Skip to main content

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

Plant Cell, Tissue and Organ Culture (PCTOC) volume 134, pages 141–151 (2018)Cite this article

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.

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

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

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

References

  • 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 154:51–56

    Article  CAS  Google Scholar 

  • Albrecht CF, Stander MA, Grobbelaar MC, Colling J, Kossmann J, Hills PN, Makunga NP (2012) LC–MS-based metabolomics assists with quality assessment and traceability of wild and cultivated plants of Sutherlandia frutescens (Fabaceae). S Afr J Bot 82:33–45

    Article  CAS  Google Scholar 

  • Allam AI, Nassar AM, Besheite SY (2001) Nitrogen fertilizer requirements of Stevia rebaudiana Bertoni, under Egyptian conditions. Egypt J Agric Res 79:1005–1018

    Google Scholar 

  • Anbazhagan M, Kalpana M, Rajendran R, Natarajan V, Dhanavel D (2010) In vitro production of Stevia rebaudiana Bertoni. Emir J Food Agric 22(3):216–222

    Google Scholar 

  • Bayraktar M, Naziri E, Akgun IH, Karabey F, Ilhan 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 Tissue Org Cult 127:289–300

    Article  CAS  Google Scholar 

  • Bondarev NI, Sukhanova MA, Semenova GA, Goryaeva OV, Andreeva SE, Nosov AM (2010) Morphology and ultrastructure of trichomes of intact and in vitro plants of Stevia rebaudiana Bertoni with reference to biosynthesis and accumulation of steviol glycosides, vol 65. Allerton Press, Incorporation, pp 12–16

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Brandle JE, Starratt AN, Gijzen M (1998) Stevia rebaudiana: Its agricultural, biological, and chemical properties. Can J Plant Sci 78:527–536

    Article  CAS  Google Scholar 

  • de Oliveira AJB, Gonçalc RAC, Chierrito TPC, dos Santos MM, 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

    Article  CAS  Google Scholar 

  • George EF, Hall MA, De Klerk GJ (2008) Plant propagation by tissue culture, 3rd edn., vol 1. Springer, New York, pp 65–113

    Google Scholar 

  • Hajihashemi S, Geuns JMC (2016) Gene transcription and steviol glycoside accumulation in Stevia rebaudiana under polyethylene glycol-induced drought stress in greenhouse cultivation. FEBS Open Bio 6:937–944

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Ibrahim IA, Nasr MI, Mohammedm BR, El-Zefzafi MM (2008) Nutrient factors affecting in vitro cultivation of Stevia rebaudiana. SugarTech 10:248–253

    CAS  Google Scholar 

  • Khalil SA, Zamir R, Ahmad N (2014) Effect of different propagation techniques and gamma irradiation on major steviol glycoside’s content in Stevia rebaudiana. J Anim Plant Sci 24:1743–1751

    Google Scholar 

  • Khan MK, Misra P, Sharma T, Shukla PK, Ramteke PW (2014) Effect of adenine sulphate on an in vitro mass propagation of Stevia rebaudiana Bertoni. J Med Plant Res 8:543–549

    Article  CAS  Google Scholar 

  • Khattab SN, Massoud MI, El-Sayed JY, Bekhit AA, El-Faham A (2015) Production and physiochemical assessment of new Stevia amino acid sweeteners from the natural stevioside. Food Chem 173:979–985

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Lemus-Mondaca R, Ah-Hen K, Vega-Gálvez A, Honores C, Moraga NO (2016) Stevia rebaudiana leaves: effect of drying process temperature on bioactive components, antioxidant capacity and natural sweeteners. Plant Foods Hum Nutr 71:49–56

    Article  PubMed  CAS  Google Scholar 

  • Michalik A, Hollinshead J, Jones L, Fleet GWJ, Yu CY, Hu XG, Van Well R, Horne G, Wilson FX, Kato A, Jenkinson SF, Nash RJ (2010) Steviamine, a new indolizidine alkaloid from Stevia rebaudiana. Phytochem Letters 3:136–138

    Article  CAS  Google Scholar 

  • Montoro P, Molfetta I, Maldini M, Ceccarini L, Piacente S, Pizza C, Macchia M (2013) Determination of six steviol glucosides of Stevia rebaudiana (Bertoni) from different geographical origin by LC-ESI-MS/MS. Food Chem 141:745–753

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  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 Tissue Org Cult 126:511–521

    Article  CAS  Google Scholar 

  • Pellny TK, Van Aken O, Dutilleul C, Wolff T, Groten K, Bor M, De Paepe R, Reyss A, Van Breusegem F, Noctor G (2008) Mitochondrial respiratory pathways modulate nitrate sensing and nitrogen-dependent regulation of plant architecture in Nicotiana sylvestris. Plant J 54:976–992

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  • Razak UNAA., Ong CB, Yu TS, Lau LK (2014) In vitro micropropagation of Stevia rebaudiana Bertoni in Malaysia. Brazil Arch Biol Technol 7:23–28

    Article  Google Scholar 

  • Singh P, Dwivedi P (2014) Two-stage culture procedure using thidiazuron for efficient micropropagation of Stevia rebaudiana, an anti-diabetic medicinal herb. J Biotechnol 4:431–437

    Google Scholar 

  • Thiyagarajan M, Venkatachalam P (2012) Large scale in vitro propagation of Stevia rebaudiana (Bert) for commercial application: pharmaceutically important and antidiabetic medicinal herb. Ind Crops Prod 37:111–117

    Article  CAS  Google Scholar 

  • Vives K, Andújar I, Lorenzo JC, Concepción O, Hernández M, Escalona M (2017) Comparison of different in vitro micropropagation methods of Stevia rebaudiana B. including temporary immersion bioreactor (BIT®). Plant Cell Tissue Org Cult 131:195–199

    Article  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • Yücesan B, Mohammed A, Büyükgöcmen R, Altus C, Kavas Ö, Gürel S, Gürel E (2016) In vitro and ex vitro propagation of Stevia rebaudiana Bertoni with high rebaudioside A content-A commercial scale application. Sci Hortic 203:20–28

    Article  CAS  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

  1. Department of Botany and Zoology, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch, 7602, South Africa

    Tandokazi P. Magangana & Nokwanda P. Makunga

  2. Department of Biochemistry, Stellenbosch University, Private Bag X1, Matieland, Stellenbosch, 7602, South Africa

    Marietjie A. Stander

Authors
  1. Tandokazi P. Magangana
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marietjie A. Stander
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nokwanda P. Makunga
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Nokwanda P. Makunga.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Additional information

Communicated by Sergio J. Ochatt.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 2402 KB)

Supplementary material 2 (PPTX 208 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. & 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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-018-1407-8

Keywords

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