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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Analysis of variance
Indole acetic acid
Honesty significance difference
Liquid chromatography-mass spectrometry
Murashige and Skoog (1962) medium
- OPLS- DA:
Orthogonal partial least squares discriminant analysis
Principal component analysis
Photo diode array
Ultra-performance liquid chromatography
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
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
Allam AI, Nassar AM, Besheite SY (2001) Nitrogen fertilizer requirements of Stevia rebaudiana Bertoni, under Egyptian conditions. Egypt J Agric Res 79:1005–1018
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
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
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
Brandle JE, Telmer PG (2007) Steviol glycoside biosynthesis. Phytochemistry 68:1855–1863
Brandle JE, Starratt AN, Gijzen M (1998) Stevia rebaudiana: Its agricultural, biological, and chemical properties. Can J Plant Sci 78:527–536
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
George EF, Hall MA, De Klerk GJ (2008) Plant propagation by tissue culture, 3rd edn., vol 1. Springer, New York, pp 65–113
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
Ibrahim IA, Nasr MI, Mohammedm BR, El-Zefzafi MM (2008) Nutrient factors affecting in vitro cultivation of Stevia rebaudiana. SugarTech 10:248–253
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
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
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
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
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
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
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
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
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
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
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
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
Ramesh K, Singh V, Megeji NW (2006) Cultivation of Stevia [Stevia rebaudiana (Bert.) Bertoni]: a comprehensive review. Adv Agron 89:137–177
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
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
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
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
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
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
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).
Conflict of interest
The authors declare no conflict of interest.
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
- Ent-kaurene diterpene glycosides
- Plant metabolomics
- Rebaudioside A