Rapid in vitro propagation of Stevia rebaudiana Bertoni and evaluation of its stevioside content

Authors

Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Tehran, Postal code: 31587-11167, Karaj, Iran.

Abstract

An efficient protocol was established for rapid in vitro propagation of Stevia rebaudiana Bertoni, using explants from mature plants. The effect of 1/2MS, MS and B5 culture media, also 27 different combinations of auxins and cytokinins was studied on in vitro growth and development of Stevia rebaudiana Bertoni. The best culture medium for shoot and root formation was 1/2MS, producing 1.30 shoots/explant, 10 leaves/explant, 6.68 roots/explant and 100% rooting. Shoot and root formation increased by using plant growth regulators. The best shoot formation (2.83 shoots/explant and 12.35 leaves) was obtained in the MS basal medium supplemented with 2.21 μM BAP and 1.42 μM IAA. The best root formation (100% rooting, 7.2 roots/explant and 2.19 cm root length) were obtained in the MS basal medium supplemented with 1.42 μM IAA. S. rebaudiana Bertoni plantlets were adapted and grown well in the growing medium containing perlite and vermiculite (1:3). After transplantation to the field. HPLC results demonstrated that stevioside content was similar between the regenerated (19.48 mg g-1 dry weight) and the mother plants (20.45 mg g-1 dry weight).

Keywords


Ahmed M. S., Dobberstein R. H., and Farnsworth N. R. (1980). Use of p-bromophenacyl bromide to enhance ultraviolet detection of water-soluble organic acids (Stevioside and Rebaudioside-B) high performance liquid chromatography analysis. Journal of Chromatography A, 192 (2): 387-393.

Aggarwal D., and Barna K. S. (2004). Tissue culture propagation of elite plant of Aloe vera Linn. Journal of Plant Biochemistry and Biotechnology, 13(1): 77-79.

Anbazhagan M., Kalpana M., Rajendran R., Natarajan V., and Dhanavel D. (2010). In vitro production of Stevia rebaudiana Bertoni. Emirates Journal of Food and Agriculture, 22 (3): 216-222.

Beike A. K., Spagnuolo V., Lüth V., Steinhart F., Ramos-Gómez J., Krebs M., and Decker E. L. (2015). Clonal in vitro propagation of peat mosses (Sphagnum L.) as novel green resources for basic and applied research. Plant Cell, Tissue and Organ Culture (PCTOC), 120(3): 1037-1049.

Bondarev N. I., Reshetnyak O. V., and Nosov A. M. (2002). Effects of medium composition on development of Stevia rebaudiana shoots cultivated in the roller bioreactor and the production of steviol glycosides. Plant Science, 165: 845–850.

Bovanova l., Brandsteterova E., and Baxa S. (1998). HPLC determination of stevioside in plant material and food samples. Zeitschrift für Lebensmittel-Untersuchung und-Forschung, 207: 352-355.

Chan l. K., Dewi P. R., and Boey P. l. (2005). Effect of plant growth regulators on regeneration of plantlets from bud cultures of Cymbopogon nardus L. and the detection of essential oils from the in vitro plantlets. Journal of Plant Biology, 48: 142-145.

Fracago F., and Echeverrigaray S. (2001). Micropropagation of Cunila galioides, a popular medicinal plant of south Brazil. Plant Cell, Tissue and Organ Culture. 64:1-4.

Ferrazzano G. F., Cantile T., Alcidi B., Coda M., Ingenito A., Zarrelli A., and Pollio A. (2015). Is Stevia rebaudiana Bertoni a Non Cariogenic Sweetener? A Review. Molecules, 21(1): 38.

Gamborg O. l., Miller R. A., and Ojima K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 50: 148-151.

Geuns J. M. C. (2003). Molecules of interest: stevioside. Phytochemistry 64: 913–921.

George E. F., and Sherrington P. D. (1984). Plant Propagation by Tissue Cultur. Exegetics Ltd., 39-71.

Harris R. E., and Stevenson J. H. (2016). In vitro propagation of Vitis. VITIS-Journal of Grapevine Research, 21(1): 22.

Hellwig S., Drossard J., Twyman R. M., and Fischer R. (2004). Plant cell cultures for the production of recombinant proteins. Nature Biotechnology, 22: 1415-1422.

Hwang S. J. (2006). Rapid in vitro propagation and enhanced stevioside accumulation in Stevia rebaudiana Bert. Journal of Plant Biology, 49(4): 267-270.

Ibrahim I. A., Nasr M. I., Mohammed B. R., and El-Zefzafi M. M. (2008). Plant growth regulators affecting in vitro cultivation of Stevia rebaudiana. Sugar Technology, 10(3): 254-259.

Lemus-Mondaca R., Ah-Hen K., Vega-Gálvez A., Honores C., and Moraga, N. O. (2016). Stevia rebaudiana Leaves: Effect of Drying Process Temperature on Bioactive Components, Antioxidant Capacity and Natural Sweeteners. Plant Foods for Human Nutrition, 71(1): 49-56.

Matsui M., Matsui K., Kawasaki Y., Oda Y., Noguchi T., Kitagawa Y., Sawada M., Hayashi M., Nohmi M., Yoshihira K., Ishidate M., and Sofuni T. (1996). Evaluation of the genotoxicity of stevioside and steviol using six in vitro and one in vivo mutagenicity assays. Mutagenesis, 11: 573–579.

Murashige T., and Skoog F. (1962). A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiologia Plantarum, 15: 473–495.

Pierik R. L. M. (1987). In vitro propagation of higher plants. Boston: Martinus, Nijhoff, 286-344.

Rajasekaran T., Giridhar P., Ravishankar G. (2007). Production of steviosides in ex vitro and in vitro grown Stevia rebaudiana Bertoni. Journal of the Science of Food and Agriculture, 87(3): 420-424.

Serfaty M., Ibdah M., Fischer R., Chaimovitsh D., Saranga Y., and Dudai, N. (2013). Dynamics of yield components and stevioside production in Stevia rebaudiana grown under different planting times, plant stands and harvest regime. Industrial Crops and Products, 50: 731-736.

Singh P., Padmanabh D., and Neelam A. (2014). In vitro shoot multiplication of Stevia and assessment of stevioside content and genetic fidelity of the regenerants. Sugar Technology, 16(4): 430-439.

Steel G. D., and Torrie J. H. (1980). Principles and procedures of statistics. Book Coy Inc, New York, 204-227.

Thiyagarajan M., and Venkatachalam P. (2012). Large scale in vitro propagation of Stevia rebaudiana (Bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb. Industrial Crops and Products, 37(1): 111-117.

Wölwer-Rieck, U. (2012). The leaves of Stevia rebaudiana (Bertoni), their constituents and the analyses thereof: a review. Journal of Agricultural and Food Chemistry, 60(4): 886-895.

Yücesan B., Mohammed A., Büyükgöçmen R., Altuğ C., Kavas Ö., Gürel, S., and Gürel, E. (2016). In vitro and ex vitro propagation of Stevia rebaudiana Bertoni with high Rebaudioside-A content-A commercial scale application. Scientia Horticulturae, 203: 20-28.