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- 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.
- 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.
- In vitro morphogenesis in zygotic embryo cultures of carob tree (Ceratonia siliqua L.)Publication . Custódio, Luísa; Romano, AnabelaIn this study, organogenic cultures were induced from zygotic embryos of carob tree. Basal medium consisted of Murashige & Tucker (1969), supplemented with 0.5 or 1 mg L-1 of 2,4-dichlorophenoxyacetic acid alone or combined with 0.5, 1, 2, 3 and 4 mg L-1 of kinetin. Calli initiation occurred in all the hormonal combinations tested. The zygotic embryo cultures differentiated two types of regenerants: somatic embryos (SEs) and shoots, with a maximum frequency of 30 and 100%, respectively. SEs exhibited morphological abnormalities, such as fusion and absence of cotyledons, and showed secondary embryogenesis. The regenerated shoots were rooted, hardened and transferred to the soil.
- Sex and developmental stage of carob flowers affects composition of volatilesPublication . Custódio, Luísa; Nogueira, José Manuel F.; Romano, AnabelaThe aim of this work was to evaluate the volatiles composition in the scent emitted in vivo by the three types of carob tree (Ceratonia siliqua L.) flowers, during the different developmental stages. Additionally, an overview of some morphological aspects of carob flowers was also done. Carob trees may be male, female, hermaphrodite or exhibit polygamous inflorescences. The androecium of male flowers consists of a group of 5-7 stamens, inserted peripherically around the receptacle. The gynoecium of female flowers is apocarpous. Hermaphrodite flowers are a combination of male and female types. During the flower development six stages were identified. The volatile compounds of flowers were analysed by headspace solid-phase microextraction followed by capillary gas chromatography and mass spectrometry detection (HS-SPME-CGC/MSD). More than 25 compounds were identified, and flowers in stages III and IV showed higher abundances. In male and hermaphrodite flowers the total volatile compounds were more abundant in stage III, and in female flowers in stage IV. Linalool and trans linalool oxide were the dominant volatiles and there were remarkable differences in those compounds between different types of flowers and during the developmental stages. The results observed were discussed in terms of their possible implication on pollination efficiency.
- Floral analysis and seasonal dynamics of mineral levels in carob tree leavesPublication . Custódio, Luísa; Correia, P. J.; Martins-Loução, M. A.; Romano, AnabelaThe mineral concentration of flowers and the seasonal fluctuation of macro- [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg)] and micronutrients [iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu)] in leaves of male, female, and hermaphroditic carob trees (Ceratonia siliqua L.) were studied. The nutrient dynamics were linked not only to phenological events, but also to the gender of the trees. The females were able to allocate more nutrients to leaves than male trees, even though male flowers were richer in particular elements such as N and Zn. The hermaphrodites supported the development of both inflorescences and fruits with a lower seasonal variation and a lower leaf nutrient concentration, as compared to the other genders, which may indicate a more efficient use of resources. Flowers had, in general, a higher concentration of N, P, and K, and a lower Mn concentration than in leaves. Flowers of the females had a lower nutrient concentration compared to males or hermaphrodites.
- Microsporogenesis and anther culture in carob tree (Ceratonia siliqua L.)Publication . Custódio, Luísa; Carneiro, M. F.; Romano, AnabelaAn in vivo study was made on male flowers of carob tree (Ceratonia siliqua L.), in order to establish a correlation between the flower and anther development, and microsporogenesis. In addition studies were conducted to find which phase is more appropriate for anther culture and haploid production. During the development of male flowers, six stages were identified. The male gametophytic cycle begins when flowers are in developmental phase 0, with the formation of the epidermis, endothecium, primary sporogencous tissue, primary parietal cells and pollen mother cells. During developmental phase I we observed the formation of pollen mother cells, the microspore tetrads, and uni- and binucleate pollen grains. At developmental phase II, uni- and binucleate microspores, and completely formed pollen grains were observed. In developmental phase III we could observe mature pollen grains ready to be released from the anthers as single binucleate pollen grains. Anthers from flowers at developmental phases I and II, with microspores at late uninucleate to early binucleate stage, were cultured in semi-solid Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) combined with one of the citokinins: N-6-benzyladenine (BA), kinetin (Kin), zeatin (Zea) and thidiazuron (TDZ). To obtain embryogenic calli anthers should be collected from flowers in developmental phase I. High frequencies of callogenesis were obtained, and the best medium for calli induction was MS supplemented with 0.5 mg l(-1) 2,4-D + 4 mg l(-1) TDZ. The frequency of haploid cells was found to be 17.2%. (C) 2004 Elsevier B.V. All rights reserved.
- Influence of sugars on in vitro rooting and acclimatization of carob treePublication . Custódio, Luísa; Martins Loução, M.; Romano, AnabelaCarob tree (Ceratonia siliqua L.) micropropagated shoots were rooted on half-strength Murashige and Skoog medium, supplemented with different types and concentrations of sugars, in order to determine the effects of sugar composition and concentration on in vitro rooting and in vivo establishment of the plantlets. Among the various sugars tested, the best rooting response was obtained with 145 mM sucrose, both in terms of rooting frequency and index of rooting. The use of filter-sterilized rather that autoclaved fructose increased root number and root length. Sugar treatment during rooting slightly influenced plantlet survival and growth during acclimatization. A reduction in the glucose concentration during rooting was beneficial for plantlet acclimatization.
- New insights on carob floral biologyPublication . Custódio, Luísa; Romano, Anabela; Carneiro, Maria FilomenaO objectivo geral deste trabalho foi o estudo de diferentes aspectos da biologia floral da alfarrobeira {Ceratonia siliqua L.), uma das árvores mais importantes da região Mediterrânica, particularmente os relacionados com o processo reprodutivo. Foi observado que o androceu das flores masculinas funcionais é composto por um grupo de 4-7 estames, inseridos na periferia do receptáculo. Cada filamento é parcialmente rodeado por uma sépala, e possui anteras dorsifixas. O gineceu das flores femininas funcionais é apocárpico, e possui um ovário unilocular superior, inserido numa depressão central no disco nectarial. As flores hermafroditas são uma combinação dos tipos anteriores, e possuem o androceu e o gineceu. Durante o desenvolvimento das flores foram identificadas seis fases. As anteras das flores masculinas e hermafroditas possuem duas tecas com quatro microsporângios posicionados lateralmente, que abrem longitudinalmente quando estão maduros. O processo de microsporogénese é sucessivo, e resulta na formação de grãos de pólen binucleados. O processo de macrogametogénese parece ser do tipo Polygonum. A formação dos óvulos e a macrosporogénese ocorrem durante as fases 0, I e II de desenvolvimento da flor, enquanto que a megagametogénese se inicia na fase III. Mais de vinte e cinco compostos voláteis foram identificados in vivo no aroma de flores inteiras e partes florais isoladas, por HS-SPME-CGC/MSD. As flores femininas possuem menos compostos voláteis do que as masculinas, e as flores nas fases de desenvolvimento III e IV apresentam maiores abundâncias. Os compostos voláteis dominantes foram o linalool e seus derivados. As diferenças na concentração de nutrientes entre os orgãos vegetativos e reprodutivos são influenciadas não só pelo estado fenológico, mas também pelo tipo sexual da árvore. As flores femininas exibem uma menor concentração de nutrientes do que as flores masculinas e hermafroditas. Para alem disso, as árvores femininas apresentam uma maior capacidade de translocação de nutrientes para as folhas, comparativamente com as masculinas. A dinâmica de partição e translocação de hidratos de carbono não estruturais parece ser bastante complexa, e dependente do tipo sexual da árvore e da cultivar feminina. A glucose foi o principal açúcar solúvel detectado nas folhas, flores e frutos. Foi observada uma maior acumulação de açúcares solúveis totais na polpa, em frutos na fase de desenvolvimento III. As galactomananas foram o único açúcar solúvel detectado nas sementes, e não foram observadas diferenças na sua concentração, entre tipos sexuais e cultivares femininas. Foi desenvolvido um protocolo de criopreservação de pólen, e os melhores resultados foram obtidos após um pré-tratamento com 0.5 M sacarose, ou com glicerol (2.5%) + DMSO (2.5%), após um período de conservação de 5 meses. Para a obtenção de calli embriogénico devem ser utilizadas anteras isoladas de flores na fase de desenvolvimento I, e inoculadas em meio basal MS suplementado com 5 mg f1 2,4-D + 4 mg f1 TDZ.
- Quantification of Polyphenols in Carob Tree (Ceratonia siliqua L.) Fruits and Leaves in Portuguese CultivarsPublication . Custódio, Luísa; Fernandes, E.; Romano, AnabelaMethanol extracts from leaves and pulps from male, female, and hermaphrodite carob trees were studied for their contents of total phenols, tannins and flavonoids. In the three genders, significantly higher amounts of total phenols, tannins and flavonoids were detected in leaf extracts. Gender significantly affected the phenolic profile with the hermaphrodites being generally richer in phenols. Our results confirm that leaves and pulps of carob tree contain high amounts of phenols, being potentially attractive as a raw material for the pharmaceutical industry.
- Cryopreservation of pollen of carob treePublication . Custódio, Luísa; Romano, Anabela; Fernandes, N.; Carneiro, M. F.In this work, anthers isolated from male flowers of carob tree (Ceratonia siliqua L.) at developmental phase II were cryopreserved by vitrification, using a fast freezing method. Different types of carbohydrates at different concentrations, namely sorbitol (0.5 M, 1 M, and 2 M), mannitol (0.5 M and 1 M), sucrose (0.5 M, 1 M, and 2 M), and glucose (0.5 M, I M, and 2 M), namely sorbitol, mannitol, sucrose and glucose, were compared with respect to their capacity of inducing freeze tolerance in pollen of carob tree. Carbohydrates were applied as a pre-treatment to anthers before cryostorage. It was also assessed if the presence of the cryoprotectant during the storage period was beneficial for pollen viability. The viability of cryopreserved pollen was evaluated after 5 and 8 months of storage. The application of the cryoprotectants generally increased pollen viability as compared with the control. The best results were obtained after 5 months storage, in the presence of the cryoprotector, with pollen pre-treated with sucrose 0.5 M. The viability of pollen decreased with increasing the duration of storage period.