The effect of chitosan on phenolic compounds, rosmarinic acid and expression of key genes involved in rosmarinic acid biosynthesis in cell suspension culture of Melissa officinalis L.

The aim of this study was the investigation of the effect of chitosan on phenolic compounds, rosmarinic acid content, and  key genes expression involved in the biosynthesis of these compounds in Melissa officinalis L. cell suspension culture.

A combination of NAA (1mg L-1), 2, 4-D (1mg L-1), and kinetin (0.5 mg L-1) and stem explants were used for callus induction. After initiation of cell suspension culture, chitosan treatment (0, 50, and100 mg L-1) was performed for one, three, and five days in a factorial experiment based on a completely randomized design with three replications. Phenylalanine ammonia-lyase (PAL) activity, soluble protein, phenolic compound, and rosmarinic acid content were measured. Also, the expressionof phenylalanine ammonia-lyase and rosmarinic acid synthase (RAS) genes was evaluated by real-time PCR.

PAL activity at 50 mg L-1 chitosan showed the highest increase after three days of treatment, which was accompanied by an increase in phenolic compounds. The relative expression of the PAL gene at 50 mg L-1 chitosan increased 2.5 fold at three days after treatment compared to control. This increase in the expression of the PAL gene resulted in an increase in enzyme activity. However, the highest increase in expression of the RAS gene was observed at 100 mg L-1 chitosan in five days after treatment, which led to an increase in rosmarinic acid content.

According to the results, chitosan treatment is recommended to increase of rosmarinic acid content in lemon balm cell culture for five days.