Impact of nutrient supply on growth and synthesis of metabolites of in vitro shoot cultures of S. rebaudiana

Iryna Smetanska, Mamdouh Diaa, Gruda Nazim, Mykola Patyka, Oksana Tonkha
Abstract

Stevia rebaudiana plants produce sweet tasting compounds, steviosides, which are 300 times sweeter as sugar and can be used as natural sweeteners. Moreover, stevia leaves contain high amount of antioxidants. The use of plant compounds as components of functional nutrition and nutraceuticals is extremely relevant today. The purpose of this study was to investigate the effect of increased nitrogen and saccharose supply on growth and accumulation of steviosides and anthocyanins as well as antioxidative activity in fast-growing shoot cultures of S. rebaudiana. The morphological changes of the leaves, the accumulation of steviosides and anthocyanins, and the antioxidant properties of the extracts were determined. It was shown that during three weeks of in vitro cultivation of shoots on MS medium with double concentration of nitrogen, the biomass increased by 71.0%, after application of double concentration of saccharose – by 133%, and after increasing nitrogen in combination with saccharose supply – by 162.0% compared to the control. Sucrose stimulated the accumulation of biomass. Anti-oxidative potential after nitrogen application was 2.4 times higher than the control, and with saccharose supply it increased 2.7 times. Shoots treated with increased nitrogen and saccharose concentration contained 50.7 and 57.8 mg/g-1 steviosides, respectively. However, the combination of nitrogen and saccharose led to accumulation of 73.4 1 mg/g-1 steviosides. Shoots grown on MS medium culture had 26.0 mg/g-1 of steviosides. The content of anthocyanins was 1.7 times greater under added nitrogen supply, and 2.3 times greater after the application of nitrogen and saccharose. Optimum cultivation media developed individually for each in vitro culture increase the production of valuable plant secondary metabolites up to 3 times

Keywords

plant biomass, cultivation, nutrient medium, secondary metabolites, steviosides, anthocyanins, antioxidative activity

Suggested citation
Smetanska, I., Diaa, M., Nazim, G., Patyka, M., & Tonkha, O. (2023). Impact of nutrient supply on growth and synthesis of metabolites of in vitro shoot cultures of S. rebaudiana. Plant and Soil Science, 14(2), 57-69. https://doi.org/10.31548/plant2.2023.57
References

 

[1] Akbari, F., Arminian, A., Kahrizi, D., Fazeli, A., & Ghaheri, M. (2018). Effect of nitrogen sources on gene expression of Stevia rebaudiana (Bertoni) under in vitro conditions. Cellular and Molecular Biology, 64(2), 11-16. doi: 10.14715/cmb/2018.64.2.3.

[2] Al-Taweel, S., Azzam C., Khaled K., & Abdel-Aziz R. (2021). Improvement of stevia (Stevia rebaudiana Bertoni) and steviol glycoside through traditional breeding and biotechnological approachesSsabrao Journal of Breeding and Genetics, 53, 88-111.

[3] Alvarez, S., & Motos, J.R. (2023). Advances in plant physiology of abiotic stresses. Retrieved from https://www.mdpi.com/books/book/6646-advances-in-plant-physiology-of-abiotic-stresses.

[4] Amien, S., Maulana., H, Ruswandi, D., & Nurjanah, S. (2022). Stevia (Stevia rebaudiana b.) genotypes assessment for leaf yield stability through genotype by environment interactions, ammi, and gge biplot analyses. SABRAO Journal of Breeding and Genetics, 54, 767-779. doi: 10.54910/sabrao2022.54.4.8.

[5] Berlin, J.,  Kukoschke, KG., & Knobloch, KH. (1981). Selection of tobacco cell lines with high yields of cinnamoyl putrescines. Planta Medica, 42(6), 173-180. doi: 10.1055/s-2007-971622.

[6] Convention on Biological Diversity (1992). Retrieved from https://zakon.rada.gov.ua/laws/show/995_030#Text.

[7] Dai, Zh., Meddar, M., Renaud, Ch., Merlin, I., Hilbert, Gh., Delrot, S., & Gomès, E. (2014). Long-term in vitro culture of grape berries and its application to assess the effects of sugar supply on anthocyanin accumulation. Journal of Experimental Botany, 65(16), 4565-4577. doi: 10.1093/jxb/ert489.

[8] El-Dawayati, M.M., & Zayed, Z.E. (2017). Controlling hyperhydricity in date palm in vitro culture by reduced concentration of nitrate nutrients. In Date palm biotechnology protocols (pp. 175-183). New York: Humana Press. doi: 10.1007/978-1-4939-7156-5_15.

[9] Emergen Research. (2022)Retrieved from https://www.emergenresearch.com/industry-report/stevia-market.

[10] Fidemann, T., Pereira, G, Heluy, T., Gallego, R., Bertão, M., Silva, R., & Fernández, E. (2018).Handling culture medium composition for optimizing plant cell suspension culture in shake flasks. Plant Cell, Tissue and Organ Culture, 133, 137-146. doi: 10.1007/s11240-017-1368-3.

[11] Hassanein, A. (2022). Application of tissue culture techniques to improve the productivity of medicinal secondary products from medicinal plants. Retrieved from https://www.intechopen.com/online-first/82048.

[12] Ibrahim, K., & Musbah, H. (2018). Increasing poly phenols in Coleus blumei at the cellular and intact plant levels using PEG stress. Research Journal of Pharmacy and Technology, 11(1), article number 321. doi: 10.5958/0974-360X.2018.00059.8.

[13] Jawad, M., Rizwan, B., Jawad, M., Khalid, F., & Ishaq, A. (2022). A nutraceutical and therapeutic potentials of stevia rebaudiana bertoni: Nutraceutical and therapeutic potentials of stevia. Pakistan BioMedical Journal, 5(1), 10-22. doi: 10.54393/pbmj.v5i1.143.

[14] Kikowska, M., Thiem, B., Szopa, A., & Ekiert, H. (2022). Accumulation of valuable secondary metabolites: Phenolic acids and flavonoids in different in vitro systems of shoot cultures of the endangered plant species Eryngium alpinum L. Plant CellTissue and Organ Culture (PCTOC), 141, 381-391. doi: 10.1007/s11240-020-01795-5.

[15] Kim, D.J., & Chang, H.N. (1990). Enhanced shikonin production from Lithospermum erythrorhizon by in situ extraction and calcium alginate immobilization. Biotechnology and Bioengineering, 36(5), 460-466. doi: 10.1002/bit.260360505.

[16] Knobloch, K.H., & Berlin, J. (1980). Influence of medium composition on the formation of secondary compounds in cell suspension cultures of Catharanthus roseus L. Naturforschung, 35(7), 551-556. doi: 10.1515/znc-1980-7-805.

[17] Król, A., Kokotkiewicz, A., Szopa, A., Ekiert, H., & Łuczkiewicz, M. (2021). Bioreactor-grown shoot cultures for the secondary metabolite production. In Plant cell and tissue differentiation and secondary metabolitesReference Series in Phytochemistry (pp. 187-247). Springer: Cham. doi: 10.1007/978-3-030-30185-9_34.

[18] Magangana, T.P., Stander, M.A., & Makunga, N.P. (2018). Effect of nitrogen and phosphate on in vitro growth and metabolite profiles of Stevia rebaudiana Bertoni (Asteraceae). Plant Cell, Tissue and Organ Culture (PCTOC), 134, 141-151. doi: 10.1007/s11240-018-1407-8.

[19] Mohdaly, A., Smetanska, I., Ramadan, F.R., Sarhanb, M.A., & Mahmoud, A. (2011). Antioxidant potential of sesame (Sesamum indicum) cake extract in stabilization of sunflower and soybean oils. Industrial Crops and Products, 34(1), 952-959. doi: 10.1016/j.indcrop.2011.02.018.

[20] Mohdaly, A.A., Sarhan, M.A., Smetanska, I., & Mahmoud, A. (2010). Antioxidant properties of various solvent extracts of potato peel, sugar beet pulp and sesame cake. Journal of the Science of Food and Agriculture, 90(2), 218-226. doi: 10.1002/jsfa.3796.

[21] Mordor Intelligence. (2022). Retrieved from https://www.mordorintelligence.com/industry-reports/stevia-market.

[22] Nguyen, T., Seo, C., Kwak, S.H., Kim, J., Kang, H.K., Kim, S.B., & Kim, D. (2019). Enzymatic production of steviol glucosides using β-glucosidase and their applications. In Enzymes in Food Biotechnology (pp. 405-418). Cambridge: Academic Press. doi: 10.1016/B978-0-12-813280-7.00023-2.

[23] Nguyen, Y., & Tan, Q.L.P. (2021). Chemical composition of dried Stevia rebaudiana Bertoni leaves and effect of ultrasound-assisted extraction on total steviosides content in extract. Herba Polonica, 67(1), 1-7.doi: 10.2478/hepo-2021-0003.

[24] Nshimiyimana, D.S., & He, Q. (2010). Radical scavenging capacity of Rwandan CTC tea polyphenols extracted using microwave assisted extraction. Pakistan Journal of Nutrition, 9(6), 589-593. doi: 10.3923/pjn.2010.589.593.

[25] Olgunsoy, P., Ulusoy, S., & Akçay, U. (2017). Metabolite production and antibacterial activities of callus cultures from Rosa damascena Mill. petals. Marmara Pharmaceutical Journal, 21(3), 590-597. doi: 10.12991/marupj.319331.

[26] Ramírez-Mosqueda, M., Iglesias, A.L., Ramírez-Madero, G., & Hernández-Rincón, E.U. (2016). Micropropagation of Stevia rebaudiana Bert. in temporary immersion systems and evaluation of genetic fidelity. South African Journal of Botany, 106, 238-243. doi: 10.1016/j.sajb.2016.07.015.

[27] Raspe, D.S., & Silvio, C.C. (2022). Compounds from Stevia rebaudiana Bertoni leaves: An overview of non-conventional extraction methods and challenges. Food Bioscience, 46, article number 101593. doi: 10.1016/j.fbio.2022.101593.

[28] Sae-Lee, N., Kerdchoechuen, O, & Laohakunjit, N. (2014). Enhancement of phenolics, resveratrol and antioxidant activity by nitrogen enrichment in cell suspension culture of Vitis vinifera. Molecules, 19(6), 7901-7912. doi: 10.3390/molecules19067901.

[29] Sakamoto, K., Iida, K., Sawamura, K., Hajiro, K., Asada, Y., Yoshikawa, T., & Furuya, T. (1994). Anthocyanin production in cultured cells of Aralia cordata Thunb. Plant Cell, Tissue and Organ Culture, 36, 21-26. doi: 10.1007/BF00048311.

[30] Sharma, N., Gauchan, D., Dhakal, A., Luitel, A., Shakya, S., & Shakya, R. (2015).Establishment of regenerative callus, cell suspension system and molecular characterization of Stevia rebaudiana Bertoni for the production of stevioside in in vitro. International Journal for Research in Applied Science & Engineering Technology, 3, 133-144

[31] Shevchenko,Y., Wendt, A., & Smetanska I. (2010). Sprout culture of Stevia rebaudiana Bertoni. In Stevia science, no fiction (pp. 5-26). Euprint Heverlee: Leuven.

[32] Shibasaki, N., Hirose, K., Yonemoto, T., & Tadaki, T. (2007). Suspension culture of Nicotiana tabacum cells in a rotarydrum bioreactor. Journal of Chemical Technology and Biotechnology, 53(4), 359-363.doi: 10.1002/jctb.280530407.

[33] Smetanska, I., Tonkha, O., Patyka, T., Hunaefi, D., Mamdouh, D., Patyka., M., Bukin, A., Mushtruk, M., Slobodyanyuk, N., & Omelian, A. (2021). The influence of yeast extract and jasmonic acid on phenolic acids content of in vitro hairy root cultures of Orthosiphon aristatus. Slovak Journal of Food Sciences, 15, 1-8. doi: 10.5219/1508.

[34] Wang, J., Zhao, H., Wang, Y., Lau, H., Zhou, W., Chen, C., & Tan, S. (2020). A review of stevia as a potential healthcare product: Up-to-date functional characteristics, administrative standards and engineering techniques. Trends in Food Science & Technology, 103, 264-281. doi: 10.1016/j.tifs.2020.07.023.

[35] WHO Guideline. (2023). Retrieved from https://www.who.int/publications/who-guidelines.

[36] Wu, T., Kerbler. S., Fernie, A., & Zhang, Y. (2021). Plant cell cultures as heterologous bio-factories for secondary metabolite production. Plant Communications, 2(5), article number 100235. doi: 10.1016/j.xplc.2021.100235.

[37] Zaheer, M., Vudem, D., & Giri, Ch. (2016). Enhanced daidzin production from jasmonic and acetyl salicylic acid elicited hairy root cultures of Psoralea corylifolia L. (Fabaceae). Natural product research, 30(13), 1542-1547. doi: 10.1080/14786419.2015.1054823.

[38] Zenk, M.H. (1978). The impact of plant cell cultures on industry. In Frontiers of plant tissue culture (pp. 1-14). Calgary: The International Association of Plant Tissue Culture.

[39] Zhong, J.J. (2001). Biochemical engineering of the production of plant-specific secondary metabolites by cell suspension cultures. Advances in Biochemical Engineering/ Biotechnology, 72, 1-26. doi: 10.1007/3-540-45302-4_1.