Steviol glycoside content and essential oil profiles of Stevia rebaudiana Bertoni in response to NaCl and polyethylene glycol as inducers of salinity and drought stress in vitro


Plants under different environmental regimes exhibit phenotypic plasticity, sometimes producing more secondary metabolites when microenvironmental conditions are manipulated but these responses may be species, cultivar and/or genotype dependent. To test the hypothesis of whether in vitro plants of S. rebaudiana Bertoni would accumulate higher amounts of steviol glycosides when plants were growing under salt and drought stress, cultivar ST2100 plants were used. We thus applied 25 to 100 mM NaCl and polyethylene glycol 6000 (PEG) at 2.5% to 10.0% (w/v) to generate different Murashige and Skoog (Physiol Plant 15:473–497, 1962) media. Microplant cultures were also profiled for stevioside, rebaudioside A and steviol via LC–MS. Essential oil chemicals and fatty acids were assessed using GC–MS. Finally, a chemometric analysis of ethanolic extracts produced from treated and control plants is presented from MSE fragmentation data and various phenolic acids were tentatively identified using ion fragmentation patterns. Increasing amounts of both NaCl and PEG led to poor growth and development in cultures of S. rebaudiana. For example, the 25 and 50 mM NaCl-treated plants had fewer roots in comparison to controls and at even higher concentrations (75 and 100 mM NaCl), plants did not to root. Poor in vitro organogenesis was more pronounced with PEG. For instance, when plants were placed on a 10% PEG-medium, the ability for shoot regeneration was lost and callus became more apparent. Increasing levels of NaCl and PEG were also correlated to lowered levels of rebaudioside A and stevioside. In relation to the control plants that had 0.054 mg g−1 FW of steviol, the 25 mM NaCl treatment group had highest levels of this compound, recorded at 0.156 mg g−1 FW. All other salt treatments led to trace amounts of this chemical (0.005–0.009 mg g−1 FW) and it was not detected in any of the PEG-treated plants, except for the controls. The PCA loadings plots exposed stevioside, rebaudioside E and a steviol glycoside derivative as the MS signals that contributed to discriminant clusters segregating controls from the NaCl-treated groups. For PEG, segregation in the PCA is mostly influenced by dicaffeoylquinic acid as a marker ion, separating the controls from the treatment groups. PEG-treatments caused more prominent changes to the essential oil chemistry of Stevia plants. This was evident when 7.5 or 10% PEG was applied as sabinene, α-terpinolene, n-amyl isovalerate, 7-octen-4-ol, α-bergamotene, junipene, (+)-calarene, α-cadinol, β-pinene, α-bergamotene, (+)-calarene and junipene became undetectable. Changes of this nature may be undesirable when aromatic oils of S. rebaudiana are targetted for commercial markets as our data suggest adjustment to stresses may negatively impact volatile compounds leading to a loss of bioactive aromatic compounds. This study reports, for the first time, the effects of salinity and drought conditions in vitro on changed essential oil profiles of S. rebaudiana, providing new insights into the effects of stress on the essential oil chemistry of S. rebaudiana.

Key message

Stevia responses to salt and drought stresses in vitro lead to lowered measured steviol glycosides (i.e stevioside, rebaudioside A and steviol) and significantly changes the essential oil terpenoid profiles

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5



Analysis of variance


Duncan’s multiple range test


Gas chromatography mass spectrometry


Headspace solid-phase micro-extraction


Liquid chromatography–mass spectrometry


Methylerythritol phosphate pathway


Murashige and Skoog (1962)


Polyetheylene glycol


Plant growth regulator


Principal component analysis


Orthogonal partial least square-discriminant analysis


Reactive oxygen species


  1. Badran AE, Abd Alhady MRA, Hassan WA (2015) In vitro evaluation of some traits in Stevia rebaudiana (Bertoni) under drought stress and their relationship on stevioside content. Am J Plant Sci 6:746–752

    CAS  Google Scholar 

  2. Bayraktar M, Naziri E, Akgun IH, Karabey F, Iihan E, Akyol B, Bedir E, Gurel A (2016) Elicitor induced stevioside production, in vitro shoot growth, and biomass accumulation in micropropagated Stevia rebaudiana. Plant Cell Tiss Org Cult 127:289–300

    CAS  Google Scholar 

  3. Bondarev N, Reshetnyak O, Nosov A (2003) Effects of nutrient medium composition on development of Stevia rebaudiana shoots cultivated in the roller bioreactor and their production of steviol glycosides. Plant Sci 165:845–850

    CAS  Google Scholar 

  4. Brandle JE, Telmer PG (2007) Steviol glycoside biosynthesis. Phytochemistry 68:1855–1863

    CAS  PubMed  Google Scholar 

  5. Braz de Oliveira AJ, Gonçalc RAC, Chierrito TPC, Müller dos Santos M, De Souza LM, Gorin PAJ, Sassaki GL, Lacomini M (2011) Structure and degree of polymerization of fructooligosaccharides present in roots and leaves of Stevia rebaudiana (Bert.) Bertoni. Food Chem 129:305–311

    CAS  Google Scholar 

  6. Cantabella D, Piqueras A, Acosta-Motos JR, Bernal-Vicente A, Hernández JA, Díaz-Vivancos P (2017) Salt-tolerance mechanisms induced in Stevia rebaudiana Bertoni: effects on mineral nutrition, antioxidative metabolism and steviol glycoside content. Plant Physiol Biochem 115:484–496

    CAS  PubMed  Google Scholar 

  7. Carakostas MC, Curry LL, Boileau AC, Brusick DJ (2008) Overview: the history, technical function and safety of rebaudioside A, a naturally occurring steviol glycoside, for use in food and beverages. Food Chem Toxicol 46:S1–S10

    CAS  PubMed  Google Scholar 

  8. Chang SS, Cook JM (1983) Stability studies of stevioside and rebaudioside A in carbonated beverages. J Agric Food Chem 31:409–412

    CAS  Google Scholar 

  9. Cioni PL, Morelli I, Andolfi L, Macchia M, Ceccarini L (2006) Qualitative and quantitative analysis of essential oils of five lines Stevia rebaudiana Bert. genotypes cultivated in Pisa (Italy). J Essential Oil Res 18:76–79

    CAS  Google Scholar 

  10. Debnath M, Ashwath N, Hill CB, Callahan DL, Dias DA, Jayasinghe NS, Midmore DJ, Roessner U (2018) Comparative metabolic and ionomic profiling of two cultivars of Stevia rebaudiana Bert. (Bertoni) grown under salinity stress. Plant Physiol Biochem 129:56–70

    CAS  PubMed  Google Scholar 

  11. Fallah F, Nokhasi F, Ghaheri M, Kahrizi D, Beheshti Ale Agha A, Ghorbani T, Kazemi E, Ansarypour Z (2017) Effect of salinity on gene expression, morphological and biochemical characteristics of Stevia rebaudiana Bertoni under in vitro conditions. J Cell Mol Biol 63:102–106

    CAS  Google Scholar 

  12. Fujita S, Taka K, Fujita Y (1977) Miscellaneous contributions to the essential oils of the plants from various territories XLI The components of the essential oil of Stevia rebaudiana. Yakugaku Zasshi 97:692–694

    CAS  PubMed  Google Scholar 

  13. Geuns JMC (2003) Stevioside. Phytochemistry 64:913–921

    CAS  PubMed  Google Scholar 

  14. Ghaheri M, Kahrizi D, Bahrami G, Mohammadi-Motlagh HR (2019) Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations. Mol Bio Rep 46:7–16

    CAS  Google Scholar 

  15. 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

    CAS  PubMed  Google Scholar 

  16. Gupta P, Sharma S, Saxena S (2015) Biomass yield and steviol glycoside production in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol. Appl Biochem Biotechnol 176:863–874

    CAS  PubMed  Google Scholar 

  17. Gupta P, Sharma S, Saxena S (2016) Effect of abiotic stress on growth parameters and steviol glycoside content in Stevia rebaudiana (Bertoni) raised in vitro. J Appl Res Med Aromat Plants 3:160–167

    Google Scholar 

  18. Hajihashemi S, Ehsanpour AA (2014) Antioxidant response of Stevia rebaudiana B. to polyethylene glycol and paclobutrazol treatment under in vitro culture. App Biochem Biotechnol 172:4038–4052

    CAS  Google Scholar 

  19. Hajihashemi S, Geuns JM (2017) Steviol glycosides correlation to genes transcription revealed in gibberellin and paclobutrazol-treated Stevia rebaudiana. J Plant Biochem Biotechnol 26:387–394

    CAS  Google Scholar 

  20. Hajihashemi S, Geuns JMC, Ehsanpour AA (2013) Gene transcription of steviol glycoside biosynthesis in Stevia rebaudiana Bertoni under polyethylene glycol, paclobutrazol and gibberellic acid treatments in vitro. Acta Physiol Plant 35:2009–2014

    CAS  Google Scholar 

  21. Hajihashemi S, Rajabpoor S, Djalovic I (2018) Antioxidant potential in Stevia rebaudiana callus in response to polyethylene glycol, paclobutrazol and gibberellin treatment. Physiol Mol Biol Plants 24:335–341

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Hossa KR, Carneiro JPC, Guedes TA, Braccini AL (2017) Stevia rebaudiana (Bert) Bertoni: influence of osmotic stress and seed priming of seed germination under laboratory conditions. Acta Sci 39:379–384

    Google Scholar 

  23. Karray-Bouraoui N, Rabhi M, Neffati M, Baldan B, Ranieri A, Marzouk B, Lachaâl M, Smaoui A (2009) Salt effect on yield and composition of shoot essential oil and trichome morphology and density on leaves of Mentha pulegium. Ind Crops Prod 30:338–343

    CAS  Google Scholar 

  24. Karimi M, Ahmadi A, Hashemi J, Abbasi A, Tavarini S, Guglielminetti L, Angelini LG (2015) The effect of soil moisture depletion on Stevia (Stevia rebaudiana Bertoni) grown in greenhouse conditions: growth, steviol glycosides content, soluble sugars and total antioxidant capacity. Sci Horticult 183:93–99

    CAS  Google Scholar 

  25. Karimi M, Ahmadi A, Hashemi J, Abbasi A, Tavarini S, Pompeiano A, Guglielminetti L, Angelini LG (2019) Plant growth retardants (PGRs) affect growth and secondary metabolite biosynthesis in Stevia rebaudiana Bertoni under drought stress. S Afr J Bot 121:394–401

    CAS  Google Scholar 

  26. Lavini A, Ricchardi M, Pulvento C, De Luca S, Scamosci M, D’andria R (2010) Yield, quality and water consumption of Stevia rebaudianaBertoni grown under different irrigation regimes in southern Italy. Ital J Agron 3:135–143

    Google Scholar 

  27. Lemus-Mondaca R, Vega-Gálvez A, Zura-Bravo L, Ah-Hen K (2012) Stevia rebaudiana Bertoni, source of a high potency natural sweetener: a comprehensive review on the biochemical, nutritional and functional aspects. Food Chem 132:1121–1132

    CAS  PubMed  Google Scholar 

  28. Lucho SR, Nogueira do Amaral M, Auler PA, Bianchi VJ, Ferrer MA, Caldero, AA, Braga EJB (2019) Salt-stress induced changes in in vitro cultured Stevia rebaudiana Bertoni: effect on metabolite contents, antioxidant capacity and expression of steviol-related biosynthetic genes. J Plant Growth Regul 38:1341–1353

    CAS  Google Scholar 

  29. Magangana TP, Stander MA, Makunga NP (2018) Effect of nitrogen and phosphate on in vitro growth and metabolite profiles of Stevia rebaudiana Bertoni (Asteraceae). Plant Cell Tiss Org Cult 134:141–151

    CAS  Google Scholar 

  30. Martelli A, Frattini C, Chialva F (1985) Unusual essential oils with aromatic properties-I. Volatile components of Stevia rebaudiana Bertoni. Flav Fragr J 1:3–7

    CAS  Google Scholar 

  31. Masondo NA, Aremu AO, Kulkarni MG, Petřík I, Plačková L, Šubrtová M, Novák O, Grúz J, Doležal K, Strnad M, Finnie JF (2018) How do different watering regimes affect the growth, chlorophyll fluorescence, phytohormone, and phenolic acid content of greenhouse-grown Ceratotheca triloba? J Plant Growth Regul 38:385–399

    Google Scholar 

  32. Muanda FN, Soulimani R, Diop B, Dicko A (2011) A study on chemical composition and biological activities of essential oil and extracts from Stevia rebaudiana Bertoni leaves. Food Sci Technol 44:1865–1872

    CAS  Google Scholar 

  33. Mubarak MH, Belal AH, El Dein TN, El Sarag EI (2012) In vitro response Stevia rebaudiana growth under salinity and drought stress. El Minia, Egypt: Minia International Conference for Agriculture and Irrigation in the Nile Basin Countries.

  34. Munns R (2002) Comparative physiology of salt and water stress. Plant Cell Environ 25:239–250

    CAS  PubMed  Google Scholar 

  35. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    CAS  Google Scholar 

  36. Musarurwa HT, Koegelenberg L, Makunga NP (2012) Chemical variation in essential oil profiles detected using headspace solid-phase microextraction gas chromatography spectrometry in response to potassium, nitrogen, and water available to micropropagated plants of Salvia stenophylla (Burch. ex Benth.). J Plant Growth Regulat 31:207–220

    CAS  Google Scholar 

  37. Pérez-López U, Robredoa A, Apodacaa JM, Lacuestab M, Munoz-Rueda A, Mena-Petitea A (2013) Carbon dioxide enrichment moderates salinity-induced effects on nitrogen acquisition and assimilation and their impact on growth in barley plants. Environ Exp Bot 87:148–158

    Google Scholar 

  38. Perrone D, Farah A, Donangelo CM, De Paulis T, Martin PR (2008) Comprehensive analysis of major and minor chlorogenic acids and lactones in economically relevant Brazilian coffee cultivars. Food Chem 106:859–867

  39. Ramesh K, Singh V, Megeji NW (2006) Cultivation of stevia [Stevia rebaudiana (Bert.) Bertoni]: a comprehensive review. Adv Agron 89:137–177

    Google Scholar 

  40. Rathore S, Singh N, Singh SK (2014) Influence of NaCl on biochemical parameters of two cultivars of Stevia rebaudiana regenerated in vitro. J Stress Physiol Biochem 10:287–296

    Google Scholar 

  41. Reis M, Coelho L, Santos G, Kienle U, Beltrão J (2015) Yield response of Stevia (Stevia rebaudiana Bertoni) to the salinity of irrigation water. Agree Water Manag 152:217–221

    Google Scholar 

  42. Richman A, Swanson A, Humphrey T, Chapman R, McGarvey B, Pocs R, Brandle J (2005) Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana. Plant J 41:56–67

    CAS  PubMed  Google Scholar 

  43. Röck-Okuyucu B, Bayraktar M, Akgun IH, Gurel A (2016) Plant growth regulator effects on in vitro propagation and stevioside production in Stevia rebaudiana Bertoni. HortSci 51:1573–1580

    Google Scholar 

  44. Sheng F, Xiuling C (1997) Using shallow saline groundwater for irrigation and regulating for soil salt-water regime. Irrig Drain Syst 11:1–14

    Google Scholar 

  45. Siddique AB, Rahman SM, Hossain MA (2016) Chemical composition of essential oil by different extraction methods and fatty acid analysis of the leaves of Stevia rebaudiana Bertoni. Arabian J Chem 1:S1185–S1189

    Google Scholar 

  46. Tuteja N (2007) Mechanisms of high salinity tolerance in plants. In: Dieter H, Helmut S (eds) Methods in Enzymology. Academic Press, London, pp 419–438

    Google Scholar 

  47. Wang L, Xu Y, Zhao G, Li J (2004) Rapid analysis of flavor volatiles in apple wine using headspace solid-phase microextraction. J Inst Brewing 110:57–65

    CAS  Google Scholar 

  48. Wölwer-Rieck U, May B, Lankes C, Wüst M (2014) Methylerythritol and mevalonate pathway contributions to biosynthesis of mono-, sesqui-, and diterpenes in glandular trichomes and leaves of Stevia rebaudiana Bertoni. J Agric Food Chem 10(62):2428–2435

    Google Scholar 

  49. Yadav AK, Singh S, Dhyani D, Ahuja PS (2011) A review on the improvement of Stevia [Stevia rebaudiana (Bertoni)]. Can J Plant Sci 91:1–27

    Google Scholar 

  50. Zeng J, Chen A, Li D, Yi B, Wu W (2013) Effects of salt stress on the growth, physiological responses, and glycoside contents of Stevia rebaudiana Bertoni. J Agric Food Chem 61:5720–5726

    CAS  PubMed  Google Scholar 

  51. Zimmerman BF (2011) Tandem mass spectrometric fragmentation patterns of known and new steviol glycosides with structure proposals. Rapid Commun Mass Spectrom 25:1575–1582

    Google Scholar 

Download references


We would like to thank the National Research Foundation [(Grant Number: 76555) Pretoria, South Africa] for financial support. Prof. Martin Kidd of the Centre for Statistical Consultation, Department of Statistics and Actuarial Sciences (Stellenbosch University) is thanked for his assistance. Technical services were provided by Mr. Fletcher Hiten, Mr. Malcolm Taylor and Mr. Lucky Mokwena (Central Analytical Facility; Stellenbosch University). Mrs Bernadette van Heerden is further thanked for language editorial improvements made to this manuscript.

Author information




TPM performed the tissue culture experiments, collected all data linked to this part of the study, conducted all the phytochemical extractions and provided the primary draft of this manuscript. NAM performed statistical analysis, wrote some sections of this work and contributed to editing the manuscript. MAS was involved in the metabolite analysis, tentative structural identification of key chemicals and writing of those parts linked to MS data. NPM was involved in the conceptual design of this study, provided intellectual inputs and coordinated the project throughout the experimental period. NPM re-drafted the manuscript and finally, all authors edited the manuscript and approved the final version of this paper.

Corresponding author

Correspondence to N. P. Makunga.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Communicated by Ali R. Alan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (PPTX 128 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Magangana, T.P., Stander, M.A., Masondo, N.A. et al. Steviol glycoside content and essential oil profiles of Stevia rebaudiana Bertoni in response to NaCl and polyethylene glycol as inducers of salinity and drought stress in vitro. Plant Cell Tiss Organ Cult 145, 1–18 (2021).

Download citation


  • Abiotic stress
  • Chemometrics
  • Essential oils
  • Steviol glycosides
  • Micropropagation
  • Rebaudioside A
  • Stevioside
  • Steviol