Abstract: ObjectiveThe present experiment was conducted to optimize the root regeneration system in sweet sorghum in order to develop an effective genetic transformation protocol.MethodDifferent basic media (MS,1/2MS) and different concentrations of growth hormones (IBA,NAA and ABT) were evaluated and their effects on various factors such as root quality,rooting rate,rooting time,growth of rooted plantlets,etc.,were determined to obtain an optimum rooting medium.ResultThe optimum rooting medium for the sweet sorghum variety BT3 and LT3 were found as 1/2MS +IBA (2.0 mg/L) and 1/2MS + IBA (3.0 mg/L),respectively.The regenerated roots were thick,and the rooted plantlets showed vigorous growth.The rooting rates varied in two varieties.ConclusionIt is concluded that best quality of roots can be regenerated on shoots using 1/2MS medium,supplemented with 2.0 or 3.0 mg/L IBA for variety BT3 and LT3,respectively.