determination of total hypericin content in callus and cell suspension cultures of Hypericum triquetrifolium Turra

The aim of this study was to investigate an efficient suspension culture allowing to improve total hypericin production from calli and cell suspension cultures in Hypericum triquetrifolium Turra. in vitro cultures. Growth parameters such as packed cell volume (PCV), fresh weight and dry weight were determined. The role of plant growth regulator treatments has been particularly studied on suspension culture development.To start with, it was determined that what is the most convenient explant type for the establishment of callogenesis. To do this, total hypericin content of leaves, flowers, stems and root of grown in natural habitat wild H. triquetrifolium was investigated. Among the different parts of H. triquetrifolium, the leaf showed the highest hypericin content with a concentration of 1.95 ± 0.0027 mg/g. The least hypericin content with a concentration of 0.08 ± 0.0015 mg/g was obtained from the roots of H. triquetrifolium. To induce callus formation, leaf explant excised from sterile germinated seedlings were cultured in MS medium containing the following PGR combinations: 1 mg/l BA + 2 mg/l NAA, BA (1 mg/l) + NAA (0.5 mg/l), 1 mg/l BA + 0.4 mg/l 2,4-D, BA (0.06 mg/l) + 2,4-D (0.1 mg/l), Kinetin (0.9 mg/l) + 2,4-D (3 mg/l) and Kinetin (0.4 mg/l) + 2,4-D (2 mg/l) The most suitable rate of calli formation (92.3 %) obtained from MS medium containing 1 mg/l BA + 2 mg/l NAA. Calli obtained from this medium was used as main material for the initiation of cell suspension cultures.After 3-5 subcultures, calli were inoculated into 100 ml flasks containing 25 ml MS medium without agar containing 3% sucrose, 1 mg/l BA + 2 mg/l NAA and they were maintained on a rotary shaker at 100 rpm. After 14 days in liquid culture cell aggregates were separated into three different size groups using the test sieves of mesh sizes 500 µm, 250 µm and 100 µm.Cell growth was measured by date determining, the packed cell volume (PCV-v/v). Two different PGR combination were used to investigate growth parameters at 4, 8, 12, 16 and 20 days. of suspension cultures. Growing cells in MS medium containing 1 mg/l BA + 2 mg/l NAA reached the highest growth rate on 20th day of culture and it was determined that there is an increase approximately 2.33-fold for PCV, 5.5-fold for wet weight and 6.30-fold for dry weight. MS medium containing 1mg/l BA + 0.4 mg/l 2, 4-D showed a similar increase in the growth parameters of cells in MS medium containing 1mg/ BA + 2 mg/g NAA.Although in the in vitro adapted cultures of H. triquetrifolium contain much lower amount of total hypericin (0.0018 ± 0.00026 mg/g) than in vivo grown leaves (1.95 ± 0.0027 mg/g), they appear to be promising system for hypericin production.Key words: Hypericum triquetrifolium Turra., Hypericin, Callus culture, Suspension culture, UV ?Spectrophotometer.