Skip to main content
SpringerLink
Log in

Photoperiod and elicitors increase steviol glycosides, phenolics, and flavonoid contents in root cultures of Stevia rebaudiana

  • Micropropagation
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

The establishment of green root cultures of Stevia rebaudiana Bertoni, and the effect of elicitors such as hydrogen peroxide (H2O2) and methyl jasmonate (MeJA), is shown in the present study. Stevioside, rebaudioside A, and the isomers steviol/isosteviol were identified through DFI-ESI-IT-MSn 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 μM H2O2. 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 H2O2 (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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.

Similar content being viewed by others

References

  • Aebi H (1984) Catalasa. In: Packer L (ed) Methods in enzymology. Pulse Academic, Orlando, pp 121–126

    Google Scholar 

  • Ahmad N, Rab A, Ahmad N (2016) Light-induced biochemical variations in secondary metabolite production and antioxidant activity in callus cultures of Stevia rebaudiana (Bert). J Photochem Photobiol B 154:51–56

    Article  CAS  PubMed  Google Scholar 

  • Ahmad N, Rab A, Ahmad N, Fazal H (2018) Differential pH-induced biosynthesis of steviol glycosides and biochemical parameters in submerge root cultures of Stevia rebaudiana (Bert.). Sugar Tech 20:100–104

    Article  CAS  Google Scholar 

  • Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399

    Article  CAS  PubMed  Google Scholar 

  • Bai L, Zhou Y, Zhang X, Song C, Cao M (2007) Hydrogen peroxide modulates abscisic acid signaling in root growth and development in Arabidopsis. Chin Sci Bull 52:1142–1145

    Article  CAS  Google Scholar 

  • Bayraktar M, Naziri E, Hakki I, Fatih A, Esra K, Begum I, Erdal A, Gurel BA (2016) Elicitor induced stevioside production, in vitro shoot growth, and biomass accumulation in micropropagated Stevia rebaudiana. Plant Cell Tiss Organ Cult 127:289–300

    Article  CAS  Google Scholar 

  • Bayraktar M, Naziri E, Karabey F, Hakki I, Bedir E, Röck-Okuyucu B, Gurel A (2018) Enhancement of stevioside production by using biotechnological approach in in vitro culture of Stevia rebaudiana. Intl J Secondary Metabol 5:362–374

  • Bondarev N, Reshetnyak O, Nosov A (2001) Peculiarities of diterpenoid steviol glycoside production in in vitro cultures of Stevia rebaudiana Bertoni. Plant Sci 161:155–163

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Ceunen S, Geuns JMC (2013) Spatio-temporal variation of diterpene steviol in Stevia rebaudiana grown under different photoperiods. Phytochem 89:32–38

    Article  CAS  Google Scholar 

  • Chen Y, Zhou B, Li J, Tang H, Tang J, Yang Z (2018) Formation and change of chloroplast-located plant metabolites in response to light conditions. Int J Mol Sci 19:654

    Article  PubMed Central  CAS  Google Scholar 

  • Dewanto V, Wu X, Adom KK, Liu RH (2002) Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem 50:3010–3014

    Article  CAS  PubMed  Google Scholar 

  • Escobar-Bravo R, Klinkhamer PG, Leiss KA (2017) Interactive effects of UV-B light with abiotic factors on plant growth and chemistry, and their consequences for defense against arthropod herbivores. Front Plant Sci 8:278

    Article  PubMed  PubMed Central  Google Scholar 

  • Faizal A, Sari AV (2019) Enhancement of saponin accumulation in adventitious root culture of Javanese ginseng (Talinum paniculatum Gaertn.) through methyl jasmonate and salicylic acid elicitation. Afr J Biotechnol 18:130–135

    Article  CAS  Google Scholar 

  • Flores HE, Dai YR, Cuello JL, Maldonado-Mendoza IE, Loyola-Vargas VM (1993) Green roots: photosynthesis and photoautotrophy in an underground plant organ. Plant Physiol 101:363–371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fu X, Yin ZP, Chen JG, Shangguan XC, Wang X, Zhang QF, Peng DY (2015) Production of chlorogenic acid and its derivatives in hairy root cultures of Stevia rebaudiana. J Agric and Food Chem 63:262–268

    Article  CAS  Google Scholar 

  • Gardana C, Scaglianti M, Simonetti P (2010) Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid chromatography-mass spectrometry. J Chromatogr A 1217:1463–1470

    Article  CAS  PubMed  Google Scholar 

  • Ghazal B, Saif S, Farid K, Khan A, Rehman S, Reshma A, Fazal H, Ai M, Rahman L, Ahmad N (2018) Stimulation of secondary metabolites by copper and gold nanoparticles in submerge adventitious root cultures of Stevia rebaudiana (Bert.). IET Nanobiotechnol 12:569–573

    Article  PubMed  PubMed Central  Google Scholar 

  • Gupta P (2013) Plant tissue culture of Stevia rebaudiana (Bertoni): a review. J Pharmacognosy Phytother 5:26–33

    Google Scholar 

  • Gupta P, Sharma S, Saxena S (2014) Effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia rebaudiana for steviol glycoside production. Appl Biochem Biotechnol 172:2894–2906

    Article  CAS  PubMed  Google Scholar 

  • Idrees M, Sania B, Hafsa B, Kumari S, Khan H, Fazal H, Ahmad I, Akbar F, Ahmad N, Ali S, Ahmad N (2018) Spectral lights trigger biomass accumulation and production of antioxidant secondary metabolites in adventitious root cultures of Stevia rebaudiana (Bert.). C R Biol 341:334–342

    Article  PubMed  Google Scholar 

  • Janarthanam B, Gopalakrishnan M, Sekar T (2010) Secondary metabolite production in callus cultures of Stevia rebaudiana Bertoni. Bangladesh J Sci Ind Res 45:243–248

    Article  CAS  Google Scholar 

  • Javed R, Yucesan B, Gurel E (2018a) Hydrogen peroxide induced steviol glycosides accumulation and enhancement of antioxidant activities in leaf tissues of Stevia rebaudiana Bertoni. Sugar Tech 20:100–104

    Article  CAS  Google Scholar 

  • Javed R, Yucesan B, Zia M, Gurel E (2018b) Elicitation of secondary metabolites in callus cultures of Stevia rebaudiana Bertoni grown under ZnO and CuO nanoparticles stress. Sugar Tech 20:194–201

    Article  CAS  Google Scholar 

  • Ladygin VG, Bondarev NI, Semenova GA, Smolov AA, Reshtnyak OV, Nosov AM (2008) Chloroplast ultrastructure, photosynthetic apparatus activities and production of steviol glycosides in Stevia rebaudiana in vivo and in vitro. Biol Plant 52:9–16

    Article  CAS  Google Scholar 

  • Libik-Konieczny M, Capeckab E, Kąkolb E, Dziurkaa M, Grabowska-Joachimiakc A, Sliwinskad E, Pistellie L (2018) Growth, development and steviol glycosides content in the relation to the photosynthetic activity of several Stevia rebaudiana Bertoni strains cultivated under temperate climate conditions. Sci Hort 234:10–18

    Article  CAS  Google Scholar 

  • Londhe SV, Nanaware SM (2013) HPTLC method for simultaneous analysis of stevioside and rebaudioside-A in Stevia rebaudiana. J AOAC Int 96:24–26

    Article  CAS  PubMed  Google Scholar 

  • Lopes SMS, Francisco MG, Higashi B, de Almeida RTR, Krausová G, Pilau EJ, Gonçalves JE, Gonçalves RAC, de Oliveira AJB (2016) Chemical characterization and prebiotic activity of fructo-oligosaccharides from Stevia rebaudiana (Bertoni) roots and in vitro adventitious root cultures. Carbohydr Polym 152:718–725

  • Lucho SR, do Amaral MN, López-Orenes A, Kleinowski AM, do Amarante L, Ferrer MA, Calderón AA, Braga EJB (2018) Plant growth regulators as potential elicitors to increase the contents of phenolic compounds and antioxidant capacity in Stevia plants. Sugar Tech 21:696–702. https://doi.org/10.1007/s12355-018-0673-4

    Article  CAS  Google Scholar 

  • Martono Y, Rohman A, Martono S, Riyanto S (2018) Degradation study of stevioside using RP-HPLC and ESI-MS/MS. Malays J Fundam Appl Sci. Chromatography and Other Analytical Techniques:138–141

  • Mehla N, Sindhi V, Josula D, Bisht P, Wani SH (2017) An introduction to antioxidants and their roles in plant stress tolerance. In: Khan M, Khan N (eds) Reactive oxygen species and antioxidant Systems in Plants: role and regulation under abiotic stress. Springer, Singapore, pp 1–23

    Google Scholar 

  • Mejía-Espejel L, Robledo-Paza A, Lozoya-Gloria E, Peña-Valdivia CB, Carrillo-Salazar JA (2018) Elicitors on steviosides production in Stevia rebaudiana Bertoni calli. Sci Hort 242:95–102

    Article  CAS  Google Scholar 

  • Michalec-Warzecha Z, Pistelli L, D’Angiolillo F, Libik-Konieczny M (2016) Establishment of highly efficient Agrobacterium rhizogenes-mediated transformation for Stevia rebaudiana Bertoni explants. Acta Biol Cracoviensia Ser Bot 56:113–118

    Article  CAS  Google Scholar 

  • Mittler R (2017) ROS are good. Trends Plant Sci 22:11–19

    Article  CAS  PubMed  Google Scholar 

  • Modi AR, Raj S, Kanani P, Patel A, Narayanan S (2014) Analysis of differentially expressed genes involved in stevioside biosynthesis in cultures of Stevia rebaudiana Bertoni treated with steviol as an immediate precursor. J Plant Growth Regul 33:481–488

    Article  CAS  Google Scholar 

  • Mohamed AA, Ceunen S, Geuns JM, Van den Ende W, De Ley M (2011) UDP-dependent glycosyltransferases involved in the biosynthesis of steviol glycosides. J Plant Physiol 168:1136–1141

    Article  CAS  PubMed  Google Scholar 

  • Momtazi-Borojeni AA, Esmaeili SA, Abdollahi E, Sahebkar A (2017) A review on the pharmacology and toxicology of steviol glycosides extracted from Stevia rebaudiana. Curr Pharm Des 23:1616–1622

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tissue culture. Physiol Plant 15:473–449

    Article  CAS  Google Scholar 

  • Neill SJ, Desikan R, Clarke A, Hurst RD, Hancock JT (2002) Hydrogen peroxide and nitric oxide as signalling molecules in plants. J Exp Botany 53:1237–1247

    Article  CAS  Google Scholar 

  • Novel Food Catalogue, European Commission (2017) https://ec.europa.eu/food/safety/novel_food/catalogue_en Accessed 02 November 2017

  • Nower AA (2014) In vitro propagation and synthetic seeds production: an efficient method for Stevia rebaudiana Bertoni. Sugar Tech 16:100–108

  • Orozco-Cárdenas ML, Narváez-Vásquez J, Ryan CA (2001) Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13:179–191

    Article  PubMed  PubMed Central  Google Scholar 

  • Pandey H, Pandey P, Pandey SS, Singh S, Banerjee S (2016) Meeting the challenge of stevioside production in the hairy roots of Stevia rebaudiana by probing the underlying process. Plant Cell Tiss Organ Cult 126:511–521

    Article  CAS  Google Scholar 

  • Pütter J (1971) Peroxidases. In: Bergmeyer HU (ed) Methods of enzymatic analysis 2. Academic Press, New York, pp 685–690

    Google Scholar 

  • Ramos-Valdivia AC, Huerta-Heredia AA, Trejo Tapia G, Cerda-García-Rojas CM (2012) Chapter 14: Secondary metabolites as no-enzymatic plant protectors from oxidative stress. In: Anjum NA, Umar S, Ahmad A (eds) Oxidative stress in plants causes, consequences and tolerance. I.K. International Publishing House Pvt. Ltd, pp 414–433

  • Reis RV, Borges APPL, Chierrito TPC, de Souto ER, de Souza LM, Iacomini M, Gonçalves RAC (2011) Establishment of adventitious root culture of Stevia rebaudiana Bertoni in a roller bottle system. Plant Cell Tiss Organ Cult 106:329–335

    Article  Google Scholar 

  • Reis RV, Chierrito TPC, Silva TFO, Albiero ALM, Souza LA, Goncalves JE, Oliveira AJB, Goncalves RAC (2017) Morpho- anatomical study of Stevia rebaudiana roots grown in vitro and in vivo. Rev Bras Farmacogn 27:34–39

  • Sanchéz-Cordova Á, Capataz-Tafur J, Barrera-Figueroa B, López-Torres A, Sanchez-Ocampo PM, García-López E, Huerta-Heredia AA (2019) Agrobacterium rhizogenes-mediated transformation enhances steviol glycosides production and growth in Stevia rebaudiana plantlets. Sugar Tech 21:398–406

    Article  CAS  Google Scholar 

  • Shafii B, Vismeh R, Beaudry R, Warner R, Jones AD (2012) Large-scale profiling of diterpenoid glycosides from Stevia rebaudiana using ultrahigh performance liquid chromatography/tandem mass spectrometry. Anal Bioanal Chem 403:2683–2690

    Article  CAS  PubMed  Google Scholar 

  • Singleton VL, Orthofe R, Lamuela-Raventos RM (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol 299:152–178

    Article  CAS  Google Scholar 

  • Smirnoff N, Arnaud D (2019) Hydrogen peroxide metabolism and functionsin plants. New Phytol 221:1197–1214. https://doi.org/10.1111/nph.15488

    Article  CAS  PubMed  Google Scholar 

  • Tada A, Ishizuki K, Iwamura J, Mikami H, Hirao Y, Fujita I, Yamazaki T, Akiyama H, Kawamura Y (2013) Improvement of the assay method for steviol glycosides in the JECFA specifications. Am J Analyt Chem 4:190–196

    Article  CAS  Google Scholar 

  • Tadhani MB, Patel VH, Subhash R (2007) In vitro antioxidant activities of Stevia rebaudiana leaves and callus. J Food Compos Anal 20:323–329

    Article  CAS  Google Scholar 

  • Vera-Reyes I, Huerta-Heredia AA, Ponce-Noyola T, Flores-Sanchez IJ, Esparza-García F, Cerda-García-Rojas CM, Trejo-Tapia G, Ramos-Valdivia AC (2013) Strictosidine-related enzymes involved in the alkaloid biosynthesis of Uncaria tomentosa root cultures grown under oxidative stress. Biotechnol Prog 29:621–630

    Article  CAS  PubMed  Google Scholar 

  • Verpoorte R, Contin A, Memelink J (2002) Biotechnology for the production of plant secondary metabolites. Phytochem Rev 1:13–25

    Article  CAS  Google Scholar 

  • Wasternack C, Strnad M (2019) Jasmonates are signals in the biosynthesis of secondary metabolites - pathways, transcription factors and applied aspects - a brief review. New Biotechnol 25:1–11. https://doi.org/10.1016/j.nbt.2017.09.007

    Article  CAS  Google Scholar 

  • Wöelwer-Rieck U, Lankes C, Wawrzun A, Wust M (2010) Improved HPLC method for the evaluation of the major steviol glycosides in leaves of Stevia rebaudiana. Eur Food Res Technol 231:581–588

    Article  CAS  Google Scholar 

  • Wu CH, Murthy HN, Hahn EJ, Paek KY (2007) Enhanced production of caftaric acid, chlorogenic acid and cichoric acid in suspension cultures of Echinacea purpurea by the manipulation of incubation temperature and photoperiod. Biochem Eng J 36:301–303

    Article  CAS  Google Scholar 

  • Yan S, Zhang T, Dong S, McLamore ES, Wang N, Shan X, Shen Y, Wan Y (2016) MeJA affects root growth by modulation of transmembrane auxin flux in the transition zone. J Plant Growth Regul 35:256–265

    Article  CAS  Google Scholar 

  • Zhang L, Xing D (2008) Methyl jasmonate induces production of reactive oxygen species and alterations in mitochondrial dynamics that precede photosynthetic dysfunction and subsequent cell death. Plant Cell Physiol 49:1092–1111

    Article  CAS  PubMed  Google Scholar 

  • Zhang W, Curtin C, Kikuchi M, Franco C (2002) Integration of jasmonic acid and light irradiation for enhancement of anthocyanin biosynthesis in Vitis vinifera suspension cultures. Plant Sci 162:459–468

    Article  Google Scholar 

Download references

Acknowledgements

We give our thanks to Cátedras-CONACyT projects 3212 and 1028 and to projects INFRA2015-01-255514, CB 284813, and INFRA-2015-01-252013. IVAO thanks CONACyT for the scholarship 571516, the complementary support for indigenous women scholarships CONACYT 2015-2, and the Mixed Scholarships for National Mobility. We thank Mittie Roger, from UNPA for the language editing of this manuscript.

Author information

Authors and Affiliations

  1. División de Estudios de Posgrado, Universidad del Papaloapan, Av. Circuito Central No. 200 Col. Parque Industrial, 68301, Tuxtepec, Oaxaca, Mexico

    I. V. Alvarado-Orea

  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

    D. Paniagua-Vega

  3. Instituto de Biotecnología, Universidad del Papaloapan, Av. Circuito Central No. 200 Col. Parque Industrial, 68301, Tuxtepec, Oaxaca, Mexico

    J. Capataz-Tafur

  4. Instituto de Química Aplicada, Universidad del Papaloapan, Av. Circuito Central No. 200 Col. Parque Industrial, 68301, Tuxtepec, Oaxaca, Mexico

    A. Torres-López

  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

    I. Vera-Reyes

  6. CONACyT-UNPA. Instituto de Biotecnología, Universidad del Papaloapan, Av. Circuito Central No. 200 Col. Parque Industrial, 68301, Tuxtepec, Oaxaca, Mexico

    E. García-López & A. A. Huerta-Heredia

Authors
  1. I. V. Alvarado-Orea
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. Paniagua-Vega
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Capataz-Tafur
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Torres-López
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. I. Vera-Reyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. E. García-López
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A. A. Huerta-Heredia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. A. Huerta-Heredia.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Additional information

Editor: Zhezhi Wang

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Alvarado-Orea, I.V., Paniagua-Vega, D., Capataz-Tafur, J. et al. Photoperiod and elicitors increase steviol glycosides, phenolics, and flavonoid contents in root cultures of Stevia rebaudiana. In Vitro Cell.Dev.Biol.-Plant 56, 298–306 (2020). https://doi.org/10.1007/s11627-019-10041-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-019-10041-3

Keywords

Search

Navigation