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

Abstract

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 µM 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 µM 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 µM 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.