Agrobacterium-mediated transformation remains challenging for economically important citrus crops because of low transformation and regeneration efficiencies. REGENERATION FACTOR1 (REF1), a plant elicitor peptide, is a local wound signal perceived by PROPEP RECEPTOR-LIKE KINASE 1 (PORK1) and transduced via WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1). Exogenous REF1 peptide application has recently been reported to improve transformation and regeneration of herbaceous plants including tomato, which was explored on Agrobacterium-mediated citrus transformation in this study. We identified REF1, PORK1, and WIND1 orthologs in sweet orange (Citrus sinensis), which encodes two REF1 isoforms, CsREF1–1 and CsREF1–2. REF1 of tomato (SlREF1) and the two CsREF1 isoforms of different concentrations were tested for their effect on Agrobacterium-mediated transformation of citrus via exogenous application. CsREF1–1 at 10 or 100 nM and CsREF1–2 at 100 nM significantly increased GFP-positive transgenic callus formation, whereas SlREF1 had no significant effect. In addition, CsREF1–1, CsREF1–2, and SlREF1 reduced shoot regeneration. To overcome this trade-off, we exposed epicotyl explants to 100 nM CsREF1–2 only during the first month and then transferred them to REF1-free regeneration medium, yielding 4.1-fold more GFP-positive shoots than the untreated control. Such a staged application of CsREF1–2 also improved GFP-positive shoot recovery in Carrizo citrange by 3.3-fold. Reverse transcription-quantitative PCR (RT-qPCR) showed that CsREF1–2 induced CsWIND1 and CsESR1, which are critical for callus formation and shoot regeneration, but continuous CsREF1–2 exposure also suppressed shoot/meristem regulators, which was mitigated by removal of CsREF1–2 in the later stage. AlphaFold3 and PRODIGY predictions indicated the highest binding affinity for CsREF1–2 with CsPORK1, consistent with its superior activity. Overall, both CsREF1 peptides enhance citrus transformation but suppress regeneration, and staged exogenous application of CsREF1 peptides improves genetic improvement of recalcitrant perennial crops such as citrus, which decouples early dedifferentiation from later organogenesis, substantially expanding the toolbox for high-throughput functional genomics in citrus.

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