The Rg-1 encoded regeneration capacity of tomato is not related to an altered cytokinin homeostasis

•  Cytokinin (CK) metabolism was analyzed in tomato (Lycopersicon esculentum) Rg-1 hybrids during in vitro shoot organogenesis from root explants. •  Data were obtained by combining physicochemical analysis with quantification and in situ detection methods. •  Although exogenous zeatin is added in all classical regeneration protocols, we show here that regenerating (Rg⁺ ) tomato explants did not require an exogenous CK source for regeneration. Irrespective of the presence or absence of exogenous zeatin, the endogenous CK levels were not affected by Rg-1 in the initial explants or in the early callus phase. In a later stage, and related to the presence of numerous shoots, the Rg⁺ explants showed much lower endogenous CK concentrations than the nonregenerating (rg^- ) explants. Cells of rg^- explants were not able to differentiate, despite their high endogenous CK content, and did not respond to exogenously applied CKs. •  We show that the insensitivity of rg^- explants to a hormonal signal, normally initiating regeneration, is not related to an altered endogenous CK metabolism. We therefore postulate that Rg-1 action involves a regeneration-specific CK receptor or a regeneration-specific CK signal transduction pathway.