There is a great opportunity to create a wide demand and expand to Jatropha plantation for produce Egyptian biodiesel in the future, so more focus is now being imposed on methods that permits commercial production of Jatropha elite material. A protocol for micropropagation has been developed by using different types of explants, medium, growth regulators with various concentrations along the two lines of Jatropha Ecuador and Madagascar. Results showed that, both lines achieved optimal number of shoots and no callus formation by culture shoot tip explants on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 6-Benzyladenine (BA) for the two lines during establishment stage. Moreover, used shoot tip with MS medium fortified with 1.0 mg/l BA gave the highest number for each of shoots and leaves than the other treatments with the two lines. Also, in the presence of 1.0 mg/l BA+ 0.5 Indole butyric acid (IBA), multiple shoot formation and elongation were observed, with 5.0, 5.4 shoot buds/explant, 3.46, and 3.70 cm shoot length in both lines, respectively. However, harvested elongated shoots were transplanted on a half-strength MS medium with various concentrations of IBA, Indole-3-acetic acid (IAA), and α-Naphthaleneacetic acid (NAA). Furthermore, the optimal concentration of 0.5 mg/l IBA on half-MS medium for Jatropha root formation was found to be 2.70 and 3.10 roots per shoot with 2.13 and 2.73 cm root length in both Ecuador and Madagascar Jatropha. Furthermore, well-developed plantlets were successfully rooted, acclimatized and produced a 40-60 percent survival rate after 6 weeks.