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  • Evaluation of the antioxidant and antimicrobial properties of in vitro cultured Drosera intermedia extracts
    Publication . Grevenstuk, Tomás; Gonçalves, Sandra; Almeida, Sara; Coelho, Natacha; Quintas, Célia; Gaspar, Maria Nelma; Romano, Anabela
    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 μmolGAE/mgextract; TEAC: 332.2 ± 29.1 μmolTE/mgextract; ORAC: 64.7 ± 7.8 μmolTE/mgextract). 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 μg/mL was scored.
  • Morphological, physiological and oxidative stress markers during acclimatization and field transfer of micropropagated Tuberaria major plants
    Publication . Osório, Maria Leonor; Gonçalves, Sandra; Coelho, N.; Osório, Júlio; Romano, Anabela
    Tuberaria major (Willk.) P. Silva and Rozeira is a critically-endangered rock rose species endemic to Portugal. Because the species needs to be preserved, this study evaluated the morphological and physiological traits of micropropagated T. major plants during acclimatization and field transfer. There were no significant differences between wild and micropropagated plants in the field, although the latter underwent significant changes during acclimatization. Leaf pubescence and leaf mass per area increased during acclimatization whereas the chlorophyll content and chlorophyll/carotenoid ratio declined to eventually match those of wild plants. Stomatal conductance (g(s)) and transpiration rates (E) also declined substantially during acclimatization, thus preventing uncontrolled wilting. Photosynthetic rate (P-N) was initially negative but increased during the later stages of acclimatization. Maximum quantum yield of PSII (F-v/F-m) remained constant at 0.78-0.85, showing that the plants were healthy and unstressed. PSII quantum efficiency (I center dot(PSII)) was initially low but increased during acclimatization along with photosynthetic performance as the energy partitioning in PSII was adjusted. This was balanced by the decline in non-regulated energy dissipation (I center dot(NO)) from an initially high value. Electrolyte leakage and malondialdehyde content remained constant at similar levels in both groups of plants, but H2O2 levels were higher in the field, perhaps indicating the early induction of antioxidant defense systems. The present study shows that T. major has enough phenotypic plasticity to adapt to changing environments and that the procedure described herein can be used for the restoration and preservation of this species.
  • Establishment of an in vitro propagation protocol for Thymus lotocephalus, a rare aromatic species of the Algarve (Portugal)
    Publication . Coelho, Natacha; Gonçalves, Sandra; González-Benito, María Elena; Romano, Anabela
    The aim of this work was to develop an in vitro propagation protocol for the endangered species Thymus lotocephalus using seedlings as explants. Several macronutrient concentrations of Murashige and Skoog medium (MS), cytokinin types and concentrations, and cytokinin/auxin combinations were tested to assess the shoots’ proliferation capacity. Although the best proliferation results were obtained with 6-benzyladenine, high percentages of hyperhidric shoots were observed. Because high proliferation of healthy shoots was observed in MS medium that was free of plant growth regulators, this medium was chosen for proliferation studies. The best rooting results were achieved in MS medium without auxins (92.00 ± 6.11%, 6.54 ± 0.52 and 1.60 ± 0.10 cm regarding rooting frequency, number of roots per shoot and longest roots, respectively) or supplemented with 0.5 mg l-1 indole-3-acetic acid (98.00 ± 2.11%, 11.14 ± 0.75 and 2.40 ± 0.24 cm, respectively). Plantlets were successfully acclimatised to ex vitro conditions with a survival rate of 93.33%. With the development of this micropropagation protocol, an important contribution has been made to the conservation of the endangered species T. lotocephalus.
  • How to monitor the acclimatization of micropropagated plants - From in vitro to the field?
    Publication . Osório, Maria Leonor; Gonçalves, Sandra; Coelho, N.; Romano, Anabela; Osório, Júlio
    The high mortality experienced by plants during ex vitro transplantation stage is the major bottleneck in large scale application of micropropagation. The abnormal physiological and anatomical characteristics of micropropagated plantlets require that they should be gradually acclimatized to the environment of the greenhouse or field. Environmental factors, especially relative humidity and irradiance, play a critical role in both physiological and biochemical functions, namely water relations, gas exchange, photosynthetic efficiency and water oxidation, during transition from in vitro to ex vitro conditions. Throughout this period of transition and acclimatization substantial changes in those characteristics are necessary to achieve a successful micropropagation. This adaptation should be accurately monitored using relevant physiological parameters including pigment content, chlorophyll a fluorescence imaging, net photosynthetic rate, transpiration rate, and stomatal conductance. Growth traits and oxidative stress markers, such as electrolyte leakage, lipid peroxidation and hydrogen peroxide content, should be also analyzed. These parameters must be assessed during the course of ex vitro acclimatization in several periods and compared with those of plants growing in their natural habitat. The performance plants of Tuberaria major, an endangered species endemic from the Algarve region (Portugal), during their acclimatization through indoor-to-outdoor conditions was monitored using several physiological and biochemical traits as indicators. In view of the results, we concluded that the transplantation protocol described can be used for restoration purposes, contributing to the preservation of the species. © ISHS 2013.
  • Thymus lotocephalus wild plants and in vitro cultures produce different profiles of phenolic compounds with antioxidant activity
    Publication . Costa, Patrícia; Gonçalves, Sandra; Valentão, Patrícia; Andrade, Paula B.; Coelho, Natacha; Romano, Anabela
    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 Fe2+ and to protect mouse brains against Fe2+-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.
  • Estimation of genetic diversity in seedlings of Plantago algarbiensis, an endangered species endemic to the south of Portugal in risk of global extinction
    Publication . Coelho, Natacha; Martin, Carmen; Gonzalez-Benito, Maria Elena; Romano, Anabela
    Plantago algarbiensis samp is an endangered species endemic to the Algarve, in southern Portugal. In the present work, the genetic diversity of three populations was assessed by RAPD markers. Samples were amplified using ten primers that generated 145 markers, 80% of which were polymorphic. Tunes population presented the highest polymorphism percentage (73.68%) and Algoz the lowest (67.67%). In the cluster analysis, two major groups were formed, one including individuals from Gambelas and the other clustered together individuals from the other populations. The highest level of genetic diversity, estimated by both Nei's gene diversity and Shannon's information measure, was found in Tunes and the lowest in Algoz. From the estimates of Shannon's index, the proportion of the diversity within populations was 86.12% and that among populations was 13.88%. Similar results were obtained by AMOVA analysis. A correlation was found between geographic distance and genetic differentiation among populations. The N-m values obtained suggest a high level of gene flow among populations, which was inversely proportional to the distance between populations. RAPDs proved to be a useful tool to determine the genetic diversity of P. algarbiensis populations. The data obtained can be used to develop effective conservation strategies to prevent the decline of populations.
  • Cryopreservation of shoot tips from the endangered endemic species Tuberaria major
    Publication . Coelho, Natacha; González-Benito, María Elena; Romano, Anabela
    Tuberaria major is an endangered endemic species from the Algarve, in the south of Portugal. We investigated two techniques for the cryopreservation of T. major shoot tips, namely vitrification and encapsulation-dehydration. Before the cryopreservation trials, shoot tips were precultured for 1 day on liquid Murashige and Skoog (MS) medium containing 0.3 M sucrose. For the vitrification method, shoots tips were exposed for 0, 30, 60, 90 and 120 min to plant vitrification solution 2 (PVS2). As for the encapsulation-dehydration method, shoot tips were dried inside a laminar air flow cabinet for 0, 1, 2, 3, 4, 5 and 6 h at room temperature. The highest regrowth percentages were approximately 60 and 67 % for vitrification and encapsulation-dehydration, respectively. The best times were 60 min exposure to PVS2 for vitrification and 3 h desiccation for encapsulation-dehydration. Though these are preliminary results, the use of the cryopreservation techniques tested here proved to be an important asset in the conservation of this endangered species and will complement the conservation strategies previously developed.
  • Germination and cryopreservation tolerance of seeds from the rare aromatic species Thymus lotocephalus
    Publication . Coelho, Natacha; Gonçalves, Sandra; González-Benito, María Elena; Romano, Anabela
    Thymus lotocephalus G. López and R. Morales is a rare aromatic species endemic from the south region of Portugal. We investigated the germination behavior and the cryopreservation tolerance of T. lotocephalus seeds collected from four populations. Seeds were set to germinate at 15 or 25 ◦C under light or darkness. The best germination temperature tested was 15 ◦C, under either light or darkness, with final germination percentages above 80% and mean germination time (MGT) below 10 days. The results showed that T. lotocephalus has non-dormant seeds. Seeds from different populations presented few differences on the final germination percentage and MGT. To assess seed tolerance to cryopreservation, they were directly immersed in liquid nitrogen for 30 days. Overall, we found that cryopreservation was non-detrimental to germination of T. lotocephalus seeds, thus being a reliable method for germplasm conservation of this endangered species.
  • Elicitation improves rosmarinic acid content and antioxidant activity in Thymus lotocephalus shoot cultures
    Publication . Gonçalves, Sandra; Mansinhos, Inês; Rodríguez Solana, Raquel; Perez-Santin, Efren; Coelho, Natacha; Romano, Anabela
    Thymus lotocephalus G. Lopez & R. Morales is an aromatic species endemic to the south of Portugal with medicinal properties. The aim of this work was to evaluate the effect of elicitors, cytokinin, and different sucrose concentrations on chemical composition and antioxidant activity of extracts from in vitro regenerated shoots. Elicitors (YE: yeast extract, salicylic acid, and AgNO3) were added directly to the medium. Phytochemical analysis include evaluation of total phenolic (TPC), flavonoid (TFC), and rosmarinic acid (RAC) contents. The TPCs and TFCs were determined by Folin-Ciocalteu and aluminum chloride calorimetric assays, respectively. Rosmarinic acid (RA), the main compound found in the studied extracts, was quantified by liquid chromatography-photodiode array detector. Results showed that the cytokinin benzyladenine has an inhibitory effect on TPC, TFC and RAC, as well as on antioxidant activity. TPC was only significantly improved by the presence of YE (from 95.49 +/- 3.34 to 118.34 +/- 6.24 mg(GAE)/g(extract)) and TFCs were not affected by elicitation. The increase in sucrose concentration, from 2 to 4%, and the three elicitors tested, particularly YE, enhanced RACs (from 48.61 +/- 0.09 in the control medium to 78.57 +/- 0.99 mg/g(extract) in YE-elicited medium). In accordance with these results, the antioxidant activity measured by 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and ferric reducing antioxidant power assays was significantly greater in extracts from elicited shoots. Overall, the results obtained suggest that shoots of T. lotocephalus are a good source of antioxidant compounds and showed that the production of RA can be promoted by altering in vitro culture conditions.
  • Rheological and microstructural features of plant culture media doped with biopolymers: Influence on the growth and physiological responses of in Vitro-Grown Shoots of Thymus lotocephalus
    Publication . Coelho, Natacha; Filipe, Alexandra; Medronho, Bruno; Magalhães, Solange; Vitorino, Carla; Alves, Luís; Gonçalves, Sandra; Romano, Anabela
    In vitro culture is an important biotechnological tool in plant research and an appropriate culture media is a key for a successful plant development under in vitro conditions. The use of natural compounds to improve culture media has been growing and biopolymers are interesting alternatives to synthetic compounds due to their low toxicity, biodegradability, renewability, and availability. In the present study, different culture media containing one biopolymer (chitosan, gum arabic) or a biopolymer derivative [hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC)], at 100 or 1000 mg L−1, were tested regarding their influence on the growth and physiological responses of Thymus lotocephalus in vitro culture. Cellulose-based biopolymers (HEC and CMC) and gum arabic were used for the first time in plant culture media. The results showed that CMC at 100 mg L−1 significantly improved shoot elongation while chitosan, at the highest concentration, was detrimental to T. lotocephalus. Concerning only the evaluated physiological parameters, all tested biopolymers and biopolymer derivatives are safe to plants as there was no evidence of stress-induced changes on T. lotocephalus. The rheological and microstructural features of the culture media were assessed to understand how the biopolymers and biopolymer derivatives added to the culture medium could influence shoot growth. As expected, all media presented a gel-like behaviour with minor differences in the complex viscosity at the beginning of the culture period. Most media showed increased viscosity overtime. The surface area increased with the addition of biopolymers and biopolymer derivatives to the culture media and the average pore size was considerably lower for CMC at 100 mg L−1. The smaller pores of this medium might be related to a more efficient nutrients and water uptake by T. lotocephalus shoots, leading to a significant improvement in shoot elongation. In short, this study demonstrated that the different types of biopolymers and biopolymer derivatives added to culture medium can modify their microstructure and at the right concentrations, are harmless to T. lotocephalus shoots growing in vitro, and that CMC improves shoot length.