Optimizing a rapid tissue culture method for steviol glycoside production from Stevia rebaudiana to address Egypt’s sugar deficit

Abstract

Egypt has encountered a significant sugar scarcity since 2023, due to a water crisis, which has reduced local sugar production, prompting a search for alternative sources. Stevia rebaudiana (Asteraceae) is a natural source of steviol glycosides, which are high-intensity, low-calorie sweeteners with increasing demand in food and pharmaceutical industries. Despite its potential as a water-efficient alternative to sugar crops, Egypt lacks optimized protocols for stevia propagation and secondary metabolite enhancement. This study aimed to develop an efficient in vitro regeneration system for a local stevia genotype using callus induction, as well as both direct and indirect micropropagation and to assess its impact on steviol glycoside accumulation by comparison with conventionally soil-grown plant. Explants were cultured on Murashige and Skoog (MS) media supplemented with varying concentrations of BAP, NAA, and kinetin to evaluate callus formation, shoot proliferation, and root development. Optimal conditions yielded significantly higher shoot regeneration frequencies (up to 93%) and shoot number per explant (up to 12.6). Regenerated plants were acclimatized with a survival rate exceeding 85%. Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) was used for metabolite profiling of the four studied stevia. 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. These findings demonstrated 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.