Jatropha is one of the most important plant species of the world valued for its biofuel property. The species is primarily propagated through seeds, and thus the seed yield and oil content varies significantly. The present work describe the reproducible and efficient in vitro regeneration method from different types of explant (Leaf, petiole, cotyledonary leaf and petiole) and source of explant (in vitro and in vivo) of toxic and non-toxic cultivar of Jatropha. Besides, providing an efficient, reproducible and genotype independent regeneration protocol from different types and source, also provides optimized protocol for successful genetic transformation of Jatropha leaf explants and molecular characterization of transformed plants which help in improvement of this important biofuel plant.