Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 134, Issue 1, pp 141–151 | Cite as

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

  • Tandokazi P. Magangana
  • Marietjie A. Stander
  • Nokwanda P. Makunga
Original Article


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.


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



Analysis of variance


Benzyl adenine


Indole acetic acid


Honesty significance difference


Liquid chromatography-mass spectrometry


Murashige and Skoog (1962) medium


Methyl erythritol-4-phosphate


Orthogonal partial least squares discriminant analysis


Principal component analysis


Photo diode array


Ultra-performance liquid chromatography



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).

Author contributions

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.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

11240_2018_1407_MOESM1_ESM.docx (2.3 mb)
Supplementary material 1 (DOCX 2402 KB)
11240_2018_1407_MOESM2_ESM.pptx (208 kb)
Supplementary material 2 (PPTX 208 KB)


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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Botany and ZoologyStellenbosch UniversityStellenboschSouth Africa
  2. 2.Department of BiochemistryStellenbosch UniversityStellenboschSouth Africa

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