Meta-Topolin-induced regeneration and ameliorated rebaudioside-A production in genetically uniform candy-leaf plantlets (Stevia rebaudiana Bert.)

Abstract

Present report ascertains the efficacy of meta-Topolin (mT) for improved in vitro mass propagation of stevia (Stevia rebaudiana Bert.) with high-frequency shoot initiation, multiplication, and proliferation over other cytokinins. Shoot tips were inoculated on Murashige and Skoog (MS) medium fortified with 6-benzyladenine, kinetin, mT, thidiazuron, and zeatin individually, at different concentrations ranged from 0.5—1.5 mg L−1. Among various combinations, MS medium + 1.5 mg L−1 mT developed the maximum number (15.33) and length (57.33 mm) of shoots with a greater number of leaves (38.67) after 4 weeks of culture. Elongated shoots were then transferred for root induction on MS medium fortified with a indole-3-butyric acid (IBA), indole-3-acetic acid, and α-naphthalene acetic acid individually, at concentrations ranged from 0.5—1.5 mg L−1. MS medium + 1 mg L−1 IBA was found as the superior rooting medium with the maximum number (10.67) and length (19.00 mm) of roots after next 4 weeks of culture. Rooted plantlets were acclimatized with 95% survival rate in sand followed by sand-soil (1:1; w w−1) mixture in next 8 weeks. Proper growth and development of acclimatized plantlets without any abnormalities was confirmed by micromorphological assessment (at cellular level), plant pigment analyses as well as leaf gas exchange measurement. Stability of ploidy level of the acclimatized plantlets was confirmed by flow cytometry. Furthermore, genetic fidelity of the acclimatized plantlets with their mother plant was confirmed by various molecular markers such as primers from directed amplification of minisatellite-region DNA, conserved DNA-derived polymorphism, inter simple sequence repeats, and start codon targeted polymorphism markers. High-performance liquid chromatography analysis indicated that acclimatized plantlets produced significantly higher (2.4-folds) rebaudioside-A (96.50 mg g−1 dry weight) in comparison to mother plant (40.20 mg g−1 dry weight). The current report, for first time, accounts the mT-mediated large-scale in vitro propagation of stevia ensuring genetic fidelity and simultaneously ameliorating the rebaudioside-A production.