Endemic Plant Species Conservation: Biotechnological Approaches.

Endemic plant species are usually more vulnerable to anthropogenic threats and natural changes and, therefore, hold a higher extinction risk. The preservation of these species is a major concern on a worldwide context and in situ protection alone will not guarantee their conservation. Ex situ conservation measures must be undertaken to support the conservation of these species, and seed banking is the more efficient and cost-effective method. However, when seed banking is not an option, alternative approaches should be considered. Biotechnological tools provide new and complementary options for plant conservation including short-, medium-, and long-term strategies, and their application for plant species conservation has increased considerably in the last years. This review provides information about the status of the use biotechnology-based techniques for the conservation of endemic plant species. Particular attention is given to cryopreservation, since is the only long-term ex situ conservation strategy that can complement and support the other conservation measures. The cryopreservation of plant genetic resources is, however, more focused on crop or economically important species and few studies are available for endemic plant species. The plant material used, the cryopreservation methods employed, and the assessment of cryogenic effects are reviewed. The reasons to explain the difficulties in cryopreserving these species are discussed and new strategies are proposed to facilitate and increase the interest on this matter. We expect that further studies on the conservation of endemic plant species will increase in a near future, thus contributing to maintain these valuable genetic resources.

The Influence of In Vitro Gastrointestinal Digestion on the Chemical Composition and Antioxidant and Enzyme Inhibitory Capacities of Carob Liqueurs Obtained with Different Elaboration Techniques.

Carob liqueur is a traditional Mediterranean alcoholic beverage obtained via a wide range of production techniques contributing to the different organoleptic attributes of the final product. The aim of this research was to evaluate the stability of the chemical composition and biological capacities (antioxidant and enzyme inhibition) under in vitro simulated gastrointestinal digestion of liqueurs prepared by flavouring the fig spirit with carob pulp by maceration, distillation, percolation, or aqueous and hydro-alcoholic infusions. For this purpose, the phenolic and furanic compositions, the total phenolic (TPC) and flavonoid (TFC) contents, antioxidant capacity (AC), and enzyme inhibitory potential against acethylcholinesterase, tyrosinase, α-glucosidase and α-amylase enzymes were evaluated. The content of gallic acid decreased after gastrointestinal digestion, while TPC, TFC, and AC significantly increased after each digestion phase. Overall, no significantly different enzyme inhibitions (p < 0.05) were observed among digested liqueurs, with moderate inhibition against acethylcholinesterase and tyrosinase (enzymes related with neurodegenerative diseases), and potent and low inhibitory capacities for α-glucosidase and α-amylase, respectively (ideal conditions employed in antidiabetic therapy). The study indicates that hydro-alcoholic infusion and maceration were the most appropriate methods to obtain liqueurs with higher values of the aforementioned parameters and safe levels of toxic furanics.

Cryopreservation of Thymus lotocephalus shoot tips and assessment of genetic stability.

BACKGROUND: Thymus lotocephalus is a rare endemic species from the Algarve, Portugal, and is legally protected by Portuguese and European legislation. OBJECTIVE: The aim is to develop a cryopreservation protocol for T. lotocephalus shoot tips, as an alternative approach for the long-term conservation of this species. METHODS: Several methods (droplet-vitrification, vitrification and encapsulation-dehydration) were tested. Conditions regarding the subculture period, cold-hardening and preculture were optimized. Cryopreserved shoot tips were also assessed for their genetic stability using RAPD markers. RESULTS: Droplet-vitrification presented the best results. The best regrowth of cryopreserved shoot tips obtained eight weeks after rewarming was 67%. This was accomplished with four weeks subculture period of in vitro-donor plants at 25 degree C, preculture of excised shoot tips for one day on MS medium containing 0.3 M sucrose, treatment in PVS2 for 60 min, and MS supplemented with 0.2 mg per L zeatin as recovery medium. The assessment using RAPD markers observed variation at a low frequency and shoots regenerated from cryopreserved apices showed normal development compared to the regular in vitro-grown shoots. CONCLUSION: Droplet-vitrification is thus a viable method for the cryopreservation of T. lotocephalus shoot tips.

Approaches for the cryopreservation of Plantago algarbiensis, a rare endemic species of the Algarve.

BACKGROUND: Plantago algarbiensis is an endangered endemic species from the Algarve, Portugal. OBJECTIVE: The main goal of this study was to investigate the viability of cryopreservation procedures in the conservation of seeds and nodal explants from this species. MATERIALS AND METHODS: Seeds were directly immersed in liquid nitrogen (LN) for 30 days. Two methods were tested for the cryopreservation of nodal explants, namely droplet-vitrification and encapsulation-dehydration. For both methods, nodal segments were precultured on Murashige and Skoog (MS) medium and recovered on MS supplemented with 0.2 mg l(-1) 6-benzyladenine (BA), after freezing. RESULTS: After 30 days in LN, the germination capacity of seeds was not affected. The regrowth percentages of cryopreserved nodal segments were approximately 60%. With the droplet-vitrification method, a regrowth percentage of 60.0+/-15.2% was obtained after 120 min exposure to PVS2 (plant vitrification solution 2) and with encapsulation-dehydration method the highest percentage, 63.3+/-9.6%, was achieved after 3 h desiccation. CONCLUSION: Seed cryopreservation and cryopreservation of nodal segments by droplet-vitrification and encapsulation-dehydration are therefore effective approaches for the conservation of P. algarbiensis.

Thymus lotocephalus wild plants and in vitro cultures produce different profiles of phenolic compounds with antioxidant activity.

We compared the phenolic metabolites and antioxidant activities of Thymus lotocephalus G. López & R. Morales wild plants and in vitro cultures using different extraction solvents. HPLC-DAD analysis allowed the identification and quantification of phenolic (caffeic and rosmarinic) acids and flavones (luteolin and apigenin) in extracts from both sources. The in vitro cultures accumulated large amounts of rosmarinic acid. However, extracts from both sources were able to neutralise free radicals in different test systems (TEAC and ORAC assays), to form complexes with Fe(2+) and to protect mouse brains against Fe(2+)-induced lipid peroxidation. The solvent significantly influenced the phenolic content and antioxidant activity of the extracts, water/ethanol being the most efficient for the extraction of antioxidant phytochemicals. We conclude that in vitro cultures of T. lotocephalus represent a promising alternative for the production of valuable natural antioxidants and an efficient tool for the in vitro biosynthesis of rosmarinic acid, therefore avoiding the need to exploit populations of wild plants.

Evaluation of the antioxidant and antimicrobial properties of in vitro cultured Drosera intermedia extracts.

Evaluation of the antioxidant activity of the methanol, water and n-hexane extracts of Drosera intermedia, determined by the Folin-Ciocalteau (F-C), trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays showed that the methanol extract had the highest antioxidant activity (F-C: 378.6 +/- 31.5 micromol(GAE)/mg(extract); TEAC: 332.2 +/- 29.1 micromol(TE)/mg(extract); ORAC: 64.7 +/- 7.8 micromol(TE)/mg(extract). Antimicrobial activity was tested against seven bacterial and eight yeast strains using the agar diffusion assay, followed by the determination of minimum inhibitory concentrations (MIC). All tested D. intermedia extracts demonstrated strong antimicrobial properties with a broad spectrum of activity. However, the n-hexane extract exhibited much greater activity than water and methanol extracts. The most susceptible microorganisms to the n-hexane extract were Staphylococcus epidermidis ATCC 12228 and Candida albicans YP0175, for which a MIC value of 13.0 microg/mL was scored.