Enhancement of stevioside production by using biotechnological approach in in vitro culture of Stevia rebaudiana

Abstract

Stevia rebaudiana Bertoni, which is an important plant for the food and health sector, contains calorie-free natural sweet-tasting steviol glycosides (SGs). In the present study, the effects of different elicitors [methyl jasmonate (MeJA), salicylic acid (SA), or chitosan (CHI)] on the in vitro production of stevioside and rebaudioside A were carried out. For this purpose, 3-week-old in vitro plantlets were transferred into 250 mL flasks containing liquid woody plant medium (WPM) supplemented with MeJA, SA, or CHI at different concentrations (0, 50, 100, or 200 µM), and were exposed to these elicitors for 2 weeks. A HPLC method was developed to quantify the aforementioned SGs in the cultivated plantlets and all of the elicitor types and concentrations resulted in an increase in stevioside production ranged between 2.87 mg/g dry weight (DW) (Control) and 50.07 mg/g DW (100 µM MeJA). The highest number of shoot, node, leaf, leaf length, and biomass accumulation and shoot length were observed with application of 100 µM CHI and control, respectively. The present findings open new perspectives for increasing the stevioside production using a plant tissue culture system.

Keywords

• Chitosan
• Elicitor
• Methyl jasmonate
• Salicylic acid
• Steviol glycosides
• Stevia rebaudiana

Enhancement of stevioside production by using biotechnological approach in in vitro culture of Stevia rebaudiana

Abstract

Stevia rebaudiana Bertoni, which is an important plant for the food and health sector, contains calorie-free natural sweet-tasting steviol glycosides (SGs). In the present study, the effects of different elicitors [methyl jasmonate (MeJA), salicylic acid (SA), or chitosan (CHI)] on the in vitro production of stevioside and rebaudioside A were carried out. For this purpose, 3-week-old in vitro plantlets were transferred into 250 mL flasks containing liquid woody plant medium (WPM) supplemented with MeJA, SA, or CHI at different concentrations (0, 50, 100, or 200 µM), and were exposed to these elicitors for 2 weeks. A HPLC method was developed to quantify the aforementioned SGs in the cultivated plantlets and all of the elicitor types and concentrations resulted in an increase in stevioside production ranged between 2.87 mg/g dry weight (DW) (Control) and 50.07 mg/g DW (100 µM MeJA). The highest number of shoot, node, leaf, leaf length, and biomass accumulation and shoot length were observed with application of 100 µM CHI and control, respectively. The present findings open new perspectives for increasing the stevioside production using a plant tissue culture system.

Keywords

• Chitosan
• Elicitor
• Methyl jasmonate
• Salicylic acid
• Steviol glycosides
• Stevia rebaudiana

References

1. [1] Brandle, J.E., Telmer, P.G. (2007). Steviol glycoside biosynthesis. Phytochemistry, 68, 1855-1863, https://doi.org/10.1016/j.phytochem.2007.02.010
2. [2] Brandle, J.E., Starratt, A.N., Gijzen, M. (1998). Stevia rebaudiana: its agricultural, biological and chemical properties. Can. J. Plant Sci., 78, 527-536, https://doi.org/10.4141/P97-114
3. [3] Lucho, S.R., do Amaral, M.N., Milech, C., Ferrer, M.Á., Calderón, A.A., Bianchi, V.J., Braga, E.J.B. (2018). Elicitor-Induced Transcriptional Changes of Genes of the Steviol Glycoside Biosynthesis Pathway in Stevia rebaudiana Bertoni. J. Plant Growth Regul., 37(3), 971-985, https://doi.org/10.1007/s00344-018-9795-x
4. [4] Bondarev, N., Reshetnyak, O., Nosov, A. (2001). Peculiarities of diterpenoid steviol glycoside production in in vitro cultures of Stevia rebaudiana Bertoni. Plant Sci., 161, 155-163, https://doi.org/10.1016/S0168-9452(01)00400-9
5. [5] Starrat, A.N., Kirby, C.W., Pocs, R., Brandle, J.E. (2002). Rebaudioside F, a diterpene glycoside from Stevia rebaudiana. Phytochemistry, 59, 367-370, https://doi.org/10.1016/S0031-9422(01)00416-2
6. [6] Kumar, H., Kaul, K., Bajpai-Gupta, S., Kaul, V.K., Kumar, S. (2012). A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni). Gene, 492, 276-284, https://doi.org/10.1016/j.gene.2011.10.015
7. [7] Lorenzo, C., Serrano-Díaz, J., Plaza, M., Quintanilla, C., Alonso, G.L. (2014). Fast methodology of analyzing major steviol glycosides from Stevia rebaudiana leaves. Food Chem., 157, 518-523, https://doi.org/10.1016/j.foodchem.2014.02.088
8. [8] Pérez-Tortosa, V., López-Orenes, A., Martínez-Pérez, A., Ferrer, M.A., Calderón, A.A. (2012). Antioxidant activity and rosmarinic acid changes in salicylic acid-treated Thymus membranaceus shoots. Food Chem., 130, 362 - 369, https://doi.org/10.1016/j.foodchem.2011.07.051

9. [9] Murthy, H.N., Lee, E.J., Paek, K.Y. (2014). Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tissue Organ Cult., 118, 1-16, https://doi.org/10.1007/s11240-014-0467-7
10. [10] Espinosa Leal, C.A., Puente Garza, C.A., García Lara, S. (2018). In vitro plant tissue culture: means for production of biological active compounds. Planta, 248, 1–18, https://doi.org/10.1007/s00425-018-2910-1
11. [11] Ramachandra Rao, S., Ravishankar, G.A. (2002). Plant cell cultures: Chemical factories of secondary metabolites. Biotechnol. Adv., 20, 101-153, https://doi.org/10.1016/S0734-9750(02)00007-1 [12] Sivanandhan, G., Arun, M., Mayavan, S., Rajesh, M., Mariashibu, T.S., Manickavasagama, M., Selvaraj, N., Ganapathia, A. (2012). Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal. Ind. Crops Prod., 37(1), 24-129, https://doi.org/10.1016/j.indcrop.2011.11.022
12. [13] Sivanandhan, G., Kapil Dev, G., Jeyaraj, M., Rajesh, M., Arjunan, A., Muthuselvam, M., Manickavasagam, M., Selvaraj, N., Ganapathi, A. (2013). Increased production of withanolide A, withanone, and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tissue Organ Cult., 114, 121-129, https://doi.org/10.1007/s11240-013-0297-z
13. [14] Singh, A., Dwivedi, P. (2018). Methyl-jasmonate and salicylic acid as potent elicitors for secondary metabolite production in medicinal plants: A review. J. Pharmacogn. Phytochem., 7(1), 750-757
14. [15] Chodisetti, B., Rao, K., Gandi, S., Giri, A. (2015). Gymnemic acid enhancement in the suspension cultures of Gymnema sylvestre by using the signaling molecules—methyl jasmonate and salicylic acid. In Vitro Cell. Dev. Biol. Plant, 51, 88-92, https://doi.org/10.1007/s11627-014-9655-8
15. [16] Rao, M.V., Lee, H., Creelman, R.A., Mullet, J.E., Davis, K.R. (2000). Jasmonic acid signaling modulates ozone-induced hypersensitive cell death. The Plant Cell, 12, 1633-1646, https://doi.org/10.1105/tpc.12.9.1633
16. [17] Vuong, T.V., Franco, C., Zhang, W. (2014). Treatment strategies for high resveratrol induction in Vitis vinifera L. cell suspension culture. Biotechnol. Rep., 1–2, 15-21, https://doi.org/10.1016/j.btre.2014.04.002
17. [18] Wiktorowska, E., Długosz, M., Janiszowska, W. (2010). Significant enhancement of oleanolic acid accumulation by biotic elicitors in cell suspension cultures of Calendula officinalis L. Enzyme Microb. Technol., 46(1), 14-20, https://doi.org/10.1016/j.enzmictec.2009.09.002
18. [19] Ladygin, V.G., Bondarev, N.I., Semenova, G.A., Smolov, A.A., Reshetnyak, O.V., Nosov, A.M. (2008). Chloroplast ultrastructure, photosynthetic apparatus activities and production of steviol glycosides in Stevia rebaudiana in vivo and in vitro. Biol. Plant., 52, 9-16, https://doi.org/10.1007/s10535-008-0002-y
19. [20] Bayraktar, M., Naziri, E., Akgun, I.H., 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 Organ Cult., 127(2), 289-300, https://doi.org/10.1007/s11240-016-1049-7
20. [21] Lloyd, G., McCown, B. (1980). Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture. Int. Plant Prop. Soc., 30, 421-427.
21. [22] Saw, N.M.M.T., Riedel, H., Cai, Z., Kütük, O., Smetanska, I. (2012). Stimulation of anthocyanin synthesis in grape (Vitis vinifera) cell cultures by pulsed electric fields and ethephon. Plant Cell Tissue Organ Cult., 108, 47-54, https://doi.org/10.1007/s11240-011-0010-z
22. [23] Mandal, S., Evelin, H., Giri, B., Singh, V.P., Kapoor, R. (2013). Arbuscular mycorrhiza enhances the production of stevioside and rebaudioside-A in Stevia rebaudiana via nutritional and non-nutritional mechanisms. Appl. Soil Ecol., 72, 187-194, https://doi.org/10.1016/j.apsoil.2013.07.003
23. [24] Richman, A., Swanson, A., Humphrey, T., Chapman, R., McGarvey, B., Pocs, R., Brandle, J. (2005). Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana. Plant J., 41, 56-67, https://doi.org/10.1111/j.1365-313X.2004.02275.x
24. [25] Tai, F.J., Wang, X.L., Xu, W.L., Li, X.B. (2008). Characterization and expression analysis of two cotton genes encoding putative UDP-Glycosyltransferases. Mol. Biol., 42(1), 44-51, https://doi.org/10.1134/S0026893308010068
25. [26] Mejía-Espejel, L., Robledo-Paz, A., Lozoya-Gloria, E., Peña-Valdiviac, C.B., Carrillo-Salazara, J.A. (2018). Elicitors on steviosides production in Stevia rebaudiana Bertoni calli. Sci. Hort., 242, 95–102, https://doi.org/10.1016/j.scienta.2018.07.023
26. [27] Álvarez-Robles, M.J., López-Orenes, A., Ferrer, M.A., Calderón, A.A. (2016). Methanol elicits the accumulation of bioactive steviol glycosides and phenolics in Stevia rebaudiana shoot cultures. Ind. Crops Prod., 87, 273–279, http://dx.doi.org/10.1016/j.indcrop.2016.04.054
27. [28] Golkar, P., Moradi, M., Garousi, G.A. (2018). Elicitation of Stevia glycosides using salicylic acid and silver nanoparticles under callus culture. Sugar Tech, https://doi.org/10.1007/s12355-018-0655-6
28. [29] Javed, R., Mohamed, A., Yücesan, B., Gürel, E., Kausar, R., Zia, M. (2017). CuO nanoparticles significantly influence in vitro culture, steviol glycosides, and antioxidant activities of Stevia rebaudiana Bertoni. Plant Cell Tissue Organ Cult., 131, 611–620, https://doi.org/10.1007/s11240-017-1312-6