Optimized In Vitro Culture Media and Plant Growth Regulators Enhance Propagation and Steviol Glycoside Yield in Stevia rebaudiana Bertoni
DOI:
https://doi.org/10.48048/tis.2026.11671Keywords:
Sweetener, Auxin, Cytokinin, Reb A, PropagationAbstract
Thailand has great potential for growing Stevia rebaudiana Bertoni, a zero-calorie sweetener. Traditional stem cutting propagation is inefficient, laborious, and susceptible to disease. Microbial contamination, hyperhydration, and poor seed germination limit tissue culture’s efficiency. This study, conducted between January 2023 and November 2024 at Kasetsart University in Thailand. The goal was to evaluate the plant growth regulators (PGRs) affected in vitro culture conditions to improve seed germination, shoot multiplication, and plant performance. Seeds cultured on Murashige and Skoog (MS) medium with 3% sucrose achieved 9.5% germination within 4 weeks, which may reflect the small seed size and the self-incompatibility commonly reported in Stevia rebaudiana. Benzyl adenine (BA) supplementation at 2 mg/L increased shoot proliferation, creating robust plantlets with higher height and biomass potential. After one month on BA-enriched medium, plantlets were moved to hormone-free MS medium for roots and greenhouse adaptation, resulting in 100% survival. Mature plants yielded an average of 12 g fresh weight and 1.78 g dry weight per plant, with favorable steviol glycoside levels (stevioside 2.37%; rebaudioside A 2.10%), analyzed by high-performance liquid chromatography (HPLC) following the FAO (2010) method. These results demonstrate that BA supplementation is critical for efficient in vitro multiplication of stevia, while subsequent hormone-free rooting ensures high survival and stable growth. The developed protocol improves propagation efficiency, enhances plant quality, and supports reliable steviol glycoside accumulation, suggesting its potential application as a scalable method for commercial stevia production.
HIGHLIGHTS
- Optimized in vitro culture media significantly improved Stevia rebaudiana propagation from seed to mature plant.
- MS medium with 3% sucrose enhanced seed germination efficiency to 9.5% within 2 weeks.
- 2 mg/L benzyl adenine (BA) promoted robust shoot multiplication and plantlet vigor.
- Rooting in hormone-free MS medium followed by acclimatization yielded a 100% survival rate.
- Mature plants exhibited high biomass yield and favorable steviol glycoside content (stevioside 2.37%; rebaudioside 2.10%).
- The protocol offers a scalable strategy for commercial stevia production with improved plant quality and sweetener profile.
GRAPHICAL ABSTRACT
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