Study of gene expression and steviol glycosides accumulation in Stevia rebaudiana Bertoni under various mannitol concentrations
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Stevia rebaudiana produces sweet steviol glycosides that are 300 times sweeter than sugar and have the beneficial effects on human health including anti-hyperglycaemic. Tissue culture is the best method with high efficacy to propagate stevia. Abiotic stress has an impact on steviol glycoside contents in stevia. Therefore, we investigated the effect of mannitol on the expression of four genes involved in the biosynthesis of stevia including UGT74G1, UGT76G1, kaurene oxidase and kaurene synthase genes and steviol glycosides accumulation in stevia under in vitro conditions. The highest expression of UGT76G1 gene occurred in the plants grown under 20 g/l mannitol. While for the kaurene synthase gene, the highest amount of gene expression was observed at 40 g/l mannitol. The results were different about kaurene oxidase gene. As the highest and lowest gene expression were seen in 50 and 30 g/l mannitol conditions respectively. There were the same results for UGT74G1 that means the most appropriate and also the most inopportune treatment for the gene expression were same as the condition for the kaurene oxidase gene. Compared with control, adding mannitol to media in all concentrations increases the expression of UGT76G1 gene. Estimation of steviol glycosides contents under different treatments of mannitol carried out by HPLC. According to the results, the highest amount of stevioside was produced under 20 g/l mannitol treatment. However, rebaudioside A was accumulated in its maximum amounts under 30 g/l mannitol. It can be concluded that adding mannitol to media in the certain concentration increases steviol glycoside contents in the stevia.
KeywordsStevia rebaudiana Bertoni Real Time PCR Rebaudioside A Stevioside HPLC
Polymerase chain reaction
High-performance liquid chromatography
4-Diphosphocytidyl-2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase
1-Hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase
1-Hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase
Geranylgeranyl diphosphate synthase
Copalyl diphosphate synthase
Kaurenoic acid 13-hydroxylase
I would like to thank Dr. Tayebeh Ghorbani for her kindly helps in doing this research. Thanks to Zagros Bioidea Co., Razi University Incubator for all supports.
MG: Execution research project, Data analysis, Manuscript preparation. DK: Experimental design, Data analysis, Manuscript preparation. GB: Data analysis, proofreading of the article. HMM: Experimental design, Data analysis, proofreading of the article.
Compliance with ethical standards
Conflict of interest
Matin Ghaheri, Danial Kahrizi, Gholamreza Bahrami, and Hamidreza Mohammadi Motlagh declares that they have no conflict of interest.
This study was approved by the Ethics Committee of Razi University, Kermanshah, Iran. All participants signed the informed consent form, in line with the principles of the Helsinki II declarations.
- 2.Crammer I, Ikan R (1986) Sweet glycosides from the stevia plant. Chem Br 22(10):815–818Google Scholar
- 20.Bhosle S (2004) Commercial cultivation of Stevia rebaudiana. Agrobios Newsletter 3(2):43–45Google Scholar
- 25.Skarla BP, Josaph R, Mathew G, Joy PP (2004) Stevia—a sweet herb. Indian J Arecanut Spices Med Plants 6:24–27Google Scholar
- 31.Saifi M, Nasrullah N, Ahmad MM, Athar A, Khan JA, Abdin MZ (2015) In silico analysis and expression profiling of miRNAs targeting genes of steviol glycosides biosynthetic pathway and their relationship with steviol glycosides content in different tissues of Stevia rebaudiana. Plant Physiol Biochem 94:57–64CrossRefGoogle Scholar
- 34.Kinghorn AD, Soejarto DD (1985) Current status of stevioside as a sweetening agent for human use. In: Wagner H, Hikino H, Farnsworth NR (Eds) Economic and medicinal plant research, vol. 1. London Academic Press, London, pp 1–52Google Scholar
- 37.Ghaheri M, Kahrizi D, Bahrami G (2015) Effect of mannitol on some morphological characteristics of in vitro Stevia rebaudiana Bertoni. Biharian Biol 11 (2): 94–97Google Scholar
- 38.Hadi SM, Ibrahim KM, Yousif ShI (2014) Effect of shock and elevated levels of mannitol on callus growth, regeneration and proline accumulation in Ruta graveolens cultures. Int J Curr Microbiol Appl Sci 3(11):479–488Google Scholar
- 39.Pandey M, Chikara SK (2014) In vitro regeneration and effect of abiotic stress on physiology and biochemical content of Stevia rebaudiana ‘Bertoni’. J Plant Sci Res 1(3):113Google Scholar
- 44.Fletcher RA, Gilley A, Sankhla N, Davis TD (2010) Triazoles as plant growth regulators and stress protectants. Hortic Rev 24:55–138Google Scholar
- 51.Tadhani MB, Jadeja RP, Rena S (2006) Micropropagation of Stevia rebaudiana Bertoni using multiple shoot culture. J Cell Tissue Res 6:545–548Google Scholar
- 52.Pandey M, Chikara SK (2015) Effect of salinity and drought stress on growth parameters, glycoside content and expression level of vital genes in steviol glycosides biosynthesis pathway of Stevia rebaudiana (Bertoni). Int J Genet 7(1):153–160Google Scholar