Biogenic synthesis, characterization and application of silver nanoparticles as biostimulator for growth and rebaudioside-A production in genetically stable stevia (Stevia rebaudiana Bert.) under in vitro conditions

In the present study, a simple, eco-friendly, biogenic protocol for synthesis of silver nanoparticles (AgNPs) using leaf extract of in vitro banana plantlets (Musa acuminata Colla cv. Grand Nain) was established. The synthesized AgNPs were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and fourier transform-infrared spectroscopy. Shoot tips of stevia (Stevia rebaudiana Bert.) were cultured on three forms of regeneration medium (Murashige and Skoog basal medium + 0.5 mg. L-1 6-benzyladenine) i.e. 'semi-solid', 'liquid', and 'semi-solid followed by liquid' supplemented with 100-400 mu M of AgNPs to study the effect of different concentrations of AgNPs along with different forms of medium on plant growth and rebaudioside-A content in stevia under in vitro conditions. Among different concentrations, 400 mu M AgNP showed the best growth response irrespective of different forms of medium. Nonetheless, the overall best growth response was recorded in 'semi-solid followed by liquid' medium. High performance liquid chromatography analysis of regenerants showed gradual incline in rebaudioside-A content with the increase in AgNP concentrations. Regenerants from medium containing 400 mu M AgNPs produced the highest rebaudioside-A of 334.8 mg. g-1 dry weight (>2-fold) while control ones produced 154.3 mg. g-1 dry weight. Genetic fidelity of AgNP-treated and control regenerants was confirmed using 20 inter simple sequence repeats and start codon targeted primers. The present study can be useful for commercial-scale propagation of stevia ensuring higher fresh and dry biomass as well as ameliorated rebaudioside-A production.