Abstract

This study describes the development of a reproducible in vitro protocol for inducing cell proliferation from transverse thin cell layer (tTCL) explants of Peumus boldus Mol., aimed at establishing a viable source of cambial meristematic cells (CMCs) for boldine production. Three in vitro-maintained genotypes (2, 7, and 8) were evaluated using tTCLs derived from the first, second, and third internodes. Explants were cultured on ½ MS medium supplemented with 2.0 mgL?1 2,4-dichlorophenoxyacetic acid and 0.1 mgL?1 kinetin under dark conditions. Macroscopic and histological analyses revealed that tTCLs from the first internode consistently exhibited enhanced friable callus proliferation, particularly in genotypes with a reduced pith-to-cortex ratio. After 35 days, cell proliferation was mainly localized in procambial zones. Cell suspension cultures derived from these calli showed genotype- and internode-dependent differences in morphogenic responses, with genotype 8 forming predominantly globular aggregates. Histochemical detection of alkaloids confirmed the presence of boldine in undifferentiated tissues. Quantification by HPLC-DAD revealed boldine levels up to 17.5 mggDW?1 in lyophilized callus from semisolid medium, and moderate levels in suspension cultures, even after UV-A elicitation. Although elicited cells did not show a marked increase in boldine content, the concentrations achieved were comparable to those found in leaves and bark of P. boldus, both in vivo and in vitro. These results highlight the biosynthetic competence of non-differentiated cell systems and lay the foundation for a more sustainable and scalable approach to boldine production. By integrating anatomical precision, hormonal control, and biochemical assessment, this study represents a significant step forward in the biotechnological valorization of P. boldus through the use of undifferentiated, cambium-derived cell cultures. © The Author(s), under exclusive licence to Springer Nature B.V. 2025.