. https://doi.org/10.1242/dev.086371
[8] Ghazal, B., Ali, F., Mirza, B., Munir, A., Mehmood, S., & Ali, M. (2024). Elicitors directed in vitro growth and production of stevioside and other secondary metabolites in Stevia rebaudiana (Bertoni) Bertoni. Scientific Reports, 14. https://doi.org/10.1038/s41598-024-65483-6
[9] Gupta, R., & Chakrabarty, S. K. (2013). Gibberellic acid in plant. Plant Signaling & Behavior, 8(9). https://doi.org/10.4161/psb.25504
[10] Izmirli, A., & Yildirim, M. (2023). Time-based changes in essential oil contents and components of English lavender (Lavandula angustifolia Mill.) after foliar application of gibberellic acid (GA3). Journal of Animal & Plant Sciences, 33(3), 708–714. https://doi.org/10.36899/JAPS.2023.3.0663
[11] Long, Y., Yang, Y., Pan, G., & Shen, Y. (2022). New insights into tissue culture plant-regeneration mechanisms. Frontiers in Plant Science, 13. https://doi.org/10.3389/fpls.2022.926752
[12] Orellana-Paucar, A. M. (2023). Steviol glycosides from Stevia rebaudiana: An updated overview of their sweetening activity, pharmacological properties, and safety aspects. Molecules, 28(3). https://doi.org/10.3390/molecules28031258
[13] Pande, S. S., & Gupta, P. (2013). Plant tissue culture of Stevia rebaudiana (Bertoni): A review. Journal of Pharmacognosy and Phytotherapy, 5(2), 26–33. https://doi.org/10.5897/JPP13.0258
[14] Patel, R. M., & Shah, R. R. (2009). Regeneration of Stevia plant through callus culture. Indian Journal of Pharmaceutical Sciences, 71(1), 46–50. https://doi.org/10.4103/0250-474X.51954
[15] Pazuki, A., Aflaki, F., Yücesan, B., & Gürel, S. (2019). Effects of cytokinins, gibberellic acid 3 and gibberellic acid 4/7 on in vitro growth, morphological traits, and content of steviol glycosides in Stevia rebaudiana. Plant Physiology and Biochemistry, 137, 154–161. https://doi.org/10.1016/j.plaphy.2019.02.009
[16] Ptak, A., Szewczyk, A., Simlat, M., Błażejczak, A., & Warchoł, M. (2023). Meta-Topolin-induced mass shoot multiplication and biosynthesis of valuable secondary metabolites in Stevia rebaudiana Bertoni bioreactor culture. Scientific Reports, 13, 1–11. https://doi.org/10.1038/s41598-023-42619-8
[17] Rodríguez-Páez, L. A., Pineda-Rodríguez, Y. Y., Pompelli, M. F., Jiménez-Ramírez, A. M., Génes-Avilez, O. J., Jaraba-Navas, J. D. D., Jarma-Orozco, A., Combatt-Caballero, E., Oviedo Zumaqué, L. E., Suárez-Padrón, I. E., Olóriz-Ortega, M. I., & Veitía Rodríguez, N. (2024). Micropropagation protocols for three elite genotypes of Stevia rebaudiana Bertoni. Horticulturae, 10(4), 1–15. https://doi.org/10.3390/horticulturae10040404
[18] Sharma, S., Walia, S., Singh, B., & Kumar, R. (2015). Comprehensive review on agro technologies of low-calorie natural sweetener stevia (Stevia rebaudiana Bertoni): A boon to diabetic patients. Journal of the Science of Food and Agriculture, 96(6), 1867–1879. https://doi.org/10.1002/jsfa.7572
[19] Singh, A., & Roychoudhury, A. (2022). Mechanism of crosstalk between cytokinin and gibberellin. Signaling and Communication in Plants, 96(6), 77–90. Springer. https://doi.org/10.1007/978-3-031-05427-3_4
[20] Sivaram, L., & Mukundan, U. (2003). In vitro culture studies on Stevia rebaudiana. In Vitro Cellular & Developmental Biology – Plant, 39(5), 520–523. https://doi.org/10.1079/IVP2003438
[21] Temanel, F., & Campos, H. C. (2025). Allelopathic effects of locally cultivated rice varieties on growth and germination of lettuce (Lactuca sativa L.) using sandwich and seed relay assays. Linker (The Journal of Emerging Research in Agriculture, Fisheries and Forestry), 5(1), 56–78. https://doi.org/10.65141/jeraff.v5i1.n4
[22] Twaij, B. M., Jazar, Z. H., & Hasan, M. N. (2020). Trends in the use of tissue culture, applications and future aspects. International Journal of Plant Biology, 11(1), 1–14. https://doi.org/10.4081/pb.2020.8385
[23] Wu, W., Du, K., Kang, X., & Wei, H. (2021). The diverse roles of cytokinins in regulating leaf development. Horticulture Research, 8(1), 1–13. https://doi.org/10.1038/s41438-021-00558-3
[24] Yoneda, Y., Nakashima, H., Miyasaka, J., Ohdoi, K., & Shimizu, H. (2017). Impact of blue, red, and far-red light treatments on gene expression and steviol glycoside accumulation in Stevia rebaudiana. Phytochemistry, 137, 57–65. https://doi.org/10.1016/j.phytochem.2017.02.002
[25] Yücesan, B., Mohammed, A., Büyükgöçmen, R., Altuğ, C., Kavas, Ö., Gürel, S., & Gürel, E. (2016). In vitro and ex vitro propagation of Stevia rebaudiana Bertoni with high Rebaudioside-A content—A commercial scale application. Scientia Horticulturae, 203, 20–28. https://doi.org/10.1016/j.scienta.2016.03.008
[26] Zainal, A., Anwar, A., Gustian, Fitriawati, & Yunita, R. (2023). The effects of several concentrations of BAP and the source of explants on gambier shoot induction (Uncaria gambier (Hunter) Roxb). IOP Conference Series: Earth and Environmental Science, 1160. https://doi.org/10.1088/1755-1315/1160/1/012021
[27] Zhang, N., & Hasenstein, K. H. (1999). Initiation and elongation of lateral roots in Lactuca sativa. International Journal of Plant Sciences, 160(3), 511–519. https://doi.org/10.1086/314147
