Effect of total nitrogen content and NH4+/NO3- ratio on biomass accumulation and secondary metabolite production in cell culture of Salvia nemorosa

Woodland sage (Salvia nemorosa) is an important native medicinal plant in Iran and is considered as a rich source of phenolic and flavonoids compounds. The present research was conducted with respect to the optimization of medium for biomass accumulation and secondary metabolite production in cell suspension culture of S. nemorosa. In this research the effects of total nitrogen content (0, 15, 30, 60, 90, and 120 mM) and NH4+/NO3- ratio (0:60, 10:50, 20:40, 30:30, 40:20, 50:10, and 60:0) were studied in the Murashige and Skoog Medium on biomass growth, total phenolic, flavonoid and rosmarinic acid contents. The maximum accumulation of fresh biomass (294.80 g/l) and total phenolic content (76.61 mg GAE/g DW) was obtained in the medium supplemented with 90 mM nitrogen. The highest rosmarinic acid content (16.41 and 16.16 mg/g DW) was recorded in the medium containing 30 and 60 mM nitrogen. Increasing total nitrogen above 30 mM resulted in a decline in rosmarinic acid production. The ammonium to nitrate ratio also affected the biomass growth and secondary products accumulation. The highest fresh biomass accumulation (296.52 g/l), total phenolic content (87.30 mg GAE/g DW) and Rosmarinic acid content (18.43 mg/ DW) were recorded in 10:50 ratio of NH4+/NO3-. Increasing the NH4+ level or complete elimination of it from culture medium reduced the rosmarinic acid and total phenolic content of S. nemorosa. Our findings revealed that Woodland sage cell suspension needs both nitrogen forms, but ammonium is required at low concentration and nitrate at high levels. The results of the current study are beneficial for medium optimization for the establishment of large scale and bioreactor assisted cell culture of woodland sage for the production of phenolic acids.