{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T09:54:43Z","timestamp":1791194083832,"version":"4.1.0"},"reference-count":41,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,10,4]],"date-time":"2026-10-04T00:00:00Z","timestamp":1791072000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Plants"],"abstract":"<jats:p>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 \u00d7 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 \u00b5M coronatine (COR). Alkaloid extracts from cellular and extracellular fractions, together with in vitro and ex vitro plant tissues, were analyzed by GC\u2013MS. 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.<\/jats:p>","DOI":"10.3390\/plants15193041","type":"journal-article","created":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T08:57:18Z","timestamp":1791190638000},"page":"3041","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["From Explant Selection to Bioreactor Cultivation: Development of a Hippeastrum \u00d7 hybridum Cell Culture Platform for Amaryllidaceae Alkaloid-Related Metabolism"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6817-6174","authenticated-orcid":false,"given":"Mar\u00eda Lenny","family":"Rodr\u00edguez-Escobar","sequence":"first","affiliation":[{"name":"Department of Biology, Healthcare and the Environment, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3598-0515","authenticated-orcid":false,"given":"Laura","family":"Torras-Claveria","sequence":"additional","affiliation":[{"name":"Department of Biology, Healthcare and the Environment, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8086-7469","authenticated-orcid":false,"given":"Diego","family":"Hidalgo-Mart\u00ednez","sequence":"additional","affiliation":[{"name":"Department of Biology, Healthcare and the Environment, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain"},{"name":"Maria de Maeztu Unit of Excellence, Institute of Nutrition and Food Safety, University of Barcelona (INSA-UB), 08921 Santa Coloma de Gramenet, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,10,4]]},"reference":[{"key":"ref_1","first-page":"113","article-title":"Chemodiversity, Chemotaxonomy and Chemoecology of Amaryllidaceae Alkaloids","volume":"Volume 83","year":"2020","journal-title":"Alkaloids: Chemistry and Biology"},{"key":"ref_2","unstructured":"Mu\u00f1oz-Torrero, D. (2011). Chemical and Biological Aspects of Amaryllidaceae Alkaloids. Recent Advances in Pharmaceutical Sciences, Transworld Research Network."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"11713","DOI":"10.3390\/ijms140611713","article-title":"Towards a Molecular Understanding of the Biosynthesis of Amaryllidaceae Alkaloids in Support of Their Expanding Medical Use","volume":"14","author":"Takos","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Koirala, M., Karimzadegan, V., Liyanage, N.S., M\u00e9rindol, N., and Desgagn\u00e9-Penix, I. (2022). Biotechnological Approaches to Optimize the Production of Amaryllidaceae Alkaloids. Biomolecules, 12.","DOI":"10.3390\/biom12070893"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1080\/07388551.2020.1731414","article-title":"Green (Cell) Factories for Advanced Production of Plant Secondary Metabolites","volume":"40","author":"Marchev","year":"2020","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Bapat, V.A., Kavi Kishor, P.B., Jalaja, N., Jain, S.M., and Penna, S. (2023). Plant Cell Cultures: Biofactories for the Production of Bioactive Compounds. Agronomy, 13.","DOI":"10.3390\/agronomy13030858"},{"key":"ref_7","first-page":"154","article-title":"Alkaloid Profiling of Hippeastrum Cultivars by GC-MS, Isolation of Amaryllidaceae Alkaloids and Evaluation of Their Cytotoxicity","volume":"14","author":"Havelek","year":"2020","journal-title":"Rec. Nat. Prod."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez-Escobar, M.L., Lara, R.F., Atahuachi, M., Fuentes, A.F., Maldonado, C., Bastida, J., Tallini, L.R., and Torras-Claveria, L. (2025). Alkaloid Profile Characterisation and Bioactivity Evaluation of Bolivian Hippeastrum Species (Amaryllidaceae) as Cholinesterase Inhibitors. Life, 15.","DOI":"10.3390\/life15050719"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1007\/s43450-021-00211-z","article-title":"Structural Diversity and Biological Potential of Alkaloids from the Genus Hippeastrum, Amaryllidaceae: An Update","volume":"31","author":"Tallini","year":"2021","journal-title":"Rev. Bras. Farmacogn."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"154223","DOI":"10.1016\/j.jplph.2024.154223","article-title":"Metabolite Profiling and Histochemical Localization of Alkaloids in Hippeastrum papilio (Ravena) van Scheepen","volume":"296","author":"Haist","year":"2024","journal-title":"J. Plant Physiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"100579","DOI":"10.1016\/j.cpb.2026.100579","article-title":"Dissecting Specialized Metabolism in Space: A MALDI-MSI Atlas of Amaryllidaceae Alkaloids in Hippeastrum papilio (Ravenna) Van Scheepen","volume":"45","author":"Liyanage","year":"2026","journal-title":"Curr. Plant Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1007\/BF02615131","article-title":"The in vitro Propagation of Amaryllis (Hippeastrum spp. Hybrids)","volume":"13","author":"Seabrook","year":"1977","journal-title":"In Vitro"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"111191","DOI":"10.1016\/j.plantsci.2022.111191","article-title":"Somatic Embryogenesis from Flower Tepals of Hippeastrum Aiming Regeneration of Virus-Free Plants","volume":"317","author":"Rivas","year":"2022","journal-title":"Plant Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.5586\/aa\/185507","article-title":"Impact of Jasmonates, Salicylic Acid, and Fungal Elicitor on the Health of Hippeastrum hybridum Bulbs Achieved by in vitro Procedure","volume":"77","author":"Trejgell","year":"2024","journal-title":"Acta Agrobot."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/j.jpba.2019.04.043","article-title":"Screening of Amaryllidaceae Alkaloids in Bulbs and Tissue Cultures of Narcissus Papyraceus and Four Varieties of N. tazetta","volume":"172","author":"Tarakemeh","year":"2019","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1007\/s11240-024-02848-9","article-title":"A Protocol for the Development and Maintenance of Coffea arabica (L.) Cell Suspension Cultures","volume":"158","author":"Marchetti","year":"2024","journal-title":"Plant Cell Tissue Organ Cult."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"734","DOI":"10.1016\/j.procbio.2006.12.006","article-title":"Galanthamine Production by Leucojum aestivum in vitro Systems","volume":"42","author":"Pavlov","year":"2007","journal-title":"Process Biochem."},{"key":"ref_18","first-page":"1934578X0900400328","article-title":"Alkaloid Synthesis and Accumulation in Leucojum aestivum in vitro Cultures","volume":"4","author":"Berkov","year":"2009","journal-title":"Nat. Prod. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1002\/pca.2865","article-title":"In vitro Micropropagation and Alkaloids Analysis by GC\u2013MS of Chilean Amaryllidaceae Plants: Rhodophiala pratensis","volume":"31","author":"Bustamante","year":"2020","journal-title":"Phytochem. Anal."},{"key":"ref_20","first-page":"75","article-title":"New Method for the Study of Amaryllidaceae Alkaloid Biosynthesis Using Biotransformation of Deuterium-Labeled Precursor in Tissue Cultures","volume":"57","author":"Boisbrun","year":"2010","journal-title":"Acta Biochim. Pol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Motolin\u00eda-alc\u00e1ntara, E.A., Castillo-araiza, C.O., Rodr\u00edguez-monroy, M., Rom\u00e1n-guerrero, A., and Cruz-sosa, F. (2021). Engineering Considerations to Produce Bioactive Compounds from Plant Cell Suspension Culture in Bioreactors. Plants, 10.","DOI":"10.3390\/plants10122762"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Verd\u00fa-Navarro, F., Moreno-Cid, J.A., Weiss, J., and Egea-Cortines, M. (2023). The Advent of Plant Cells in Bioreactors. Front. Plant Sci., 14.","DOI":"10.3389\/fpls.2023.1310405"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Titova, M., Popova, E., and Nosov, A. (2024). Bioreactor Systems for Plant Cell Cultivation at the Institute of Plant Physiology of the Russian Academy of Sciences: 50 Years of Technology Evolution from Laboratory to Industrial Implications. Plants, 13.","DOI":"10.3390\/plants13030430"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e8688","DOI":"10.7717\/peerj.8688","article-title":"Carbohydrates Stimulated Amaryllidaceae Alkaloids Biosynthesis in Leucojum aestivum L. Plants Cultured in RITA\u00ae Bioreactor","volume":"2020","author":"Ptak","year":"2020","journal-title":"PeerJ"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.jplph.2012.09.004","article-title":"Coronatine, a More Powerful Elicitor for Inducing Taxane Biosynthesis in Taxus Media Cell Cultures than Methyl Jasmonate","volume":"170","author":"Onrubia","year":"2013","journal-title":"J. Plant Physiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1016\/j.plaphy.2021.03.047","article-title":"Improved Biotechnological Production of Paclitaxel in Taxus Media Cell Cultures by the Combined Action of Coronatine and Calix[8]Arenes","volume":"163","author":"Escrich","year":"2021","journal-title":"Plant Physiol. Biochem."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Perez-Matas, E., Garcia-Perez, P., Miras-Moreno, B., Lucini, L., Bonfill, M., Palazon, J., and Hidalgo-Martinez, D. (2023). Exploring the Interplay between Metabolic Pathways and Taxane Production in Elicited Taxus baccata Cell Suspensions. Plants, 12.","DOI":"10.3390\/plants12142696"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Malik, M., Mazur, J., and Kapczy\u0144ska, A. (2025). Optimizing Somatic Embryogenesis and Biomass Proliferation in Narcissus L. \u2018Carlton\u2019 Callus Lines Using Solid and Liquid Media. Agronomy, 15.","DOI":"10.3390\/agronomy15112460"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Li, J., Wu, K., Li, L., Ma, G., Fang, L., and Zeng, S. (2023). Identification of HpMYB1 Inducing Anthocyanin Accumulation in Hippeastrum hybridum Tepals by RNA-Seq. BMC Plant Biol., 23.","DOI":"10.1186\/s12870-023-04582-4"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1016\/j.phytochem.2015.06.013","article-title":"Changes in Gene Transcription and Taxane Production in Elicited Cell Cultures of Taxus \u00d7 Media and Taxus Globosa","volume":"117","author":"Osuna","year":"2015","journal-title":"Phytochemistry"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"155966","DOI":"10.1016\/j.cej.2024.155966","article-title":"Mass Transfer Characteristics in Disposable Rocking Bioreactors: A Critical Review and Quantitative Data Catalogue","volume":"499","author":"Wierzchowski","year":"2024","journal-title":"Chem. Eng. J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1007\/s11240-022-02368-4","article-title":"Effect of Plant Growth Regulators on Alkaloid the Production in Cell Cultures of Chilean Amaryllidaceae: Rhodophiala pratensis, Rhodophiala splendens, Rhodophiala advena, and Rhodolirium speciosum","volume":"151","author":"Correa","year":"2022","journal-title":"Plant Cell Tissue Organ Cult."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1016\/j.bjp.2019.02.001","article-title":"Influence of in vitro Micropropagation on Lycorine Biosynthesis and Anticholinesterase Activity in Hippeastrum goianum","volume":"29","author":"Costa","year":"2019","journal-title":"Rev. Bras. Farmacogn."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1988","DOI":"10.1111\/pbi.70026","article-title":"Elucidating the Enzyme Network Driving Amaryllidaceae Alkaloids Biosynthesis in Leucojum aestivum","volume":"23","author":"Lamichhane","year":"2025","journal-title":"Plant Biotechnol. J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1093\/jxb\/erae187","article-title":"Navigating Amaryllidaceae Alkaloids: Bridging Gaps and Charting Biosynthetic Territories","volume":"76","author":"Liyanage","year":"2025","journal-title":"J. Exp. Bot."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"107649","DOI":"10.1016\/j.jbc.2024.107649","article-title":"Kinetic and in Silico Structural Characterization of Norbelladine O-Methyltransferase of Amaryllidaceae Alkaloids Biosynthesis","volume":"300","author":"Koirala","year":"2024","journal-title":"J. Biol. Chem."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Marasek-Ciolakowska, A., Sochacki, D., and Marciniak, P. (2021). Breeding Aspects of Selected Ornamental Bulbous Crops. Agronomy, 11.","DOI":"10.3390\/agronomy11091709"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"114619","DOI":"10.1016\/j.indcrop.2022.114619","article-title":"Evaluation of Hippeastrum papilio (Ravenna) Van Scheepen Potencial as a New Industrial Source of Galanthamine","volume":"178","author":"Berkov","year":"2022","journal-title":"Ind. Crops Prod."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1455","DOI":"10.1016\/j.talanta.2010.11.029","article-title":"Development and Validation of a GC-MS Method for Rapid Determination of Galanthamine in Leucojum aestivum and Narcissus ssp.: A Metabolomic Approach","volume":"83","author":"Berkov","year":"2011","journal-title":"Talanta"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez-Escobar, M.L., Tallini, L.R., Lisa-Molina, J., Berkov, S., Viladomat, F., Meerow, A., Bastida, J., and Torras-Claveria, L. (2023). Chemical and Biological Aspects of Different Species of the Genus Clinanthus Herb. (Amaryllidaceae) from South America. Molecules, 28.","DOI":"10.3390\/molecules28145408"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez-Escobar, M.L., Mart\u00ednez-Franc\u00e9s, V., R\u00edos, S., Feresin, G.E., Borges, W.D.S., Bastida, J., Torras-Claveria, L., and Tallini, L.R. (2025). Alkaloid Profile of Fifteen Different Species of Narcissus L. (Amaryllidoideae) Collected in Spain. Plants, 14.","DOI":"10.3390\/plants14172793"}],"container-title":["Plants"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2223-7747\/15\/19\/3041\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T09:25:43Z","timestamp":1791192343000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2223-7747\/15\/19\/3041"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,10,4]]},"references-count":41,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["plants15193041"],"URL":"https:\/\/doi.org\/10.3390\/plants15193041","relation":{},"ISSN":["2223-7747"],"issn-type":[{"value":"2223-7747","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,10,4]]}}}