Responses of antioxidant enzymes, photosynthetic pigments and carbohydrates in micropropagated Pitcairnia encholirioides L.B. Sm. (Bromeliaceae) under ex vitro water deficit and after rehydration / Respostas de enzimas antioxidantes, pigmentos fotossintéticos e carboidratos em Pitcairnia encholirioides L.B. Sm. (Bromeliaceae) micropropagadas, sob déficit hídrico ex vitro e após reidratação

Abstract In this study, the activities of antioxidant enzymes, photosynthetic pigments, proline and carbohydrate contents in Pitcairnia encholirioides under ex vitro conditions of water deficit were evaluated. Results show that plants under progressive water stress, previously in vitro cultured in media supplemented with 30 g L-1 sucrose and GA3, accumulated more proline and increased peroxidase (POD) activity and the contents of photosynthetic pigments and carbohydrates. For plants previously in vitro cultured with 15 g L-1 sucrose and NAA, no differences were found for proline content and there were reductions in activities of peroxidase (POD), catalase (CAT) and poliphenoloxidase (PPO), and in contents of carbohydrates, with progress of ex vitro water deficit. After rehydration, plants showed physiological recovery, with enzymatic activities and contents of metabolites similar to those found in the controls not submitted to dehydration, regardless of the previous in vitro culture conditions. These results show that micropropagated P. encholirioides has high tolerance to dehydration once in ex vitro conditions, which can ensure the survival of plants from tissue culture when transferred to its natural environment, emphasizing the importance of such biotechnology for the propagation of endangered species.

Resumo Neste estudo, foram avaliadas as atividades de enzimas antioxidantes, pigmentos fotossintéticos, conteúdo de prolina e carboidratos em Pitcairnia encholirioides sob déficit hídrico em condições ex vitro. Os resultados mostraram que as plantas sob estresse hídrico progressivo, previamente cultivadas in vitro em meio de cultura suplementado com 30 g L-1 de sacarose e GA3 acumularam mais prolina e aumentaram a atividade da peroxidase (POD) e os teores de pigmentos fotossintéticos e carboidratos. Para plantas previamente cultivadas in vitro com 15 g L-1 de sacarose e ANA, não foram encontradas diferenças nos conteúdos de prolina e houve reduções nas atividades da peroxidase (POD), catalase (CAT) e polifenoloxidase (PPO), e no conteúdo de carboidratos, com o progresso do déficit hídrico ex vitro. Após a reidratação, as plantas apresentaram recuperação fisiológica, com atividades enzimáticas e conteúdo de metabólitos semelhantes aos encontrados nos controles não sujeitos à desidratação, independentemente das condições de cultivo in vitro. Estes resultados mostram que P. encholirioides micropropagada tem alta tolerância à desidratação uma vez em condições ex vitro, o que pode garantir a sobrevivência de plantas provenientes da cultura de tecidos quando transferidas para seu ambiente natural, enfatizando a importância desta biotecnologia para a propagação de espécies ameaçadas.

Pollen morphology and viability in Bromeliaceae

ABSTRACT Pollen morphology characterization is important in taxonomy, conservation and plant breeding, and pollen viability studies can support breeding programs. This study investigated pollen morphology and male fertility in 18 species of Bromeliaceae with ornamental potential. For morphological characterization, pollen grains were acetolyzed and characterization of exine was done using scanning and transmission electron microscopy. Pollen viability was investigated by in vitro germination and histochemical tests. Species belonging to Aechmea and Ananas genera presented medium size pollen, except for Ae. fasciata, with large pollen. Al. nahoumii, P. sagenarius and the Vriesea species analyzed showed large pollen, except for V. carinata, with very large pollen. Pollen of Aechmea, Ananas and P. sagenarius presented bilateral symmetry, diporate, exine varying from tectate to semitectate. Al. nahoumii and Vriesea species presented pollen with bilateral symmetry, monocolpate; exine was semitectate, reticulate and heterobrochate. Germination percentage and tube growth were greater in SM and BKM media. Histochemical tests showed pollen viability above 70% for all species, except for Ananas sp. (40%). Pollen morphology is important for the identification of species, especially in this family, which contains a large number of species. High rates of viability favor fertilization and seed production, essential for efficient hybrid production and conservation.

Viabilidad y germinación de semillas de Hechtiaperotensis (Bromeliaceae) / Viability and germination of Hechtia perotensis (Bromeliaceae) seed

ResumenPoblaciones endémicas de Hechtiaperotensis se han descrito en Puebla y Veracruz, México. La recolección de semillas de buena calidad permite su uso en conservación, investigación y restauración ecológica. Algunos compuestos para evaluar la calidad de las semillas silvestres y endémicas, como nitrato de potasio (KNO3) y ácido giberílico (AG3), se utilizan para incrementar la germinación de las semillas y disminuir la latencia. La prueba de cloruro de trifeniltetrazolio (tetrazolio) se correlaciona con la viabilidad de las semillas porque se basa en la actividad de las deshidrogenasas de tejidos vivos que catalizan la respiración mitocondrial. El objetivo de este estudio fue conocer el tamaño y el peso de las cápsulas y las semillas y la germinación y viabilidad de las semillas de H. perotensis recolectadas en Veracruz en 2012 y 2015. Las hipótesis fueron (1) que la germinación y la viabilidad de las semillas son independientes del año de recolecta, (2) que hay una concentración adecuada de tetrazolio para identificar la viabilidad de las semillas y (3) que el pretratamiento con KNO3 o AG3 incrementa la germinación de las semillas. La germinación se evaluó con un diseño completamente al azar con tres tratamientos (testigo y promotores de la germinación KNO3 0.2 % y AG3 500 mg/L), cuatro tratamientos para la prueba de viabilidad (testigo, 0.2, 0.5 y 1.0 % de tetrazolio) y seis repeticiones de cada tratamiento. La germinación se evaluó en 100 semillas y la viabilidad en 25. Los resultados entre y dentro de años se analizaron con ANDEVA y prueba de comparación múltiple de medias de Tukey. La proporción de semillas no germinadas se cuantificó junto con el número de plántulas normales y anormales, semillas con embrión viable o sin él, y con viabilidad baja o sin viabilidad. En promedio la muestra recolectada en 2012 tuvo 36 % de semillas con embrión viable, 7 con viabilidad baja, 24 % no viables y 33 % sin embrión. Este resultado fue significativamente diferente al de 2015, que presentó 87 % de embriones viables, 10 % con viabilidad baja, 0 % no viables y 3 % sin embrión. La germinación también fue significativamente diferente entre los años (22 y 92 %). Los tratamientos pregerminativos no modificaron la germinación. La germinación y la viabilidad de las semillas de H. perotensis varían significativamente entre los años de recolecta.

Abstract:Endemic populations of Hechtiaperotensis have been described in Puebla and Veracruz, Mexico. Good quality seed collections can be used in conservation, research and ecological restoration. To evaluate seed quality of wild and endemic species, some compounds are used as effective promoters of germination, such as potassium nitrate (KNO3) and gibberellic acid (AG3), because they increase seed germination capacity and reduce latency. The triphenyl tetrazolium chloride (tetrazolium) test correlates seed viability because it is based on the activity of dehydrogenases in live tissues that catalyze mitochondrial respiration. The objective of this study was to obtain information on size and weight of capsules and seeds and seed germination and viability of H.perotensis, collected in Veracruz in the year 2012 and 2015. The hypotheses were 1) that seed germination and viability are independent of the year of collection, 2) that there is a tetrazolium concentration that can identify seed viability better than others, and 3) that pretreatment with KNO3 or AG3 improves seed germination. Seed germination was assessed using a completely randomized design with three treatments (control and the germination promoters 0.2 % KNO3 and 500 mg/L AG3), four treatments for the viability test (control, 0.2, 0.5 and 1.0 % of tetrazolium) and six replicates for each treatment. A total of one hundred seeds for germination experiments, and 25 seeds for the viability test were used. The results between and within years were analyzed with ANOVA and multiple comparison with the Tukey test. The proportion of non-germinated seeds was quantified along with the number of normal and abnormal seedlings, seeds with viable embryo, seeds without embryo, and seeds with low or no viability. On average, for the 2012 collected sample, 36 % had viable embryos, 7 % had low viability, 24 % were not viable and 33 % had no embryo. This result was significantly different from the 2015 sample, for which 87 % of seed showed viable embryos, 10 % had low viability, 0 % was not viable and 3 % had no embryo. Seed germination was also significantly different between years (22 and 92 %) Pregerminative treatments did not improve germination. Seed germination and viability of H. perotensis significantly varied between years of seed collection. Rev. Biol. Trop. 65 (1): 153-165. Epub 2017 March 01.

Effects of climate and forest structure on palms, bromeliads and bamboos in Atlantic Forest fragments of Northeastern Brazil / Efeitos do clima e da estrutura da vegetação em palmeiras, bromélias e bambus em fragmentos de Mata Atlântica no nordeste brasileiro

Abstract Palms, bromeliads and bamboos are key elements of tropical forests and understanding the effects of climate, anthropogenic pressure and forest structure on these groups is crucial to forecast structural changes in tropical forests. Therefore, we investigated the effects of these factors on the abundance of these groups in 22 Atlantic forest fragments of Northeastern Brazil. Abundance of bromeliads and bamboos were assessed through indexes. Palms were counted within a radius of 20 m. We also obtained measures of vegetation structure, fragment size, annual precipitation, precipitation seasonality and human population density. We tested the effects of these predictors on plant groups using path analysis. Palm abundance was higher in taller forests with larger trees, closed canopy and sparse understory, which may be a result of the presence of seed dispersers and specific attributes of local palm species. Bromeliads were negatively affected by both annual precipitation and precipitation seasonality, what may reflect adaptations of these plants to use water efficiently, but also the need to capture water in a regular basis. Bamboos were not related to any predictor variable. As climate and forest structure affected the abundance of bromeliads and palms, human-induced climatic changes and disturbances in forest structure may modify the abundance of these groups. In addition, soil properties and direct measurements of human disturbance should be used in future studies in order to improve the predictability of models about plant groups in Northeastern Atlantic Forest.

Resumo Palmeiras, bromélias e bambus são importantes elementos das florestas tropicais e, entender os efeitos do clima, pressão antrópica e estrutura da floresta nesses grupos é crucial para prever alterações estruturais em florestas tropicais. Portanto, nós investigamos os efeitos desses fatores na abundância desses grupos vegetais em 22 fragmentos de Mata Atlântica no nordeste brasileiro. Abundâncias de bromélias e bambus foram registradas através de índices. Palmeiras foram contadas em um raio de 20 m. Nós também obtemos medidas da estrutura da vegetação, tamanho de fragmento, precipitação anual, sazonalidade na precipitação e densidade populacional humana. Nós testamos os efeitos desses preditores nos grupos vegetais através de análises de caminhos. A abundância de palmeiras foi maior em florestas mais altas, com árvores mais grossas, dossel fechado e sub-bosque aberto, o que deve refletir a presença de dispersores de sementes e atributos específicos das espécies de palmeiras locais. Bromélias foram negativamente afetadas pela precipitação anual e pela sazonalidade na precipitação, o que deve refletir adaptações dessas plantas para o uso eficiente da água, mas também a necessidade de captar água regularmente. Bambus não estiveram relacionados com nenhum dos preditores avaliados. Dado que clima e estrutura florestal afetaram a abundância de bromélias e palmeiras, as mudanças climáticas e distúrbios na estrutura das matas causados por ações antrópicas podem alterar a abundância desses grupos. Adicionalmente, propriedades do solo e medidas diretas de distúrbios antrópicos devem ser usadas em estudos futuros para melhorar o poder preditivo dos modelos sobre a abundância de plantas na Mata Atlântica do nordeste brasileiro.

Proline levels, oxidative metabolism and photosynthetic pigments during in vitro growth and acclimatization of Pitcairnia encholirioides L.B. Sm. (Bromeliaceae) / Niveis de prolina, do metabolismo oxidativo e dos pigmentos fotossintéticos durante o crescimento in vitro e aclimatização de Pitcairnia encholirioides L.B. Sm. (Bromeliaceae)

Abstract This study aimed to evaluate the variation in the levels of proline, oxidative metabolism and photosynthetic pigments in plants of Pitcairnia encholirioides grown in vitro under different conditions and after acclimatization. The analyses were performed after 150 days of in vitro cultivation in MS media supplemented with 10 µM GA3 or 0.2 µM NAA, sucrose at 15 or 30 g L–1, in test tubes which allowed gas exchange or in a hermetically sealed system, and 180 days after acclimatization. The in vitro maintenance in hermetically sealed flasks, with GA3 and 15 g L–1 sucrose had adverse metabolic effects, which was demonstrated by the lower proline and photosynthetic pigments accumulation and by the increase in antioxidant enzymes activities. After acclimatization, differences for proline and photosynthetic pigments were no longer found and the enzymatic activities ranged unevenly. The results suggest that the in vitro cultivation in media with 0.2 µM NAA and 30 g L–1 sucrose, in test tubes capped with closures which allowed gas exchange, is more suitable for micropropagation of P. encholirioides, providing a prolonged maintenance of in vitro cultures and plantlets with superior quality for ex vitro development.

Resumo Este trabalho objetivou avaliar a contribuição da prolina, do metabolismo oxidativo e dos pigmentos fotossintéticos na propagação in vitro e aclimatização de Pitcairnia encholirioides, uma bromélia criticamente ameaçada de extinção. As análises foram realizadas após 150 dias de cultivo in vitro em meio MS suplementado com 10 µM de GA3 ou 0,2 µM de ANA, 15 ou 30 g L–1 de sacarose, em tubos de ensaio que permitiam trocas gasosas ou em sistema hermeticamente vedado, e também 180 dias após aclimatização. A manutenção in vitro em frascos hermeticamente fechados, com GA3 e 15 g L–1 de sacarose apresentou efeito metabólico adverso, demonstrado pelo menor acúmulo de prolina e pigmentos fotossintéticos e também pelo aumento das atividades de enzimas antioxidantes. Após aclimatização, as diferenças para prolina e pigmentos fotossintéticos não foram mais encontradas e as atividades enzimáticas variaram de maneira desuniforme. Os resultados sugerem que o cultivo in vitro em meio com 0,2 µM de ANA e 30 g L–1 de sacarose, em tubos fechados com tampas que permitem trocas gasosas, é mais adequado para a micropropagação de P. encholirioides, proporcionando uma manutenção prolongada das culturas in vitro e plântulas com qualidade superior para o desenvolvimento ex vitro.

Reproductive biology of Hechtia schottii, a dioecious Bromeliaceae, in Mexico

Hechtia schottii is a terrestrial, rosetofilous, dioecious, polycarpic succulent herb, that grows mainly in shrubby associations, and less frequently, in secondary low caducifolious forests, both on calcareous soils or limestone outcrops in Yucatan and Campeche States, Mexico. We studied phenology, floral and pollination biology, and breeding system at Calcehtok, Yucatan, during two flowering seasons. Plants bloom mainly during the dry season (November-April) and disperse seeds during the rainy season (May-October). Both floral morphs have diurnal anthesis; pollen is removed ca. 1 h after anthesis starts and both floral morphs are visited by several insect species, especially bees, but results suggest that the introduced honey bee, Apis mellifera, is the pollinator. Controlled crossings show that the species is functionally dioecious and requires to be serviced by pollinators based on fruit setting only in unassisted cross pollination crosses.

Hechtia schottii es una hierba terrestre, suculenta, rosetófila, dioica y policárpica, que crece en asociaciones arbustivas y selva baja caducifolia secundaria, ambos en suelos calcáreos o limosos. Estudiamos la fenología, la biología floral, reproductiva y de la polinización en una población en Calcehtok, Yucatán, México, durante dos estaciones de floración. Las plantas florecen principalmente en la época de secas (noviembre-abril) y la dispersión de semillas es durante la estación de lluvias (mayo-octubre). Ambas formas (morphs) florales tienen antesis diurna; el polen es removido ca. 1 h después del comienzo de la antesis y ambas formas florales son visitadas por varias especies de insectos, especialmente abejas, pero los resultados sugieren que la abeja introducida, Apis mellifera, es el polinizador. Cruces controlados muestran que la especie es funcionalmente dioica y que requiere de un polinizador, ya que solo produce frutos por polinización no asistida entre formas.

Spatial distribution by Canistropsis microps (E. Morren ex Mez) Leme (Bromeliaceae: Bromelioideae) in the Atlantic rain forest in Ilha Grande, Southeastern Brazil

Canistropsis microps (Bromeliaceae: Bromelioideae) is an endemic species of Atlantic rain forest areas in Rio de Janeiro State, which are very abundant in not very disturbed forests in Ilha Grande, on the southern coast of the State. In this study, we analyzed the vertical and horizontal distribution patterns of the species in an area of rain forest with little evidence of disturbance at Vila Dois Rios, Ilha Grande, relating the patterns to sunlight in the microhabitat. We also identified the types of substrate used by the species and the rate of asexual reproduction. Canistropsis microps had high densities (estimated at 84,425 rosettes/ha), and has an aggregated distribution (Id = 2.86). About 80 percent of the rosettes were generated by clonal growth, whereas less than 20 percent were produced from seedlings. Most of the rosettes were found on straight tree trunks (DBH > 50 cm). There was a significant inverse correlation between the incidence of sunlight in the habitat and the abundance of individuals. Rosettes were found up to a maximum height of 9.5 m, but most occured between 1.5 and 5.5 m, where light varied from 25 to 50 µmol.s-1.m-2. We conclude that vertical and horizontal distribution patterns in C. microps may be partially explained by the occurrence of appropriate substrate, an intensity of sunlight favorable to the development of the species and to a high rate of vegetative reproduction.

Canistropsis microps (Bromeliaceae: Bromelioideae) é uma espécie endêmica das áreas de Mata Atlântica do Estado do Rio de Janeiro, sendo muito abundantes em florestas pouco perturbadas da Ilha Grande, na costa sudeste do Estado. Nesse estudo, nós analisamos o padrão de distribuição vertical e horizontal da espécie em uma área de floresta com pouca evidência de distúrbio na Vila Dois Rios, Ilha Grande, relacionando os padrões à incidência de luz no microhabitat. Nós também identificamos os tipos de substratos usados pela espécie e a taxa de reprodução assexuada. Canistropsis microps possui elevadas densidades (estimada em 84.425 rosetas/ha), e uma distribuição agregada (Id = 2,86). Cerca de 80 por cento das rosetas são geradas pelo crescimento clonal, enquanto menos que 20 por cento são produzidos a partir de sementes. Muitas das rosetas foram encontradas em troncos retos de árvores (DAP > 50 cm). Houve uma correlação significativa inversa entre a incidência da luz solar no habitat e abundância de de indivíduos. As rosetas foram encontradas até uma altura máxima de 9,5 m, mas a maioria ocorreu entre 1,5 e 5,5 m, onde a luz variou de 25 a 50 µmol.s-1.m-2. Nós concluímos que o padrão de distribuição vertical e horizontal em C. microps pode ser parcialmente explicada pela ocorrência de um substrato apropriado, uma intensidade de luz solar favorável para o desenvolvimento da espécie e a alta taxa de reprodução vegetativa.

Heterogeneity of terrestrial bromeliad colonies and regeneration of Acacia praecox (Fabaceae) in a humid-subtropical-Chaco forest, Argentina

In several tropical and subtropical forests, plants of the understorey act as an ecological filter that differentially affects woody species regeneration. In convex sectors of the Schinopsis balansae (Anacardiaceae) forests of the Southeastern Chaco there are dense colonies of terrestrial bromeliads. These may influence forest regeneration by intercepting rain water and propagules in their tanks. Within colonies, the spatial distribution of bromeliads is clumped because their clonal growth leaves numerous internal gaps. In this study we describe the internal heterogeneity of three bromeliad colonies (plots) and analyze how this heterogeneity affects Acacia praecox regeneration (i.e. seedling recruitment and survival). In January 1996, we randomly placed three transects with 150 contiguous quadrats of 100 cm2 in each plot. For each quadrat we recorded the type of floor cover (i.e. bromeliads, herbs, litter, or bare soil) and the presence of A. praecox seeds or seedlings. In July 1996 we relocated the transects and recorded seedling survival. Bromeliad colonies showed a high internal heterogeneity. Almost half of the 450 quadrats were covered by two terrestrial bromeliads. Aechmea distichantha was recorded in 81% of all quadrats with bromeliads, and Bromelia serra in the others. All quadrats with bromeliads were covered by litter. Half of them were occupied by the bases of bromeliads and the others were covered by their leaves. In contrast, where bromeliads were not present, soil surface was covered by litter in 83% and by herbaceous vegetation in 11% of the quadrats; very few quadrats were covered by bare soil. In January 1996, we recorded 127 seeds and 176 seedlings of A. praecox. Seed and seedling densities of A. praecox were similar in quadrats with and without bromeliads, but variability in seedling density of A. praecox was higher within than among plots. Seed density was higher in quadrats covered by bromeliad leaves than inside the tanks.

En varios bosques tropicales y subtropicales, las plantas del sotobosque actúan como filtro ecológico que afecta diferencialmente la regeneración de las especies leñosas. En los bosques de Schinopsis balansae (Anacardiaceae) del Chaco Oriental existen densas colonias de bromeliáceas terrestres que afectarían su regeneración al interceptar el agua de lluvia y los propágulos dentro de sus tanques. Estas bromeliáceas tienen distribución agrupada dejando espacios libres entre ellas. Describimos la heterogeneidad de tres colonias y analizamos como afectan la regeneración de Acacia praecox. En enero 1996, colocamos en cada colonia tres transectos con 150 parcelas de 100 cm2. En cada parcela registramos el tipo de cobertura (i.e. bromeliáceas, hierbas, hojarasca ó suelo desnudo) y la presencia de semillas o plántulas de A. praecox. En julio 1996, registramos la supervivencia de plántulas. Las colonias de bromiliáceas mostraron una alta heterogeneidad interna. Casi la mitad de las 450 parcelas estuvo cubierto por dos bromeliáceas terrestres. Aechmea distichantha se registró en 81% de las parcelas con bromeliáceas y Bromelia serra en las otras. Todas las parcelas estuvieron cubiertas por hojarasca. La mitad de ellas estuvieron cubiertas por hojas de bromeliáceas y el resto ocupado por sus bases. Donde no había bromeliáceas, el suelo estuvo cubierto por mantillo (83%), herbáceas (11%), u otros. En enero registramos 127 semillas y 176 plántulas de A. praecox. La densidad de ambas fue similar en parcelas con y sin bromeliáceas, pero su variabilidad fue mayor dentro de cada colonia que entre ellas. La densidad de semillas fue mayor debajo de las hojas de bromeliáceas que dentro de las plantas. La supervivencia de plántulas fue superior en parcela con bromeliáceas en sólo una de las tres colonias. Ninguna plántula sobrevivió dentro de las bromeliáceas. Aparentemente...