Establishment of Bidens pilosa L. ‘hairy’ root culture

  • N. A. Matvieieva Institute of Cell Biology and Genetic Engineering, NAS of Ukraine, Ukraine, 03143, Kyiv, Zabolotnogo str, 148
  • A. M. Shakhovsky Institute of Cell Biology and Genetic Engineering, NAS of Ukraine, Ukraine, 03143, Kyiv, Zabolotnogo str, 148


Aim. The aim of the work was the establishment of Bidens pilosa L. “hairy” root culture using Agrobacterium rhizogenes-mediated transformation. Methods. A. rhizogenes strain А4 carried pCB161 and pCB124 vectors with the selective nptII (nos promoter and terminator) and ifn-α2b (Mll or 35S promoter respectively, and ocs terminator) genes were used for genetic transformation. Explants were cocultivated with bacterial suspension for 30 min and transferred to 1/2MS solidified medium. PCR analysis with primers specific for rolB, nptII, and ifn-α2b genes was used to confirm the transgenic nature of obtained roots. Results. The growth of roots started in 7-10 days after the transformation. The number of roots generated on the leaf explants was significantly lower than of those formed on the stem explants (2-8 and 12-22, respectively). The frequency of root formation for leaf and stem explants was 56.7±6.5% and 84.4±5.8%, respectively. The roots demonstrated the typical phenotype with significant branching, negative geotropism and were able to grow without growth regulators in the nutrient medium. The presence of rolB, ifn-α2b, and nptII genes was confirmed by PCR analysis. Conclusions. An effective system for genetic transformation of B. pilosa plants using A. rhizogenes-mediated transformation method was developed and "hairy" root culture was established. The presence of transferred genes was confirmed by PCR analysis. The obtained "hairy" root lines differed in growth rate.

Keywords: Bidens pilosa L., Agrobacterium rhizogenes, transformation, "hairy" root culture.


Tobinaga S., Sharma M.K., Aalbersberg W.G. et al. Isolation and identification of a potent antimalarial and antibacterial polyacetylene from Bidens pilosa. Planta Med. 2009. Vol. 75(6). Р.624–628. doi: 10.1055/s-0029-1185377

Silva Junior J., Cerdeira C.D., Chavasco J.M. et al. In vitro screening antibacterial activity of Bidens pilosa Linné and Annona crassiflora Mart. against oxacillin resistant Staphylococcus aureus (ORSA) from the aerial environment at the dental clinic. Rev. Inst. Med. Trop. Sao Paulo. 2014. Vol. 56(4). Р. 333–340. doi: 10.1590/s0036-46652014000400011

Nakama S., Tamaki K., Ishikawa C. et al. Efficacy of Bidens pilosa extract against Herpes Simplex Virus infection in vitro and in vivo. Evid. Based Complement. Alternat. Med. 2012. Article ID 413453, doi: 10.1155/2012/413453

Mbogning T. G., Poné J. W., Komtangi M. C. et al. In vitro anthelmintic activity of Bidens pilosa Linn. (Asteraceae) leaf extracts against Haemonchus contortus eggs and larvae. Europ. J. of Med. Plants. 2014. Vol. 4(11). Р. 1282–1292.

Hsu Y.J., Lee T.H., Chang C.L. et al. Anti-hyperglycemic effects and mechanism of Bidens pilosa water extract. J. Ethnopharmacol. 2009. Vol. 122(2). Р. 379–383. doi: 10.1016/j.jep.2008.12.027

Yang W.C. Botanical, pharmacological, phytochemical, and toxicological aspects of the antidiabetic plant Bidens pilosa L. Evid. Based Complement. Alternat. Med. 2014. Article ID 698617. doi: 10.1155/2014/698617.-

Chang S.L., Chang C.L., Chiang,Y.M. et al. Polyacetylenic compounds and butanol fraction from Bidens pilosa can modulate the differentiation of helper T cells and prevent autoimmune diabetes in non-obese diabetic mice. Planta Med. 2004. Vol. 70(11). Р. 1045–1051. doi: 10.1055/s-2004-832645

Chien S.C., Young P.H., Hsu Y.J. et al. Anti-diabetic properties of three common Bidens pilosa variants in Taiwan. Phytochem. 2009. Vol. 70(10). Р. 1246–1254. doi: 10.1016/j.phytochem.2009.07.011

Sundararajan P., Dey A., Smith A. et al. Studies of anticancer and antipyretic activity of Bidens pilosa whole plant. African Health Sci. 2005. Vol. 6(1). Р. 27-30.

Kviecinski M.R., Benelli P., Felipe K.B. et al. SFE from Bidens pilosa Linné to obtain extracts rich in cytotoxic polyacetylenes with antitumor activity. J. of Supercritical Fluids. 2011. Vol. 56(3). P. 243–248. doi: 10.1016/j.supflu.2010.12.011

Chang J.S., Chiang L.C., Chen C.C. et al. Antileukemic activity of Bidens pilosa L. var. minor (Blume) Sherff and Houttuynia cordata Thunb. Amer. J. of Chinese Med. 2001. Vol. 29. P. 303–312. doi: 10.1142/S0192415X01000320

Fotso A.F., Longo F., Djomeni P.D., et al. Analgesic and antiinflammatory activities of the ethyl acetate fraction of Bidens pilosa (Asteraceae). Inflammopharmacol. 2014. Vol. 22(2). Р. 105–114. doi: 10.1007/s10787-013-0196-2

Yang H. L., Chen S. C., Chang N.W. et al. Protection from oxidative damage using Bidens pilosa Extracts in normal human erythrocytes. Food and Chem Toxicol. 2006. Vol. 44. Р. 1513–1521. doi: 10.1016/j.fct.2006.04.006

Dimo T., Rakotonirina S.V., Tan P.V. et al. Leaf methanol extract of Bidens pilosa prevents and attenuates the hypertension induced by high-fructose diet in Wistar rats. J. Ethnopharmacol. 2002. Vol. 83. Р. 183–191. doi: 10.1016/S0378-8741(02)00162-9

Oliveira F.Q., Andrade-Neto V., Krettli A.U. et al. New evidences of antimalarial activity of Bidens pilosa roots extract correlated with polyacetylene and flavonoids. J. Ethnopharmacol. 2004. Vol. 93(1). Р. 39–42. doi: 10.1016/j.jep.2004.03.026

Andrade-Neto V.F., Brandão M.G., Oliveira F.Q. et al. Antimalarial activity of Bidens pilosa L. (Asteraceae) ethanol extracts from wild plants collected in various localities or plants cultivated in humus soil. Phytother Res. 2004. Vol. 18(8). Р. 634–639. doi: 10.1002/ptr.1510

Chang S.L., Chiang Y.M., Chang Cicero L.T. et al. Flavonoids, centaurein and centaureidin, from Bidens pilosa, stimulate IFN-expression. J. Ethnopharmacol. 2007. Vol. 112. Р. 232–236. doi: 10.1016/j.jep.2007.03.001

Silva F.L., Fischer D.C., Tavares J.F. et al. Compilation of secondary metabolites from Bidens pilosa L. Molecules. 2011. Vol. 16(2). Р. 1070–1102. doi: 10.3390/molecules16021070

Wang C.K., Hsu S.Y., Chen P.Y., To K.Y. Transformation and characterization of transgenic Bidens pilosa L. Plant Cell Tiss. Organ. Cult. 2012. Vol. 109(3). Р. 457–464. doi: 10.1007/s11240-011-0110-9

Murashige T., Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plantarum. 1962. Vol. 15. Р. 473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x

Draper J., Scott R. The isolation of plant nucleic acids. plant genetic transformation and gene expression: a laboratory manual. Oxford, Great Britain: Blackwell Sci. Publ., 1988. 355 p.