Callus browning is a major problem in Boerhaavia diffusa. This phenomenon was investigated in present study by evaluating major reason for callus browning, develop a strategy for the survivals of callus and study the accumulation of secondary metabolites. Torpedo shaped embryos were cultured on semisolid MS basal medium supplemented with n various combinations of hormones, with and without adjuvants. After a particular time callus used for cytological, fresh viz dry weight studies and later used for the secondary metabolite study by HPTLC method. Cytological studies of the callus were performed to understand the reason for low survival of the callus. Over a culture period of 30 days revealed that the callus was made up of three types of cells: small isodiametric cells, elongated cells and elongated enucleated cells. The isodiametric cells were meristematic and predominant during the initial days of the culture and subsequently their number decreased and elongated nucleated and enucleated cells increased. Towards the latter part of the culture period the enucleated cells were predominant. The increase in elongated cells coincided with increased browning of the callus and peroxidase activity. The HPTLC of extracted callus with different precursors confirmed the presence of some flavonoids likes kaempferol, quercetin, myrecetin. A strategic subculturing method was developed where in the small cells were isolated and subcultured every three weeks and the life of callus could thus be prolonged to almost 30-36 weeks. Based on these studies conclude that the life of callus could be prolonged to almost 30-36 weeks by strategic subculturing method. This study is important because as plant has various medicinal properties so its secondary metabolites can be collected by in vitro callus production at particular time period.