In order to establish a highly efficient and sustainable regeneration system, we systematically researched the key factors affecting direct shoot regeneration from Jatropha curcas leaves that were collected from Hainan (HN1-1), Lijiang (LJ3-1), and Yuxi (YX2-12) provinces in China. The L9(34) orthogonal test of thidiazuron (TDZ), kinetin (Kn), and gibberellic acid (GA3) were studied, and the explant type, growth age, and cultivar of leaves were subsequently investigated. Simultaneously, the combinations of plant growth regulators (PGRs) promoting shoot bud proliferation, elongation, and root establishment were examined. The results showed that the best medium for shoot bud induction was Murashige and Skoog (MS) medium supplemented with 1.0 mg/L TDZ, 0.5 mg/L Kn, and 0.5 mg/L GA3. TDZ was the key PGR, while Kn and GA3 played an important role in shoot bud elongation and the number of shoots per leaf disk, respectively. The induced shoot buds proliferated and readily elongated in MS medium with 0.3 mg/L 6-benzylaminopurine and 0.01 mg/L indole-3-butyric acid (IBA) and established roots in half-strength MS medium supplemented with 2.0 mg/L IBA. Using the previously described methods, the third to fifth leaves were found to be the best explant source for shoot bud induction, with a high induction rate, large shoot numbers per disk, excellent proliferation, and consistent rooting. With the use of this regeneration system, the shoot bud induction rate increased from the reported rate of 53.5% to more than 90% using different explants and cultivars, and the shoot number per leaf disk (shoot length ≥ 0.5 cm) increased from 1.6 to 3.5. Thus, this optimized regeneration system will effectively promote the propagation and genetic transformation of J. curcas.
