ORIGINAL PAPER Sucrose-induced osmotic stress affects biomass, metabolite, and antioxidant levels in root suspension cultures of Hypericum perforatum L. Xi-Hua Cui • Hosakatte Niranjana Murthy • Chun-Hua Wu • Kee-Yoeup Paek Received: 30 December 2009 / Accepted: 12 April 2010 / Published online: 14 May 2010 Ó Springer Science+Business Media B.V. 2010 Abstract In this study, we investigated the influence of initial sucrose concentration on the accumulation of bio- mass, phenols, flavonoids, chlorogenic acid, and hypericin in adventitious root cultures of Hypericum perforatum L. Cultures were initiated in shake flasks by using half- strength Murashige and Skoog (MS) medium, 1.0 mg l -1 indolebutyric acid (IBA), 0.1 m g l -1 kinetin, and different concentrations 0, 1, 3, 5, 7, or 9% in w/v) of sucrose and were maintained in darkness. The medium supplemented with 3% (w/v) sucrose resulted in the optimum biomass accumulation, but higher sucrose concentrations (5, 7, and 9%) inhibited biomass accumulation due to the relatively higher osmotic pressure. However, the amount of total phenols, flavonoids, chlorogenic acid, and total hypericin was increased with the roots grown in the medium sup- plemented with 5, 7, and 9% (w/v) sucrose. The antioxidant potential of methanolic extract [1,1-diphenyl-2-picrylhy- drazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline- 6-sulfonic acid; ABTS) radical scavenging activities] of H. perforatum adventitious roots was also assessed and correlated with the metabolite accumulation. Cultures maintained with higher initial sucrose concentration (5, 7, and 9% w/v) showed increased accumulation of phenols, flavonoids, chlorogenic acid, and total hypericin, and this might be due to the osmotic stress at elevated sucrose concentrations. To verify the effect of osmotic stress on lipid peroxidation, the levels of hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA), and proline were determined in the adventitious roots and the results revealed a marked increase in the concentrations of these compounds. These results suggest that optimal adventitious root biomass could be achieved in the MS medium with 3% (w/v) sucrose and increased sucrose concentration resulted in osmotic stress and, in turn, induces the accumulation of secondary metabolites. Keywords Adventitious root cultures Á Antioxidant activity Á Biomass Á Flavonoids Á Hypericum perforatum Á Phenol Abbreviations ABTS 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) DPPH 1,1-diphenyl-2-picrylhydrazyl DW Dry weight FW Fresh weight H 2 O 2 Hydrogen peroxide IAA Indole-3-acetic acid IBA Indolebutyric acid KI Potassium iodide MDA Malondialdehyde MS Murashige and Skoog O 2 Á- Superoxide radical . OH Hydroxyl radical RO . Alkoxyl radical TBA Thiobarbituric acid TCA Trichloracetic acid X.-H. Cui Á H. N. Murthy Á C.-H. Wu Á K.-Y. Paek (&) Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Korea e-mail: paekky@chungbuk.ac.kr H. N. Murthy Department of Botany, Karnatak University, Dharwad 580 003, India 123 Plant Cell Tiss Organ Cult (2010) 103:7–14 DOI 10.1007/s11240-010-9747-z