Research article Changes in apolar metabolites during in vitro organogenesis of Pancratium maritimum Strahil Berkov a, b, * , Atanas Pavlov c , Vasil Georgiev c , Jost Weber d , Thomas Bley d , Francesc Viladomat a , Jaume Bastida a , Carles Codina a a Departament de Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona. Av. Joan XXIII s/n, 08028 Barcelona, Catalonia, Spain b AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164- Sofia, Bulgaria c Department of Microbial Biosynthesis and Biotechnologies e Laboratory in Plovdiv, Institute of Microbiology, Bulgarian Academy of Sciences, 26 “Maritza” Blvd., 4002 Plovdiv, Bulgaria d Institute of Food Technology and Bioprocess Engineering, Technische Universität Dresden, 01069 Dresden, Germany article info Article history: Received 17 November 2009 Accepted 12 July 2010 Keywords: Amaryllidaceae alkaloids Apolar metabolites Lipids Pancratium maritimum Tissue differentiation abstract Calli, shoot-clumps and regenerated plants were initiated from young fruits of Pancratium maritimum L. Their genetic stability was monitored by flow cytometry before chemical studies. Apolar metabolites (alkaloids extracted at pH > 7, free fatty acids and fatty alcohols, sterols etc.) were qualitatively and quantitatively analyzed by GCeMS. The results clearly demonstrated that alkaloid synthesis in P. mar- itimum is closely related with tissue differentiation. The highest amounts of alkaloids and presence of homolycorine and tazettine type compounds (end products of the biosynthetic pathway of the Amar- yllidaceae alkaloids) were found in highly differentiated tissues. Galanthamine accumulated in the leaves of plantlets. The amount of hordenine, a protoalkaloid, is related with the ability of tissues to synthesize alkaloids. Saturated fatty acids were found in considerably higher levels in undifferentiated callus cultures and partially differentiated shoot-clumps than in regenerated plants. Mono- and dienoic fatty acids were found at higher levels in non-photosynthesizing tissues e calli, and in vitro and intact bulbs, while a-linolenic acid (trienoic acid) was found in higher amounts in the photosynthesizing leaves of shoot-clumps and regenerated plants than in bulbs and calli. Fatty alcohols were found mainly in leaves, while sterols tended to accumulate in photosynthesizing and undifferentiated tissues. Ó 2010 Elsevier Masson SAS. All rights reserved. 1. Introduction Differentiation of cells into plant tissues and organs leads to metabolic changes, as well as to excretion and/or storage of specific metabolites. The level of metabolites - end products of cellular regulatory processes - can be regarded as the ultimate response of biological systems to genetic or environmental changes. Physio- logical and chemical changes during plant growth and development are usually investigated by analysing a limited number of target metabolites. A multi-target profiling analysis is a valuable approach to the study of complex biological systems, leading to a better understanding of their biological role or functions at different developmental stages or under specific environmental factors [13]. Apolar plant metabolites such as lipids and alkaloids are involved in the mechanisms of plant defense and adaptation. Surface plant lipids, deposited in the cuticule, form a functional barrier preventing excessive water loss and entry of pathogens into the plant [25]. One of the main functions of the alkaloids is that of chemical defense against herbivores. Some alkaloids are antibac- terial, antifungal, and antiviral; and these properties may extend to toxicity towards animals [28]. Recently, in vitro cultures of amar- yllidaceous plants have attracted attention as an alternative source of alkaloids, including galanthamine - an AChE inhibitor marketed for the treatment of Alzheimer’s disease [19]. It was found that galanthamine is synthesized and accumulated in shoot-clumps at considerably higher levels than in unorganised callus cultures of N. confusus and Leucojum aestivum [4,8,10,14,23]. Fewer alkaloids have been found in the alkaloid profiles of calli in comparison with in vitro shoot-clumps of L. aestivum [4]. Pancratium maritimum L. (sea daffodil) is an amaryllidaceaous species growing in extreme environmental conditions - in salty and Abbreviations: FAs, fatty acids; SFAs, saturated fatty acids; MFAs, monoenoic fatty acids; DFAs, dienoic fatty acids; TFAs, trienoic fatty acids; DW, Dry weight; 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, a-naphthylacetic acid; BAP, 6-benzy- laminopurine; TIC, total ion current; IS, Internal standard. * Corresponding author at: Departamentde Productes Naturals, Biologia Vegetal i Edafologia, Facultat de Farmàcia, Universitat de Barcelona. Av. Joan XXIII s/n, 08028 Barcelona, Catalonia, Spain. Tel.: þ34 93 402 02 68; fax: þ34 93 402 90 43. E-mail address: berkov_str@yahoo.com (S. Berkov). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2010.07.002 Plant Physiology and Biochemistry xxx (2010) 1e9 Please cite this article in press as: S. Berkov, et al., Changes in apolar metabolites during in vitro organogenesis of ..., Plant Physiology and Biochemistry (2010), doi:10.1016/j.plaphy.2010.07.002