MICROPROPAGATION Synergism of polyamines and plant growth regulators enhanced morphogenesis, stevioside content, and production of commercially important natural antioxidants in Stevia rebaudiana Bert Shahid Akbar Khalil 1 & Nasir Kamal 2 & Muhammad Sajid 2 & Nisar Ahmad 3 & Roshan Zamir 1 & Naveed Ahmad 2 & Shahid Ali 3 Received: 27 June 2015 /Accepted: 26 January 2016 / Editor: Ewen Mullins # The Society for In Vitro Biology 2016 Abstract 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 combina- tions 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 organogen- esis. Vigorous plantlets having optimum roots were success- fully 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 con- trast, the Spd and BA (2.0 mg L -1 ) increased antioxidant ac- tivity (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. Keywords Morphogenesis . Putrescine . Stevia . Stevioside . Spermidine . Phenolics Introduction Stevia rebaudiana (Stevia) belongs to family Asteraceae with a long history of ethnomedicinal uses (Aman et al. 2013). Traditionally, the leaves of Stevia have been used for more than 1500 yr by Guarani tribes in Brazil and Paraguay to sweeten local teas and medicines (Ahmad et al. 2011). Stevia leaves naturally synthesize the zero caloric sweet com- pound known as stevioside, which is considered as an attrac- tive sugar alternative because of its negligible effects on blood glucose. Due to its increasing applications in food, drugs, and pharmaceutical industries, Stevia is now regularly cultivated in several countries like Canada, China, Japan, Mexico, South America, UK, USA, Korea, Malaysia, Brazil, and Paraguay (Ahmad et al. 2011; Dey et al. 2013). Traditionally, seeds and stem cuttings have been exploited for Stevia plantations (Khalil et al. 2014a, b). But traditional * Nisar Ahmad ahmadn@uswat.edu.pk; nisarbiotech@gmail.com Shahid Akbar Khalil shahidakhalil@yahoo.com 1 Nuclear Institute for Food and Agriculture (NIFA), Peshawar 25120, Pakistan 2 Department of Horticulture, Faculty of Crop Production Sciences, The University of Agriculture, Peshawar 25120, Pakistan 3 Center for Biotechnology and Microbiology, University of Swat, Swat 19200, Pakistan In Vitro Cell.Dev.Biol.—Plant DOI 10.1007/s11627-016-9749-6