Synergism of polyamines and plant growth regulators enhanced morphogenesis, stevioside content, and production of commercially important natural antioxidants in Stevia rebaudiana Bert

This study was aimed to evaluate the synergistic effects of polyamines (PAs) and plant growth regulators (PGRs) on in vitro propagation and stevioside production in Stevia rebaudiana (Stevia). A large-scale in vitro propagation protocol was established for leaf explants on Murashige and Skoog medium (MS) supplemented with various combinations of PAs and PGRs. The synergistic combination of spermidine (Spd, 2.0 mg L-1) with 2, 4-dichlorophenoxy acetic acid (2, 4-D, 1.5 mg L-1) and 6-benzyleadenine (BA, 1.5 mg L-1) induced maximum callogenic response (91.7%). The combination of Spd (1.0 mg L-1) and BA (1.0 mg L-1) was found most effective for shoot regeneration (94.4%), mean number of shoots (14. 7), and leaves per explant (88.3). However, the combination of putrescine (Put, 2.0 mg L-1) and kinetin (Kn, 2.0 mg L-1) promoted mean shoot length (6.6 cm). Incorporation of either Spd or Put in combination with naphthalene acetic acid (NAA) or indole butyric acid (IBA) to culture media improved root organogenesis. Vigorous plantlets having optimum roots were successfully acclimatized in soil. Chromatographic data revealed that the synergism of Spd, BA, and Kn (2.0 mg L-1) enhanced stevioside content in shoots (10.20 mg/g DSB) as compared to control (3.02 mg/g DW). Furthermore, application of Put and BA (2.0 mg L-1) enhanced fresh (57.5 g L-1 FSB) and dry shoot biomass (9.03 g L-1 DSB) compared to control. In contrast, the Spd and BA (2.0 mg L-1) increased antioxidant activity (80.6%) as compared to control (55.3%). Combination of Spd, BA, and GA(3) (2.0 mg L-1) enhanced the production of phenolic and flavonoid contents. This is the first successful report on the application of polyamines for large-scale in vitro propagation and accumulation of higher stevioside content, a potential step towards industrial production.