Qualitative and Quantitative Estimation of Glycosides in Stevia rebaudiana Bertoni Callus Cultures under the Influence of Various Growth Regulators

Stevia rebaudiana Bertoni is a novel natural sweetener and hypoglycemic plant. It has many diterpenoid glycosides with no negative effect on the sugar levels of the blood and no calories, although its sweetness is 300 times more than sugar. Traditional propagation methods for S. rebaudiana include seed planting and stem cutting but are limited by small seed size, poor seed viability, and limited availability of stem cuttings. The current research assessed the impact of plant growth regulators (PGRs) on the formation and proliferation of callus and quantified glycosides produced in callus tissues, aiming to maximize yields for medicinal, industrial, and commercial applications. Different combinations and concentrations of PGRs: 2,4-dichlorophenoxyacetic acid (2,4-D) with kinetin (Kin), naphthalene acetic acid (NAA) with 6-benzyl adenine (BA), and 0.1, 0.5, and 1.0 mg/L of thidiazuron (TDZ) in combination with 4.0 mg/L NAA and 1.0 mg/L BA, were evaluated to determine the most effective treatment for inducing callus formation from leaf explants. High-performance liquid chromatography was used to analyze the glycosides that were extracted from the tissues of the leaves and calli. The interaction of 2, 4-D and Kin exhibited a weak response in inducing callus formation. In contrast, the combination of 4.0 mg/L NAA and 1.0 mg/L BA was the most effective for callus development and rebaudiosides accumulation. Moreover, supplementation with 1.0 mg/L TDZ alongside 4.0 mg/L NAA and 1.0 mg/L BA resulted in the highest accumulation of stevioside. The study’s findings revealed that effective PGR combinations enhanced callus formation and glycoside accumulation in Stevia.