Establishment of Regenerative Callus, Cell Suspension System and Molecular Characterization of Stevia Rebaudiana Bertoni for the Production of Stevioside in In Vitro
Journal Title: International Journal for Research in Applied Science and Engineering Technology (IJRASET) - Year 2015, Vol 3, Issue 8
Abstract
An efficient and standardized medium for callus induction was developed using leaf explants cultured on MS medium supplemented with 1.0mgl-1 1-Naphthaleneacetic acid (NAA) and 1.0 mgl-1 2,4-dichlorophenoxyacetic acid (2,4-D),which is a noble approach to produce maximum amount of callus within short time period. Similarly, for the optimization of suspension culture, callus cultured on Murashige and Skoog basal liquid media supplemented with 2, 4-D, 6-Benzylaminopurine (BAP) and ascorbic acid along with various concentrations of macro salts; the media supplemented with ammonium nitrate (NH4NO3) in addition of 4.5% sucrose showed the highest cell growth response on twentieth day. For the enhancement of the optimal cell growth 100 µm methyl jasmonate was added, which showed the highest cell growth as well as stevioside content on twentieth days at 4.5% sucrose concentration.HPLC (High-performance liquid chromatography) analyses for the estimation of the stevioside were carried out using C18 column. The highest (40.0 mg/g) steviol glycosides was in leaf of the Russian population as compared with Indian population (21.2 mg/g), followed by the callus (2.997 mg/g) and (0.325 mg/g) in suspension culture. The highest biomass yield (15.23gm/l) and stevioside content was observed in the media supplemented with NH4NO3 in addition of 4.5% sucrose on twentieth day. The maximum stevioside content was observed indicating the supportive role of biomass and stevioside content in the callus. Random Amplified Polymorphic DNA Markers were used to study the genetic variation between the Russian and Indian population of Stevia rebaudiana and callus. Using RAPD analysis, we confirmed that mother plants of Russian and Indian stevia populations showed genetic variation but, as their calluses were grown they came to show genetic similarity to some extent.
Authors and Affiliations
Nisha Sharma, Dhurva P. Gauchan, Ashna Dhakal, Anup Luitel
Keywords
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