Changes in biochemical constituents, enzyme activities and protein profiles during root-shoot differentiation in callus culture of Dioscorea alata

Abstract

The changes in biochemical constituents and polypeptide (PP) bands were studied during root-shoot differentiation in Dioscorea alata (greater yam). Among different explants used - leaf, node, internode and tuber, node explants were found to be the best for induction and growth of callus. Depending on weights of fresh and dry callus, high growth and best callusing were observed on MS medium supplemented with 2.5 μM NAA. This high growth value callus was subsequently supplemented with various concentrations and combinations of growth regulators to identify the suitable regeneration media. Regeneration of roots occurred in 12-16 days and was the best on MS medium having 2.0 μM NAA and 0.5 μM IBA. Shoots regenerated in 16 days in MS medium supplemented with 2.0 μM BAP and 0.5 μM NAA. The biochemical constituents such as total soluble sugars, reducing sugars, total soluble proteins and total phenols decreased whereas free amino acids increased during root and shoot differentiation of D. alata. Activities of enzymes, viz. α-amylase, acid invertase, acid phosphatase, acid protease and peroxidase decreased during callus differentiation. While four PP bands (25.56, 24.35, 19.13 and 18.2 kDa) appeared during root differentiation, only three PP bands (53.7, 25.12 and 19.13 kDa) were noted during shoot differentiation. One common PP band (19.13 kDa) appeared during both root and shoot differentiation. There was disappearance of four common bands (89.13, 69.8, 36.3 and 27.43 kDa) during differentiation of root and shoot. To conclude, changes in biochemical constituents and expression of root/shoot specific PP may be used as markers to characterize differentiation pathway and to augment the selection of regenerating potential callus for rapid in vitro propagation.