Remedial effect of ascorbic acid and citric acid on oxidative browning of Glycyrrhiza glabra callus cultures

Oxidative browning is a common and severe problem in plant tissue cultures including callus cultures of Glycyrrhiza glabra , where browning is caused by accumulation and oxidation of phenolic compounds. Brown callus results in decreased biosynthetic potential and poor growth, eventually leading to cell death. In this study, the effect of different concentrations and combinations of antioxidants – ascorbic acid, and citric acid on browning of G.glabra callus was studied. Morphological and ultra-structural changes related to browning and antioxidant treatment were also studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to evaluate the cellular characteristics and metabolic activity of control and treated callus. Intensity of browning in callus remarkably decreased with increasing concentrations of ascorbic acid; the browning was completely overcome at a concentration of 20 mg/l ascorbic acid, in combination with 10 mg/l citric acid. An electron microscopy analysis showed that brown callus was found to have ruptured cell surface and a disorganized cell structure with the absence of cell organelles that affected callus development and lead to cell death. Non-brown callus had all the characteristics typical for a non-embryogenic callus cells with high-level activity showing all vital cell organelles including abundant and elongated mitochondria. Therefore, we conclude that ascorbic acid and citric acid have wide applications in tissue cultures of Glycyrrhiza glabra where browning restricts the callus development, and lead to the establishment of non-embryogenic callus with high metabolic activity for further use in secondary metabolite production.