In cuttings, detached leaves or stems are exposed to many stresses during the root regeneration process. Here, we show that the detached Arabidopsis thaliana leaf can tolerate mild osmotic stress and still regenerate roots. Under stress conditions, wounding and stress upregulate the jasmonate (JA) signaling pathway transcription factor gene MYC2 and the abscisic acid (ABA) signaling pathway transcription factor gene ABA INSENSITIVE5 (ABI5). The MYC2-ABI5 complex upregulates the expression of β-GLUCOSIDASE18 (BGLU18), which releases ABA from ABA glucose ester, resulting in ABA accumulation in the detached leaf. Mutations in MYC2, ABI5, and BGLU18 lead to the loss of stress tolerance and defects in root regeneration under osmotic stress. The successive application of JA and ABA can enhance the root regeneration ability in Arabidopsis and poplar cuttings. Overall, the JA-mediated wound signaling pathway and the ABA-mediated stress signaling pathway collaboratively amplify ABA signals to protect root regeneration under stress conditions.