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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.
- Field performance of seedlings and microproppagated plants of carob treePublication . Osório, Maria Leonor; Osório, Júlio; David, Maria Manuela; Romano, AnabelaMicropropagated plantlets of cv. Mulata and cv. Galhosa growing in pots, after acclimatization in the glasshouse and growth for several months under natural conditions, were transferred to a field trial. Carob seedlings of ‘Mulata’ were also transferred under the same conditions. The field trial has been established with 100% of success in micropropagated plants and 97% in seedlings. Three months after transfer to the field, plants showed good growth but micropropagated plants of ‘Mulata’ exhibited more vigour than ‘Galhosa’ and than seedlings, with the greatest number of branches and a larger length increase in the main stem. Before transference to the field, net photosynthetic rates (PN), water use efficiency (PN/gs) and quantum yield of PSII (fPSII) of the potted plants were higher in seedlings than in micropropagated plants. Three months after transplantation all gas exchange parameters were improved for all types of plants, particularly net photosynthetic rate, and no significant differences were observed between plants either micropropagated or seedlings. On the other hand, fPSII decreased significantly in seedlings. The results suggest that the pre-acclimation, in pots, to external environmental conditions might have improved field survival, plant vigour and enhanced the endurance to adverse conditions of micropropagated carob trees.
- Antioxidant activity and in vitro inhibition of tumor cell growth by leaf extracts from the carob tree (Ceratonia siliqua)Publication . Custódio, Luísa; Fernandes, Eliana; Escapa, Ana Luísa; López-Avilés, Sandra; Fajardo, Alba; Aligué, Rosa; Alberício, Fernando; Romano, AnabelaThe methanol leaf extracts of female cultivars of the carob tree [Ceratonia siliqua L. (Fabaceae)] and of hermaphrodite and male trees were investigated for their contents of phenolic compounds, their in vitro antioxidant activity, measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and linoleic acid system assays, and their in vitro tumor growth inhibition on HeLa cells. The different cultivars and trees showed high levels of phenols, and considerable variations in the amount of these compounds. The extracts showed significant radical scavenging activity (RSA), which was not significantly affected by the gender of the tree. From the female cultivars tested, Galhosa exhibited the highest RSA. Gender significantly affected the antioxidant activity of the extracts measured by the linoleic acid system assay, and males and hermaphrodites showed the highest activities. The extracts displayed a remarkable ability to inhibit tumor cell proliferation, and their bioactivity varied with different cultivars or trees tested. Extracts from male and hermaphrodite trees exhibited higher capacity to inhibit the proliferation of HeLa cells than the female cultivars.
- In vitro spore germination of Polystichum drepanum, a threatened fern from Madeira IslandPublication . Chá-Chá, R.; Fernandes, F.; Romano, AnabelaPolystichum drepanum (Sw) C. Presl is a threatened fern endemic to a few forest areas in the north-west of Madeira Island. The aims of this work were to establish suitable culture conditions for in vitro germination of spores, and to evaluate short-term storage conditions for P drepanum spores. The highest frequency of germination was obtained in Murishage and Skoog (MS) liquid medium, without agitation. However, gametophytes maintained in MS liquid medium did not grow and, after 4 weeks, became anoxic and died. Thus, after germination in liquid medium, gametophytes were transferred to an MS double-phase culture system for further growth. The effects of storage period, temperature, and relative humidity during storage on in vitro spore germination were studied. Spore viability was assessed after 2, 4 and 6 months, and high viability (> 94%) was observed in all the assays. However, germination capability decreased with increased storage periods. The number of sporophytes obtained also decreased with prolonged storage periods. The results indicate that spores of R drepanum stored for 4 months at 21 degrees C maintain high viability and high germination frequency. ne sporophytes obtained were acclimatised in a mixture of peat and vermiculite [2:1 (v/v)] under high relative humidity (90-95%). Seventy-five sporophytes were successfully acclimatised to ex vitro conditions and showed active growth in the glasshouse.
- 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.
- In vitro mass clonal propagation of Dittrichia viscosa subsp revoluta and analysis of its secondary metabolitesPublication . Costa, M.; Nogueira, J. M. F.; Miguel, Maria Graça; Romano, AnabelaIn vitro clonal propagation of native Portuguese Dittrichia viscosa subsp. revoluta was obtained from mature field-grown plants. High numbers of shoots (8-12 shoots per culture) were obtained for all clones and benzyladenine (BA) concentrations (0.44, 0.89 and 1.33 muM) tested. All clones developed numerous roots after auxin treatment, with the root length strongly dependent on auxin type (IAA, IBA, NAA). All plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. The essential oils of D. viscosa were analysed by capillary GC and GC-MS using three types of plant material from the same clone: field-grown plant (FP), in vitro shoot-cultures (InV) and micropropagated plants (MP). The 24 identified components represented 39.9, 42.0 and 49.5% of the total oils in FP, InV and MP samples, respectively. The major constituents of the essential oils were 1,8-cineole (11.0, 5.1 and 5.1%), delta-cadinene (5.0,7.0 and 6.6%); E-nerolidol (4.4,9.7 and 11.9%); T-cadinol (7.1,14.4 and 17.4%) and a-cadinol (6.7, 8.4 and 9.4%), for FP, InV and MP, respectively. Efficient separation of the flavonoids was achieved by RP-HPLC. Quercetin was the major component whose concentrations ranged from 0.3 mg per 100 mg FW, in FP, to 48.5 mg per 100 mg FW, in InV samples.
- 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.