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

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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


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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.

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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.

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Correspondence to N. P. Makunga.

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Communicated by Ali R. Alan.

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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 (2021).

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