Establishment of Stevia rebaudiana Cell Cultures and Phytochemical Profiling via GC-MS and HPLC

Abstract

Stevia rebaudiana, a plant renowned for its high content of non-caloric steviol glycosides, is widely used as a natural sweetener. This study focused on the establishment of Stevia cell suspension cultures from in vitro-derived and the comparative phytochemical analysis of leaves, calli, and cell suspension cultures using Gas Chromatography–Mass Spectrometry (GC-MS) and High-Performance Liquid Chromatography (HPLC). Six-week-old leaf disc-derived calli (~5.0g) were transferred into 150 mL of liquid Murashige and Skoog (MS) medium supplemented with 2.0 mg/L BAP and 1.0 mg/L NAA, and they were agitated at different speeds (50, 100, 150 rpm) on a rotary shaker at 25±1°C and kept under both light and dark conditions. Phytochemical analysis via GC-MS and HPLC was conducted on three-monthold plant leaves, six-week-old calli, and two-week-old cell cultures. Phytochemical extraction of leaves, callus, and cell pellet obtained by centrifugation was performed using methanol as the solvent. The optimal agitation speed for successful culture establishment was 100 rpm, which ensured effective cell separation. Lower speeds resulted in cell clustering, while higher speeds caused cell damage. HPLC analysis showed that plant leaves accumulated significantly higher levels of steviol glycosides (Rebaudioside A: 8.234±0.04 mg/mL, Stevioside: 10.132±0.03 mg/mL, Rebaudioside C: 1.585±0.02mg/mL, Dulcoside A: 0.477±0.01 mg/mL) compared to callus (Rebaudioside A: 1.793±0.01 mg/mL, Stevioside: 2.228±0.03 mg/mL, Rebaudioside C: 0.365±0.03 mg/mL, Dulcoside A: 0.118±0.01 mg/mL) and cell cultures (Rebaudioside A: 0.272±0.02 mg/mL, Stevioside: 0.384±0.01 mg/mL). This disparity is likely due to the higher levels of photosynthetic pigments in leaf tissues, which positively correlated with steviol glycoside content. The callus exhibited lower glycoside levels, and cell cultures produced minimal amounts, underscoring the essential role of light in glycoside biosynthesis. GC-MS identified distinct phytochemical profiles across the different culture systems (22 in leaves, 16 in callus, and 12 in cell culture), revealing bioactive compounds with pharmacological properties. The results indicate that in vitro systems represent promising platforms for producing bioactive phytochemicals, including steviol glycosides, although further optimization is required.