Because of the increased interest in the potential of Jatropha as a biofuel plant, more attention is given to methods that allow the mass production of elite material. A simple and efficient protocol has been developed for high frequency regeneration using nodal explants of Madagascar and Ecuador Jatropha curcas lines. The optimum shoot growth with high number and without callus formation was observed using MS medium supplemented with 1.0 mg/l BA in both lines within a period of 30-40 days which shows 3.8 and 3.4 shoot buds in Madagascar and Ecuador lines, respectively. Healthy elongated shoots were harvested and cultured on ½ strength MS basal salts without vitamins medium containing different concentrations of IBA individual or in combination with IAA or 2,4-D. Moreover, the optimum rooting medium with good root formation (46.6 and 40%) in both Jatropha lines and (3.0 – 2.4) roots per shoot containing half-strength MS supplemented with 0.5 mg/l IBA. In the present study, different light sources like cool white fluorescent, warm white fluorescent and different high- brightness LEDs (red – blue – white) were used to monitor the root initiation of in vitro Jatropha shoots. Red LED light provided the most favorable growth conditions inducing a rooting response in 66.6% of the shoots in Madagascar Jatropha and 56.5% in Ecuador Jatropha which produced on average 4.2 and 3.5 roots per shoot, respectively in both lines. Thus, white LED light was more effective on root elongation than all studied light treatments that recorded (5.6 and 4.4 cm) root length in both lines. Therefore, well developed healthy in vitro rooted plantlets after 6 weeks were successfully acclimatized and the survival rate of in vitro propagated plantlets after transplanting was 40-60% and they grew as normal plants in the greenhouse.