Establishment and optimization of cell suspension cultures of juvenile mastic tree (Pistacia lentiscus L.)

In this study, a protocol was developed for the initiation and optimization of cell suspension cultures from in vitro germinated seeds of Pistacia lentiscus L. (mastic tree). Callus tissue was first obtained from axenic leaves and root explants of mastic seedlings germinated in vitro and then cell suspension cultures were initiated from these callus lines. To initiate suspension cultures, P. lentiscus L. seeds were first germinated in Murashige and Skoog (MS) medium containing 1 mg / l IBA. Root and leaf explants were cultured in MS medium containing BAP, Kin and 2,4-D (1 mg/l each) to produce callus. The best plant growth regulator (BBD) combination for callus formation was determined as an agar-free liquid MS medium containing 1 mg/l Kin and 1 mg/l 2,4-D and suspension cultures were initiated. In order that the suspension culture conditions can be optimized, the combinations of different BBD (BAP, Kin (1.0 and 0.5 mg/l) and 2,4-D (1 mg/l)), different shaking speeds (90, 95, 100 and 110 rpm), different light intensities (dark and light), different temperature grades (4, 25, 37 ° C), different pH media (4,5, 5 and 7.0), different types of sugar (sucrose and glucose) and their different combinations (15, 30, 50 mg / l) were separately tested by cultured on a MS medium. The most effective medium among the different BBD combinations tested was determined as MS medium supplemented with 1 mg/l BAP and 1 mg/l 2,4-D in terms of packed cell volume (PCV, ml/l) and fresh and dry weight (g/l). However, in order not to cause somaclonal variations at the same time in terms of sustainability of cultures, optimization studies were continued with the combination of 1 mg/l Kin and 1 mg/l 2,4-D, which is no statistical difference between the combination of 1 mg/l BAP and 1 mg/l 2,4-D. In terms of the parameters tested on the optimization of root and leaf derived suspension cultures; the highest PCV, fresh and dry weight results were obtained from MS medium applied 25 °C temperature, 95 rpm agitation speed, pH 5 and supplemented with 15g/l or 30 g/l sucrose (for root and leaf derived cultures, respectively). In the light of the data obtained, a growth curve showing PCV depending on the time for each of the growth phases (lag phase, exponential or log phase, linear phase, deceleration phase and stationary phase) of the mastic cell suspension cultured was created. Cell suspension cultures were maintained regularly by subcultured every 28 days in MS medium supplemented with 3% sucrose, 1 mg/l 2,4-D and 1 mg/l Kin. These findings from our studies suggest that cell suspension cultures of mastic tree may be suitable for large-scale production of fine chemicals in bioreactors.