MICROPROPAGATION Photoperiod and elicitors increase steviol glycosides, phenolics, and flavonoid contents in root cultures of Stevia rebaudiana I. V. Alvarado-Orea 1 & D. Paniagua-Vega 2 & J. Capataz-Tafur 3 & A. Torres-López 4 & I. Vera-Reyes 5 & E. García-López 6 & A. A. Huerta-Heredia 6 Received: 27 March 2019 /Accepted: 9 December 2019 / Editor: Zhezhi Wang # The Society for In Vitro Biology 2020 Abstract The establishment of green root cultures of Stevia rebaudiana Bertoni, and the effect of elicitors such as hydrogen peroxide (H 2 O 2 ) and methyl jasmonate (MeJA), is shown in the present study. Stevioside, rebaudioside A, and the isomers steviol/ isosteviol were identified through DFI-ESI-IT-MS n and UPLC-TOFMS spectrometric systems, in combination with solid- phase extraction. The accumulation of steviol glycosides increased by 2.4 times (compared to the control value of 22.35 μgSG per gDW), with the addition of 250 μMH 2 O 2 . The non-enzymatic antioxidant response, which resulted from production of phenolic and flavonoid compounds, was modified based on the elicitor and the dose used. The maximum accumulation of flavonoids was induced on the third day with the addition of H 2 O 2 (250 or 500 μM), and with MeJA (250 or 500 μM); the increase was observed on the fifth day. The enzymatic antioxidant response of the catalase and peroxidase from the roots under elicitation confirmed the stress conditions. Keywords Stevia rebaudiana . Green root culture . Sweetener . DFI-ESI-IT-MS n . LC/MS Introduction Stevia rebaudiana Bertoni is a plant that is native to Paraguay, which has become widely distributed in the world for its abil- ity to produce non-caloric steviol glycosides (SG) with high sweetening power. There are different SG identified in stevia leaves; however, the most important are stevioside (St) and rebaudioside A (Reb A). SG are mainly used to sweeten soft drinks, soy sauce, yogurt, and other foods in Japan, Korea, and Brazil (Tadhani et al. 2007; Modi et al. 2014), and Europe (Novel Food Catalog, European Commission 2017 ). Additionally, it has been reported that SG have glucoregulatory, hypotensive, anti-inflammatory, and anticancer biological ac- tivities (Momtazi-Borojeni et al. 2017). Due to the importance of the production of SG from S. rebaudiana, several studies have been reported using in vitro cultures as synthetic seeds (Nower 2014), callus, cells in suspension, and seedlings (Gupta 2013), transformed roots (Fu et al. 2015; Pandey et al. 2016; Michalec-Warzecha et al. 2016), and adventitious roots (Reis et al. 2011; Lopes et al. 2016; Reis et al. 2017; Ahmad et al. 2018; Ghazal et al. 2018). The culture of adventitious roots as a differentiated system enables the accumulation of secondary metabolites in a higher quantity than the undifferentiated systems (Verpoorte et al. * A. A. Huerta-Heredia aahuertahe@conacyt.mx; ahuerta@unpa.edu.mx 1 División de Estudios de Posgrado, Universidad del Papaloapan, Av. Circuito Central No. 200 Col. Parque Industrial, 68301 Tuxtepec, Oaxaca, Mexico 2 CONACyT-UANL. Departamento de Química Analítica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Av.Fco. I. Madero y Dr. E. Aguirre Pequeño s/n, 64460 Nuevo León, Mexico 3 Instituto de Biotecnología, Universidad del Papaloapan, Av. Circuito Central No. 200 Col. Parque Industrial, 68301 Tuxtepec, Oaxaca, Mexico 4 Instituto de Química Aplicada, Universidad del Papaloapan, Av. Circuito Central No. 200 Col. Parque Industrial, 68301 Tuxtepec, Oaxaca, Mexico 5 CONACyT-CIQA. Departamento de Biociencias y Agrobiotecnología, Centro de Investigación en Química Aplicada, Blvd. Enrique Reyna Hermosillo 140, Col. San José de los Cerritos, 25294 Saltillo, Coahuila, Mexico 6 CONACyT-UNPA. Instituto de Biotecnología, Universidad del Papaloapan, Av. Circuito Central No. 200 Col. Parque Industrial, 68301 Tuxtepec, Oaxaca, Mexico In Vitro Cellular & Developmental Biology - Plant https://doi.org/10.1007/s11627-019-10041-3