Enhanced in vitro production of Ruta graveolens L. coumarins and rutin by mannitol and ventilation

Ruta graveolens shoot cultures were maintained on static medium supplemented with 0, 1, 2 and 3 % mannitol. The cultures were grown in vessels that ensured a ventilation rate of 7.44, 10.82 or 62.83 air exchanges per day (V1, V2 or V3, respectively). The growth index and fresh weight were significantly increased at 1 % mannitol and decreased with increasing mannitol concentrations, whereas the dry weight (DW) and DW % increased at higher concentrations of mannitol. Improving the culture ventilation significantly increased all of these parameters. A higher concentration of mannitol resulted in a higher proline content and percentage of coumarins and rutin, but the final accumulation of these bioactive molecules decreased. The coumarins, calculated as xanthotoxin, were increased from 8.15 to 13.60 mg g(-1) DW using (V1 and mannitol-free medium) and (V2 with medium enriched with 2 % mannitol), respectively. Rutin was linearly increased by raising the mannitol concentrations, achieving the highest content of 54.87 mg g(-1) DW using V2 and medium supplemented with 3 % mannitol. The lowest accumulation of coumarins and rutin (32, 144 mg vessel(-1), respectively) were found on mannitol-free medium using V1, whereas the highest rutin contents were found on medium with 1 % mannitol using V3. A GC analysis revealed the presence of five main compounds in all of the cultures, coumarin, 7-hydroxucoumarin, scopoletin, xanthotoxin and bergapten, whereas pasoralen was not detected when the cultures were maintained on mannitol-free medium, regardless of the type of vessel. Moreover, the concentrations of these compounds varied according to the mannitol concentration and ventilation.