Effect of nitrogen and phosphate on in vitro growth and metabolite profiles of Stevia rebaudiana Bertoni (Asteraceae)

Abstract

The commercialization of Stevia rebaudiana Bertoni (Asteraceae) extracts as a natural sweetener is driving interest in the use of in vitro propagation systems as an alternative source of steviol glycosides. Out of this suite of chemicals, stevioside is the most abundant but rebaudioside A is the sweetest. We established an in vitro propagation method from germinated seedlings on a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium with aims to study the effects of nitrogen and phosphate on the growth and metabolite profiles of S. rebaudiana plants. Generally, NH4NO3 is supplied at a concentration of 20.61 mM in MS medium and together with 18.79 mM KNO3, provide nitrogen to in vitro growing plants. In this study, we used a range of 0.3–72.1 mM NH4NO3 and 9.4–65.8 mM KNO3 and generated six different media with altered nitrogen. Similarly, six different concentrations of KH2PO4, ranging from 0.6 to 4.4 mM were tested for the phosphate treatments and the control medium had 1.25 mM KH2PO4. By reducing the nitrogen and phosphate levels to half, respectively, this led to the tallest plants. Increasing concentrations of nitrogen in the medium significantly lowered the amount of rebaudioside A as plants on the control medium accumulated 270 mg g−1 rebaudioside A compared to those that were on a medium with 3.5 times the nitrogen supply (30 mg g−1 rebaudiose A). Steviol increased with increasing nitrogen available to the microplants. The highest levels of stevioside (740 mg g−1) quantified was linked to microplants on a medium with half the phosphate concentration. To further assess changes to the metabolomic profiles of treated microplants, LC–MS/MS was used in combination with multivariate statistical analyses. Two distinct clusters were revealed after principal component analysis. Steviol hydrate, stevioside hydrate and rebaudioside A contributed significantly to the separation of phosphate-treated plants from those with variable nitrogen concentrations. Chlorogenic acid and its derivatives were linked to changing phosphate concentrations. The clustering suggests different molecular mechanisms at play that are affected by nitrogen and phosphate supply which serve to alter secondary metabolic flux, resulting in different chemical profiles.

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Abbreviations

ANOVA:

Analysis of variance

BA:

Benzyl adenine

IAA:

Indole acetic acid

HSD:

Honesty significance difference

LC–MS:

Liquid chromatography-mass spectrometry

MS:

Murashige and Skoog (1962) medium

MEP:

Methyl erythritol-4-phosphate

OPLS- DA:

Orthogonal partial least squares discriminant analysis

PCA:

Principal component analysis

PDA:

Photo diode array

UPLC:

Ultra-performance liquid chromatography

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Acknowledgements

The National Research Foundation (NRF) (Grant Number: 76555) Pretoria, South Africa and Stellenbosch University Division of Research Development (Sub-committee B incentive funding) are thanked for financial support. Ms TP Magangana received a NRF Grant-holder linked scholarship (Grant Number: 10871). Mr Fletcher Hiten is thanked for technical assistance with LC–MS/MS analysis (Central Analytical Facility of Stellenbosch University).

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This work is based on the MSc thesis of TPM under the supervision of NPM. Experiments were conducted by TPM before data analysis and she wrote the first draft of the paper. MAS was responsible for the LC–MS/MS analysis and data interpretation linked to this part of the study. This project was conducted in the laboratories of NPM. She acted as the principal investigator of this study, provided intellectual inputs to the data analysis and wrote significant parts of the manuscript. All authors saw and approved the final version of this paper.

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Correspondence to Nokwanda P. Makunga.

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The authors declare no conflict of interest.

Additional information

Communicated by Sergio J. Ochatt.

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Magangana, T.P., Stander, M.A. & Makunga, N.P. Effect of nitrogen and phosphate on in vitro growth and metabolite profiles of Stevia rebaudiana Bertoni (Asteraceae). Plant Cell Tiss Organ Cult 134, 141–151 (2018). https://doi.org/10.1007/s11240-018-1407-8

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Keywords

  • Stevia
  • Ent-kaurene diterpene glycosides
  • Micropropagation
  • Plant metabolomics
  • Rebaudioside A
  • Stevioside
  • Steviol