Research article INFLUENCE OF LIGHT, GROWTH REGULATORS, NITRATE AND SUGARS ON THE PRODUCTION OF STEVIOSIDE AND REBAUDIOSIDE-A IN THE LEAF CALLUS CULTURES OF STEVIA REBAUDIANA BERTONI

Stevioside and rebaudioside-A are diterpenes steviol glycosides which are estimated to be 250-400 times sweeter than cane sugar. These natural sweeteners with zero calories are produced in green leaves of perennial shrub Stevia rebaudiana Bertoni, family- Asteraceae. In the present study, production of Stevioside and rebaudioside-A in callus cultures under the influence of light, growth regulators, carbohydrates and nitrate levels was investigated using LC-MS method. Stevia leaf explants showed 10.2 - 96.52% callus induction on Murashige and Skoog medium with different, 36 single and 28 combinations of growth regulators. Callus accumulated about 0.47 to 2.90μg/gdc (gram dry callus) Stevioside and 0.35 to1.91 μg/gdc rebaudioside-A on eight different media i.e. one concentration of each growth regulator 2,4-dichlorophenoxy acetic acid; α-naphthalene acetic acid; Indole- 3-acetic acid; Indole-3butric acid; Kinetin and 6-benzyl amino purine and two combinations (6-benzyl amino purine + 2,4-dichlorophenoxy acetic acid and 6-benzyl amino purine + Indole-3-acetic acid) under dark conditions. A clear and significant increase in the accumulation of both Stevioside (132.9 μg/gdc) and rebaudioside-A (148.7 μg/gdc) was observed on exposure to 12 hours light conditions and sucrose (3%) containing medium with MS nitrates. Supplementation of glucose or fructose as carbon source, has further promoted production of both Stevioside (158.1 and 232.65μg/gdc) and rebaudioside-A (98.0 and 186.05 μg/gdc). Whereas increase in the total nitrate levels showed drastic reduction in the steviol glycosides accumulation in leaf derived callus culture s.