Optimization on In vitro production of multiple shoots of Marsilea quadrifolia (L.) was established by using various carbon sources and silver nitrate. In this study the carbon sources used are sucrose, fructose, maltose and glucose by using rhizome explants of Marsilea quadrifolia (L.).The data was collected with respective to growth rate and regeneration frequency. The multiple shoots are developed on MS media of various carbon sources along with 0.5µM concentration of NAA. After testing various carbon sources, Fructose has given more number of shoots (9.00 ± 1.00) with 88% of regeneration frequency and least number of shoots are found with maltose (1.66 ± 0.57) and glucose (1.33 ± 0.57). Compare to fructose, more number of shoots (17.00 ± 2.00) and 96% regeneration frequency was found on MS Media with silver nitrate (60µM).