Effects of explant age and strain of Agrobacterium rhizogenes on hairy roots induction in Fenugreek (Trigonella foenum– graecum L.)

Document Type : Research paper


Department of Horticultural science, Faculty of Agriculture and Natural resources, University of Mohaghegh Ardabili, P. O. Box: 56199-11367, Ardabil, Iran


Induced hairy roots by Agrobacterium rhizogenes are suitable organs for the production of secondary metabolites, due to the stability and high production of roots without phyhormones in a short time. Fenugreek (Trigonella foenum– graecum L.) is one of the most important medicinal plants with valuable medicinal compounds. For optimizing the hairy root induction of T. foenum-graecum L., six strains of A. rhizogenes ATCC15834, ATCC11325, K599, A7, A4 and A13 (MAFF-02-10266) and three explant ages (7, 14 and 21 day old seedlings) were tested. The explants were cultured in B5 in a completely randomized design with 4 replications. Hairy roots appeared after 3 to 4 weeks. Transgenic status of the roots was confirmed by PCR using rolB specific primers. The results revealed that all strains were able to induce hairy roots after 3-4 weeks. The maximum percentage of hairy root induction (87.5%) was obtained from A7 and ATCC11325 strains and 7-day-old explants. Among the strains, ATCC11325 strain showed the highest hairy root number (4.25) and root length (2.1 cm) using 14-day-old explant. Also, the highest fresh (0.203 g) and dry weights (0.029 g) were observed in the A4, A7 and ATCC11325 strains. The highest total phenolic and flavonoid contents were found at 74.05 mg g-1 DW and 11.80 µg g-1 DW in hairy roots induced by A7 strain.


Aasim M., Hussain N., Umer2 E. M., Zubair M., Hussain S. B., Saeed S. H.,.Rafique T. S., and Sancak C. (2010). In vitro shoot regeneration of fenugreek (Trigonellafoenum-graecum L.) using different cytokinins. African Journal of Biotechnology, 9( 42): 7174-7179.
Akbarian R., Hasanloo T., and Khosroshahi M. (2011). Evaluation of trigonelline production in Trigonella foenum-graecum hairy root cultures of two Iranian masses. Plant Omics Journal, 4: 408-412.
Al-Mahdawe M. M., Al-Mallah M. K., and Al-Attrakchii A. O. (2013). Genetically transformed hairy roots producing agropine induced on Trigonella foenum-graecum L. plant by Agrobacterium rhizogenes 1601. Journal of Biotechnology Research Center, 7: 91-98.
Balasundram N., Sundram K., and Sammar S. (2006). Phenolic compounds in plants and agroindustrial by-products: antioxidant activity, occurrence, and potential uses. Food Chemistry, 1: 191–203.
Banihashemi O., Khavari R. A., Yassa N., and Najafi F. (2015). Induction of hairy roots in Atropa komarovii using Agrobacterium rhizogenes. Indian Journal of Fundamental and Applied Life Sciences, 5(3): 2014-2020.
Batra J., Ajaswrata D., Singh D., Kumar S., and Sen J. (2004). Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration. Plant Cell Reports, 23: 148–154.
Bel-Rhlid R., Chabot S., Piche Y., and Chenevert T. (1993). Isolation and identification of flavonoids from Ri T-DNA transformed roots (Daucus carota) and their significance in vesicular–arbuscular mycorrhiza. Phytochemistry, 35: 381–383.
Bhagyasri Y., Lavakumar V., Divya Sree M. S., and Ashok Kumar C. K. (2015). An overview on anti-inflammatory activity of Indian herbal plants. International Journal of Research in Pharmaceutical and Nano Sciences, 4(1): 1-9.
Billaud C., and Adrian J. (2001). Fenugreek composition, nutritional value and Physiological properties. Sciences des Aliments, 21: 3–26.
Cao D., Hou W., Song S., Sun H., Cao Y., and Han T. (2009). Assessm ent of conditions affecting Agrobacterium rhizogenes mediated transformation of soybean. Plant CellTissue and Organ Culture, 96: 45-52.
Chang C., Yang M., Wen H., and Chern J. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis, 10: 178-182.
Charlwood B. V., and Charlwood K. A. (1991). Terpenoid production in plant cell culture. In: Harborne J. B., and Tomas-Barberan F. A. Ecological chemistry and biochemistry of plant terpenoids. Clarendon Press, Oxford, 95–132.
Doyle J. J., and Doyle J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19: 11-15.
Gamborg O. L., Miller R. A., and Ojima K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 50: 151–158.
Giri A., Dhingra V., Giri C. C., Singh A., Ward O. P., and Narasu M. L. (2001). Biotransformations using plant cells, organ cultures and enzyme systems: current trends and future prospects. Biotechnology Advances, 19: 175-199.
Huang S. H., Vishwakarma R. K., Lee T. T., Chan H. S., and Tsay H. S. (2014). Establishment of hairy root lines and analysis of iridoids and secoiridoids in the medicinal plant Gentiana scabra. Botanical Studies, 55: 1-17.
Kabirnotaj S., Zolalla J., Nematzadeh G., and SHokri E. (2013). Optimization of hairy root culture establishment in Chicory plants (Cichorium intybus) through inoculation by Agrobacterium rhizogenes. Journal of Agriculture Biotechnology, 4(2): 61-75.
Lucia C., Calogero P., Maurizio Z., Antonella C., Silvia G., Franco S., Sabrina T., and Luciano G. (2003). Effects of γ-Irradiation on the Free Radical and Antioxidant Contents in Nine Aromatic Herbs and Spices. Journal of Agricultural and Food Chemistry, 51: 927-934.
Majumdar S., Garai S., and Jha S. (2011). Genetic transformation of Bacopa monnieri by wild type strains of Agrobacterium rhizogenes stimulates production of bacopa saponins in transformed calli and plants. Plant Cell Reports, 30: 941–954.
Meda A., Lamien C. E., Romito M., Millogo J., and Nacoulma O. G. (2005). Determination of the total phenolic,flavonoid and pralin contents in Burkina Fasan honey, as well as their scavenging activity. Food Chemistry, 91: 571-577.
Merkli A., Christen P., and Kapetanidis I. (1997). Production of diosgenin by hairy root cultures of Trigonella foenum.graecum L. Plant Cell Reports, 16: 632-636.
Murashige Toshio., and Skoog Folke. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum,15: 473- 497.
Ooi C. T., Syahida A., Stanslas J., and Maziah M. (2013). Efficiency of different Agrobacterium rhizogenes strains on hairy roots induction in Solanum mammosum. World Journal of Microbiology and Biotechnology, 29(3):421-30.
Peraza-Luna F., Rodrı´guez-Mendiola M., Arias-Castro C., Bessiere J. M., and Calva-Calva g. (2001). Sotolone production by hairy root cultures of Trigonella foenum-graecum in Airlift with Mesh Bioreactors. Journal of Agricultural and Food Chemistry, 49: 6012-6019.
Qaderi A., Akbari Z., Kalateh-jari S, Fatehi F., Tolyat M., Jalali Moghadam M., and Naghdi Badi H. (2014). Improving Trigonelline Production in Hairy Root Culture of Fenugreek (Trigonella foenum-graecum). Journal of Medicinal Plants, 15: 73-80.
Rashidi Asl K., Hosseini B., Sharafi A., And Palazon J. (2018). Influence of Nano-Zinc Oxide on Tropane Alkaloid Production, h6h Gene Transcription and Antioxidant Enzyme Activity in Hyoscyamus reticulatus L. Hairy Roots. Engineering in Life Sciences.
Shahabzadeh Z., Heidari B., and Faramarzi Hafez R. (2013). Induction of Transgenic Hairy Roots in Trigonella foenumgraceum Co-cultivated with Agrobacterium Rhizogenes Harboring a GFPGene. Journal of Crop Science and Biotechnology, 16(4): 263- 268.
Sharafi A., Hashemi Sohi H., Azadi P., and Sharafi A. A. (2014a). Hairy root induction and plant regeneration of medicinal plant Dracocephalum kotschyi. Physiology and Molecular Biology of Plants, 20(2): 257–262.
Sharafi A., Hashemi Sohi H., Mirzaee H., and Azadi P. (2014b). In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss. Physiology and Molecular Biology of Plants, 20(4): 487–494.
Sharafi A., Hashemi Sohi H., Mousavi A., Azadi P., Dehsara B., and Hosseini Khalifani B. (2013a). Enhanced morphinan alkaloid production in hairy root cultures of Papaver bracteatum by over-expression of salutaridinol 7-o-acetyltransferase gene via Agrobacterium rhizogenes mediated transformation. World Journal of Microbiology and Biotechnology, 29: 2125–2131.
Sharafi A., Hashemi Sohi H., Mousavi A., Azadi P., Hosseini Khalifani B., and Razavi K. (2013b). Metabolic engineering of morphinan alkaloids by over expression of codeinone reductase in transgenic hairy root of Papaver bracteatum. Biotechnology Letters, 35: 445–453.
Sharafi A., Hashemi. S. H., Mousavi. A., Azadi. P., Razavi. K., and Ntui V. O. (2012). A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum. Plant Cell Tissue and Organ Culture, 113: 1-9.
Thiruvengadam M., and Chung I. M. (2014). Optimization of factors influencing in vitro flowering of gherkin (Cucumis anguria L.). Acta Biologica Hungarica, 65: 72–84.
Tiwari R. K., Trivedi M., Guang Z. C., Guo G. Q., and Zheng G. C. (2007). Genetic transformation of Gentiana macrophylla with Agrobacterium rhizogenes: growth and production of secoiridoid glucoside gentiopicroside in transformed hairy root cultures. Plant Cell Reports, 26: 199–210.
Tzfira T., Li J., Lacroix B., and Citovsky V. (2004). A. grobacterium T-DNA integration: molecules and models. Trends in Genetics, 20: 375-383
Valimehr S., Sanjarian F., Hashemi sohi H., Sharafi A., and Sabouni F. (2014). A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma. Physiology and Molecular Biology of Plants, 20(3): 351–356.
Vinterhalter B., Ninkovi´ c. s., Cingel A., and Vinterhalter D. (2006). Shoot and root culture of Hsypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS. Biologia Plantarum, 50: 767–770.
Zhou M. L., Zhu X. M., Shao J. R., Tang Y. X., and Wu Y. M. (2011). Production and metabolic engineering of bioactive substances in plant hairy root culture. Applied Microbiology and Biotechnology, 90: 1229-1239.