In recent years, the market for succulent plants has experienced rapid growth. To increase the yield and value of cultivar Echeveria ‘Pretty in Pink’, a member of the Crassulaceae family, a tissue culture method via indirect organogenesis was established using leaf explants. The highest callus induction rate (26.7%) was achieved when explants were cultured on Murashige and Skoog (MS) medium supplemented with 3 mg/L 6-benzylaminopurine (6-BA), 1 mg/L \(\alpha\) -naphthaleneacetic acid (NAA), and 0.1 mg/L thidiazuron (TDZ). The highest callus proliferation coefficient (4.26) was observed on medium containing 3 mg/L 6-BA and 1 mg/L NAA. Plantlets were regenerated when calli were cultured on MS or 1/2 MS medium without plant growth regulators (PGRs). During proliferation, three distinct types of calli were identified and analyzed using morphological observations and widely targeted metabolomics. Morphological analysis revealed that regenerable calli (GCs) presented numerous granular buds on their surface, and the cells were regular and complete, contributing to their regenerability. Metabolomic analysis revealed that the differentially accumulated metabolites (DAMs) were primarily amino acids and their derivatives, with deoxyuridine monophosphate (dUMP) identified as a biomarker for distinguishing among the three callus types. Additionally, changes in phytohormone levels were observed across the different callus types. These findings provide a valuable reference for optimizing culture systems, minimizing undesirable tissue changes, and enhancing propagation efficiency.
