From Explant Selection to Bioreactor Cultivation: Development of a Hippeastrum × hybridum Cell Culture Platform for Amaryllidaceae Alkaloid-Related Metabolism

Amaryllidaceae alkaloids are pharmaceutically relevant specialized metabolites whose variable accumulation in planta has driven the development of controlled biotechnological production systems. This study developed an integrated Hippeastrum × hybridum cell-culture platform encompassing explant selection, callus induction, suspension culture establishment, bioreactor cultivation, and elicitation. Different explants were evaluated for callogenic and morphogenic competence, selected callus lines were adapted to suspension culture. B1- and BS-derived cultures were progressively expanded and transferred to 2 L bioreactors as an intermediate scale-up step and subsequently exposed to 1 µM coronatine (COR). Alkaloid extracts from cellular and extracellular fractions, together with in vitro and ex vitro plant tissues, were analyzed by GC–MS. Explant origin strongly influenced developmental responses, and B1- and BS-derived lines were selected based on sustained callus proliferation, biomass availability, suitable friability, and successful adaptation to liquid culture. Both lines maintained growth after transfer to 2 L bioreactors; in the bioreactor cultures evaluated, B1 showed greater estimated net biomass accumulation than BS. Their alkaloid-related profiles differed markedly between lines and sampling times during the elicitation phase. Notably, an extracellular signal assigned to O-methylnorbelladine was detected in B1 at D7, consistent with retention of metabolic competence related to Amaryllidaceae alkaloid biosynthesis. The developed system should therefore be regarded as a proof-of-concept laboratory-scale platform rather than an optimized alkaloid-production process, providing a basis for future studies with biological replication, quantitative process optimization, and application to Hippeastrum species or genotypes preselected for pharmaceutically relevant alkaloid profiles.