Establishment and Comparative Secondary Metabolite Production of Callus and Cambial Meristematic Cells of Coffea arabica and Coffea canephora

Coffee is a globally important agricultural commodity whose production is increasingly challenged by climate change and sustainability constraints, necessitating alternative production strategies independent of traditional cultivation. Plant cell culture represents a promising cellular agriculture approach for producing valuable coffee secondary metabolites, including purine alkaloids (e.g., caffeine and theobromine) and chlorogenic acids, under controlled conditions. This study aimed to establish cambial meristematic cells (CMCs) of Coffea arabica and evaluate their biotechnological performance relative to dedifferentiated cells (DDCs) with respect to growth characteristics and metabolite production. For the first time, stable C. arabica CMC lines were successfully established and cultivated alongside DDCs in shake-flask systems under heterotrophic and mixotrophic conditions. Cellular morphology, growth kinetics, and secondary metabolite accumulation were systematically analysed. CMCs exhibited characteristic morphology with small vacuoles and significantly smaller cell size (59.51 ± 14.22 µm) compared with DDCs (82.34 ± 20.21 µm). Under heterotrophic cultivation, DDCs achieved higher intracellular caffeine yields (0.285 ± 0.067 mg/g _CDW ) than CMCs (0.041 ± 0.005 mg/g _CDW ). In contrast, mixotrophic conditions markedly enhanced CMC metabolic performance, resulting in increased caffeine accumulation (0.447 ± 0.015 mg/g _CDW ) and substantial chlorogenic acid production (0.314 ± 0.057 mg/g _CDW ), whereas DDCs showed reduced productivity and increased stress sensitivity. Comparative analysis of CMCs from C. canephora revealed species-specific differences, with caffeine concentrations nearly twice those observed in C. arabica (0.85 ± 0.15 vs. 0.45 ± 0.02 mg/g _CDW ). These findings identify CMCs as a promising platform for light-driven coffee cell biotechnology and provide a foundation for developing sustainable, cell culture–derived coffee production systems.