In vitro and ex vitro propagation of Stevia rebaudiana Bertoni with high Rebaudioside-A content-A commercial scale application

Stevia rebaudiana Bertoni is a self-incompatible and the pollination is an entomophilous short-day plant. Rebaudioside A, as an individual steviol glycoside, is of particular interest in global sweetener market due to its most desirable flavour profile as compared to stevioside having aftertaste bitterness. In the present investigation, regenerant formation was performed for 6 weeks, in which randomly selected nodes excised from in vitro germinated seedlings were cultured on solid Murashige and Skoog medium (MS) with or without growth regulators (6-benzylaminopurine- BAP or kinetin- KIN) at various concentrations (ranging from 0.1 to 2.0 mg/L). Irrespective of the medium composition, the results showed that all treatments were effective for shoot induction, producing an average of 2 shoots per explant after three weeks of culture. Following subsequent sub-culturing on MS medium at 3-week intervals, all the shoots regenerated from the nodes were transferred to MS medium with or without indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) at two different concentrations (0.25 or 0.50 mg/L) for root formation for 3 weeks. IAA was more effective for root formation, producing 7.6 roots per shoot with 100% rooting frequency. All the regenerants (~13.5 cm in length) were potted and successfully acclimatized in a greenhouse for 2 weeks, and then transferred to the field for 14 weeks with a high survival rate (>99%). Similarly, seedlings (8 weeks-old) derived from seed germination in pots were also transferred to the same field in a different plot. There was no significant difference in terms of morphological, yield performance and steviol glycoside compositions between regenerants and seedlings sampled from two developmental periods (late vegetative and flowering period). It was clearly shown that clonal propagation using nodal explants was effective for superior stock plant production with high Reb-A content (11.7% w/w).