Jump to ContentJump to Main Navigation
Show Summary Details
More options …


More options …
Volume 69, Issue 7


A reliable and efficient protocol for induction of hairy roots in Agastache foeniculum

Elnaz Nourozi / Bahman Hosseini / Abbas Hassani
Published Online: 2014-08-19 | DOI: https://doi.org/10.2478/s11756-014-0382-8


Hairy root culture system is a valuable tool to study the characteristics of gene expression, gene function, root biology, biochemical properties and biosynthesis pathways of secondary metabolites. In the present study, hairy roots were established in Anise hyssop (Agastache foeniculum) via Agrobacterium rhizogenes. Three strains of Agrobacterium rhizogenes (A4, A7 and 9435), were used for induction of hairy roots in four various explants (hypocotyl, cotyledon, one-month-old leaf and five-month-old leaf) of Anise hyssop. The highest frequency of transformation was achieved using A4 strain in one-month-old leaves (51.1%). The transgenic states of hairy root lines were confirmed by PCR (Polymerase chain reaction) method. High performance liquid chromatography analysis revealed that the production of rosmarinic acid (RA) in transformed roots of A. foeniculum was almost 4-fold higher than that of the non-transformed roots. In a separate experiment, hairy roots obtained from one-month-old leaves inoculated with A4 strain, were grown in liquid medium and the effects of different concentrations of salicylic acid (0.0, 0.01, 0.1 and 1 mM) and chitosan (0, 50, 100 and 150 mg L−1) (as elicitor) and sucrose (20, 30, 40 and 50 g L−1) on the growth of hairy roots were evaluated. The results showed that, 30 g L−1 sucrose and 100 mg L−1 chitosan increased the biomass of hairy root cultures and application of salicylic acid reduced the growth of hairy roots compared with control roots.

Keywords: Agastache foeniculums; Agrobacterium rhizogenes; elicitation; hairy roots; sucrose concentration

  • [1] Ahmadian N., Sharifi M., Karimi F. & Rahnema H. 2010. Comparison of tropane alkaloids production in hairy roots and seedlings in Atropa belladonna L. effect by salicylic acid. Iranian J. Plant Biol. 1: 63–76. Google Scholar

  • [2] Ali M., Kiani B.H., Mannan A., Ismail T. & Mirza B. 2012. Enhanced production of artemisinin by hairy root culture of Artemisia dubia. J. Med. Plants Res. 6: 1619–1622. Google Scholar

  • [3] Aoki S. 2004. Resurrection of an ancestral gene: functional and evolutionary analyses of the Ng rol genes trans-ferred from Agrobacterium to Nicotiana. J. Plant. Res. 117: 329–337. Google Scholar

  • [4] Bais H.P., Walker T.S., Schweizer H.P. & Vivanco J.M. 2002. Root specific elicitation and antimicrobial activity of rosmarinic acid in hairy rot cultures of Ocimum basilicum. Plant Physiol. Biochem. 40: 983–995. CrossrefGoogle Scholar

  • [5] Bensaddek L., Villarreal M.L. & Fliniaux M.A. 2008. Induction and growth of hairy roots for the production of medicinal compounds. Electron. J. Integr. Biosci. 3: 2–9. Google Scholar

  • [6] Bertani G. 1952. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62: 293–300. Google Scholar

  • [7] Bulgakov P. 2008. Functions of rol genes in plant secondary metabolism. Biotech. Adv. 23(4): 318–324. http://dx.doi.org/10.1016/j.biotechadv.2008.03.001CrossrefGoogle Scholar

  • [8] Bulgakov V.P., Veselova M.V. & Tchernoded G.K. 2005. Inhibitory effect of the Agrobacterium rhizogenes rol gene on rabdosiin and RA production in Eritrichium sericeum and Lithospermum erythrorhizon transformed cell cultures. Planta. 221: 471–478. http://dx.doi.org/10.1007/s00425-004-1457-5CrossrefGoogle Scholar

  • [9] Cao D., Hou W., Song S., Sun H., Wu C., Gao Y. & Han T. 2009. Assessment of condition affecting Agrobacterium rhizogenes — mediated transformation of soybean. Plant Cell. Tiss. Org. Cult. 96: 45–52. http://dx.doi.org/10.1007/s11240-008-9458-xCrossrefGoogle Scholar

  • [10] Cardarelli M., Mariotti D. & Pomponi M. 1987. Agrobacterium rhizogenes TDNA genes capable of inducing hairy root phenotype. Mol. Gen. Genet. 209: 475–480. http://dx.doi.org/10.1007/BF00331152CrossrefGoogle Scholar

  • [11] Chichana N. & Dheeranupattana S. 2012. Effect of methyl jasmonate and salicylic acid on alkaloid production from in vitro culture of Stemona sp. Inter. J. Biosci. Biochem. Bioinforma. 2: 146–150. Google Scholar

  • [12] Choudhary R.K., Saroha A.E. & Swarnkar P.L. 2011. Radical scavenging activity of phenolics and flavonoids in some medicinal plants of India. J. Pharmacy. Res. 4: 712–713. Google Scholar

  • [13] Christey M.C. & Braun R.H. 2005. Production of hairy root cultures and transgenic plants by Agrobacterium rhizogenes — mediated transformation. Methods. Mol. Biol. 286: 47–60. Google Scholar

  • [14] Chtikadachanarong k., Dheeranupattana S., Jatisatienr A., Wangkarn S., Mungkornasawakul P., Pyne S.G., Ung A.T. & Sastraruju T. 2011. Influence of salicylic acid on alkaloid production by root culture of Stemona curisii Hook. F. J. Biol. Sci. 3: 322–325. Google Scholar

  • [15] Danphitsanuparn P., Boonsnongcheep P., Boribonkaset T., Chintapakorn Y. & Prathanturarug S. 2012. Effect of Agrobacterium rhizogenes strains and other parameters on production of isoflavonoids in hairy roots of Pueraria condollei Grah. Plant Cell. Tiss. Org. Cult. 111: 315–322. http://dx.doi.org/10.1007/s11240-012-0196-8CrossrefGoogle Scholar

  • [16] Doma M., Abhayankar G., Reddy V.D. & Kavikishor P.B. 2012. Carbohydrate and elicitor enhanced withanolied (withaferin A and withanolide A) accumulation hairy root culture of Withania somnifera (L). Indian. J. Exp. Biol. 50: 484–490. Google Scholar

  • [17] Eghdami A., Hashemi Sohi S.M., Eradatmand Asli D. & Houshmandfar A. 2011. Antioxidant activity of methanolin and hydroalcohlic extracts of Garlic plant. Adv. Environ. Biol. 5: 1575–1578. Google Scholar

  • [18] Fesen M.R., Kohn K.W., Leteutre F. & Pommier Y. 1993. Inhibitores of human immunodeficiency virus integrase. Proc. Natl. Acad. Sci. U.S.A. 90: 507–511. http://dx.doi.org/10.1073/pnas.90.6.2399CrossrefGoogle Scholar

  • [19] Gangopadhyay M., Chakraborty D. & Bhattacharyya S. 2010. Regeneration of transformed plants from hairy root of Plumbago indica. Plant Cell. Tiss. Org. Cult. 120: 109–114. http://dx.doi.org/10.1007/s11240-010-9702-zCrossrefGoogle Scholar

  • [20] Gorgiev M., Pavlov A. & Bley T. 2007. Hairy root type plant in vitro systems as sources of bioactive substances. Appl. Microbiol. Biotechnol. 74: 1175–1185. http://dx.doi.org/10.1007/s00253-007-0856-5CrossrefGoogle Scholar

  • [21] Guillun S., Tremouillaux-Guiller J., Pati P.K., Rideau M. & Ganten P. 2006. Hairy root research: recent scenario and exciting prospects. Curr. Opin. Plant Biol. 9: 341–346. http://dx.doi.org/10.1016/j.pbi.2006.03.008CrossrefGoogle Scholar

  • [22] Hamill J.D. 1993. Alterations in auxin and cytikinin metabolism of higher plants due to expression of specific genes from pathogenic bacteria. a review”. Aust. J. Plant Physiol. 20: 405–423. CrossrefGoogle Scholar

  • [23] Hu Z.B. & Du M. 2006. Hairy root and its application in plant genetic engineering. J. Integr. Plant Biol. 48: 121–127. http://dx.doi.org/10.1111/j.1744-7909.2006.00121.xCrossrefGoogle Scholar

  • [24] Jin J.H., Shin J.H., Kim J.H. & Lee H.J. 1999. Effect of chitosan elivitation and media components on the production of Anthraquinone colorants in Madder (Rubia akane Nakaki) cell culture. Biotechnol. Bioprocess. Eng. 4: 300–304. http://dx.doi.org/10.1007/BF02933757CrossrefGoogle Scholar

  • [25] Kayser O. & Quax W.G. 2007. Medicinal plant biotechnology. Vol. 1, WILEY-VCH Verlag GmbH & Co., Weinheim, 604 pp. Google Scholar

  • [26] Khan S., Irfan Q.M., Kamaluddin A.T. & Abdin M.Z. 2007. Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. Afr. J. Biotechnol. 6: 175–178. Google Scholar

  • [27] Komariah P., Naga Amrutha R., Kavi Kishor P.B. & Ramakrishna S.V. 2002. Elicitor enhanced production of plumbagin in suspension cultures of Plumbago rosea L. Enzyme. Microb. Tech. 31: 634–639. http://dx.doi.org/10.1016/S0141-0229(02)00159-XCrossrefGoogle Scholar

  • [28] Lee S.Y., Lee Ch.Y., Eom S.H., Kim Y.K., Park N. & Park S.U. 2010. Rosmarinic acid production from transformed root cultures of Nepeta cataria L. Sci. Res. Essays. 5(10): 1122–1126. Google Scholar

  • [29] Lee S.Y., Xu H., Kim Y.K. & Park S.U. 2008. Rosmarinic acid production in hairy root cultures of Agastache rugosa Kuntze. World J. Microbial. Biotechnol. 24: 969–972. http://dx.doi.org/10.1007/s11274-007-9560-yCrossrefGoogle Scholar

  • [30] Lemcke K. & Schmulling T. 1998. Gain of function assays identify non rol genes from Agrobacterium rhizogenes TLDNA that alter plant morphogenesis or hormone sensitivity. Plant. J. 15: 423–433. http://dx.doi.org/10.1046/j.1365-313X.1998.00223.xCrossrefGoogle Scholar

  • [31] Luo C., Peng Z. & Pu L. 2004. Transformation of medicinal plants mediated by Agrobacterium rhizogenes. Biotechnology. 14: 58–61. Google Scholar

  • [32] Matei C.F. 2012. Researches regarding the biology and crop technology of the Agastache foeniculum (Pursh) Kuntze species in the conditions of Transylvania plane. Dissertation, University of agricultural sciences and veterinary medicine Cluj-Napoca Faculty of agriculture. Google Scholar

  • [33] Morgan J.A., Bamey C.S., Penn A.H. & Shnks J.V. 2000. Effects of buffered media upon growth and alkaloid production of Catharanthus roseus hairy roots. Appl. Microbiol. Biotechnol. 53: 205–210. http://dx.doi.org/10.1007/s002530050018CrossrefGoogle Scholar

  • [34] Murashige T. & Skoog F. 1962. A revised medium for rapid growth and bioassays with Tobacco tissue cultures. Physiol. Plant. 15: 473–497. http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.xCrossrefGoogle Scholar

  • [35] Nguyen C., Bourgaud F., Forlot P. & Guckert A. 1992. Establishment of hairy root cultures of Psoralea species. Plant. Cell. Rep. 11: 424–427. http://dx.doi.org/10.1007/BF00234375CrossrefGoogle Scholar

  • [36] Omidbaigi R. 2007. Production and processing of medicinal plants. Astane Quds, Mashhed, 424 pp. Google Scholar

  • [37] Ono N.N. & Tain L. 2011. The multiplicity of hairy root cultures: prolific possibilities. Plant Sci. 180: 439–446. http://dx.doi.org/10.1016/j.plantsci.2010.11.012CrossrefGoogle Scholar

  • [38] Palazon J., Cusido R.M., Bontill M., Mallol A., Moyano E., Morales C. & Pinol M.T. 2003a. Elicitation of different Panax ginseng transformed root phenotypes for an improved ginsenoside production. Plant Physiol. Biochem. 41: 1019–1025. CrossrefGoogle Scholar

  • [39] Palazon J., Cusido R.M., Roig C. & Pinol M.T. 1997. Effect of rol genes from Agrobacterium rhizogenes TL-DNA on nicotine production in tobacco root cultures. Plant. Physiol. Bioch. 35: 155–162. Google Scholar

  • [40] Palazon J., Mallol A., Eibl R., Lettenbauer C., Cusido R.M. & Pinol M.T. 2003b. Growth and ginsenoside production in hairy root cultures of Panax ginseng using a novel bioreactor. Planta. Med. 69: 344–349. http://dx.doi.org/10.1055/s-2003-38873CrossrefGoogle Scholar

  • [41] Pawar P.K. & Matheshwari V.L. 2003. Agrobacterium rhizogenes mediated hairy root induction in two medicinally important of family. Indian J. Biotechnol. 3: 414–417. Google Scholar

  • [42] Pirian K., Piri Kh. & Ghiyasvand T. 2012. Hairy roots induction from Protulaca oleracea using Agrobacterium rhizogenes to noradenaline’s production. Int. Res. J. Appl. Basic. Sci. 3: 642–649. Google Scholar

  • [43] Porter J.R. 1991. Host range and implication of plainftion by Agrobacterium rhizogenes. Crit. Rev. Plant Sci. 10: 387–421. http://dx.doi.org/10.1080/07352689109382318CrossrefGoogle Scholar

  • [44] Putalun W., Pimmeuangkao S., De-Eknamkul W., Tanaka H. & Shoyama Y. 2006. Sennosides A and B production by hairy roots of Senna alata (L.) Roxb. Z. Naturforsch. C.Biosci. 61: 367–371. Google Scholar

  • [45] Samadi A., Carapetian J., Heidary R., Gafari M. & Hssanzadeh A. 2012. Hairy root induction in Linum mucronatum ssp. mucronatum an anti-tumor lignans production plant. Not. Bot. Hort. Agrobot. Cluj 40: 125–131. Google Scholar

  • [46] Sanford R.A. & Hutchings G.P. 1987. Chitosan-a natural, cationic biopolymeras commercial application, pp. 363–376. In: Rapalma M. (ed.) Industrial Polysaccharides. Genetic Engineering, Structure/Property relations and Application, Amsterdum, The Netherlands. Google Scholar

  • [47] Sevon N., Hiltunen R. & Oksman-Caldentey K.M. 1992. Chitosan increases hyoscyamine content in hairy root cultures of Hyoscyamus muticus. Pharm. Pharmacol. Lett. 2: 96–99. Google Scholar

  • [48] Sharafi A., Hashemisohi H., Mousavi A., Azadi P., Razavi Kh. & Otang Nuti V. 2013. A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum. Plant Cell. Tiss. Org. Cult 113: 1–9. http://dx.doi.org/10.1007/s11240-012-0246-2CrossrefGoogle Scholar

  • [49] Smetanska I. 2008. Production of secondary metabolites using plant cell cultures. Adv. Biochem. Eng. Biotechnol. 111: 187–228. Google Scholar

  • [50] Soleimani T., Keyhanfer M., Piri K.H. & Hsanloo T. 2012. Morphological evaluation of hairy roots induced in Artemisia annua L. and investigating elicitation effects on hairy roots biomass production. Intl. J. Agric. Res & Rev. 2: 1005–1013. Google Scholar

  • [51] Weber R.L.M. & Bodanese-Zanettini M.H. 2011. Induction of transgenic hairy roots in soybean genotypes by Agrobacterium rhizogenes-mediated transformation. Pesqui. Agropecu. Bras. 46: 1070–1075. Google Scholar

  • [52] Yoshikawa M. 1978. Diverse modes of action of biotic and abiotic phytoalexin elicitors. Nature. 275: 546–547. http://dx.doi.org/10.1038/275546a0CrossrefGoogle Scholar

  • [53] Xu H., Park J.H., Kim Y.K., Park N., Lee S.Y. & Un S. 2009. Optimization of growth and pyranocoumarins production in hairy root culture of Angelica gigas Nakai. J. Med. Plants. Res. 3: 978–981. Google Scholar

About the article

Published Online: 2014-08-19

Published in Print: 2014-07-01

Citation Information: Biologia, Volume 69, Issue 7, Pages 870–879, ISSN (Online) 1336-9563, DOI: https://doi.org/10.2478/s11756-014-0382-8.

Export Citation

© 2014 Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

Elnaz Nourozi, Bahman Hosseini, Ramin Maleki, and Babak Abdollahi Mandoulakani
Journal of the Science of Food and Agriculture, 2019
Siel Desmet, Ellen De Keyser, Johan Van Vaerenbergh, Steve Baeyen, Johan Van Huylenbroeck, Danny Geelen, and Emmy Dhooghe
Applied Microbiology and Biotechnology, 2019, Volume 103, Number 16, Page 6657
Elnaz Nourozi, Bahman Hosseini, Ramin Maleki, and Babak Abdollahi Mandoulakani
Industrial Crops and Products, 2019, Volume 133, Page 435
Izabela Weremczuk-Jeżyna, Ewa Skała, Monika A. Olszewska, Anna K. Kiss, Ewa Balcerczak, Halina Wysokińska, and Agnieszka Kicel
Industrial Crops and Products, 2016, Volume 91, Page 125
Shivani Srivastava, Xavier A. Conlan, Alok Adholeya, and David M. Cahill
Plant Cell, Tissue and Organ Culture (PCTOC), 2016, Volume 126, Number 1, Page 19

Comments (0)

Please log in or register to comment.
Log in