Key Points
- Researchers from Egypt developed a time-efficient protocol for Stevia rebaudiana regeneration, with direct micropropagation yielding the highest levels of stevioside (13.17%) and rebaudioside A (5.71%), along with the fastest maturation time (140 days), making it ideal for commercial-scale production in resource-limited regions.
- Prompted by decreasing sugar production due to climate change, water scarcity, and agricultural disruption, the research supports stevia as a viable low-water alternative to sugarcane and beet, using only a third and half of the water, respectively.
- Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis identified 18 key compounds in stevia samples, confirming that direct micropropagation maintains metabolic integrity.
A study conducted by Egyptian Russian University (Cairo, Egypt), Heliopolis University (Cairo, Egypt), and Beni-suef University (Beni-suef, Egypt) set out to establish a time-efficient protocol for callus induction and regeneration of Stevia rebaudiana via both direct and indirect micropropagation by optimizing certain variables. Specifically, the researchers explored the type and concentration of plant growth regulators, the type of explants used, and the culture conditions. Qualitative and quantitative assessments of the differences in metabolite profiles between the propagation-derived lines from a single Egyptian genotype and the soil-grown plant was performed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) coupled with multivariate data analysis. These investigations were conducted to achieve the ultimate objective of defining the optimum approach that yields the highest steviol glycoside content within the shortest cultivation period. A paper based on this research was published in Scientific Reports (1).
This research was begun in response to recent sugar shortages in Egypt, as climate change poses a major threat to the agricultural sector and the stability of food security in the country as a result to rising temperatures, an alteration of precipitation patterns, and an increase in water scarcity (2). A crucial factor in the country’s economy, agriculture, and food security, sugar is derived from sugarcane and beet (3). U.S. Department of Agriculture (USDA) statistics for the Marketing Year 2024/2025 reveal that Egypt’s sugar production decreased by 110,000 compared to the previous marketing year (4).
Stevia has been found to be a less water-intensive crop than sugarcane or beets, requiring roughly a third of the amount consumed by the former, and approximately half required by the latter) (5). While native to South America particularly Paraguay and Brazil, Stevia rebaudiana is now grown in many regions throughout the world, including Asia, Europe, and North America, owing to its importance in food and pharmaceutical industries as a natural substitute for sugar (6). Known as honey leaf, sweet leaf, and sweet herb due to its steviol glycosides, which are 100 to 300 times sweeter than sucrose without causing dental caries, stevia compounds have gained international regulatory approval for use as natural sweeteners in over 60 countries. This includes approval by the FAO/WHO Joint Expert Committee on Food Additives (JECFA), the European Food Safety Authority (EFSA), and the U.S. FDA (7,8). There have been many biological activities of stevia reported, such as anti-caries, antimicrobial, antioxidant, anti-diabetic, anti-obesity, anti-hypertensive, and anti-tumor properties (9). In addition, stevia’s natural origin holds strong appeal with health-conscious consumers looking to avoid artificial sweeteners (10). Stevia has been registered and used as a safe natural sweetener in soft drinks, baked goods, dairy products and herbal teas in Egypt since 1994 (11).
A total of 18 compounds were detected across the four studied stevia samples, including 11 phenolic compounds, and 7 diterpenoids, primarily stevioside, rebaudioside A, and rebaudioside C. Metabolite quantification based on relative peak areas revealed that the direct micropropagation strategy yielded the highest levels of stevioside and rebaudioside A (13.17 and 5.71%, respectively), surpassing those in soil-grown plants, callus-derived and indirectly propagated samples. Multivariate data analysis was conducted to identify relationships among metabolite markers in the four studied stevia samples. The metabolite profiles of both soil-grown and regenerated through direct micropropagation stevia was found to be similar, with both being rich in steviol glycosides. Notably, the growth duration varied among the four studied stevia. The soil-grown and indirectly micropropagated stevia took 180 and 196 days to reach maturity, respectively while stevia regenerated via direct micropropagation took 140 days, demonstrating a more rapid development (1).
The researchers state that their findings demonstrate that direct micropropagation not only enhances growth but also conserves metabolic integrity and highlights it as an ideal strategy for scalable production of sweetener under resource-restricted settings in arid and semi-arid regions (1).
References
- Abouelela, M.B.; Eid, M.; Ali, F.M. et al. Optimizing a Rapid Tissue Culture Method for Steviol Glycoside Production from Stevia rebaudiana to Address Egypt's Sugar Deficit. Sci Rep. 2025, 15 (1), 25495. DOI: 10.1038/s41598-025-10491-3
- Abou-Hadid, A. F. Impact of Climate Change on Egyptian Agriculture, Challenges, and Opportunities in Climate. Changes Impacts on Aquatic Environments; Springer, 2025, 171–182. DOI:10.1007/978-3-031-74897-4_7
- Abdrabbo, M. A. A; Farag, A. A.; Radwan, H. A. et al. Climate Change Impact on Economic and Irrigation Requirements for Sugarcane Crop in Egypt. FOFJ 2021,9 (2). DOI: 10.17170/kobra-202011192212.
- Report No. EG2024-0011, Egypt Sugar Annual Report & Washington, D. C. 2024. USDA Foreign Agricultural Service, United States Department of Agriculture. https://apps.fas.usda.gov/newgainapi/api/Report/DownloadReportByFileName?fileName=Sugar+Annual_Cairo_Egypt_EG2024-0011.pdf (accessed 2025-01).
- Hamed, A. E.;Salem, A. Z. M.; Abou El-Nour, H. M. Water Footprint and Productivity of Stevia Compared to Sugar Crops Under Egyptian Conditions. Irrig. Drain. Syst. Eng. 2021, 10, 1–7. DOI: 10.4172/2168-9768.1000255
- Ahmad, J.; Khan, I.; Blundell, R. et al. Stevia rebaudiana Bertoni.: An Updated Review of its Health Benefits, Industrial Applications and Safety. Trends Food Sci. Technol. 2020, 100, 177–189. DOI: 10.1016/j.tifs.2020.04.030
- Sharma, S. et al. Exploring Plant Tissue Culture and Steviol Glycosides Production in Stevia rebaudiana (Bert.) Bertoni: A Review. Agriculture 2023, 13, 475. DOI: 10.3390/agriculture13020475
- Additives, E. P. et al. Safety Evaluation of the Food Additive Steviol Glycosides, Predominantly Rebaudioside M, Produced by Fermentation Using Yarrowia lipolytica VRM. Efsa J. 2023, 21, e8387. DOI: 10.2903/j.efsa.2023.8387 (2023).
- Schiatti-Sisó, I. P., Quintana, S. E.; García-Zapateiro, L. A. Stevia (Stevia rebaudiana) as a Common Sugar Substitute and its Application in Food Matrices: An Updated Review. J. Food Sci. Technol. 2023, 60, 1483–1492. DOI: 10.1007/s13197-022-05396-2 (2023).
- Prakash, I.; Chaturvedula, V. S. P. Steviol Glycosides: Natural Noncaloric Sweeteners. In Reference Series in Phytochemistry; Merillon, J. M., Ramawat, K., eds.; Springer, 2018. 101–128. DOI: 10.1007/978-3-319-26478-3_9-1
- Ghonema, M. A. Biosafety of Stevia Extract Employing a Variety of Short-Term Genotoxic Bioassays. J. Adv. Agric. Res. 2014, 19, 722–737. DOI: 10.21608/jalexu.2014.160559
