Navegando por Autor "Silva, Luciano Coutinho"

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Aspectos fisiológicos, bioquímicos, biofísicos e ultraestruturais associados à criopreservação em sementes de Coffea arabica L.
(Universidade Federal de Lavras, 2019-04-26) Souza, Ana Cristina de; Rosa, Sttela Dellyzete Veiga Franco da; Silva, Luciano Coutinho
Tolerância à dessecação é a capacidade de recuperar as atividades metabólicas depois da remoção parcial da água celular em sementes, possibilitando sua sobrevivência. A conservação a longo prazo, temperatura em nitrogênio líquido, se mostra economicamente viável em comparação com a conservação de plantas a campo. Pesquisas sobre tolerância à dessecação e métodos de criopreservação contribuem para o entendimento do comportamento destas sementes em relação a sensibilidade, à dessecação e ao resfriamento. Neste contexto, estudos foram realizados objetivando-se investigar os aspectos fisiológicos, bioquímicos, biofísicos e ultraestruturais em sementes de Coffea arabica L. No primeiro estudo, o objetivo foi investigar metodologias de armazenamento em longo prazo, de sementes de café, realizando estudos fisiológicos, bioquímicos e ultraestruturais relacionados com os efeitos da desidratação, pré-resfriamento e reaquecimento após exposição ao nitrogênio líquido. No segundo estudo, foram investigadas as propriedades calorimétricas dos vidroscitoplasmáticos associados a criopreservação. A qualidade fisiológica de sementes de Coffea arabica L. é reduzida à medida que perdem água durante a secagem. O reaquecimento em temperatura de 25 °C ou em banho-maria a 40 °C de sementes de Coffea arabica L. após pré-resfriamento, em taxa de 1°C min-1 até -40 °C, não altera a qualidade fisiológica e o perfil eletroforético das enzimas catalase e superóxido dismutase. As enzimas polifenol oxidase e peroxidase apresentam menor expressão em gel de eletroforese em sementes de Coffea arabica L. reaquecidas em temperatura de 25 °C. do que em banho-maria a 40 °C, após pré-resfriamento, em taxa de 1°C min-1 até -40 °C. Identificou-se que danos nas membranas celulares são menos drásticos nas sementes que são crioarmazenadas após a secagem. Sementes de Coffea arabica L. secadas em sílica gel até 20 ou 17% (bu), pré-resfriadas em biocongelador ou diretamente imersas em nitrogênio líquido, apresentam alta sobrevivência. Em sementes de Coffea arabica L. com teores de água, de (43 e 35% bu) a entalpia de fusão e a porcentagem de água congelável são maiores do que em sementes com teores de água de 20, 17 ou 12% bu. O ponto de fusão da água congelada em sementes hidratadas ocorre em temperaturas inferiores à temperatura de fusão da água pura de 0 ºC. A formação de vidros intracelulares em sementes hidratadas foi detectada em temperaturas de -18 e -22 °C, sementes com 17 e 20% (bu) entre -20 e -23 °C e em sementes crioarmazenadas em -18 °C.
Cryopreservation in Coffea canephora Pierre seeds: slow and fast cooling
(Editora UFLA, 2018-11) Coelho, Stefânia Vilas Boas; Rosa, Sttela Dellyzete Veiga Franco da; Fantazzini, Tatiana Botelho; Baute, Júlia Lima; Silva, Luciano Coutinho
Coffee is one of the main agricultural commodities in the country, and it is important to conservation of plant material for breeding programs. Cryopreservation is a promising alternative for preserving in the long-term the germplasm of species considered recalcitrant. However, studies should be performed to achieve maximum survival of seedlings after immersion in liquid nitrogen. The objective of this work was to find a cryopreservation protocol for storing seeds of Coffea canephora, studying two methods of cryopreservation, slow and fast cooling. Seeds were subjected to drying in silica gel up to the water content of 0.25 g g -1 . In the first experiment, dried seeds were subjected to treatments of slow cooling at speeds of -1 oC min -1 ,-3 oC min -1 and -5 oC min -1 until the end temperatures of -40 oC, -50 oC and -60 oC, by means of a bio freezer and subsequently immersed in liquid nitrogen. In the second experiment, the best result was selected of the first experiment and compared with the rapid cooling, in which dried seeds, with 0.25 g g -1 of water content, were immersed directly into liquid nitrogen. Physiological and biochemical alterations occurring in the seeds after cryopreservation were evaluated. Coffea canephora seeds respond better to cryopreservation by rapid cooling, when compared to slow cooling. Drying, one of the cryopreservation steps does not affect the viability of Coffea canephora Pierre seeds, when these seeds are dried to 0.25 g g -1 of water content. Catalase and esterase enzymes are good biochemical markers for cryopreserved coffee seeds and their activity is greater in larger seed physiological quality.
Cryopreservation of coffee zygotic embryos: dehydration and osmotic rehydration
(Editora UFLA, 2016-07) Pinto, Maísa de Siqueira; Paiva, Renato; Silva, Diogo Pedrosa Corrêa da; Santos, Paulo Augusto Almeida; Freitas, Rodrigo Therezan de; Silva, Luciano Coutinho
Conservation of plant genetic resources is important to prevent genetic erosion. Seed banks are the most common method of ex situ conservation; however, coffee seeds can not be stored by conventional methods. Cryopreservation is a viable alternative for long-term conservation of species that produce intermediate or recalcitrant seeds, as coffee. The aim of this work was to cryopreserve Coffea arabica L. cv Catuaí Vermelho IAC 144 zygotic embryos, and analyse the effects of dehydration prior cryopreservation and osmotic rehydration after thawing, in embryos germination and seedlings formation after cryopreservation. Prior to cryopreservation, different dehydration times (0, 15, 30, 60 and 120 min) were tested. Dehydrated embryos were cryopreserved in liquid nitrogen for 1 hour, and after thawing were rehydrated by osmotic solutions. Dehydrated and non-cryopreserved embryos were also analysed. The test with 2,3,5 triphenyl tetrazolium chloride (TTC) was used to evaluate the embryos viability. Non-dehydrated embryos did not survive after freezing. Embryos that were dehydrated until 20% of the moisture content did not germinate when osmotic rehydration was not performed. In contrast, cryopreserved embryos with the same moisture content presented 98% germination when they were rehydrated slowly in osmotic solution. According to tetrazolium tests, embryos presented maximum viability (75%) after dehydration for 60 minutes (23% moisture content). Therefore, coffee zygotic embryos (Coffea arabica L. cv. Catuaí Vermelho) can be successfully cryopreserved using physical dehydration in silica gel for 60 minutes (23% moisture content), followed by osmotic rehydration after thawing. This method allowed a germination of 98% of cryopreserved zygotic embryos.
Exploratory studies for cryopreservation of Coffea arabica L. seeds
(Associação Brasileira de Tecnologia de Sementes - ABRATES, 2017) Figueiredo, Madeleine Alves de; Coelho, Stefania Vilas Boas; Rosa, Sttela Dellyzete Veiga Franco da; Vilela, Amanda Lima; Silva, Luciano Coutinho
Cryopreservation is a viable option for conservation of coffee germplasm. However, for this technique to be completely successful, it is of fundamental importance to carry out studies that ensure maintenance of cell integrity before and after immersion in liquid nitrogen (LN). Therefore, the aim in this study was to investigate the water content, cooling rate, and final temperature most suitable for cryopreservation of Coffea arabica L. seeds. The seeds were dried by silica gel to water contents of 5, 10, 15, 20, 30 and 40 % wb, subjected to slow cooling treatments at speeds of -1, -3 and -5 °C min.-1 to final temperatures of -40, -50 and -60 °C and then directly immersed in LN. After storage, seeds were rewarmed at 40 °C for two minutes. The survival rate and viability of the seeds and embryos were evaluated by the tetrazolium and germination tests. Results of the tetrazolium test indicate that embryos excised from cryopreserved seeds are less sensitive to cryopreservation than whole seeds are. The water content of 20% wb and the use of zygotic embryos led to the highest survival rate of the coffee seeds, depending on the cooling rate and the final temperature of precooling.
Indução de calos embriogênicos em Coffea arabica L. cv. Catuaí Amarelo
(Embrapa Café, 2015) Souza, Ana Cristina de; Paiva, Renato; Reis, Michele Valquíria dos; Carvalho, Milene de Figueiredo; Silva, Luciano Coutinho; Silva, Diogo Pedrosa Corrêa da; Nery, Fernanda Carlota; Sales, Thais Silva; Rosa, Stella Veiga Franco da
O cafeeiro (Coffea sp.) é um arbusto pertencente à família Rubiaceae e ao gênero Coffea. O grande valor comercial do Coffea arábica é devido ao fato de seus grãos apresentarem sabor mais pronunciado e refinado. A embriogênese somática é um importante método de multiplicação em larga escala de plantas in vitro e pode maximizar a propagação do cafeeiro. Além disso, apresenta aplicações práticas para estudos de desenvolvimento embriológico e como técnica conjunta aos trabalhos de transformação genética de plantas. Várias pesquisas de cultivo in vitro já foram desenvolvidas com a espécie, entretanto, existe uma grande variedade de cultivares carentes deste tipo de estudo. O objetivo deste trabalho foi de indução de calos embriogênicos em Coffea arábica L. cv Catuaí Amarelo. Plântulas de cafeeiro Catuaí Amarelo cultivadas in vitro foram utilizadas como fonte de explantes. As folhas foram excisadas (1 cm2), pequenos cortes foram feitos na nervura central para indução de calos e os explantes foram inoculados com o lado abaxial voltado para o meio de cultivo MS com metade da concentração dos sais, 10 μM de Cinetina e diferentes concentrações (0, 2,5, 5, 10 e 20μM) do ácido 2,4-diclorofenoxiacético (2,4-D). Os explantes foram mantidos em sala de crescimento, no escuro e com temperatura de 25 ± 2 °C. Após 45 dias de cultivo, a porcentagem de formação de calos e formação de raízes foram avaliados e os dados submetidos à ANAVA. A formação de calos e de rizogênese foi significativa (p<0.001). O uso de 2,5 μM e 10 μM de 2,4-D promoveram maiores porcentagens de formação de calos em 51% dos explantes. Ocorreu a formação de rizogênese quando utilizado 2,5 μM ou ausência de 2,4 D. O tratamento com 10 μM de 2,4-D é o mais indicado para a indução de calos em explantes foliares da cultivar Catuaí Amarelo.
Physiological, biochemical, and ultrastructural aspects of Coffea arabica L. seeds under different cryopreservation protocols
(Editora UFLA, 2021) Figueiredo, Madeleine Alves de; Rosa, Sttela Dellyzete Veiga Franco da; Ricaldoni, Marcela Andreotti; Pereira, Cristiane Carvalho; Coelho, Stefânia Vilas Boas; Silva, Luciano Coutinho
Cryopreservation is a technique that may potentially conserve the germplasm of species of the Coffea genus for an indeterminate time. The aim of this study was to evaluate the physiological, biochemical and ultrastructural characteristics of cryopreserved seeds of Coffea arabica L., cultivar Catucaí amarelo IAC 62, which was subjected to different protocols regarding dehydration, precooling, cooling, rewarming and cathode water use. According to each protocol, the seeds were subjected to fast or slow drying to moisture contents of 17 or 20% (wet basis), cooled in different ways, and then immersed in liquid nitrogen for 24 hours. Different rewarming times in a water bath were also used. Physiological, biochemical and ultrastructural analyses were performed on the seeds after the cryopreservation steps. Moisture content at a 17% wb is the key factor for the cryopreservation of Coffea arabica L. seeds, which have better physiological quality and better preserved cell structures. Precooling of coffee seeds before immersion in liquid nitrogen does not provide advantages compared to direct immersion. The rewarming times tested (2, 4, and 6 minutes) and cathode water use did not cause changes in the physiological and biochemical quality or in the cell structures of Coffea arabica L. cryopreserved seeds. The pattern of cell structure observed in all seeds indicates that the damage from cryopreservation is less drastic in the cells of the embryos than in those of the endosperm, with the latter less tolerant to the stresses of dehydration, precooling, and rewarming.
Ultrastructural damage in coffee seeds exposed to drying and to subzero (°C) temperatures
(Editora UFLA, 2020) Coelho, Stefânia Vilas Boas; Rosa, Sttela Dellyzete Veiga Franco da; Clemente, Aline da Consolação Sampaio; Lacerda, Laura Nardelli Castanheira; Silva, Luciano Coutinho; Fantazzini, Tatiana Botelho; Ribeiro, Fernando Augusto Sales; Castro, Elisa de Melo
During drying and freezing, protective mechanisms act to maintain seed physiological quality. Some of these mechanisms contribute to the integrity of cell membranes. The damage caused to cell membranes due to cell stress can be seen in ultrastructural studies, comparing these results to those of physiological evaluation. The aim of this study was to investigate ultrastructural changes in endosperm cells of coffee seeds brought about by drying and by exposure to low temperatures. Seeds of Coffea arabica were dried in silica gel to moisture contents of 40, 20, and 5 % (wb) and brought to equilibrium at temperatures of 10, -20, and -86 oC. Germination, vigor, and tetrazolium tests were performed for evaluation of seed physiological quality. Ultrastructural damage was analyzed by scanning electron microscopy. Coffee seeds with 40% moisture content have whole, swollen, and expanded cells, with a filled lumen and without signs of damage. The physiological and ultrastructural quality of seeds exposed to below zero temperatures with 40% moisture content is compromised. They have null germination and empty cells, indicating leakage of cell content. Drying of coffee seeds leads to uniform contraction of inner cell content. Drying of coffee seeds to 5% moisture content leads to intense contraction of cell volume, with physiological and ultrastructural damage.