Feasibility of hairy root induction in two Hyssopus species ‎‎(H. officinalis and H. angustifolius) ‎

Document Type : Full Paper


1 Ph.D. Candidate, Faculty of Agriculture, Tabriz University, Tabriz, Iran‎

2 Associate Professor, Agricultural and Natural Resources University, Sari, Iran

3 Assistant Professor, Genetics Research Center, Agricultural and Natural Resources University, Sari, Iran

4 Associate Professor, Faculty of Agriculture, Tabriz University, Tabriz, Iran‎


In the present study, hairy root induction from leaf and stem explants in two Hyssopus species (H. officinalis and H. angustifilius) was assessed using four Agrobacterium rhizogenes strains including A4, ATCC15834, LB9404 and 2656 on three culture media (MS, ½ MS, B5) as a factorial experimental based on a completely randomized design with three replicates. Almost all leaf and stem explants produced hairy roots. A fragment of transferred rol B was amplified through PCR from the genomic DNA extracted from transformed roots. Different A. rhizogenes strains and culture media and Hyssopus species had significant effects on hairy root percentage and root length. The highest rooting percentage (%71) and root length (4.7 cm) were resulted from stem explants of H. angustifolius co-cultivated with the strain ATCC15834 on MS culture medium. While, the leaf explantsof H. officinalis co-cultivated with the strain A4 on MS culture medium showed the rooting percentage of 55% and the root length of 1.6 cm at most. The MS medium was the best culture medium for both species. These results can be useful in genetic manipulation of hyssop and its hairy root culture to produce high-value secondary metabolites.


  1. Akramian, M., Fakhr Tabatabaei, S.M. & Mirmasoumi, M. (2008). Virulence of different strains of Agrobacterium rhizogenes on genetic transformation of four Hyoscyamus species. American-Eurasian Journal of Agricultural and Environmental Science, 3(5), 759-763.
  2. Asghari, Gh. & Salimian Rizi, T. (2008). The influence of fructose, glucose and sucrose on flavolignans formation in Silybum marinum callus culture. Journal of Medicinal Plants, 7, 16-23.
  3. Bonhomme, V., Laurain-Mattar, D. & Fliniaux, M.A. (2000). Effects of the rol C gene on hairy root: induction development and tropane alkaloid production by Atropa belladonna. Journal of Natural Products, 63, 1249-1252.
  4. Brijwal, L. & Tamta, S. (2015). Agrobacterium rhizogenes mediated hairy root induction in endangered Berberis aristata DC. Springer Plus, 4, 443-453.
  5. Chandran, R.P. & Potty, V. (2008). Induction of hairy roots through the mediation of four strains of Agrobacterium rhizogenes on five host plants. Indian Journal of Biotechnology, 7, 129-132.
  6. Chen, H. & Chen F. (2000). Induction ofInfluence of different strains of Agrobacterium phytoalexin formation in crown gall and hairy rootrhizogenes on induction of hairy roots and culture of Salvia miltiorrhiza by methyl viologen. Biotechnology Letters, 22, 715-20.
  7. Chilton, Md., Tepfer, Da., Petit, A., David, C. Casse-Delbart & Tepe, G. (1982), Agrobacterium rhizogenes insert T-DNA into the genomes of the host plant root cells. Nature, 295, 432-4.
  8. Christensen, B., Sriskandarajah, S. & Müller, R. (2009). Transformation of Hibiscus rosa-sinensis L. by Agrobacterium rhizogenes. Journal of Horticultural Science and Biotechnology, 84, 204-208.
  9. Combard, A. & Baucher, M.F. (1988). A common organization of the T-DNA genes expressed in plant hairy roots induced by different plasmids of Agrobacterium rhizogenes. Plant Molecular Biology, 10, 499-509.
  10. Dessaux, Y., Petit, A. & Tempe, J. (1993). Chemistry and biochemistry of opines, chemical mediators of parasitism. Phytochemistry, 34, 31-38.
  11. Do, CB. & Cormier, F. (1990). Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions. Plant Cell Reports, 9, 143-146.
  12. Doyle, J.J. & Doyle, J.L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bull, 19, 11-15.
  13. Dzhumaev, Kh.K. (1986). Dynamics of essential oil accumulation in Hyssopus seravschanicus. Uzbekskii Biologicheskii Zhurnal, 6, 31-33.
  14. Ercan, A.G., Taşkin, K.M., Turgut, K. & Yüce, S. (1999). Agrobacterium rhizogenes-mediated hairy root formation in some Rubia tinctorum L. populations grown in Turkey. Turkish Journal of Botany, 23, 373-378.
  15. Garg, S., Naqvi, A.A., Singh, A., Ram, G. & Kumar, S. (1999). Composition of essential oil from an annual crop of Hyssopus officinalis grown in Indian plains. Flavour and Fragrance Journal, 14, 170-172.
  16. George, E.F., Hall, M.A. & Klerk, G.J.D. (2008). Plant propagation by tissue culture. Volume 1. Springer, Netherlands.
  17. Gollapudi, S., Shara, H.A., Aggarval, S., Byers, L.D., Ensley, H.E. & Gupta S. (1995). Isolation of a previously unidentified polysacharide (MAR-10) from Hyssopus officinalis that exhibits strong activity agains human imunodeficiency virus type 1. Biochemical and Biophysical Research Communications, 210, 145-151.
  18. Gorunovic, M., Bogavac, P., Chalchat, J. & Chabardi J. (1995). Essential oil of Hyssopus officinalis L. Lamiaceae of Montenegro original. Journal of Essential Oil Research, 7, 39-43.
  19. Hasanloo, T. (2006). Study of some secondary metabolites in Silybum marianum from different area of Iran and its cell and tissue culture for production of silymarin. Ph.D. Thesis. Iranshahr University Iran.
  20. Hu, Z.B. & Du, M. (2006). Hairy root and its application in plant genetic engineering. Journal of Integrative Plant Biology, 48, 121-127.
  21. Ishida, J.K., Yoshida, S., Ito, M., Namba, S. & Shirasu, K. (2011). Agrobacterium rhizogenes- mediated transformation of the parasitic plant Phtheirospermum japonicum. PloS One, 6(10), e25802.
  22. Kittipongpatana, N., Darryl, L., Davis & John, R. Porter. (2002). Methyl jasmonate increases the production of valepotriates by transformed root cultures of Valerianella locusta. Plant Cell Tissue and Organ Culture, 71, 65 - 75.
  23. Kizil, S., Toncer, O., Ipek, A., Arslan, N., Saglam, S. & Khawar, K.M. (2008). Blooming stages of Turkish hyssop (Hyssopus officinalis L.) affect essential oil composition. Acta Agriculture Scandinavia, Section B-Soil and Plant Science, 58(3), 273-279.
  24. Kizil, S., Hasimi, N., Tolan, V., Kilin, E. & Karatas H. (2010). Chemical composition, antimicrobial and antioxidant activities of hyssop (Hyssopus officinalis L.) essential oil. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 38(3), 99-103.
  25. Kochan, E., Wysokinska, H. & Chmiel A. (1999). Rosmarinic acid and other phenolic acids in hairy roots of Hyssopus officinalis. Der Naturforscher. 54c, 11.16182-16185.
  26. Kuzovkina, I.N. & Schneider, B. (2006). Progress in botany. Springer, Berlin, Heidelberg. (pp: 275-314).
  27. Lee, S.Y., Xu, H., Kim, Y.K. & Park, S.U. (2007). Rosmarinic acid production in hairy root cultures of Kuntze. World Journal of Microbiology & Biotechnology, 24, 969-972.
  28. Li, W., Koike, K., Asada, Y., Yoshikawa, T. & Nikaido, T. (2005). Rosmarinic acid production by Coleus forskohlii hairy root cultures. Plant Cell, Tissue and Organ Culture, 80, 151-155.
  29. Mahesh, A. & Jeyachandran, R. (2011). Agrobacterium rhizogenes-mediated hairy root induction in Taraxacum officinale and analysis of sesquiterpene lactones. Plant Biosystems, 145, 620-626.
  30. Mitic, V. & Dordevic, S. (2000). Essential oil composition of Hyssopus officinalis L. cultivated in Serbia. Facta Universitatis- Series: Physics Chemistry and Technology, 2, 105-108.
  31. Mojab, F., Mosadegh, M., Monsefesfahani, H.R. & Najari, A. (2002). Examination of retail journalism and identification of components essential oil Hyssopus officinalis. Journal of Pazhohandeh, 8(2), 9-15. (In Farsi)
  32. Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473-497.
  33. Nanova, Z., Slavova, Y., Nenkova, D. & Ivanova, I. (2007). Microclonal propagation of Hyssopus officinalis. Bulgarian Journal of Agricultural Science, 13, 213- 219.
  34. Nguyen, C., Bourgaud, F., Forlot, P. & Guckert, A. (1992). Establishment of hairy root cultures of Psoralea species. Plant Cell Reports, 11, 424-427.
  35. Omidbagi, R., Borna, F., Borna, T. & Inotai, K. (2009). Sowing dates affecting on the essential oil content of dragonhead (Dracocephalum moldavica l) and its constituents. Journal of Bearing Plants, 15(5), 580-5.
  36. Ooi, C.T., Syahida, A., Stanslas, J. & Maziah, M. (2013). Efficiency of different Agrobacterium rhizogenes strains on hairy roots induction in Solanum mammosum. World Journal of Microbiology & Biotechnology, 29(3), 421-430.
  37. Ozer, H., Sahin, F., Kilic, H. & Gulluce, M. (2005). Essential oil composition of Hyssopus officinalis L. subsp. Angustifolius (Bieb.) Archangelic from Turkey. Flavour and Fragrance Journal, 20, 42 44.
  38. Pawar, P.K. & Maheshwari, V.L. (2004). Agrobacterium rhizogenes mediated hairy root induction in two medicinally important members of family Solanaceae. Indian Journal of Biotechnology, 3, 414-417.
  39. Petersen, S.G., Stummann, B.M., Olesen, P. & Henningsen, K.W. (1989). Structure and function of rootinducing (Ri) plasmids and their relation to tumorinducing (Ti) plasmids. Physiologia Plantarum. 77, 427-435.
  40. Pirian, K., Piri, K. & Ghiyasvand, T. (2012). Hairy roots induction from Portulaca oleracea using Agrobacterium rhizogenes to noradrenaline’s production. International Journal of Basic and Applied Sciences, 3, 642-649.
  41. Rechinger, K.H. (1982). Labiatae. Flora Iranica. Akademische Drucku-Verlagsanstalt, Graz, (598 pp).
  42. Rechinger, K.H. (1984). Alliaceae. Flora Iranica, Akademische Drucku-Verlagsanstalt, Graz, (85 pp).
  43. Said-Al Ahl, H., Abbas, Z., Sabra, A. & Tkachenko, K. (2015). Essential oil composition of Hyssopus officinalis cultivated in Egypt. International Journal of Plant Research, 1 (2), 49-53
  44. Sambrook, J. & Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, (2344 pp).
  45. Slightom, J.L., Durand-Tardif, M., Jouanin, L. & Tepfer, D. (1986). Nucleotide sequence analysis of TLDNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading frames.  Journal of Biological Chemistry, 261, 108-121.
  46. Soleimani, T., Keyhanfar, M., Piri, K.H. & Hasanloo, T. (2012). Hairy root induction in burdock (Arctium lappa L.). Journal of Medicinal Plants, 44, 176-184.
  47. Sreeramanan, S., Vinod, B., Sashi, S. & Xavier, R. (2008). Optimization of the transient Gusa gene transfer of Phalaenopsis Violacea orchid via Agrobacterium tumefaciens: an assessment of factors influencing the efficiency of gene transfer mechanisms. Advances in Natural & Applied Sciences, 2, 77-89.
  48. Srivastava, S. & Srivastava, A.K. (2007) Hairy root culture for mass-production of high-value secondary metabolites. Critical Reviews in Biotechnology, 27, 29-43.
  49. Tada, H., Murakami, Y., Omoto, T., Shimomura, K. & Ishimaru, K. (1996). Rosmarinic acid and related phenolics in hairy root cultures of Ocimum basilicum. Phytochemistry, 42, 431-434.
  50. Tao, J. & Li, L. (2006). Genetic transformation of Torenia fournieri L. mediated by Agrobacterium rhizogenes. South African Journal of Botany, 72, 211-216.
  51. Tepfer, D. (1990). Genetic transformation using Agrobacterium rhizogenes. Plant Physiology, 79, 140-146.
  52. Vallejo, M., Herraiz, J., Perez-Alonso, M. & Velasco Negueruela, A. (1995). Volatile oil of Hyssopus officinalis L. from Spain. Journal. Essential Oil Research, 7, 567-568.
  53. Vanhala, L., Hiltunen, R. & Oksman-Caldentey, K.M. (1995). Virulence of different Agrobacterium strains on hairy root formation of Hyoscyamus muticus. Plant Cell Reports, 14, 236-240.
  54. Wang, B., Zhang, G., Zhu, L., Chen, L. & Zhang, Y. (2006). Genetic transformation of Echinacea purpurea with Agrobacterium rhizogenes and bioactive ingredient analysis in transformed cultures. Colloids and Surfaces B: Biointerfaces, 53, 101-104.
  55. Wang, Q.J., Zheng, L.P., Yuan, H.Y. & Wang, J. (2013). Propagation of Salvia miltiorrhiza from hairy root explants via somatic embryogenesis and tanshinone content in obtained plants. Industrial Crops and Products, 50, 648-653.
  56. Wesołowska, A., Jadczak, D. & Grzeszczuk, M. (2010). Essential oil composition of hyssop (Hyssopus officinalis L.) cultivated in north-western Poland. Herba Polonica, 56 (1), 57-65.