Agrobacterium rhizogenes-mediated hairy root induction and plant regeneration from transgenic roots in Ficus carica L.

Document Type : Original research paper


1 Department of Horticulture Sciences, University of Mohaghegh Ardabili, Ardabil, Iran

2 Cellular and Molecular Research Center & Department of Medical Parasitology and Mycology, Urmia University of Medical Sciences, Urmia, Iran

3 Department of Plant Breeding and Biotechnology, Urmia University, Urmia, Iran


One of the most effective biotechnological techniques for producing plant metabolites is the hairy roots (HRs) culture system. HRs are genetically and biologically stable and able to produce secondary metabolites in a short time. Ficus carica L. is one of the most important plant sources of valuable medicinal compounds, especially polyphenolic compounds. The aim of this study was to investigate the growth and morphological features of HRs, methyl jasmonate (MeJA) elicitation and plant regeneration potential of HRs induced by different strains of Agrobacterium rhizogenes on Ficus carica cv. Siah. Four bacterial strains (A4, A7, A13 and ATCC 15834) were used for HR induction in leaf and shoot samples. The MS medium containing 2 mg/l of 2,4-D in combination with 1 mg/l of TDZ or BAP was used to induce callus from HRs, and then the callus regeneration ability was evaluated in MS medium containing TDZ and NAA. Depending on explant type and bacterial strain, the roots were induced directly or indirectly (via callus formation) from the wound sites, and exhibited different morphology. The HRs showed high levels of phenolic compounds. A 4-day elicitation with MeJA, in dependence on the concentration, enhanced the phenolic capacity and antioxidant capacity of HRs. The calli obtained from HRs showed root (70-80%) and bud (23.33%) regeneration potential. The current study described that the HR culture systems, in addition to providing the possibility of plant regeneration from transgenic roots, could be a promising in vitro technique for high production of secondary metabolites through elicitation.


[1]Aljane, F., Essid, A., and Nahdi, S. 2018. Improvement of Fig (Ficus carica L.) by Conventional Breeding and Biotechnology, in Advances in Plant Breeding Strategies: Fruits. Springer. p. 343-375.
[2]Amessis Ouchemoukh, N., Ouchemoukh, S., Meziant, N., Idiri, Y., Hernanz, D., Stinco, C.M., Rodriguez Pulido, F.J., Heredia, F.J., Madani, K., and Luis, J. 2017. Bioactive metabolites involved in the antioxidant, anticancer and anticalpain activities of Ficus carica L., Ceratonia siliqua L. and Quercus ilex L. extracts. Ind Crops Prod, 95: 6-17. DOI:
[3]Argolo, A., Sant'Ana, A., Pletsch, M., and Coelho, L. 2004. Antioxidant activity of leaf extracts from Bauhinia monandra. Bioresour Technol, 95(2): 229-233. DOI:
[4]Bakar, M.F.A., Mohamed, M., Rahmat, A., and Fry, J. 2009. Phytochemicals and antioxidant activity of different parts of bambangan (Mangifera pajang) and tarap (Artocarpus odoratissimus). Food Chem, 113(2): 479-483. DOI:
[5]Bandyopadhyay, M., Jha, S., and Tepfer, D. 2007. Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera. Plant Cell Rep, 26(5): 599-609. DOI:
[6]Barolo, M.I., Mostacero, N.R., and López, S.N. 2014. Ficus carica L.(Moraceae): an ancient source of food and health. Food Chem, 164: 119-127. DOI:
[7]Benzie, I.F. and Strain, J.J. 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem, 239(1): 70-76. DOI:
[8]Caliskan, O. and Polat, A.A. 2011. Phytochemical and antioxidant properties of selected fig (Ficus carica L.) accessions from the eastern Mediterranean region of Turkey. Sci Hortic, 128(4): 473-478. DOI:
[9]Cardarelli, M., Mariotti, D., Pomponi, M., Spano, L., Capone, I., and Costantino, P. 1987. Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype. Mol Gen Genet, 209(3): 475-480. DOI:
[10]  Chandra, S. 2012. Natural plant genetic engineer Agrobacterium rhizogenes: role of T-DNA in plant secondary metabolism. Biotechnol Lett, 34(3): 407-415. DOI:
[11]  Chaudhuri, K.N., Ghosh, B., Tepfer, D., and Jha, S. 2006. Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes. Plant Cell Rep, 25(10): 1059-1066. DOI:
[12]  Choi, D.-W., Jung, J., Im Ha, Y., Park, H.-W., In, D.S., Chung, H.-J., and Liu, J.R. 2005. Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites. Plant Cell Rep, 23(8): 557-566. DOI:
[13]  Choi, P., Kim, Y., Choi, K., Chung, H., Choi, D., and Liu, J.R. 2004. Plant regeneration from hairy-root cultures transformed by infection with Agrobacterium rhizogenes in Catharanthus roseus. Plant Cell Rep, 22(11): 828-831. DOI:
[14]  Christey, M.C. and Braun, R.H. 2005. Production of hairy root cultures and transgenic plants by Agrobacterium rhizogenes-mediated transformation, in Transgenic plants: methods and protocols. Springer. p. 47-60.
[15]  Crane, C., Wright, E., Dixon, R.A., and Wang, Z.-Y. 2006. Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens-transformed roots and Agrobacterium rhizogenes-transformed hairy roots. Planta, 223(6): 1344-1354. DOI:
[16]  Duenas, M., Perez-Alonso, J.J., Santos-Buelga, C., and Escribano-Bailon, T. 2008. Anthocyanin composition in fig (Ficus carica L.). J Food Compos Anal, 21(2): 107-115. DOI:
[17]  Fattahi, M., Nazeri, V., Torras-Claveria, L., Sefidkon, F., Cusido, R.M., Zamani, Z., and Palazon, J. 2013. A new biotechnological source of rosmarinic acid and surface flavonoids: Hairy root cultures of Dracocephalum kotschyi Boiss. Ind Crops Prod, 50: 256-263. DOI:
[18]  Gai, Q.-Y., Jiao, J., Luo, M., Wang, W., Gu, C.-B., Fu, Y.-J., and Ma, W. 2016. Tremendous enhancements of isoflavonoid biosynthesis, associated gene expression and antioxidant capacity in Astragalus membranaceus hairy root cultures elicited by methyl jasmonate. Process Biochem, 51(5): 642-649. DOI:
[19]  Gai, Q.-Y., Jiao, J., Luo, M., Wei, Z.-F., Zu, Y.-G., Ma, W., and Fu, Y.-J. 2015. Establishment of hairy root cultures by Agrobacterium rhizogenes mediated transformation of Isatis tinctoria L. for the efficient production of flavonoids and evaluation of antioxidant activities. PLoS One, 10(3): e0119022. DOI:
[20]  Gai, Q.-Y., Jiao, J., Wang, X., Zang, Y.-P., Niu, L.-L., and Fu, Y.-J. 2019. Elicitation of Isatis tinctoria L. hairy root cultures by salicylic acid and methyl jasmonate for the enhanced production of pharmacologically active alkaloids and flavonoids. Plant Cell Tissue Organ Cult, 137(1): 77-86. DOI:
[21]  Georgiev, M.I., Agostini, E., Ludwig-Müller, J., and Xu, J. 2012. Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotechnol, 30(10): 528-537. DOI:
[22]  Giri, A. and Narasu, M.L. 2000. Transgenic hairy roots recent trends and applications. Biotechnol Adv, 18(1): 1-22. DOI:
[23]  Habibi, P., de Sa, M.F.G., da Silva, A.L.L., Makhzoum, A., da Luz Costa, J., Borghetti, I.A., and Soccol, C.R. 2016. Efficient genetic transformation and regeneration system from hairy root of Origanum vulgare. Physiol Mol Biol Plants, 22(2): 271-277. DOI:
[24]  Hao, X., Shi, M., Cui, L., Xu, C., Zhang, Y., and Kai, G. 2015. Effects of methyl jasmonate and salicylic acid on tanshinone production and biosynthetic gene expression in transgenic Salvia miltiorrhiza hairy roots. Biothechnol Appl Biochem, 62(1): 24-31. DOI:
[25]  Henzelyova, J. and Cellarova, E. 2018. Modulation of naphthodianthrone biosynthesis in hairy root-derived Hypericum tomentosum regenerants. Acta Physiol Plant, 40(5): 82. DOI:
[26]  Ho, T.-T., Lee, J.-D., Ahn, M.-S., Kim, S.-W., and Park, S.-Y. 2018. Enhanced production of phenolic compounds in hairy root cultures of Polygonum multiflorum and its metabolite discrimination using HPLC and FT-IR methods. Appl Microbiol Biotechnol, 102(22): 9563-9575. DOI:
[27]  Jouanin, L., Tourneur, J., Tourneur, C., and Casse-Delbart, F. 1986. Restriction maps and homologies of the three plasmids of Agrobacterium rhizogenes strain A4. Plasmid, 16(2): 124-134. DOI:
[28]  Kang, H., Anbazhagan, V., You, X., Moon, H., Yi, J., and Choi, Y. 2006. Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots. Plant Cell Tissue Organ Cult, 85(2): 187-196. DOI:
[29]  Kim, O.T., Bang, K.H., Kim, Y.C., Hyun, D.Y., Kim, M.Y., and Cha, S.W. 2009. Upregulation of ginsenoside and gene expression related to triterpene biosynthesis in ginseng hairy root cultures elicited by methyl jasmonate. Plant Cell Tissue Organ Cult, 98(1): 25-33. DOI:
[30]  Koike, Y., Hoshino, Y., Mii, M., and Nakano, M. 2003. Horticultural characterization of Angelonia salicariifolia plants transformed with wild-type strains of Agrobacterium rhizogenes. Plant Cell Rep, 21(10): 981-987. DOI:
[31]  Lansky, E.P., Paavilainen, H.M., Pawlus, A.D., and Newman, R.A. 2008. Ficus spp.(fig): Ethnobotany and potential as anticancer and anti-inflammatory agents. J Ethnopharmacol, 119(2): 195-213. DOI:
[32]  Li, C., Wang, P., Menzies, N.W., Lombi, E., and Kopittke, P.M. 2018. Effects of methyl jasmonate on plant growth and leaf properties. J Plant Nutr Soil Sc, 181(3): 409-418. DOI:
[33]  Lloyd, G. and McCown, B. 1980. In: proceedings of the international plant propagation society. 30: 421-427.
[34]  Loizzo, M.R., Bonesi, M., Pugliese, A., Menichini, F., and Tundis, R. 2014. Chemical composition and bioactivity of dried fruits and honey of Ficus carica cultivars Dottato, San Francesco and Citrullara. J Sci Food Agr, 94(11): 2179-2186. DOI:
[35]  Mahmoudi, S., Khali, M., Benkhaled, A., Benamirouche, K., and Baiti, I. 2016. Phenolic and flavonoid contents, antioxidant and antimicrobial activities of leaf extracts from ten Algerian Ficus carica L. varieties. Asian Pac J Trop Biomed, 6(3): 239-245. DOI:
[36]  Mallol, A., Cusido, R.M., Palazon, J., Bonfill, M., Morales, C., and Pinol, M.T. 2001. Ginsenoside production in different phenotypes of Panax ginseng transformed roots. Phytochemistry, 57(3): 365-371. DOI:
[37]  Marsh, Z., Yang, T., Nopo-Olazabal, L., Wu, S., Ingle, T., Joshee, N., and Medina-Bolivar, F. 2014. Effect of light, methyl jasmonate and cyclodextrin on production of phenolic compounds in hairy root cultures of Scutellaria lateriflora. Phytochemistry, 107: 50-60. DOI:
[38]  Mirjalili, H.M., Fakhr‐Tabatabaei, S.M., Bonfill, M., Alizadeh, H., Cusido, R.M., Ghassempour, A., and Palazon, J. 2009. Morphology and withanolide production of Withania coagulans hairy root cultures. Engineering in Life Sciences, 9(3): 197-204. DOI:
[39]  Nadeem, M. and Zeb, A. 2018. Impact of maturity on phenolic composition and antioxidant activity of medicinally important leaves of Ficus carica L. Physiol Mol Biol Plants, 24(5): 881-887. DOI:
[40]  Oliveira, A.P., Baptista, P., Andrade, P.B., Martins, F., Pereira, J.A., Silva, B.M., and Valentao, P. 2012. Characterization of Ficus carica L. cultivars by DNA and secondary metabolite analysis: Is genetic diversity reflected in the chemical composition? Food Res Int, 49(2): 710-719. DOI:
[41]  Ono, N.N. and Tian, L. 2011. The multiplicity of hairy root cultures: prolific possibilities. Plant Sci, 180(3): 439-446. DOI:
[42]  Otani, M., Shimada, T., Kamada, H., Teruya, H., and Mii, M. 1996. Fertile transgenic plants of Ipomoea trichocarpa Ell. induced by different strains of Agrobacterium rhizogenes. Plant Sci, 116(2): 169-175. DOI:
[43]  Pirttila, A.M., Hirsikorpi, M., Kamarainen, T., Jaakola, L., and Hohtola, A. 2001. DNA isolation methods for medicinal and aromatic plants. Plant Mol Biol Rep, 19(3): 273-273. DOI:
[44]  Prior, R.L., Wu, X., and Schaich, K. 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem, 53(10): 4290-4302. DOI:
[45]  Roychowdhury, D., Majumder, A., and Jha, S. 2013. Agrobacterium rhizogenes-mediated transformation in medicinal plants: prospects and challenges, in Biotechnology for Medicinal Plants. Springer. p. 29-68.
[46]  Sarkar, S., Ghosh, I., Roychowdhury, D., and Jha, S. 2018. The Effects of rol genes of Agrobacterium rhizogenes on morphogenesis and secondary metabolite accumulation in medicinal plants, in Biotechnological approaches for medicinal and aromatic plants. Springer. p. 27-51.
[47]  Sedaghat, S. and Rahemi, M. 2018. Effects of physio-chemical changes during fruit development on nutritional quality of fig (Ficus carica L. var.‘Sabz’) under rain-fed condition. Sci Hortic, 237: 44-50. DOI:
[48]  Sharafi, A., Sohi, H.H., Mousavi, A., Azadi, P., Razavi, K., and Ntui, V.O. 2013. A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum. Plant Cell Tissue Organ Cult, 113(1): 1-9. DOI:
[49]  Singleton, V.L. and Rossi, J.A. 1965. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Viticult, 16(3): 144-158.
[50]  Skała, E., Picot, L., Bijak, M., Saluk-Bijak, J., Szemraj, J., Kicel, A., Olszewska, M.A., and Sitarek, P. 2019. An efficient plant regeneration from Rhaponticum carthamoides transformed roots, enhanced caffeoylquinic acid derivatives production in pRi-transformed plants and their biological activity. Ind Crops Prod, 129: 327-338. DOI:
[51]  Sujatha, G., Zdravkovic-Korac, S., Calic, D., Flamini, G., and Kumari, B.R. 2013. High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia vulgaris: hairy root production and essential oil analysis. Ind Crops Prod, 44: 643-652. DOI:
[52]  Tsuro, M. and Ikedo, H. 2011. Changes in morphological phenotypes and essential oil components in lavandin (Lavandula× intermedia Emeric ex Loisel.) transformed with wild-type strains of Agrobacterium rhizogenes. Sci Hortic, 130(3): 647-652. DOI:
[53]  Vallejo, F., Marin, J., and Tomas-Barberan, F.A. 2012. Phenolic compound content of fresh and dried figs (Ficus carica L.). Food Chem, 130(3): 485-492. DOI:
[54]  Wang, J., Qian, J., Yao, L., and Lu, Y. 2015. Enhanced production of flavonoids by methyl jasmonate elicitation in cell suspension culture of Hypericum perforatum. Bioresour Bioprocess, 2(1): 5. DOI:
[55]  Wang, Y.M., Wang, J.B., Da, L., and Jia, J.F. 2001. Regeneration of plants from callus cultures of roots induced by Agrobacterium rhizogenes on Alhagi pseudoalhagi. Cell Res, 11(4): 279. DOI:
[56]  Wasternack, C. and Parthier, B. 1997. Jasmonate-signalled plant gene expression. Trends Plant Sci, 2(8): 302-307. DOI:
[57]  Wu, J., Wang, Y., Zhang, L.-X., Zhang, X.-Z., Kong, J., Lu, J., and Han, Z.-H. 2012. High-efficiency regeneration of Agrobacterium rhizogenes-induced hairy root in apple rootstock Malus baccata (L.) Borkh. Plant Cell Tissue Organ Cult, 111(2): 183-189. DOI:
[58]  Yancheva, S.D., Golubowicz, S., Yablowicz, Z., Perl, A., and Flaishman, M.A. 2005. Efficient Agrobacterium-mediated transformation and recovery of transgenic fig (Ficus carica L.) plants. Plant Sci, 168(6): 1433-1441. DOI:
[59]  Zhang, L., Yang, B., Lu, B., Kai, G., Wang, Z., Xia, Y., Ding, R., Zhang, H., Sun, X., and Chen, W. 2007. Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing putrescine N-methyltransferase is methyl jasmonate-dependent. Planta, 225(4): 887-896. DOI:
Zhou, J., Ran, Z.-F., Liu, Q., Xu, Z.-X., Xiong, Y.-H., Fang, L., and Guo, L.-P. 2019. Jasmonic acid serves as a signal role in smoke-isolated butenolide-induced tanshinones biosynthesis in Salvia miltiorrhiza hairy root. S Afr J Bot, 121: 355-359. DOI:
Volume 7, Issue 1
June 2019
Pages 10-21
  • Receive Date: 03 August 2019
  • Revise Date: 23 November 2019
  • Accept Date: 21 December 2019
  • First Publish Date: 21 December 2019