Callus Induction, Artificial Seed Production, and Extraction of Bioactive Compounds from Stevia rebaudiana under In Vitro Conditions

This study aimed to examine the influence of plant growth regulators on the initiation of callus formation in various parts of the Stevia rebaudiana Bertoni plant, specifically in nodal segments and shoot tips taken from the multiplication stage, under in vitro conditions.The explants were cultured on Murashige and Skoog (MS) medium was supplemented with various combination of naphthalene acetic acid NAA (0.5, 1.0, and 1.5 mg/L) and benzyladenine (BA) at 0.1, 0.2, and 0.3 mg/L. The results revealed that the treatment with 0.3 mg/L BA combined with 1.5 mg/L NAA led to the highest callus induction rate in nodal segments, with a maximum fresh weight of 0.1017 g. Similarly, the same treatment produced the highest fresh weight of callus in shoot tips, reaching 0.1491 g. In this study, the callus segments derived from both nodes and shoot tips were encapsulated using a sodium alginate solution at a concentration of 2% (w/v), then transferred into a 2% (w/v) calcium chloride solution, which led to the formation of a transparent gel matrix around the callus. This encapsulation process achieved a 100% success rate, with complete survival when stored at 4°C for duration of 28 days. Furthermore, callus samples were collected and analyzed for active compounds using high-performance liquid chromatography (HPLC), yielding the highest concentrations of 1704 ppm of stevioside and 98.7 ppm of rebaudioside A, extracted from apical meristem-derived callus. The aim of this study was to optimize callus induction, somatic embryogenesis and artificial seed production in Stevia rebaudiana.