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In vitro seed germination, callus induction and naphthodianthrones production in Hypricumm triquetrifolium Turra

Hoshyar A. Azeez, Kadhim M. Ibrahim

Department of Biology, College of Science, University of Sulaimani, Sulaimani, Kurdistan Region, Iraq.
College of Biotechnology, Al-Nahrain University, Baghdad, Iraq,


Original:1 August 2016, Revised:15 October 2016, Accepted:20 November 2016, Published online: 20 June 2017


The present study was designed to investigate seed germination and callus induction in vitro and to produce (naphthodianthrones) hypericin and psudohypericin in Hypericum triquetrifolium Turra tissue cultures. Seeds were germinated aseptically on MS medium and water agar medium. Seeds germinated on water agar were better than those germinated on ½ MS medium. Germination percentage increased after treatment with GA3 at 100 mg L-1. Leaf, stem and root explants were excised from in vitro grown seedlings. Callus was initiated on the leaf discs which were previously cultured on MS medium supplemented with Thidiazuron (TDZ) at different concentrations included 1.0, 1.25, 1.5, 2.0, or 2.5 mg L-1 and indole-3-acetic acid (IAA) at 0.5 mgL-1. It was observed that the combination of TDZ at 2 mg L-1 with 0.5 mg L-1 IAA supplemented to MS medium, yielded the highest fresh and dry weights of callus initiated on leaf explants. The same combined mixture was used for callus maintenance, The same components described for callus maintenance medium devoid agar, was used for initiating cell suspension cultures. To define the type and quantity of the secondary metabolites, HPLC was depended. Hypericin and pseudo-hypericin were not detected in calli, and cell suspension cultures initiated on leaf explants, while they were detected in calli and cell suspension cultures initiated on stem and root explants.

Key Words:
Hypericum triquetrifolium, Callus, Hypericin and Psudohypericin, HPLC 



[1] Yadava RNS, and Agarwala, M. "Phytochemical analysis of some medicinal plants". Journal of Phytology, Vol. 3, No. 12, pp. 10-14, (2011).

[2] Robeson, N.K.B. "Studies in the genus Hypericum L. (Clusiaceae) 1. Section 9. Hypericum sensu lato (part 3): subsection 1. Hypericum series 2. Senanensia, subsection 2. Erecta and section 9b. Graveolentia". Syst. Biodivers, Vol. 4, pp.19-98, (2006).

[3] Al-Mukhtar, J.A.H. "Hypericum plant. Directorate plant". Bulletin No. 231. Ministry of Agriculture and Agrarian Reform, Iraq. pp. 2-15, (1975).

[4] Akgoz, Y. "The effects of Hypericum (Hypericacea) species on microorganisns: A review". Int. Res. J. Pharm. Vol. 6, No. 7, (2015).

[5] Ghosian Moghaddam MH., Roghani, M., and Malek, M. "Effect of Hypericum perforatum aqueous extracts on serum lipids, Aminotransferases, and Lipid Peroxidation in Hyperlipidemic Rats". Res Cardiovasc Med. Vol. 5, No. 2, e31326, (2016).

[6] Hosni, K., Msaada, K., Taarit, M.B. and Marzouk, B. "Phonological variations of secondary metabolites from Hypericum triquetrifolium Turra". Biochemical Systematics and Ecology, Vol. 39, pp. 43-50, (2011).

[7] Ghavamaldin, A., Aptin, R., Khalil, P., Mansour, G. and Mariamalsadat, T. "Study of variation of biochemical components in Hypericum perforatum L. growm in north of Iran". J. of Medicinal Plants Res., Vol. 6, No. 3, pp. 366-372, (2012).

[8] Asghari, M., FallahAmoli, H., and Niknezhad, Y. "Comparative Study on the Effect of Elevation Variation in North of Iran on Hypericum perforatum Essence". Biological Forum – An International Journal Vol. 7, No. 1, pp. 973-978, (2015).

[9] Sajjadi, SE., Mehregan, I., and Taheri, M. "Essential oil composition of Hypericum triquetrifolium Turra growing wild in Iran". Res Pharm Sci. Jan-Feb, Vol. 10, No. 1, pp. 90-94, (2015).

[10] Al-Rawi, A. and Chakravarty, H.L. "Medicinal Plants of Iraq". 2nd edition. Ministry of Agriculture and Irrigation. Iraq, Baghdad, Vol. 54, pp. 67-78, (1988).

[11] Vijaya Sree, N., Udayasri, P.V.V., Aswani, K.Y., Ravi, B.B., Phani, K.Y. and Vijay, V.M. "Advancements in the production of secondary metabolites". J. of Natural Products, Vol. 3, pp. 112-123, (2010).

[12] Oluk, E.A., Orhan, S., Karakas, O., Cakir, A. and Gonuz, A. "High efficiency indirect shoot regeneration and hypericin content in ebryogenic callus of Hypericum triqutrifolium Turra". Afric. J. of Biotechnology, Vol. 9, No. 15, pp. 2229-2233, (2010).

[13]Cirak, C. "Seed germination protocols for Ex situ conservation of some Hypericum species from Turkey". Am. J. Plant Physiol., Vol. 2, pp. 287-294, (2007).

[14] Oluk, E.A. and Orhan, S. "Thidiazuron induced micropropagation of Hypericum triqutrifolium Turra". Afric. J. of Biotechnology, Vol. 8, No. 15, pp. 3506-3510, (2009).

[15] Murashige, T. and Skoog, F. "A revised medium for rapid growth and bioassays with tobacco tissue cultures". Physiol Plantarum, Vol. 15, pp. 473-497, (1962).

[16] Karim, Z.J. "In vivo and in vitro studies on (Rosmarinus officinalis L.) secondary metabolites". M.Sc. Thesis. Sulaimani University, College of Science. Biology Dept. pp.28, (2008).

[17] Gopi, G. and Vatsala, T.M. "In vitro studies on effects of plant growth regulators on callus and cell suspension culture biomass yield from Gymnema sylvestre R.Br". African J. of Biotech., Vol. 5, No. 12, pp. 1215-1219, (2006).

[18] Smelcerovic, A., Spiteller, M. and Zuehlke, S. "Comparison of methods for the exhaustive extraction of hypericin, flavonoids, and hyperforin from Hypericum perforatum L". J. Agric. Food. Chem., Vol. 54. pp. 2750-2753, (2006).

[19] Gulcin, I., Kirecci, F. and Akkemik, E. "Antioxidant and antimicrobial activities of an aquatic plant: Duckweed (Lemna minor L)". Turk. J. Biol., Vol. 34, pp. 175-188, (2010).

[20] Alali, F., Tawaha, K. and Al-Elemat, T. "Determination of hypericin content in Hepericum triqutrifolium Turra. (Hypericaceae) growing wild in Jordan". Natural Product Res., Vol. 18, pp.147-151. (2004).

[21] Tasis, E.C., Boeren, S., Exarchou, V., Troganis, A.N., Vervoort, J. and Gerothanassis, I.P. "Identification of the major constituents of Hypericum perforatum by LC\SPE\NMR and \or LC\MS". Phytochemistry, Vol. 68, pp. 383-393, (2007).

[22] Danova, K. "Production of polyphenolic compounds in shoot cultures of Hypericum species characteristic for the Balkan flora". Botanica Serbica., Vol. 34, No. 1, pp. 29-36, (2010).

[23] Duncan, D.B. "Multiple range and multiple F-test", Biometrics., Vol. 11, pp.1-42, (1995).

[24] Baskin, C.C. "A classification system for seed dormancy". Seed Sci. Res., Vol. 14, 1-11.

[25] Campbel, l.M.H."Germination, emergence and seedling growth of Hypericum perforatum". Weed Res., Vol. 25, pp. 259-266, (2004).

[26] Ash, J.E., Groves, R.H. and Willis, A.J. "Seed Ecology of Hypericum gramineum, an Australian forb". Aust .J. Bot., Vol. 45, pp. 1009-1022, (1998).

[27] Bertelle, F.M.L., Beatriz, P.M. and Augusto, L.A. "Light, temperature and potassium nitrate in the germination of Hypericum perforatum L. and H. Brasiliense Choisy seeds". Bragantia, Vol. 63, pp. 193-199, (2004).

[28] Cirak, C., Radusiene, J., Janulis, V. and Ivanauskas, L. "Secondary metabolites in Hypericum perforatum: variation among plant parts and phonological stages". Bot. Helv., Vol. 117, pp. 29-36, (2007).

[29] Camas, N. and Caliskan, O. "Breaking seed dormancy in Hypericum Leptophyllum Hochst., an endemic Turkish species". J. Med. Plants Res., Vol. 5, No. 32, pp. 6968-6971, (2011).

[30] Cirak, C., Ayan, A. and Kevseroglu, K. "The effect of light and some pre-soaking treatments on germination rate of St. John's wort (Hypericum perforatum L.) seeds". Pak. J. Biol. Sci., Vol. 7, pp. 182-186, (2004a).

[31] Verma, S.K. "A textbook of Plant Physiology and Biochemistry". 5th ed. Chand & Company Ltd. Ram Nagar, New Delhi, India, pp. 336-343, (2006).

[32] Kyauk, H., Hopper, N.W. and Brigham, R.D. "Effects of temperature and pre-soaking on germination, root length of sesame (Sesamum indicum L)". Environ. Exp. Bott., Vol. 35, pp. 345-351, (1995).

[33] Ali, A. and Helal, A. "Studies on germination of Rumex dentatus L. seeds". J. Arid. Environ. Vol. 33, pp. 39-47, (1996).

[34] Wang, Q., Feng, Z. and Smith, D. "Application of GA and Kinetin to improve corn and soybean seedling emergence at low temperature". Environ. Exp. Bott., Vol. 36, pp. 377-383, (1996).

[35] Carrillo, Y.O., Guzman, J.M.B., Victor, S.C., Esther, O. and Alma, S. "Germination of the hard seed coated Opuntia tomentosa, a cacti from the Mexico valley". J. Arid. Environ., Vol. 55, pp. 29-42, (2003).

[36] Cerabolini, B., Rossella, A., Roberta, M., Ceriani, S.P. and Barbara, R. "Seed germination and conservation of endangered species from the Italian Alps, Physoplexis comosa and Primula glaucescens". Biol. Conserv., Vol. 117, pp. 351-356, (2004).

[37] Booth, D.T. and Sowa, S. "Respiration in dormant and non-dormant bitterbrush seeds". J. Arid. Environ., Vol. 48, pp. 35-39, (2001).

[38] Cirak, C., Ayan, A., Kevseroglu, K., and Caliskan, O. "Germination rate of St. John’s wort (Hypericum perforatum L.) seeds exposed to different light intensities and illumination periods". J. Biol. Sci., Vol. 4, pp. 279-282, (2004b).

[39] Yazaki, K. and Okuda, T." Procyanins in callus and multiple shoots of Hypericum erectum". Planta Med., Vol. 56, pp. 490-491, (1990).

[40] Cardoso, M.A. and Oliveira, D.E. "Tissue culture of Hypericum brasiliense Choosy: shoot multiplication and callus induction". Cell Tiss. and Org. Cult., Vol. 44, pp. 91-94, (1996).

[41] Ferrari, F., Monacelli, B. and Messana, I. "Comparison between in vivo and in vitro metabolite production of Morus nigra". Planta. Med., Vol. 65, pp. 85-87, (1999).

[42] Schimdt, W., Peters, S. and Beerhues, L. "Xanthone 6-hydroxylase from cell cultures of Centaurium erythraea and Hypericum androsaemum L". Phytochemistry, Vol. 53, pp. 427-431, (2000).

[43] Guo, B., Abbasi, B.H. Zeb, A., Xu, L.L. and Wei, Y.H. "Thidiazuron: A multi- dimensional plant growth regulator". African J. of Biotech. Vol. 10, No. 45, 8984-9000, (2011).

[44] Murthy, B.N.S., Murch, S.J. and Saxena, P.K. "Thidiazuron: a potent regulator of in vitro plant morphogenesis In Vitro Cell". Dev. Biol. Plant, Vol. 34, pp. 267–275, (1998).

[45] Rani, N.S., Balaji, K. and Veeresham, C. "Production of hypericin from tissue culture of Hypericum perforatum", Indian J. Parm. Sci., Vol. 63, No. 5, pp. 431-433, (2001).

[46] Dias, A.C.P., Seabra, R.M., Andrade, P.B. and Fernandes-Ferria, M. "The development and evaluation of an HPLC-DAD method for the analysis of the phenolic fractions from in vivo and in vitro biomass of Hypericum species". J. of Liquid Chromatography & Related Techn. Vol. 22, No. 2, pp. 215-227, (2007).

[47] Karakas, O., Cetin, H., and Onay, A. "Determination of Hypericin Content in Callus and Cell Suspension Cultures of Hypericum triquetrifolium Turra". Advances in Zoology and Botany Vol. 3, No. 4, pp. 184-189, (2015).

[48] Gadzovska-Simic, S., Tusevski, O., Antevski, S., Atanasova-pancevska, N., Petreska, J., Stefova, M., Kungulovskim, D. and Spasenoski, M. "Secondary metabolite production in Hypericum perforatum L. cell suspension upon elicitation with fungal mycelia from Aspergillus flavus". Arch. Biol. Sci., Belgrade, Vol. 64, No. 1, pp. 113-121, (2012).

[49] Karakas, O., Toker, Z., Tilkat, E., Ozen, H.C. and Ony, A. "Effects of different concentration of benzylaminopyrine on shoot regeneration and hypericin content in Hypericum triquetrifolium Turra". Natural Prod. Res. part B: Bioactive natural product, Vol. 23, pp. 1459-1465, (2009).

[50] Pasqua, G., Avoto, p., Monacelli, B., Santamaria, A.R. and Argentieri, M.P. "Metabolites in cell suspension cultures, calli, and in vitro regenerated organs of Hypericum perforatum CV. Topas". Plant Science, Vol. 165, pp. 977-982, (2003).

[51] Gadzovska, S., Maury, S., Ounnar, S., Righezza, M., Kascakova, S. and Refregiers, M. "Identification and quantification of hypericin and psudohypericin in different Hypericum perforatum L. in vitro cutures". Plant physiology and Biochemistry, Vol. 43, pp. 591-601, (2005).

[52] Ayan, A.k., and Cirak, C. "Hypericin and psudohypericin contents in some Hypericum species growing in Turkey". Pharmaceutical Biology, Vol. 46, No. 4, pp. 288–291, (2008 a).

[53] Murch, S.J., Choffe, K.L., Victor, J.M.R., Slimmon, T.Y., Krishna, R. and Saxena, P.K. "Thidiazuron-induced plant regeneration from hypocotly cultures of St. John 's wort (Hypericum perforatum L)". Plant Cell Rep., Vol. 19, pp. 576-581, (2000).