Crescimento in vitro e caracterização bioquímica, morfológica e histoquímica de culturas embriogênicas de Ocotea catharinensis Mez. (Lauraceae)

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas.Ocotea catharinensis Mez. é uma espécie florestal nativa da Mata Atlântica, de importância econômica e em risco de extinção. Um sistema de embriogênese somática foi desenvolvido para esta espécie. Entretanto, a taxa de germinação dos embriões somáticos é baixa. O objetivo deste trabalho foi a avaliação do crescimento in vitro das culturas embriogênicas, em diferentes condições de cultura e realizar análises bioquímicas, morfológicas e histoquímicas de embriões somáticos em diferentes estágios de desenvolvimento. O aumento em matérias fresca e seca nas culturas embriogênicas foi lento nas duas primeiras semanas, as quais apresentaram crescimento máximo na quarta semana. Nas duas primeiras semanas, estas culturas apresentaram níveis superiores de proteínas totais e açúcares solúveis totais enquanto que níveis superiores de amido foram observados nas três últimas semanas. Embriões somáticos no estágio globular acumularam maiores níveis de proteínas totais e menores níveis de açúcares solúveis totais e amido. Os embriões somáticos maduros apresentaram cotilédones grandes, com a protoderme bem formada e feixes vasculares diferenciados. O eixo embrionário não apresentou organização completa, mas foi observado um meristema apical da raiz bem formado. Também foram observadas células meristemáticas na região do meristema apical do ramo

Polyamine patterns in haploid and diploid tobacco tissues and in vitro cultures

The aim of this work was to determine PAs levels in pith tissues and callus cultures from haploid and diploid tobacco plants, explanted from the apical and basal regions of the stem. These explants were cultured in an RM-64 medium supplied with IAA and kinetin, under light or in the dark, during successive subcultures. PAs levels followed a basipetal decrease in diploid and an increase in haploid, pith tissues. A similar pattern of total PAs (free + conjugated) was observed for the callus of diploid and haploid plants maintained in the light, and for the haploid callus in the dark, whereas the diploid callus in the dark showed a constant increase in total PAs levels until the end of culture. The PA increase in the diploid callus in the dark was related to free Put levels increase. The ploidy status of the plants could express different PA gradients together with the plant pith and in vitro callus cultures.O objetivo deste trabalho foi determinar os níveis de PAs em tecidos de medula e cultura de calos de plantas haplóides e diplóides de tabaco, obtidas da região apical e basal do caule. Estes explantes foram cultivados em meio RM-64 suplementado com AIA e cinetina, na luz e no escuro, durante vários subcultivos. Nos tecidos medulares, os níveis de PAs apresentam um decréscimo basípeto em diplóides e um aumento em haplóides.Um padrão similar nos níveis de PAs totais (livres+ conjugadas) foi observado em calos haplóides e diplóides mantidos na luz, e haplóides no escuro, enquanto os diplóides cultivados no escuro mostraram um aumento constante até o final do cultivo. O aumento no conteúdo de PAs nos calos diplóides no escuro, foi devido ao aumento do conteúdo de Put livre. Foi observado que a ploidia da planta pode expressar diferentes gradientes de PA ao longo do tecido medular e nas culturas de calos in vitro.(FAPESP) São Paulo Research Foundation(CNPq) National Council for Scientific and Technological Developmen

Alterações no perfil proteômico e de poliaminas induzido pelo envelhecimento de sementes de Cariniana legalis (Martius) O. Kuntze

Studies on metabolic processes and seed germinability of native endangered species are essential for its conservation. The objective of this work was to evaluate the effect of accelerated aging on germination and on the proteomic and polyamine (PAs) profile in seeds of Cariniana legalis. The seeds were aged at 41, 43, 45 and 47ºC for 24, 48, 72 and 96 h, under 100 % relative humidity using a completely randomized factorial design. The data were submitted to analysis of variance followed by Tukey’s test. The temperature of 47ºC was efficient in simulating seed aging, with no germination after 72 h of incubation. At 41ºC, the germination did not change significantly until 96 h. The reduction in the concentration of total free PAs, mainly spermidine, and the down-accumulation of heat shock proteins in seeds aged at 47°C were associated with induction of seed aging. Furthermore, the down-accumulation of the aconitate hydratase protein and the up-accumulation of the alcohol dehydrogenase protein were associated with loss of seed germinability and rapid deterioration in seeds aged at 47°C. The aging of C. legalis seeds is related to alteration in the contents of PAs and accumulation of some proteins involved with the loss of germinability.Estudos sobre processos metabólicos e germinabilidade de sementes de espécies nativas ameaçadas de extinção são essenciais para sua conservação. O objetivo deste trabalho foi avaliar o efeito do envelhecimento acelerado na germinação e no perfil proteômico e de poliamina (PAs) em sementes de Cariniana legalis. As sementes foram envelhecidas a 41, 43, 45 e 47°C por 24, 48, 72 e 96 h, sob 100% de umidade relativa, usando um delineamento fatorial inteiramente casualizado. Os dados foram submetidos à análise de variância seguida de teste Tukey. A temperatura de 47°C foi eficiente em simular o envelhecimento das sementes, que não germinaram após 72 h de incubação. Em 41°C, a germinação não mudou significativamente até 96h. A redução na concentração de PAs livres totais, principalmente espermidina e a diminuição no acúmulo de proteínas de choque térmico em sementes envelhecidas à 47°C foram associados à indução do envelhecimento das sementes. Além disso, a diminuição no acúmulo da proteína aconitato hidratase e o aumento no acúmulo da proteína álcool desidrogenase foram associados à perda de germinabilidade das sementes e rápida deterioração em sementes envelhecidas à 47°C. O envelhecimento de sementes de C. legalis está relacionado à alteração no conteúdo de PAs e a alteração de acúmulo de algumas proteínas envolvidas na perda da germinabilidade

Citrus rootstocks modify scion antioxidant system under drought and heat stress combination

Climate change is altering weather conditions such as mean temperatures and precipitation patterns. Rising temperatures, especially in certain regions, accelerates soil water depletion and increases drought risk, which affects agriculture yield. Previously, our research demonstrated that the citrus rootstock Carrizo citrange (Citrus sinensis × Poncirus trifoliata) is more tolerant than Cleopatra mandarin (C. reshni) to drought and heat stress combination, in part, due to a higher activation of the antioxidant system that alleviated damage produced by oxidative stress. Here, by using reciprocal grafts of both genotypes, we studied the importance of the rootstock on scion performance and antioxidant response under this stress combination. Carrizo rootstock, under stress combination, positively influenced Cleopatra scion by reducing H2O2 accumulation, increasing superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzymatic activities and inducing SOD1, APX2 and catalase (CAT) protein accumulations. On the contrary, Cleopatra rootstock induced decreases in APX2 expression, CAT activity and SOD1, APX2 and CAT contents on Carrizo scion. Taken together, our findings indicate that the activation of the antioxidant system under stress combination is a transmissible trait from the rootstock to the scion and highlight the importance of the rootstock selection to improve crop performance and maintain citrus yield under the current scenario of climate change.Funding for open access charge: CRUE-Universitat Jaume

Reduction of heat stress pressure and activation of photosystem II repairing system are crucial for citrus tolerance to multiple abiotic stress combination

Drought, heat and high irradiance are abiotic stresses that negatively affect plant development and reduce crop productivity. The confluence of these three factors is common in nature, causing extreme situations for plants that compromise their viability. Drought and heat stresses increase the saturation of the photosystem reaction centers, increasing sensitivity to high irradiance. In addition, these stress conditions affect photosystem II (PSII) integrity, alter redox balance of the electron transport chain and decrease the photosynthetic rate. Here, we studied the effect of the stress combinations on the photosynthetic apparatus of two citrus genotypes, Carrizo citrange (Citrus sinensis × Poncirus trifoliata) and Cleopatra mandarin (Citrus reshni). Results obtained showed that physiological responses, such as modulation of stomatal aperture and transpiration rate, aimed to reduce leaf temperature, are key to diminishing heat impact on photosynthetic apparatus and increasing tolerance to double and triple combinations of drought, high irradiance and high temperatures. By using transcriptomic and proteomic analyses, we have demonstrated that under these abiotic stress combinations, Carrizo plants were able to increase expression of genes and proteins related to the photosystem repairing machinery (which better maintained the integrity of PSII) and other components of the photosynthetic apparatus. Our findings reveal crucial physiological and genetic responses in citrus to increase tolerance to the combination of multiple abiotic stresses that could be the basis for breeding programs that ensure a sustainable citrus production.Funding for open access charge: CRUE-Universitat Jaume

Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.O objetivo deste trabalho foi a caracterização dos perfis protéicos expressos durante o desenvolvimento da semente de O. catharinensis, uma espécie recalcitrante, via eletroforese bidimensional. A extração de proteínas foi realizada utilizando tampão uréia/tiouréia seguido de uma etapa de precipitação com TCA 10%. A análise comparativa durante o desenvolvimento da semente mostrou um grande número de proteínas exclusivas em cada estádio do desenvolvimento. O estádio cotiledonar, que representa a fase de transição entre a embriogênese e o começo do metabolismo relacionado a maturação, apresentou o maior número de proteínas estádio-específicas. Proteínas relacionadas ao metabolismo oxidativo e a síntese de reservas foram identificadas via MS/MS. Estes resultados contribuem para o melhor entendimento do metabolismo de proteínas durante o desenvolvimento de sementes recalcitrantes, além de prover informações no estabelecimento de parâmetros para protocolos de embriogênese somática.(FAPESP) São Paulo Research Foundation(CNPq) National Council for Scientific and Technological Developmen

Ecophysiological and biochemical parameters for assessing Cr+6 stress conditions in Pterogyne nitens Tul. : new and usual methods for the management and restoration of degraded areas.

Heavy metals, such as Cr (chromium), have received attention during recent years due to their disposal in soil and water. Brave peanut (Pterogyne nitens Tul.), a tropical tree, was cultivated in 500 ?mol L-1 Cr+6 to observe how this species responds to this stress, with a view to its possible application in the restoration of degraded areas. In the present study, we report the use of a new method, photoacoustic spectroscopy, to evaluate the emission of CO2 and ethylene and the use of other standard techniques for assessing stress (chlorophyll a fluorescence, photosynthetic pigment determination, leaf relative water, specific leaf area, NO (nitric oxide) and polyamines. Photoacoustic spectroscopy appears to provide an innovative and efficient technique for detecting stress induced by heavy metals soon after their contact with plants. Polyamine content, as well as ethylene and NO, were seen to be sensitive to Cr+6; polyamines were decreased in leaves and increased in roots, ethylene was increased in the whole plant and NO was increased in the roots. Our results demonstrating alterations in ethylene in response to Cr+6 are, to date, unreported and the technique used for these measurements is novel. With regard to the polyamines, modulations in their concentrations may have protected the photosystem II, since no photosynthetic alterations were observed for pigments and chlorophyll a fluorescence

Morphological and polyamine content changes in embryogenic and non-embryogenic callus of sugarcane

Differences in competence acquisition and subsequent embryo maturation in embryogenic and non-embryogenic callus of sugarcane var. SP79-1011 were evaluated using histomorphological analysis, growth curves, numbers of somatic embryos, and polyamine contents. Embryogenic callus was formed by cells with embryogenic characteristics such as a rounded shape, prominent nuclei, a high nucleus: cytoplasm ratio, small vacuoles and organized globular structures. However, non-embryogenic callus presented dispersed, elongated and vacuolated cells with a low nucleus: cytoplasm ratio; these characteristics did not allow for the development of somatic embryos even upon exposure to a maturation stimulus. These results suggest that non-embryogenic callus does not acquire embryogenic competence during induction and that maturation treatment is not sufficient to promote somatic embryo differentiation. The use of activated charcoal (AC; 1.5 g L−1) resulted in a higher somatic embryo maturation rate in embryogenic callus but did not yield success in non-embryogenic callus. Embryogenic callus incubated with control (10 μM 2,4-dichlorophenoxyacetic acid) and maturation (1.5 g L−1 AC) treatments for 28 days showed similar patterns of total free polyamines; these results differed from the results observed with non-embryogenic callus, suggesting that embryogenic callus already exhibits a characteristic pattern of endogenous polyamine levels. At 28 days of culture with maturation treatment, embryogenic callus exhibited significantly higher levels of free Spm than embryogenic callus incubated with control treatment and non-embryogenic callus incubated with both treatments. This result suggests that Spm could be important for the acquisition of embryogenic competence and somatic embryo maturation in sugarcane var. SP79-1011.Funding for this work was provided by the CNPq (403015/2008-1 and 480142/2010-6) and FAPERJ (E-26/101.513/2010) to VS. AMV thanks CAPES for her fellowship

Caracterização histomorfológica e bioquímica de calos induzidos em Cenostigma pyramidale [Tul.] Gagnon & G.P. Lewis

Cenostigma pyramidale [Tul.] has timber, forage and medicinal uses, and due to the exploitation of its natural resources, it may become endangered. This work aimed to induce callus in Cenostigma pyramidale and to identify the embryogenic potential by histomorphological and biochemical analysis. Leaf segments were inoculated in MS culture medium containing different concentrations (0.0; 2.5; 5.0 and 10.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), combined with 6-benzylaminopurine (BAP; 0.0; 2.5 and 5.0 μM) and glutamine (0.0; 0.342 and 0.684 mM). The growth curve was performed at 7-day intervals until day 77, and samples were collected for histomorphological analysis and determination of reducing sugars (RA), sucrose, and total soluble sugars (TSS) content. Callus induction is possible using leaf segments in treatment with 5.0 μM of BAP, 7.0 µM of 2.4-D and 0.684 mM of glutamine. The growth curve shows sigmoidal behavior with five distinct phases: lag, exponential, linear, deceleration and decline. It was verified the presence of cells with meristematic characteristics from the 7th day of induction, suggesting that the calli have embryogenic potential. However, differentiation of somatic embryos was not observed. There was a reduction in RA content and increase in AST and sucrose during the lag phase and beginning of the exponential phase, followed by degradation in the exponential phase until the decline phase, showing the involvement of these carbohydrates with callus growth. These results are unprecedented for the species and suggest future studies related to callus maturation aiming the differentiation of somatic embryos.Cenostigma pyramidale [Tul.] possui utilização madeireira, forrageira e medicinal e, em virtude da exploração de seus recursos naturais, pode acabar em via de extinção. Este trabalho objetivou induzir calos em Cenostigma pyramidale e identificar o potencial embriogênico por análises histomorfológica e bioquímica. Segmentos foliolares foram inoculados em meio de cultura MS contendo diferentes concentrações (0,0; 2,5; 5,0 e 10,0 μM) de ácido 2,4-diclorofenoxiacético (2,4-D), combinadas com 6-benzilaminopurina (BAP; 0,0; 2,5 e 5,0 μM) e glutamina (0,0; 0,342 e 0,684 mM). A curva de crescimento foi realizada em intervalos de 7 dias, até o 77º dia, sendo coletadas amostras para análise histomorfológica e determinação do conteúdo de açúcares redutores (AR), sacarose e açúcares solúveis totais (AST). É possível a indução de calos utilizando segmentos foliolares no tratamento com 5,0 μM de BAP, 7,0 µM de 2,4-D e 0,684 mM de glutamina. A curva de crescimento apresenta comportamento sigmoidal com cinco fases distintas: lag, exponencial, linear, desaceleração e declínio. Verificou-se a presença de células com características meristemáticas a partir do 7º dia de indução, sugerindo que os calos possuem potencial embriogênico. Porém, não se observou a diferenciação de embriões somáticos. Verificaram-se redução no conteúdo de AR e aumento de AST e sacarose durante a fase lag e início da fase exponencial, seguido de degradação na fase exponencial até a fase de declínio, mostrando o envolvimento destes carboidratos com o crescimento dos calos. Esses resultados são inéditos para a espécie e sugerem estudos futuros relacionados à maturação dos calos visando a diferenciação de embriões somáticos