Embriogênese somática indireta em genótipos de Coffea arabica e de C. canephora
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2002
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Universidade Federal de Viçosa
Resumo
Explantes foliares dos canéforas Apoatã e Clone 100, dos arábicas Catuaí Vermelho, Catimor UFV 395-141 e dos híbridos 337 (Catuaí Vermelho X Híbrido de Timor) e 447 (Mundo Novo X Híbrido de Timor) foram utilizados para obtenção de calos embriogênicos friáveis em oito meios de cultura: A (BAP 20 µM), B (BAP 5 µM), C (2iP 5 µM), D1 (2,4-D 4,5 µM e Kin 18,6 µM), D2 (ANA 0,54 µM e Kin 4,6 µM), E1 (2,4-D 1,5 µM e BAP 7,5 µM), E2 (2iP 25 µM e AIB 5 µM) e E3 (BAP 5 µM), relativos aos tratamentos A, Aag, A/B, B, C, D1/D2, E1/E2 e E1/E3. Os calos embriogênicos friáveis obtidos foram utilizados para o estabelecimento de culturas líquidas em Erlenmeyers contendo o mesmo meio de sua origem, porém sem Gelrite TM. A densidade inicial foi mantida em 10 g L-1 de meio, sendo as suspensões celulares subcultivadas a intervalos de 14 dias. Para diferenciação de agregados celulares e embriões, a biomassa na densidade de 1 g L-1 foi transferida para os meios de diferenciação. Amostras de calos obtidos em cultura semi-sólida e agregados celulares de culturas líquidas foram coletadas e fixadas em FAA50, por 24 horas, e armazenadas em etanol 70%, com os objetivos de caracterizar, histologicamente, os tipos de calos existentes e diferenciar, em nível celular, as culturas líquidas embriogênicas daquelas não-embriogênicas. As amostras foram incluídas em historesina glicolmetacrilato (HISTORESIN, LEICA) e os blocos, cortados em micrótomo rotativo com 5 µM de espessura. Os cortes foram corados com Azul de Toluidina, P.A.S. e Xylidine Ponceau e as lâminas, montadas com resina Permount. Aos cinco meses pós-cultivo, o canéfora Clone 100 apresentou calo embriogênico friável em todos os meios utilizados, exceto na seqüência D1D2. Aos sete meses pós-cultivo, os genótipos canéforas produziam calos embriogênicos friáveis em quase todos os meios testados, enquanto o Catuaí Vermelho só o fez aos 10 meses no meio Aag1, que continha como agente gelificante ágar em vez de Gelrite TM, como nos outros meios utilizados. Aos 10 meses, todos os meios utilizados para o genótipo Apoatã haviam apresentado calo embriogênico friável em diferentes proporções. Os demais genótipos não apresentaram reação favorável aos meios de cultura utilizados. O estudo da cinética de crescimento das suspensões celulares revelou, imediatamente após a subcultura, uma fase exponencial de crescimento celular até um ponto de inflexão (ti), a partir do qual as taxas de crescimento reduziram progressivamente para atingir um peso de matéria fresca de agregados celulares assintótico. Os ti's das culturas líquidas de Apoatã, Clone 100 e Catuaí Vermelho foram 14, 11 e 10 dias, respectivamente. A diferenciação embriogênica de agregados celulares de Apoatã, em cultura líquida, rendeu cerca de 55.000 embriões g-1 de matéria fresca. O calo iniciou-se com a proliferação das células perivasculares nos genótipos canéforas e também no parênquima clorofiliano no arábica Catuaí. As células do calo possuíam dois padrões de divisão: o primeiro, unidirecional, dava origem a células que se tomavam vacuolizadas e, posteriormente, degeneravam; no segundo, as células se dividiam em três dimensões, originando as células embriogênicas. Nas culturas líquidas de manutenção, observaram-se agregados celulares contendo células meristemáticas de núcleo grande com nucléolo proeminente e citoplasma denso. Nas culturas de diferenciação embriogênica, os agregados celulares formavam nódulos periféricos, de onde se diferenciavam células embriônicas, as quais se dividiam intensamente, formando o embrião. As culturas do Catuaí e Clone 100 podem ser consideradas embriogênicas, pois possuíam embriões globulares, apesar de não terem completado seu desenvolvimento normalmente. As células do genótipo Catuaí apresentaram depósitos de proteínas tanto na cultura de manutenção quanto na cultura de diferenciação. A presença de amido de reserva foi bem maior nas culturas de diferenciaçi4o em relação às de manutenção, em todos os genótipos analisados.
Leaf explants from the canephora and Apoatã and Clone 100, from the arabica Catuaí Vermelho cv. and Catimor UFV 395-141 cv. and from hybrids 337 (Catuaí Vermelho X Timor Hybrid) and 447 (Mundo Novo X Timor Hybrid) were used to obtain the embryogenic friable calli in eight culture media: A (BAP 20 µM), B (BAP 5 µM), C (2iP 5 µM), D1 (2,4-D 4.5 µM and Kin 18.6 µM), D2 (ANA 0.54 µM and Kin 4.6 µM), E1 (2,4-D 1.5 µM and BAP 7.5 µM), E2 (2iP 25 µM and AIB 5 µM) and E3 (BAP 5 µM) relative to the treatments A, Aag, A/B, B, C, D1/D2, E1/E2 and E1/E3. The obtained embryogenic friable calli were used for establishing the liquid cultures in Erlenmeyers flasks containing the same medium of their origin, except for Gelrite TM. The initial density was maintained at 10 g L-1 medium, and the cellular suspensions were subcultured at 14-day intervals. For differentiation of the cellular aggregates into embryos, the biomass at 1 g L-1 density was transferred to the differentiation media. Some callus samples obtained from semisolid culture and cell aggregates from liquid cultures were collected and fixed in FAA50 for 24 hours and stored in 70% ethanol in order to histologically characterize the types of the existent calli and to differentiate, at cellular level, the liquid embryogenic cultures from the nonembryogenic ones. The samples were included into historesin glycolmetacrylate (HISTORESIN, LEICA) and the blocks were cut by a 5mM-thick rotary microtome. The cuts were dyed with Toluidine Blue, P.A.S. and Xylidine Ponceau, whereas the slides were mounted with resin Permount. At five months after culture, the canephora Clone 100 exhibited friable embryogenic calli in all tested media, except for the sequence D1D2. At seven months after culture, the canephora genotypes were already producing friable embryogenic calli in almost all tested media, while in Catuaí Vermelho these calli appeared only at 10 months in the Aag medium, that contained agar as the gelling agent instead of Gelrite TM, such as in the other media. At 10 months, all media used for the genotype Apoatã had already presented the friable embryogenic calli at different proportions. The other genotypes did not present any reactions favorable to the culture media. Just after subculture, the study on the growth kinetics of the cell suspensions revealed an exponential phase up to an inflection point (ti), from which the growth rates began to reduce progressively until the cellular aggregates reached an asymptotic fresh matter weight. The ti’s for the liquid cultures of Apoatã, Clone 100 and Catuaí Vermelho were 14, 11 and 10 days, respectively. The embryogenic differentiation in the cellular aggregates of Apoatã, in liquid culture, yielded about 55.000 embryos g-1 fresh matter. The callus began with proliferation of the perivascular cells in canephora genotypes, as well as in the leaf parenchyma in arabica Catuaí. The calli cells had two division patterns: the first one (unidirectional) gave rise to the cells that became vacuolated and subsequently degenerated; in the second one, the cells divided into three dimensions originating the embryogenic cells. In the liquid maintenance cultures, a number of cellular aggregates containing meristematic cells exhibiting a large nucleus with prominent nucleolus and dense cytoplasm were observed. In the embryogenic differentiation cultures, the cellular aggregates formed peripheral nodules, from which the embryonic cells were differentiated and divided intensively forming the embryo. The cultures of Catuaí and Clone 100 might be considered as embryogenic ones, since they exhibited globular embryos, in spite of having not normally completed their development. The cells of the Catuaí genotype presented deposits of proteins in both the maintenance and differentiation cultures. In all analyzed genotypes, the amount of the stored starch was much higher in the differentiation cultures as compared to the maintenance ones.
Leaf explants from the canephora and Apoatã and Clone 100, from the arabica Catuaí Vermelho cv. and Catimor UFV 395-141 cv. and from hybrids 337 (Catuaí Vermelho X Timor Hybrid) and 447 (Mundo Novo X Timor Hybrid) were used to obtain the embryogenic friable calli in eight culture media: A (BAP 20 µM), B (BAP 5 µM), C (2iP 5 µM), D1 (2,4-D 4.5 µM and Kin 18.6 µM), D2 (ANA 0.54 µM and Kin 4.6 µM), E1 (2,4-D 1.5 µM and BAP 7.5 µM), E2 (2iP 25 µM and AIB 5 µM) and E3 (BAP 5 µM) relative to the treatments A, Aag, A/B, B, C, D1/D2, E1/E2 and E1/E3. The obtained embryogenic friable calli were used for establishing the liquid cultures in Erlenmeyers flasks containing the same medium of their origin, except for Gelrite TM. The initial density was maintained at 10 g L-1 medium, and the cellular suspensions were subcultured at 14-day intervals. For differentiation of the cellular aggregates into embryos, the biomass at 1 g L-1 density was transferred to the differentiation media. Some callus samples obtained from semisolid culture and cell aggregates from liquid cultures were collected and fixed in FAA50 for 24 hours and stored in 70% ethanol in order to histologically characterize the types of the existent calli and to differentiate, at cellular level, the liquid embryogenic cultures from the nonembryogenic ones. The samples were included into historesin glycolmetacrylate (HISTORESIN, LEICA) and the blocks were cut by a 5mM-thick rotary microtome. The cuts were dyed with Toluidine Blue, P.A.S. and Xylidine Ponceau, whereas the slides were mounted with resin Permount. At five months after culture, the canephora Clone 100 exhibited friable embryogenic calli in all tested media, except for the sequence D1D2. At seven months after culture, the canephora genotypes were already producing friable embryogenic calli in almost all tested media, while in Catuaí Vermelho these calli appeared only at 10 months in the Aag medium, that contained agar as the gelling agent instead of Gelrite TM, such as in the other media. At 10 months, all media used for the genotype Apoatã had already presented the friable embryogenic calli at different proportions. The other genotypes did not present any reactions favorable to the culture media. Just after subculture, the study on the growth kinetics of the cell suspensions revealed an exponential phase up to an inflection point (ti), from which the growth rates began to reduce progressively until the cellular aggregates reached an asymptotic fresh matter weight. The ti’s for the liquid cultures of Apoatã, Clone 100 and Catuaí Vermelho were 14, 11 and 10 days, respectively. The embryogenic differentiation in the cellular aggregates of Apoatã, in liquid culture, yielded about 55.000 embryos g-1 fresh matter. The callus began with proliferation of the perivascular cells in canephora genotypes, as well as in the leaf parenchyma in arabica Catuaí. The calli cells had two division patterns: the first one (unidirectional) gave rise to the cells that became vacuolated and subsequently degenerated; in the second one, the cells divided into three dimensions originating the embryogenic cells. In the liquid maintenance cultures, a number of cellular aggregates containing meristematic cells exhibiting a large nucleus with prominent nucleolus and dense cytoplasm were observed. In the embryogenic differentiation cultures, the cellular aggregates formed peripheral nodules, from which the embryonic cells were differentiated and divided intensively forming the embryo. The cultures of Catuaí and Clone 100 might be considered as embryogenic ones, since they exhibited globular embryos, in spite of having not normally completed their development. The cells of the Catuaí genotype presented deposits of proteins in both the maintenance and differentiation cultures. In all analyzed genotypes, the amount of the stored starch was much higher in the differentiation cultures as compared to the maintenance ones.
Descrição
Tese de Doutorado defendida na Universidade Federal de Viçosa
Palavras-chave
Café Embriogênese somática Histologia Cultura in vitro Cultura de tecidos Coffea arabica Coffea canephora, Coffee Somatic embryogenesis Hystology In vitro culture Tissue culture Coffea arabica Coffea canephora
Citação
Santos, Aparecida Célia Paula dos. Embriogênese somática indireta em genótipos de Coffea arabica e de C. canephora. Viçosa : UFV, 2002. 90p. : il. (Tese - Doutorado em Genética e Melhoramento) Orientador: Wagner Campos Otoni T 633.733 S237e 2002