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- Evaluation of the antioxidant and antimicrobial properties of in vitro cultured Drosera intermedia extractsPublication . Grevenstuk, Tomás; Gonçalves, Sandra; Almeida, Sara; Coelho, Natacha; Quintas, Célia; Gaspar, Maria Nelma; Romano, AnabelaEvaluation 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.
- Effects of CO2 concentration on acclimatization and physiological responses of two cultivars of carob treePublication . Osório, Maria Leonor; Gonçalves, Sandra; Osório, Júlio; Romano, AnabelaThis study reports survival and physiological responses of micropropagated Ceratonia siliqua L. cvs. Galhosa and Mulata plants during ex vitro acclimatization under ambient (AC; 330 μmol mol-1) or elevated (EC; 810 μmol mol-1) CO2 concentration and a photosynthetic photon flux density of 125 μmol m-2 s-1. CO2 enrichment during acclimatization did not improve survival rate that was around 80 % for both treatments. Eight weeks after ex vitro transplantation, photosynthetic capacity and apparent quantum yield in acclimatized leaves were higher in comparison with those in in vitro-grown leaves, without any significant difference between CO2 treatments. Chlorophyll content increased after acclimatization. However, EC led to a decrease in the total amount of chlorophyll in new leaves of both cultivars, compared to those grown at AC. Soluble sugars and starch contents were not markedly affected by growth EC, although starch had significantly increased after transfer to ex vitro conditions. EC induced an increase in the stem elongation and in the effective life of leaves, and a decrease in the number of new leaves.
- In vitro minimum growth for conservation of Drosophyllum lusitanicumPublication . Gonçalves, Sandra; Romano, AnabelaThe present paper reports a protocol for minimum growth conservation of Drosophyllum lusitanicum (L.) Link. in vitro. Double-node cuttings were maintained for 4, 8 and 12 months at 5 or 25 ºC in the dark. The effects of sucrose either alone at 5, 20, 30, 40 and 60 g dm-3 or at 20, 40 and 60 g dm-3 in combination with 20 g dm-3 mannitol, on survival and post-storage shoot multiplication efficiency were investigated. The cultures could effectively be conserved under minimum growth at 5 ºC for 8 months on Murashige and Skoog’s medium supplemented with 60 g dm-3 sucrose, 20 g dm-3 mannitol and 0.91 μM zeatin. Following extended conservation, the cultures could be successfully regenerated into new shoots, and they were morphologically similar to those of non-stored controls.
- Analysis of the volatiles emitted by whole flowers and isolated flower organs of the carob tree using HS-SPME-GC/MSPublication . Custódio, Luísa; Serra, Hugo; Nogueira, José Manuel F.; Gonçalves, Sandra; Romano, AnabelaThe volatiles emitted by fresh whole flowers and isolated flower organs of male, female, and hermaphrodite carob trees (Ceratonia siliqua L.; Leguminosae)were analyzed by headspace solid-phase microextraction followed by capillary gas chromatography and mass spectrometry. The headspace of carob flowers is mainly constituted of high amounts of monoterpenes and sesquiterpenes, and more than 25 compounds were identified. The gender and cultivar affected both the qualitative profile and the relative abundances of the volatiles of whole flowers and isolated floral organs. Linalool and its derivatives (cis-linalool furan oxide, 2,2,6-trimethyl-3-keto-6-vinyltetrahydropyran, cis-linalool pyran oxide, and trans-linalool furan oxide), a-pinene, and a-farnesene were the dominant volatiles. Female flowers had a higher diversity of volatile compounds than males and hermaphrodites, but a lower abundance of the major ones. Similarly, the floral scent of female flowers of cv. Mulata had a higher content of volatiles but a lower abundance of the major ones, when compared to cv. Galhosa. In each of the three gender types of flowers, the nectary disks seemed to be the major source of volatiles.
- Phytotoxic properties of Drosophyllum lusitanicum leaf extracts and its main compound plumbaginPublication . Gonçalves, Sandra; Ferraz, Marco; Romano, AnabelaThe aim of this work was to evaluate the phytotoxic properties of aqueous and hexane extracts from the insectivorous plant Drosophyllum lusitanicum (L.) Link using lettuce and bread wheat as model species. The results obtained confirmed that both germination and seedling growth bioassays were sensitive and able to detect the heterotoxicity potential of D. lusitanicum extracts. Aqueous and hexane extracts at several concentrations significantly inhibited the seed germination of lettuce and wheat, although wheat was less sensitive. The inhibitory effects of plumbagin, the major compound found in D. lusitanicum hexane extracts, were also evaluated. Comparing the results of the assays obtained with extracts and plumbagin it was postulated that plumbagin is the principal compound responsible for the phytotoxic effects of the extracts on lettuce but not on wheat. Therefore, although the phytotoxic potential of D. lusitanicum was demonstrated, further studies are needed to clearly specify the compounds responsible for the inhibitory effects and to ensure if the results obtained with themodel species are reproducible to weed species in field conditions.
- Insecticidal activity of leaf extracts from Drosophyllum lusitanicum against Liriomyza trifolii (Burgess) (Diptera: Agromyzidae)Publication . Gonçalves, Sandra; Gonçalves, Maria; Ameixa, Olga; Nogueira, José Manuel F.; Romano, AnabelaThe present study was carried out to evaluate the insecticidal properties of Drosophyllum lusitanicum (L.) Link, an insectivorous plant that is endemic to the western Iberian Peninsula and northwest Morocco. The insecticidal capacities of extracts from field-grown D. lusitanicum plants were evaluated using contact and feeding bioassays against female adults of Liriomyza trifolii (Burgess), one of the major pests of several floricultural and vegetable crops. In addition, the toxicity of the extract from in vitro cultures of D. lusitanicum was evaluated in contact bioassays. The results showed that extracts of D. lusitanicum have considerable insecticidal potential, particularly through contact application (100% mortality 1 d after treatment). Moreover, extracts from in vitro cultures had similar toxicity to those from field-grown plants. Extracts were analysed by gas chromatography coupled to mass spectrometry (GC-MS). The major constituent was 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), and its level was similar in extracts from field-grown plants (11.0 mg ml–1) and from in vitro cultures (8.2 mg ml–1). Although the results suggest that the insecticidal activity of extracts of D. lusitanicum is probably due to the presence of this naphthoquinone, further detailed research is required to identify other compounds that may be responsible for the activity.
- 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.
- Micropropagation of Drosophyllum lusitanicum (Dewy pine), an endangered West Mediterranean endemic insectivorous plantPublication . Gonçalves, Sandra; Romano, AnabelaIn this work, in vitro clonal propagation of Drosophyllum lusitanicum (Dewy pine) was obtained from seedlings germinated in vitro. Seeds were collected in various populations identified in the Algarve region and germinated in vitro on MS medium supplemented with 0.5 mg l 1 BA (6-benzyladenine) and 0.1 mg l 1 GA3 (gibberellic acid). The obtained shoots were used in several multiplication assays. The best results were observed in MS medium supplemented with 0.2 or 0.5 mg l 1 zeatin. The highest rooting frequency (83%) was observed on 1/4MS medium supplemented with 0.2 mg l 1 IBA (indole-3-butyric acid). Fifty percent of the plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. Plans are underway to reintroduce the in vitro produced plants from this study in selected locations in their natural habitat.
- A new medium formulation for in vitro rooting of carob tree based on leaf macronutrients concentrationsPublication . Gonçalves, Sandra; Correia, P. J.; Martins-Loução, M. A.; Romano, AnabelaExperiments were performed to optimize the macronutrients concentrations for in vitro rooting of Ceratonia siliqua micropropagated shoots. Several dilutions of Murashige and Skoog (MS) medium were tested: full-strength MS, halfstrength MS (½MS), and ½MS + full N. The frequency of in vitro rooting was enhanced when the ½MS was used (50 % rooted shoots). Mature leaves from 20 - 30 year-old carob trees and from 2 year-old micropropagated plants were collected and the concentrations of macronutrients (N, P, K, Ca, Mg) assessed. Based on the mineral composition of the leaves a new medium was formulated and compared with the previous ones showing an increment of the rooting frequency to 80 %. Moreover, shoots rooted in the new medium did not show symptoms of apical necrosis that occurred in the other tested media.
- Ex vitro rooting of Drosophyllum lusitanicum micropropagated shoots improves acclimatizationPublication . Gonçalves, Sandra; Romano, AnabelaHigh frequency survival of in vitro propagated shoots of Drosophyllum lusitanicum, an endangered insectivorous plant native of the southern part of the Iberian Peninsula and northern Morocco, through ex vitro rooting was accomplished. All the ex vitro rooting treatments performed in the present study favoured high acclimatization rates compared to that of in vitro rooting. It was observed that ex vitro rooting was strongly influenced by auxin type and concentration. Of the various treatments, ex vitro rooting after dipping the basal end of in vitro derived shoots in 1 g L-1 indole-3-butyric acid for 2 min facilitated the best rooting with a mean of 5 roots in 85% shoots and highest survival percentage. Rooted shoots exhibited lateral roots and presence of root hairs. Ex vitro rooting of D. lusitanicum favours rooting and hardening in a simple step with reduction of micropropagation cost.