Study the effect of heavy metals and Nano TiO2 on stevioside and Rebadioside A production in Stevia rebaudiana Bertoni hairy roots

Document Type : Research paper


Biotechnology Department, Faculty of Agriculture and Natural Sciences, Imam Khomeini International University, P. O. Box: 34149-16818, Qazvin, Iran.


Stevia rebaudiana Bert. is a medicinal plant with anti-oxidant, antimicrobial, antifungal and antiviral drug properties which contains two valuable secondary metabolites, stevioside and Rebadioside A, with very high sweetness capacity. In this research hairy roots were produced from S. rebaudiana leaf disks using two strains of 15834 and A4 A. rhizogenes. Then, the best hairy root line was selected and propagated. The effect titanium dioxide nanoparticles at 20, 40 and 60 mg/l after 0, 24, 48 and 72 h of elicitation and cobalt chloride and cadmium chloride each at 100 and 200 μmol after 2 and 8 h of elicitation were studied on the production and accumulation of stevioside and Rebadioside A. The results demonstrated that most treatments of elicitors at different times had significant effects on the production and accumulation of target metabolites, but titanium dioxide nanoparticles caused the highest production and accumulation of stevioside (96.88 mg/l and 24.28 mg/g DW), respectively at 40 mg/l after 24 h. However, at 60 mg/l and 48 h it had the highest effect on production and accumulation of Rebadioside A after 48 h (89.39 mg/l and 21.28 mg/g DW), respectively. Also the results demonstrated that treatment of cobalt chloride and cadmium chloride both at 100 μmol after 8 h had a strong effect on the production (114.19 and 183.98 mg/l) and accumulation (29.54 and 42.87 mg/g DW ) of stevioside, respectively. Cadmium chloride at 200 μmol had the highest effect on the production and accumulation of Rebadioside A after 2 h (105.51 mg/l and 25.42 mg/g DW), respectively. Meanwhile, no production of Rebadioside A was observed at 100 and 200 μmol in 8 h treatment.


Abdelhamid H. N., and Wu H.-F. (2015). Proteomics analysis of the mode of antibacterial action of nanoparticles and their interactions with proteins. TrAC Trends in Analytical Chemistry, 65: 30–46.
Bota C., and Deliu C. (2011). The effect of copper sulphate on the production of flavonoids in digitalis lanata cell cultures. Farmacia, 59(1): 113–118.
Ceunen S., and Geuns J. M. (2013). Influence of photoperiodism on the spatio-temporal accumulation of steviol glycosides in Stevia rebaudiana (Bertoni). Plant Science, 198: 72–82.
Ch B., Rao K., Gandi S., and Giri A. (2012). Abiotic elicitation of gymnemic acid in the suspension cultures of Gymnema sylvestre. World Journal of Microbiology and Biotechnology, 28(2): 741–747.
Chilton M.-D., Tepfer D. A., Petit A., David C., Casse-Delbart F., and Tempé J. (1982). Agrobacterium rhizogenes inserts t-DNA into the genomes of the host plant root cells. Nature, 295(5848): 432.
Chung I.-M., Rajakumar G., and Thiruvengadam M. (2018). Effect of silver nanoparticles on phenolic compounds production and biological activities in hairy root cultures of Cucumis anguria. Acta Biologica Hungarica, 69(1): 97–109.
Fu C.-X., Zhao D.-X., Xue X.-F., Jin Z.-P., and Ma F. S. (2005). Transformation of saussurea involucrata by Agrobacterium rhizogenes: Hairy root induction and syringin production. Process Biochemistry, 40(12): 3789–3794.
Gangopadhyay M., Dewanjee S., and Bhattacharya S. (2011). Enhanced plumbagin production in elicited plumbago indica hairy root cultures. Journal of Bioscience and Bioengineering, 111(6): 706–710.
Georgiev M., Kuzeva S., Pavlov A., Kovacheva E., and Ilieva M. (2006). Enhanced rosmarinic acid production by lavandula vera mm cell suspension culture through elicitation with vanadyl sulfate. Zeitschrift für Naturforschung C, 61(3–4): 241–244.
Ghorbanpour, M., Hatami M., and Hatami M. (2015). Activating antioxidant enzymes, hyoscyamine and scopolamine biosynthesis of Hyoscyamus niger L. Plants with nano-sized titanium dioxide and bulk application. Acta Agriculturae Slovenica, 105(1): 23–32.
Golkar P., Moradi M., and Garousi G. A. (2018). Elicitation of stevia glycosides using salicylic acid and silver nanoparticles under callus culture. Sugar Technology, 21(4): 569–577.
Hézode C., Fontaine H., Dorival C., Larrey D., Zoulim F., Canva V., de Ledinghen V., Poynard T., Samuel D., Bourlière M., Zarski J. P., Raabe J. J., Alric L., Marcellin P., Riachi G., Bernard P. H., Loustaud-Ratti V., Métivier S., Tran A., Serfaty L., Abergel A., Causse X., Di Martino V., Guyader D., Lucidarme D., Grando-Lemaire V., Hillon P., Feray C., Dao T., Cacoub P., Rosa I., Attali P., Petrov-Sanchez V., Barthe Y., Pawlotsky J. M., Pol S., Carrat F., Bronowicki J. P., and CUPIC Study Group (2013). Triple therapy in treatment-experienced patients with hcv-cirrhosis in a multicentre cohort of the french early access programme (anrs co20-cupic)–nct01514890. Journal of Hepatology, 59(3): 434–441.
Japelaghi R. H., Haddad R., and Garoosi G.-A. (2011). Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides. Molecular Biotechnology, 49(2): 129–137.
Javed R., Mohamed A., Yücesan B., Gürel E., Kausar R., and Zia M. (2017). Cuo nanoparticles significantly influence in vitro culture, steviol glycosides, and antioxidant activities of Stevia rebaudiana Bertoni. Plant Cell, Tissue and Organ Culture (PCTOC), 131(3): 611–620.
Kahila M. M. H., Najy A. M., Rahaie M., and Mir-Derikvand M. (2018). Effect of nanoparticle treatment on expression of a key gene involved in thymoquinone biosynthetic pathway in Nigella sativa L. Natural Product Research, 32(15): 1858–1862.
Kolb H. C., Finn M., and Sharpless K. B. (2001). Click chemistry: Diverse chemical function from a few good reactions. Angewandte Chemie International Edition, 40(11): 2004–2021.
Kołodziejczak-Radzimska A., and Jesionowski T. (2014). Zinc oxide—from synthesis to application: A review. Materials, 7(4): 2833–2881.
Lok C.-N., Ho C.-M., Chen R., He Q.-Y., Yu W.-Y., Sun H., Tam P. K., Chiu J. F., and Che C.-M. (2007). Silver nanoparticles: Partial oxidation and antibacterial activities. JBIC Journal of Biological Inorganic Chemistry, 12(4): 527–534.
Mohammadi H., Esmailpour M., and Gheranpaye A. (2016). Effects of TiO2 nanoparticles and water-deficit stress on morpho-physiological characteristics of dragonhead (Dracocephalum moldavica L.) plants. Acta agriculturae Slovenica, 107(2): 385–396.
Moradi F., Mehrjerdi M. Z., Vahdati K., and Hasanloo T. (2019). Effect of different factors on induction of hairy roots in iranian garlic. Journal of Plant Productions (Scientific Journal of Agriculture), 41(4): 43–54.
Nikravesh F., Khavari-Nejad R., Rahimian H., and Fahimi H. (2012). Study of antioxidant enzymes activity and isozymes pattern in hairy roots and regenerated plants in Nicotiana tabacum. Acta Physiologiae Plantarum, 34(2): 419–427.
Putalun W., Luealon W., De-Eknamkul W., Tanaka H., and Shoyama Y. (2007). Improvement of artemisinin production by chitosan in hairy root cultures of Artemisia annua L. Biotechnology Letters, 29(7): 1143–1146.
Raei M., Angaji S. A., Omidi M., and Khodayari M. (2014). Effect of abiotic elicitors on tissue culture of aloe vera. International Journal of Biosciences, 5(1): 74–81.
Sambrook J., Fritsch E. F., and Maniatis T. (1989). Molecular cloning: A laboratory manual. Cold spring harbor laboratory press,  pp. 1546.
Sivanandhan G., Dev G. K., Jeyaraj M., Rajesh M., Arjunan A., Muthuselvam M., Manickavasagam M., Selvaraj N.,  and 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 and Organ Culture (PCTOC), 114(1): 121–129.
Takallu S., Davodi D., Omidi M., Ebrahimi M., Rouzbeh F., and Rasulnia A. (2013). The effect of titanium dioxide nanoparticles on barley cytogenetical index. Journal of Agricultural Biotechnology, 5(1): 13–25.
Tavassoli P., and Afshar A. S. (2018). Influence of different Agrobacterium rhizogenes strains on hairy root induction and analysis of phenolic and flavonoid compounds in marshmallow (Althaea officinalis L.). 3 Biotech, 8(8): 351.
Tripath L., and Tripathi J. N. (2003). Role of biotechnology in medicinal plants. Tropical Journal of Pharmaceutical Research, 2(2): 243–253.
Vermeersch M., Foubert K., Luz R. I.d., Puyvelde L. V., Pieters L., Cos P., and Maes L. (2009). Selective antileishmania activity of 13, 28-epoxy-oleanane and related triterpene saponins from the plant families Myrsinaceae, Primulaceae, Aceraceae and Icacinaceae. Phytotherapy Research: An International Journal Devoted to Pharmacological and Toxicological Evaluation of Natural Product Derivatives, 23(10): 1404–1410.
Vinterhalter B., Savić J., Zdravković-Korać S., Banjac N., Vinterhalter D., and Krstić-Milošević D. (2019). Agrobacterium rhizogenes-mediated transformation of Gentiana utriculosa L. And xanthones decussatin-1-o-primeveroside and Decussatin accumulation in hairy roots and somatic embryo-derived transgenic plants. Industrial Crops and Products, 130: 216–229.
Wang J. W., and Wu J. Y. (2013). Effective elicitors and process strategies for enhancement of secondary metabolite production in hairy root cultures. Biotechnology of Hairy Root Systems, Springer, 55–89.
Wang W., Cang L., Zhou D. M., and Yu Y. C. (2017). Exogenous amino acids increase antioxidant enzyme activities and tolerance of rice seedlings to cadmium stress. Environmental Progress and Sustainable Energy, 36(1): 155–161.
Wu J., Wong K., Ho K., and Zhou L. (2005). Enhancement of saponin production in panax ginseng cell culture by osmotic stress and nutrient feeding. Enzyme and Microbial Technology, 36(1): 133–138.
Zahir A., Nadeem M., Ahmad W., Giglioli-Guivarc’h N., Hano C., and Abbasi B. H. (2019). Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L. Plant Cell, Tissue and Organ Culture (PCTOC), 136(3): 589–596.
Zhao J.-L., Zhou L.-G., and Wu J.-Y. (2010). Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures. Applied Microbiology and Biotechnology, 87(1): 137–144.
Zhao J., Davis L. C., and Verpoorte R. (2005). Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances, 23(4): 283–333.
Zheng Z., and Wu M. (2004). Cadmium treatment enhances the production of alkaloid secondary metabolites in Catharanthus roseus. Plant Science, 166(2): 507–514.