Effect of elicitation on antioxidant activity and production of tropane alkaloids in Hyoscyamus reticulatus hairy root cultures

Document Type: Original paper


1 Department of Horticulture, Faculty of Agriculture, Urmia University, Urmia, Iran.

2 Horticulture Department, Agriculture Faculty, Urmia University, 165 Urmia, Iran.

3 Agronomy Department, Agriculture Faculty, Urmia University, Iran.


Background and objectives: Hyoscyamus reticulatus contains two distinguished tropane alkaloids, hyoscyamineand scopolamine and both of the compounds possesspotential acute or chronic toxicity. In the present study, a simple and efficient transformation system was established for in vitro hairy roots induction in Hyoscyamus reticulatus.
Methods: Effect of different factors including Agrobacterium rhizogenes strains (A7, 15834, A13 and D7), various explant types (cotyledon, hypocotyl, two weeks old leaf, four weeks old leaf, two weeks old internode and four weeks old internode), two inoculation methods (immersion and injection) and four types of culture media (MS, ½ MS, ¼ MS and B5) on hairy roots induction efficiency in Hyoscyamus reticulatus were tested. In the second part of the experiments, elicitations with different concentrations of colchicine (0, 0.01, 0.03 and 0.05% w/v) and different UV-B exposure time (0, 3, 6 and 9 min) were used to analyze hyoscyamineand scopolamine production.  Transgenic status of hairy roots was confirmed by PCR using specific primers of the rolB gene. The total antioxidant activity was evaluated by DPPH) method. Results: Induction of hairy roots in H. reticulatus was affected by bacterial strain and explant type. A7 strain and cotyledon explants were detected as the best strain and explants for induction of hairy root in H. reticulatus. Hairy roots growth was significantly affected by medium type. The highest fresh weight was produced in MS and B5 medium.Fresh and dry weight of hairy root reached 1.44 and 0.134 mg at 0.05 percentage of colchicine after 48 h, respectively but in UV-B treatment fresh weight was decreased. In addition, antioxidant activity of hairy root samples treated with colchicine and UV-B increased to 27% (0.05 colchicine) and 26% (UV-B 9 min), respectively compared to the antioxidant activity level in non-transgenic roots (12%) and transgenic roots (18%). The highest amount of hyoscyamine and scopolamine (0.58% and 1.9 %) found in elicited hairy root cultures was 3.2 and 5.1 folds higher than the non-transformed roots (0.18% and 0.37%), respectively. B5 and MS medium were detected as the best appropriate medium for growth of H. reticulatus hairy roots. Antioxidant activity in elicited hairy roots with elicitors increased in comparison to the antioxidant activity level in transgenic and non-transgenic hairy roots. Conclusion: Hairy root lines developed and elicited in this study can be used to investigate the production of pharmaceutically important metabolites of H. reticulatus.


[1] Sajeli B, Sahai M, Suessmuth R, Asai T, Hara N, Fujimoto Y. Hyosgerin, a new optically active coumarinolignan, from the seeds of Hyoscyamus niger. J Chem Pharm Bull. 2006; 54(4): 538-541.

[2] Winston D. An introduction to herbal medicine. Nat Med. 1994; 64: 346-353.

[3] Li GQ, Duke CC, Roufigalis BD. The quality and safety of Traditional Chinese Medicines. Aust Prescr. 2003; 26(6): 128-130.

[4] Vike GM, Ufberg JW, Harrigan RA, Chan TC. Evaluation and treatment of acute urinary retention. J Emerg Med. 2008; 35(2): 193-198.

[5] Hashimoto T, Hayashi A, Amano Y, Kohno J, Iwanari H, Usuda S, Yamada Y. Hyoscyamine 6β-hydroxylase, an enzyme involved in tropane alkaloid biosynthesis, is localized at the pericycle of the root. J Biol Chem. 1991; 266(7): 4648-4653.

[6] Hashimoto T, Yamada Y. Hyoscyamine 6α-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures.  Plant Physiol. 1986; 81(2): 619-625.

[7] Hartmann T, Witte L, Oprach F, Toppel G. Reinvestigation of the alkaloid composition of Atropa belladonna plants, root cultures and cell suspension cultures. Planta Med. 1986; 52(5): 390-395.

[8] Maldonado-Mendoza IE, Ayora-Talavera T, Loyola-Vargas VM. Tropane alkaloid production in root cultures of Datura stramonium. In vitro Cell Dev Biol Plant. 1992; 28(2): 67-72.

[9] Sevón N, Oksman-Caldentey KM. Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids. Planta Med. 2002; 68: 859-868.

[10] Guillon S, Temouillaux-Guiller J, Pati PK, Rideau M, Gantet P. Hairy root research: recent scenario and exciting prospects. Curr Opin Plant Biol. 2006; 9(3): 341-346.

[11] Gangopadhyay M, Chakraborty D, Bhattacharya S. Regeneration of transformed plants from hairy root of Plumbago indica. Plant Cell Tiss Org. 2010; 102(1): 109-114.

[12] Giri A, Narasu ML. Transgenic hairy roots: recent trends and applications. Biotechnol Adv. 2000; 18(1): 1-22.

[13] Banerjee S, Singh S, Rahman LU. Biotransformation studies using hairy root cultures, a review. Biotechnol Adv. 2012; 30(3): 461-468.

[14] Doran PM. Properties and applications of hairy-root cultures. In: Okasman-Caldenty KM, Barz WH Eds. Plant biotechnology and transgenic plants. New York: Marcel Dekker Inc., 2002.

[15] Cho HJ, Widholsm JM, Tanaka N, Nakanishi Y, Murooka Y. Agrobacterium rhizogenes mediated transformation and regeneration of the legume Astragalus sinicus (Chinese milk vetch). Plant Sci. 1998; 138(1): 53-65.

[16] Pirian K, Piri Kh, Ghiyasvand T. Hairy roots induction from Protulaca oleracea using Agrobacterium rhizogenes to noradenaline production. Intl Res J Appl Basic Sci. 2012; 3(3): 642-649.

[17] Sharafi A, Hashemi Sohi H, Mousavi A, Azadi P, Razavi Kh, Otang Nuti V. A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum. Plant Cell Tiss Org. 2013; 113(1): 1-9.

[18] Sharafi A, Hashemi Sohi H, Mirzaee H, Azadi P. In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss.  Physiol Mol Biol Plants. 2014; 20(4): 487-494.

[19] Sharafi A, Hashemi Sohi H, Mousavi A, Azadi P, Dehsara B, Hosseini B. Increasing morphinan alkaloid production by over-expressing salutaridinol 7- acetyltransferase in Iranian poppy hairy roots. World J Microb Biot. 2013; 29(11): 2125-2131.

[20] Sharafi A, Sohi HH, Azadi P, Sharafi AA. Hairy root induction and plant regeneration of medicinal plant Dracocephalum kotschyi. Physiol Mol Biol Plants. 2014; 20(2): 257-262.

[21]  Samadi A, Carapetian J, Heidary R, Gafari M, Hssanzadeh A. Hairy root induction in Linum mucronatum spp. mucronatum an anti-tumor lignans production plant. Not Bot Hort Agrobot Cluj. 2012; 40(1): 125-131.

[22] Nourozy E, Hosseini B, Hassani A. A reliable and efficient protocol for induction of hairy roots in Agastache foeniculum. Biologia. 2014; 69(7): 870-879.

[23] Valimehr S, Sanjarian F, Sohi HH, Sharafi A, Sabouni F. A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma. Physiol Mol Biol Plants. 2014; 20(3): 351-356.

[24] Shinde AN, Malpathak N, Fulzele DP. Impact of nutrient components on production of the phytoestrogens daidzein and genistein by hairy roots of Psoralea corylifolia. J Nat Med. 2010; 64(3): 346-353.

[25] Ebel J, Scheel D. Signals in host-parasite interactions. In: Carroll GC, Tudzynski P. eds. The Mycota. Plant relationships, part A. Berlin: Springer-Verlag, 1997.

[26] Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum. 1962; 15(3): 473-497.

[27] Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 1997; 50(1): 151-158.

[28] Khan S, Irfan QM, Kamaluddin AT, Abdin MZ. Protocol for isolation of genomic DNA from dry and fresh roots of medicinal plants suitable for RAPD and restriction digestion. Afr J Biotechnol. 2007; 6(3): 175-178.

[29] Chiou A, Karathanos VT, Mylona A, Salta FN, Preventi F, Andrikopoulos NK. Currants (Vitis vinifera L.) content of simple phenolics and antioxidant activity. Food Chem. 2007; 102(2): 516-522.

[30] Petit A, David C, Dhal GA, Ellis JG, Guyon P, Casse Debart F, Tempe J. Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol Gen Genet. 1983;190(2):204-214.

[31] Isogai A, Fukuchi N, Hayashi M, Kamada H, Harada H, Suzuki A. Mikimopine, an opine in hairy roots of tobacco induced by Agrobacterium rhizogenes. Phytochemistry. 1990; 29(10): 3131-3134.

[32] Vergauwe A, Van Geldre E, Inze D, Vanmontagu M, Van Deneeckhout E. Factors influencing A. tumefaciens mediated transformation of Artemisia annua L. Plant Cell Rep. 1998; 18(1): 105-110.

[33] Sujatha G, Zdravkovic-Korac S, Calic D, Flamini G, Ranjitha Kumari BD. High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia vulgaris: hairy root production and essential oil analysis. Ind Crop Prod. 2013; 44: 643-652.

[34] Yonemitsu H, Shimomura K, Satake M, Mochida S, Tanaka M, Endo T, Kaji A. Lobeline production by hairy root culture of Lobelia inflate L. Plant Cell Rep. 1990;9(6): 307-310.

[35] Trypsteen M, Van Lijsebettens M, Van Severen R, Van Montagu M. Agrobacterium rhizogenes mediated transformation of Echinacea purpureaPlant Cell Rep. 1991;10(2): 85-89.

[36] Pawar PK, Matheshwari VL. Agrobacterium rhizogenes mediated hairy root induction in two medicinally important of family. Ind J Biotechnol. 2003; 3(3): 414-417.

[37] Wolter KE, Skoog F. Nutritional requirements of Fraxinus callus cultures. Am J Bot. 1966; 53(3): 263-269.

[38] Boitel CM, Gontier E, Laberche JC, Ducrocq C, Sangvan-Norreel BS. Inducer effect of Tween 20 permeabelization treatment used for release of stored alkaloids in Datura innoxia Mill. hairy root cultures. Plant Cell Rep. 1996; 16(3), 241-244.

[39] Drewes F, Staden E, Van J. Initiation of and solasodine production in hairy root cultures of Solanum mauritianum Scop. Plant Growth Regulation. 1995; 17(1): 27-31.

[40] Sudha CG, Reddy BO, Ravishankar GA, Seeni S. Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f., a rare and endemic medicinal plant. Biotechnol Lett. 2003; 25(8): 631-636.

[41] Sivakumar G, Yu KW, Hahn EJ, Paek KY. Optimization of organic nutrients for ginseng hairy roots production in large-scale bioreactors. Curr Sci. 2005; 89(4): 641-649.

[42] Palazon J, Mallol A, Eibl R, Lettenbauer C, Cusido RM, Pinol MT. Growth and ginsenoside production in hairy root cultures of Panax ginseng using a novel bioreactor. Planta Med. 2003; 69(4): 344-349.

[43] Tambong JT, Sapra VT, Garton S. In vitro induction of tetraploids in colchicine-treated cocoyam plantlets. Euphytica. 1998; 104(3): 191-197.

[44] Dhawan OP, Lavania UC. Enhancing the productivity of secondary metabolites via induced polyploidy: A review. Euphytica. 1996; 87(2): 81-89.

[45] Adaniya S, Shirai D. In vitro induction of tetraploid ginger (Zinger officinalis Roscoe) and its pollenfertility germinability. Sci Hort. 2001; 88(4): 277-287.

[46]  Bornman JF, Reuber S, Cen YP, Weissenbck G. Ultraviolet radiation as a stress factor and the role of protective pigments. In: Lusden P, Ed. Plants and UV-B. Responses to environmental change, society for experimental biology seminar series 64. Cambridge: Cambridge University Press, 1997.

[47] Katerova Z, Todorova D, Tasheva K, Sergiev I. Influence of ultraviolet radiation on plant secondary metabolite production. Genet Plant Physiol. 2012; 2(3-4): 113-144.

[48] Takahashi Y, Hitaka Y, Kino-oka M, Taya M, Tone S. Evaluation of growth property of red beet hairy roots depending on condition of inoculation and its application to culture control with fuzzy logic theory. Biochem Eng J. 2001; 8(2): 121-127.

[49] Dijkestra H, Speckmann GJ. Autotetraploidy in caraway (Carum carvi L.) for the increase of aetheric oil content of the seed. Euphytica. 1980; 29(1): 89-96.

[50] Saharkhiz MJ. The effects of some environmental factors and ploidy level on morphological and physiological characteristics of feverfew (Tanacetum parthenium L.) medicinal ornamental plant. Ph.D. thesis, Tarbiat Modarres University, Tehran, Iran, 2007.