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- An efficient in vitro propagation protocol for Pinguicula lusitanica, a rare insectivorous plantPublication . Gonçalves, Sandra; Escapa, Ana Luísa; Grevenstuk, Tomás; Romano, AnabelaIn this study, an efficient protocol was developed for in vitro propagation of Pinguicula lusitanica L., a rare insectivorous plant with pharmacological value and limited reproductive capacity. The effects of two concentrations (0.1 and 0.5 mg l-1) of a range of plant growth regulators, including cytokinins (BA, KIN, and ZEA) and auxins (IAA, IBA, and NAA), and three concentrations of MS medium macronutrients (total, 1/2 and 1/4MS) on proliferation and rooting, were investigated. P. lusitanica shoots showed abundant proliferation and rooting capacity, both of which were significantly influenced by MS medium strength. The use of 1/2MS supplemented with 0.5 mg l-1 BA or KIN ensure a 29-fold rate of proliferation. Best rooting frequency and higher root number and length were attained in 1/4MS medium containing 0.2 mg l-1 IAA. Sixty percent of the plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. These plantlets can be used to replenish declining populations in the wild and/or for the extraction of bioactive compounds, reducing pressure on wild stocks.
- Phytochemical studies and biological activity of carnivorous plants from the Mediterranean regionPublication . Grevenstuk, Tomás; Romano, AnabelaIn this thesis several studies were conducted with four carnivorous plant species which occur on Portuguese territory: Pinguicula lusitanica, Pinguicula vulgaris, Drosera intermedia and Drosera rotundifolia. Most habitats of these plants are threatened and natural populations are scarce, therefore micropropagation protocols were developed to produce biomass for the subsequent studies. Efficient micropropagation protocols were developed for P. lusitanica and D. intermedia enabling large scale biomass production, while protocols for the other two species have still to be optimized (in Chapter 2). The in vitro established cultures represent active germplasm collections of Portuguese natural populations and contribute therefore for their conservation. In Chapter 3 extracts prepared from micropropagated plant material were analyzed using state of the art HPLC-ESI-MS and HPLC-SPE-NMR equipment which enabled the identification of the major secondary metabolites produced by P. lusitanica and D. intermedia, directly from essentially crude extracts. The metabolites identified in P. lusitanica belong to the iridoid glucosides and caffeoyl phenylethanoid glycosides and D. intermedia was shown to produce mainly flavonoid glucosides, ellagic acid derivatives and the naphthoquinone plumbagin. The evaluation of the biological activities of these extracts, compiled in Chapter 4, showed that the methanol extract of P. lusitanica has considerable antioxidant activity and that the n-hexane extract of D. intermedia has high antimicrobial potential. In Chapter 5 a method for the extraction of plumbagin from micropropagated D. intermedia plants was optimized and its potential as an alternative for bioprospection evaluated. It was shown that the commercial exploitation of plumbagin from D. intermedia cultures might be viable and that UAE with n-hexane followed by an SPE purification step is an efficient procedure for obtaining large quantities of high purity plumbagin. It is hoped that this study represents an enrichment of the knowledge on these plants and contributes to their conservation and valorisation.
- In vitro plantlet production of the endangered Pinguicula vulgarisPublication . Grevenstuk, Tomás; Romano, AnabelaThis study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free A1/4MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.