Effects of buffer materials and antioxidants on growth rate and biochemical characteristics of Dracocephalum kotschyi Boiss. hairy roots

Rosmarinic acid (RA) is considered as one of the most important polyphenols. It is an ester of 3, 4-dihydroxyphenyl actic and caffeic acids and as an antioxidant significantly reduces the DNA damage induced by the anticancer compound doxorubicin. Hairy roots of Dracocephalum kotschyi Boiss contain rosmarinic acid with biological and anticancer activities. However, due to the high production of phenolic compounds in liquid medium, the hairy roots encounter with tissue browning and death problem. In this study, the effect of antioxidant compounds on growth of D. kotschyi hairy roots and inhibition of tissue browning has been surveyed. In vitro Hairy roots were induced by inoculation of two-weeks-old cotyledons of D. kotschyi via Agrobacterium rhizogenes (15834 strain). Gene transformation confirmed by PCR using rol B gene specific primers. In order to optimize the growth and prevent the tissue browning, hairy roots were cultured in ½MS liquid medium containing different buffers and antioxidants including ascorbic acid (0.05 and 0.1 g/L), MES buffer (2.66 and 5.33 g/L), activated charcoal (1.5 and 3 g/L) and polyvinyl pyrrolidone (0.5 and 1.5 g/L) in two concentrations. In order to record the quality of hairy roots under the influence of different chemicals, the phytochemical properties of samples (total phenol and flavonoid, antioxidant activity and antioxidant enzymes activity) were analyzed by spectrophotometer. Results after 21 days showed that the maximum biomass (1.64 g) and the lowest tissue browning was obtained in ½MS media containing activated charcoal (1.5 g/L). And after AC the highest biomass was observed on both PVP concentrations (1.36 g) and the highest browning and growth loss was observed in the control samples. The maximum and minimum amount of total phenol and flavonoid were observed in control samples and 1.5 g/L of activated charcoal treatment, respectively. The highest and the lowest levels of total antioxidant activity and antioxidant enzymes were related to 1.5 g/L of activated charcoal and control samples, respectively. It is considerable that the inhibition of PAL enzyme by using antioxidants such as activated charcoal and PVP has a potential to reduce biosynthesis of phenolic compounds and prevent the browning.