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Embriogênese somática em pupunheira (Bactris gasipaes), butiá-da-serra (Butia eriospatha) e açai (Euterpe oleracea)
2015Co-Authors: Ree, Joseph FrancisAbstract:Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Recursos Genéticos Vegetais, Florianópolis, 2015.A embriogênese somática (ES) é um métedo eficiente à propagaçãomassiva de plantas e à conservação de germoplasma. Em nível básico elase configura como um modelo biológico para o aprofundamento deestudos de morfogênese, fisiologia, bioquímica e genética de plantas.Muitos organismos apresentam características similares durante a ES, taiscomo a expressão de genes homólogos, o desenvolvimento de tecidosespecializados semelhantes ao observado na embriogênese zigótica e asrespostas a certos reguladores de crescimento. Para abordarprofundamente estas características associadas à ES de palmeirasneotropicais foi elaborada uma ampla revisão sobre este tema no primeirocapítulo desta dissertação. De uma forma geral esta revisão mostrou queembriões zigóticos e/ou meristemas apicais têm sido os explantes maisempregados, mostrando-se responsivos ao meio de cultura baseado naformulação salina de Murashige e Skoog suplementado com vitaminas deMorel, 3% de sacarose, carvão ativado e concentrações elevadas deauxinas, principalmente 2,4-D ou picloram. Em seguida, odesenvolvimento embrionário normalmente é estimulado em meios decultura suplementados com teores reduzidos destas auxinas e com oemprego de citocininas, notadamente BAP e 2-ip. Por fim, os embriõessão convertidos em plântulas em meios isentos de reguladores decrescimento. Além da micropropagação, muitos estudos concentraram-sena expressão de genes e na bioquímica do desenvolvimento de embriõessomáticos de palmeiras. Estudos histológicos sugeriram tendênciascomuns em diferentes espécies durante a morfogênese in vitro, tais comomorfologia semelhante dos ápices caulinares meristemáticos, calos ecélulas epidérmicas. No segundo capítulo estudou-se o emprego deculturas de Protoplastos em três espécies de palmeiras, pupunha (Bactrisgasipaes), butiá-da-serra (Butia eriospatha) e açaí (Euterpe oleracea).Protoplastos são células com suas paredes celulares removidas eapresentam interesse tanto pela sua capacidade regenerativa quanto pelapossibilidade da geração de híbridos através da fusão somática de doisProtoplastos diferentes. Esta tecnologia tem sido investigada em váriasespécies de palmeiras, tais como dendê (Elaeis guineensis) e tamareira(Phoenix dactyilifera). Na presente dissertação, culturas embriogênicaspreviamente estabelecidas de pupunha, butiá-da-serra e açaí foramtratadas com uma solução enzimática composta por 2% de celulase, 0,5% de hemicelulase e 0,5 % de pectinase e em seguida elas foram23incubadas no escuro a 25 ± 2 ° C durante 6, 12, 18, ou 24 horas semagitação ou num agitador orbital a 45 rpm para o isolamento deProtoplastos. As maiores quantidades de Protoplastos foram obtidasatravés da incubação em agitador orbital, sendo 5.50±0.68x105 e1.22±0.13x106 Protoplastos / grama de peso fresco de pupunha e açaí,respectivamente, depois de seis horas de incubação, e 5,36 ± 2.23x105células / grama de peso fresco para butiá após 24 horas incubação.Culturas de pupunha e açaí mostraram diminuição de rendimento deProtoplastos após seis horas de incubação, enquanto a quantidade deresíduos celulares visíveis aumentou. A viabilidade de Protoplastosmanteve-se elevada (> 70%), com exceção de Protoplastos de açaí, cujaviabilidade diminuiu rapidamente após seis horas na incubação orbital.Os Protoplastos foram cultivados usando o método de gotas de agarose oude alginato, sendo submetidos a seis tipos de meios líquidos contendo 0,1, ou 10 µM de picloram com ou sem 2µM de 2-ip. Picloram não semostrou essencial para as divisões celulares, as quais, no entanto forammais frequentes e ocorreram mais cedo em resposta a meios com níveiscrescentes do mesmo. Foram observadas microcolônias se formando nasgotas de alginato com 10 de µM picloram e 2 µM de 2-ip, no entantocolonias visíveis foram formados apenas em duas esferas de agarose. Noterceiro capítulo desta dissertação o incremento de massa seca e os teoresde proteínas, açúcares e amido foram investigados em culturas depupunha com diferentes capacidades para ES: alta capacidadeembriogênica (ACE), baixa capacidade embriogênica (BCE), e nãoembriogênica(NE). Culturas de ACE e NE apresentaram incrementos demassa seca semelhantes e que foram maiores queo incremento de massaseca em culturas de BCE. Culturas de ACE apresentaram teoresligeiramente mais elevados de proteínas do que as culturas NE, contudoambas continham maiores teores de proteínas do que as culturas de BCE.Níveis de amido foram semelhantes entre culturas de ACE e NE e queforam maiores que os níveis de amido em culturas de BCE. Níveis deaçúcares foram demasiadamente baixos para terem seus valorescalculados utilizando uma curva padrão de glicose, porém as leituras deabsorbância mostraram que as culturas de ACE e NE apresentaramvalores médios semelhantes e superiores aos níveis encontrados emculturas de BCE. Estes dados sugerem que os diferentes comportamentosde crescimento das diferentes culturas podem não refletir as quantidadestotais de proteínas, e sim os tipos de proteínas a serem expressas. A grandequantidade de amido nas culturas NE, juntamente com a morfologia24distinta destas culturas, pode sugerir uma rota regenerativa baseada naorganogênese. Além disso, é possível que a falta de reservas de energianas culturas de BCE esteja relacionada com o seu rápido crescimento.Formações de tecidos organizados também foram observadas nas culturade BCE, isto pode sugerir que a morfogênese in vitro não inclui apenasos embriões somáticos, calos, raiz e plântulas, mas pode incluir outrostipos de tecidos. No quarto capítulo buscou-se a optimização da ES emculturas de pupunha, butiá e açaí. Culturas de pupunha 'G3' foramcultivadas em meios de cultura compostos pelos sais de MS ou MSmodificado, suplementados com 1 mM de espermidina, 1 mM deespermina, ou sem poliaminas. Não houve diferença no número deembriões somáticos regenerados a partir dos meios de MS ou MSmodificado, no entanto, ambas as poliaminas apresentaram efeitosnegativos para a formação de embriões. Culturas de pupunha ?G2? foraminoculadas em meio de cultura contendo 3% de sacarose ou uma misturaigual de sacarose, glicose, frutose, e sorbitol, não revelando diferenças emtermos de números totais de embriões regenerados. Embriões zigóticos debutiá inoculados em meio MS contendo 3% sacarose, 2,5 g / L de carvãoativado e 300 µM ou 450 µM de picloram responderam com a formaçãode calos com aparência embriogênica similares aos observados empupunha, 40% e 27,5% respectivamente. No entanto, culturas friáveis debutiá foram submetidas a vários tipos de meios de cultura, incluindomeios sólidos e líquidos; diferentes concentrações de auxinas, citocininas,ABA, GA3, sacarose e carvão ativado,e ausência ou presença de luzdurante o cultivo, porém, em nenhum dos ambientes proporcionadosocorreu a formação de embriões somáticos. Além disso, desidrataçãoparcial não induziu melhorias na multiplicação das culturasde butiá, omesmo foi observado em culturas de pupunha. A multiplicação dasculturas de açaí foi melhorada com a adição 2,5 g / L de carvão ativado eaumento nos teores de picloram de 10 µM até 50 µM. O carvão ativadolevou à redução de 400% no número de explantes oxidados, um aumentono número de explantes produzindo embriões, número deembriõesformados por explante, e numero de massas poliembriogênicas porexplante. No entanto, os casos de crescimento de raizes organogênicas ede calogênese observados a partir de explantes de açaí colocadas emmeios com carvão ativado sugerem que o efeito da auxina diminui napresença de carvão ativado. Tomados em conjunto, os resultados dapresente dissertação contribuem para uma melhor compreensão dosestudos da morfogênese in vitro de palmeiras, notadamente aqueles25associados aos fatores envolvidos na tecnologia de Protoplastos, àscaracterísticas bioquímicas de culturas de pupunha com diferentespotenciais embriogênicos e à otimização dos protocolos regenerativosestudados, especialmente o de açaí.Abstract : Somatic embryogenesis (SE) is a powerful technology that is useful for commercial propagation, scientific study, germplasm conservation, and has potential applications in reforestation attempts in damaged ecosystems. Many organisms show similar traits during SE, such as the expression of commonly-found gene homologs, development of specialized tissues similar to zygotic embryogenesis, and response to certain growth regulators. To summarize these trends, a review article was written (Chapter 1 of this thesis). A literature analysis of SE in the palm family, Arecaceae, suggests similar trends across palm species. Both zygotic embryos or palm shoot meristems tended to respond well to Culture media composed of Murashige and Skoog salts, vitamins, 3% sucrose, exogenous activated carbon, and high concentrations of auxins, such as 2,4-D or picloram. Embryo development was usually stimulated through subCulture onto media with reduced auxins and addition of cytokinin, such as BAP and 2-ip and then embryos could be frequently converted on media without growth regulators. In addition to micropropagation, many studies focused on the biochemistry and gene expression of developing palm somatic embryos. Histological studies revealed common trends, such as similar morphology of meristematic vs. callus and epidermal cells. One of the technologies that several research groups had evaluated was Protoplast Culture. Protoplasts, cells with their cell walls removed, are of scientific interest due to both being able to regenerate into larger Cultures and the ability to create hybrids through somatic fusion of two different Protoplasts. This technology has only been investigated in several large-scale economic species, such as oil palm (Elaeis guineensis) or date palm (Phoenix dactyilifera), and increased understanding of trends might lead to further development of Protoplast Culture as a whole (Chapter 2). To understand Protoplast isolation and Culture, previously-established peach palm (Bactris gasipaes), butiá-da-serra (Butia eriospatha), and açaí (Euterpe oleracea) Cultures were treated with an enzyme solution composed of 2% w/v cellulase, 0.5% hemicellulase, and 0.5% pectinase and then either incubated stationary for 6, 12, 18, or 24 hours or on an orbital shaker at 45 rpm for 3, 6, 12, or 24 hours in the dark at 25±2 °C. Stationary incubation did not provide large amounts of Protoplasts in comparison to incubation done on an orbital shaker. The greatest numbers of Protoplasts per species were 5.50±0.68x105 and 1.22±0.13x106 Protoplasts/ gram FW for peach palm and açaí after six hours of orbital shaking and 5.36±2.23x105 cells/gram FW for butiá after 24 hours of orbital shaking. Both peach palm and açaí saw dramatic decreases in Protoplast yield after later incubations while the amount of visible cellular debris increased, possibly showing a potential reason for the decreased cell yield as cellular debris might lyse cells in motion. Protoplast viability remained high (>70%), except for açaí Protoplasts, which decreased rapidly during orbital incubation. Protoplasts were Cultured either using the agarose bead or the alginate bead method. Protoplasts Cultured in alginate beads were subjected to six types of liquid media containing 0, 1, or 10µM picloram either with or without 2µM 2-ip. Auxin was not shown to be essential for causing cell division in several occasions, however cell division was more frequent and occurred earlier in media with increasing levels of picloram. 2-ip was not found to have a major impact. Microcolonies were observed to form in alginate beads with 10µM picloram and 2µM 2-ip, however visible colonies were only formed twice in agarose beads. Further optimization would be required to achieve regeneration of whole plants, however there are many trends in other species, such as use of nurse Cultures, higher cell density during Culture, and type of Culture method, which can be pursued. The biochemistry of peach palm Cultures with different capacities for SE, high embryogenic (HE), low embryogenic (LE), and non-embryogenic (NE), were investigated for dry weight, protein, sugar, and starch content (Chapter 3). It was found that both HE and NE Cultures had similar dry weights, but water made up a larger proportion of LE Cultures. Because of this difference, protein, sugar, and starch amounts were evaluated in terms of both fresh weight and dry weight. HE contained slightly higher amounts of protein than NE Cultures, but both tissues contained higher proteins contents than LE Cultures, even after adjusting for dry weight. Starch levels were comparable between HE and NE Cultures, but LE Cultures contained significantly fewer starch reserves. Sugar levels were too low to have their amounts calculated using a glucose standard curve, however their absorbance readings showed HE and NE Cultures had the about the same values, while once again LE Cultures contained fewer reserves. These data suggest that the different growth behavior of the different tissues may not reflect overall protein amounts, but rather the types of proteins being expressed. Additionally, the large amounts of starch growth, along with the distinct morphology of NE tissues might suggest a type of organogenesis. Additionally, it is possible that the lack of energy reserves found in LE tissue is related to its rapid growth. Organized tissue formations were also observed in LE tissue, possibly suggesting that in vitro organogenesis extends beyond simply somatic embryos, roots, and shoots. Additionally, SE optimization was investigated in peach palm, butiá, and açaí Cultures. Peach palm tissue lines  G3 were placed on media containing either MS or modified MS salts and media containing 1mM spermidine, spermine, or no polyamines. No difference was detected in the number of somatic embryos recovered from MS or modified MS Cultures, however either polyamine had a negative effect. Peach palm  G2 Cultures were placed on media containing either 3% sucrose or an equal mix of sucrose, glucose, or sorbitol with no difference in total numbers of recovered embryos. Fast-growing friable butiá Cultures were subjected to numerous types of media, including both solid and liquid media, media containing different concentrations of auxins, cytokinins, ABA, GA3, sucrose, and activated charcoal in both light and dark, but no somatic embryos were induced. Additionally, partial dehydration did not induce improved peach palm multiplication nor butiá SE. However, 40% and 27.5% placed on MS media containing 2.5g/L activated charcoal and 300µM or 450µM, respectively, picloram responded with tissue growth similar to that found in peach palm highly embryogenic tissue. Açaí multiplication was greatly improved through optimizing multiplication media with addition 2.5g/L activated charcoal and increased picloram up to 50µM from 10µM. The effects of the activated charcoal led to 400% reduction in number of oxidized explants, an increase in number of explants producing embryos, embryos per explant, number of embryos producing polyembryogenic masses, and number of polyembryogenic masses per explant. However, instances of organogenic root growth and increased callogenesis were observed on açaí explants placed on media with activated charcoal, which suggests that the decreased effect of auxin caused by activated charcoal can reduce embryogenic potential. Together, these works contribute to better understanding the trends found in multiple palms, factors involved in the valuable technology of Protoplast Culture, the biochemical characteristics of several types of peach palm tissue with different embryogenic potential, and cultural optimization, especially for the economically valuable açaí palm
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Embriogênese somática em pupunheira (Bactris gasipaes), butiá-da-serra (Butia eriospatha) e açai (Euterpe oleracea): isolamento e cultura de Protoplastos, avaliações bioquímicas e otimização das condições de cultura
2015Co-Authors: Ree, Joseph FrancisAbstract:Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Recursos Genéticos Vegetais, Florianópolis, 2015.A embriogênese somática (ES) é um métedo eficiente à propagaçãomassiva de plantas e à conservação de germoplasma. Em nível básico elase configura como um modelo biológico para o aprofundamento deestudos de morfogênese, fisiologia, bioquímica e genética de plantas.Muitos organismos apresentam características similares durante a ES, taiscomo a expressão de genes homólogos, o desenvolvimento de tecidosespecializados semelhantes ao observado na embriogênese zigótica e asrespostas a certos reguladores de crescimento. Para abordarprofundamente estas características associadas à ES de palmeirasneotropicais foi elaborada uma ampla revisão sobre este tema no primeirocapítulo desta dissertação. De uma forma geral esta revisão mostrou queembriões zigóticos e/ou meristemas apicais têm sido os explantes maisempregados, mostrando-se responsivos ao meio de cultura baseado naformulação salina de Murashige e Skoog suplementado com vitaminas deMorel, 3% de sacarose, carvão ativado e concentrações elevadas deauxinas, principalmente 2,4-D ou picloram. Em seguida, odesenvolvimento embrionário normalmente é estimulado em meios decultura suplementados com teores reduzidos destas auxinas e com oemprego de citocininas, notadamente BAP e 2-ip. Por fim, os embriõessão convertidos em plântulas em meios isentos de reguladores decrescimento. Além da micropropagação, muitos estudos concentraram-sena expressão de genes e na bioquímica do desenvolvimento de embriõessomáticos de palmeiras. Estudos histológicos sugeriram tendênciascomuns em diferentes espécies durante a morfogênese in vitro, tais comomorfologia semelhante dos ápices caulinares meristemáticos, calos ecélulas epidérmicas. No segundo capítulo estudou-se o emprego deculturas de Protoplastos em três espécies de palmeiras, pupunha (Bactrisgasipaes), butiá-da-serra (Butia eriospatha) e açaí (Euterpe oleracea).Protoplastos são células com suas paredes celulares removidas eapresentam interesse tanto pela sua capacidade regenerativa quanto pelapossibilidade da geração de híbridos através da fusão somática de doisProtoplastos diferentes. Esta tecnologia tem sido investigada em váriasespécies de palmeiras, tais como dendê (Elaeis guineensis) e tamareira(Phoenix dactyilifera). Na presente dissertação, culturas embriogênicaspreviamente estabelecidas de pupunha, butiá-da-serra e açaí foramtratadas com uma solução enzimática composta por 2% de celulase, 0,5% de hemicelulase e 0,5 % de pectinase e em seguida elas foram23incubadas no escuro a 25 ± 2 ° C durante 6, 12, 18, ou 24 horas semagitação ou num agitador orbital a 45 rpm para o isolamento deProtoplastos. As maiores quantidades de Protoplastos foram obtidasatravés da incubação em agitador orbital, sendo 5.50±0.68x105 e1.22±0.13x106 Protoplastos / grama de peso fresco de pupunha e açaí,respectivamente, depois de seis horas de incubação, e 5,36 ± 2.23x105células / grama de peso fresco para butiá após 24 horas incubação.Culturas de pupunha e açaí mostraram diminuição de rendimento deProtoplastos após seis horas de incubação, enquanto a quantidade deresíduos celulares visíveis aumentou. A viabilidade de Protoplastosmanteve-se elevada (> 70%), com exceção de Protoplastos de açaí, cujaviabilidade diminuiu rapidamente após seis horas na incubação orbital.Os Protoplastos foram cultivados usando o método de gotas de agarose oude alginato, sendo submetidos a seis tipos de meios líquidos contendo 0,1, ou 10 µM de picloram com ou sem 2µM de 2-ip. Picloram não semostrou essencial para as divisões celulares, as quais, no entanto forammais frequentes e ocorreram mais cedo em resposta a meios com níveiscrescentes do mesmo. Foram observadas microcolônias se formando nasgotas de alginato com 10 de µM picloram e 2 µM de 2-ip, no entantocolonias visíveis foram formados apenas em duas esferas de agarose. Noterceiro capítulo desta dissertação o incremento de massa seca e os teoresde proteínas, açúcares e amido foram investigados em culturas depupunha com diferentes capacidades para ES: alta capacidadeembriogênica (ACE), baixa capacidade embriogênica (BCE), e nãoembriogênica(NE). Culturas de ACE e NE apresentaram incrementos demassa seca semelhantes e que foram maiores queo incremento de massaseca em culturas de BCE. Culturas de ACE apresentaram teoresligeiramente mais elevados de proteínas do que as culturas NE, contudoambas continham maiores teores de proteínas do que as culturas de BCE.Níveis de amido foram semelhantes entre culturas de ACE e NE e queforam maiores que os níveis de amido em culturas de BCE. Níveis deaçúcares foram demasiadamente baixos para terem seus valorescalculados utilizando uma curva padrão de glicose, porém as leituras deabsorbância mostraram que as culturas de ACE e NE apresentaramvalores médios semelhantes e superiores aos níveis encontrados emculturas de BCE. Estes dados sugerem que os diferentes comportamentosde crescimento das diferentes culturas podem não refletir as quantidadestotais de proteínas, e sim os tipos de proteínas a serem expressas. A grandequantidade de amido nas culturas NE, juntamente com a morfologia24distinta destas culturas, pode sugerir uma rota regenerativa baseada naorganogênese. Além disso, é possível que a falta de reservas de energianas culturas de BCE esteja relacionada com o seu rápido crescimento.Formações de tecidos organizados também foram observadas nas culturade BCE, isto pode sugerir que a morfogênese in vitro não inclui apenasos embriões somáticos, calos, raiz e plântulas, mas pode incluir outrostipos de tecidos. No quarto capítulo buscou-se a optimização da ES emculturas de pupunha, butiá e açaí. Culturas de pupunha 'G3' foramcultivadas em meios de cultura compostos pelos sais de MS ou MSmodificado, suplementados com 1 mM de espermidina, 1 mM deespermina, ou sem poliaminas. Não houve diferença no número deembriões somáticos regenerados a partir dos meios de MS ou MSmodificado, no entanto, ambas as poliaminas apresentaram efeitosnegativos para a formação de embriões. Culturas de pupunha ?G2? foraminoculadas em meio de cultura contendo 3% de sacarose ou uma misturaigual de sacarose, glicose, frutose, e sorbitol, não revelando diferenças emtermos de números totais de embriões regenerados. Embriões zigóticos debutiá inoculados em meio MS contendo 3% sacarose, 2,5 g / L de carvãoativado e 300 µM ou 450 µM de picloram responderam com a formaçãode calos com aparência embriogênica similares aos observados empupunha, 40% e 27,5% respectivamente. No entanto, culturas friáveis debutiá foram submetidas a vários tipos de meios de cultura, incluindomeios sólidos e líquidos; diferentes concentrações de auxinas, citocininas,ABA, GA3, sacarose e carvão ativado,e ausência ou presença de luzdurante o cultivo, porém, em nenhum dos ambientes proporcionadosocorreu a formação de embriões somáticos. Além disso, desidrataçãoparcial não induziu melhorias na multiplicação das culturasde butiá, omesmo foi observado em culturas de pupunha. A multiplicação dasculturas de açaí foi melhorada com a adição 2,5 g / L de carvão ativado eaumento nos teores de picloram de 10 µM até 50 µM. O carvão ativadolevou à redução de 400% no número de explantes oxidados, um aumentono número de explantes produzindo embriões, número deembriõesformados por explante, e numero de massas poliembriogênicas porexplante. No entanto, os casos de crescimento de raizes organogênicas ede calogênese observados a partir de explantes de açaí colocadas emmeios com carvão ativado sugerem que o efeito da auxina diminui napresença de carvão ativado. Tomados em conjunto, os resultados dapresente dissertação contribuem para uma melhor compreensão dosestudos da morfogênese in vitro de palmeiras, notadamente aqueles25associados aos fatores envolvidos na tecnologia de Protoplastos, àscaracterísticas bioquímicas de culturas de pupunha com diferentespotenciais embriogênicos e à otimização dos protocolos regenerativosestudados, especialmente o de açaí.Abstract : Somatic embryogenesis (SE) is a powerful technology that is useful for commercial propagation, scientific study, germplasm conservation, and has potential applications in reforestation attempts in damaged ecosystems. Many organisms show similar traits during SE, such as the expression of commonly-found gene homologs, development of specialized tissues similar to zygotic embryogenesis, and response to certain growth regulators. To summarize these trends, a review article was written (Chapter 1 of this thesis). A literature analysis of SE in the palm family, Arecaceae, suggests similar trends across palm species. Both zygotic embryos or palm shoot meristems tended to respond well to Culture media composed of Murashige and Skoog salts, vitamins, 3% sucrose, exogenous activated carbon, and high concentrations of auxins, such as 2,4-D or picloram. Embryo development was usually stimulated through subCulture onto media with reduced auxins and addition of cytokinin, such as BAP and 2-ip and then embryos could be frequently converted on media without growth regulators. In addition to micropropagation, many studies focused on the biochemistry and gene expression of developing palm somatic embryos. Histological studies revealed common trends, such as similar morphology of meristematic vs. callus and epidermal cells. One of the technologies that several research groups had evaluated was Protoplast Culture. Protoplasts, cells with their cell walls removed, are of scientific interest due to both being able to regenerate into larger Cultures and the ability to create hybrids through somatic fusion of two different Protoplasts. This technology has only been investigated in several large-scale economic species, such as oil palm (Elaeis guineensis) or date palm (Phoenix dactyilifera), and increased understanding of trends might lead to further development of Protoplast Culture as a whole (Chapter 2). To understand Protoplast isolation and Culture, previously-established peach palm (Bactris gasipaes), butiá-da-serra (Butia eriospatha), and açaí (Euterpe oleracea) Cultures were treated with an enzyme solution composed of 2% w/v cellulase, 0.5% hemicellulase, and 0.5% pectinase and then either incubated stationary for 6, 12, 18, or 24 hours or on an orbital shaker at 45 rpm for 3, 6, 12, or 24 hours in the dark at 25±2 °C. Stationary incubation did not provide large amounts of Protoplasts in comparison to incubation done on an orbital shaker. The greatest numbers of Protoplasts per species were 5.50±0.68x105 and 1.22±0.13x106 Protoplasts/ gram FW for peach palm and açaí after six hours of orbital shaking and 5.36±2.23x105 cells/gram FW for butiá after 24 hours of orbital shaking. Both peach palm and açaí saw dramatic decreases in Protoplast yield after later incubations while the amount of visible cellular debris increased, possibly showing a potential reason for the decreased cell yield as cellular debris might lyse cells in motion. Protoplast viability remained high (>70%), except for açaí Protoplasts, which decreased rapidly during orbital incubation. Protoplasts were Cultured either using the agarose bead or the alginate bead method. Protoplasts Cultured in alginate beads were subjected to six types of liquid media containing 0, 1, or 10µM picloram either with or without 2µM 2-ip. Auxin was not shown to be essential for causing cell division in several occasions, however cell division was more frequent and occurred earlier in media with increasing levels of picloram. 2-ip was not found to have a major impact. Microcolonies were observed to form in alginate beads with 10µM picloram and 2µM 2-ip, however visible colonies were only formed twice in agarose beads. Further optimization would be required to achieve regeneration of whole plants, however there are many trends in other species, such as use of nurse Cultures, higher cell density during Culture, and type of Culture method, which can be pursued. The biochemistry of peach palm Cultures with different capacities for SE, high embryogenic (HE), low embryogenic (LE), and non-embryogenic (NE), were investigated for dry weight, protein, sugar, and starch content (Chapter 3). It was found that both HE and NE Cultures had similar dry weights, but water made up a larger proportion of LE Cultures. Because of this difference, protein, sugar, and starch amounts were evaluated in terms of both fresh weight and dry weight. HE contained slightly higher amounts of protein than NE Cultures, but both tissues contained higher proteins contents than LE Cultures, even after adjusting for dry weight. Starch levels were comparable between HE and NE Cultures, but LE Cultures contained significantly fewer starch reserves. Sugar levels were too low to have their amounts calculated using a glucose standard curve, however their absorbance readings showed HE and NE Cultures had the about the same values, while once again LE Cultures contained fewer reserves. These data suggest that the different growth behavior of the different tissues may not reflect overall protein amounts, but rather the types of proteins being expressed. Additionally, the large amounts of starch growth, along with the distinct morphology of NE tissues might suggest a type of organogenesis. Additionally, it is possible that the lack of energy reserves found in LE tissue is related to its rapid growth. Organized tissue formations were also observed in LE tissue, possibly suggesting that in vitro organogenesis extends beyond simply somatic embryos, roots, and shoots. Additionally, SE optimization was investigated in peach palm, butiá, and açaí Cultures. Peach palm tissue lines  G3 were placed on media containing either MS or modified MS salts and media containing 1mM spermidine, spermine, or no polyamines. No difference was detected in the number of somatic embryos recovered from MS or modified MS Cultures, however either polyamine had a negative effect. Peach palm  G2 Cultures were placed on media containing either 3% sucrose or an equal mix of sucrose, glucose, or sorbitol with no difference in total numbers of recovered embryos. Fast-growing friable butiá Cultures were subjected to numerous types of media, including both solid and liquid media, media containing different concentrations of auxins, cytokinins, ABA, GA3, sucrose, and activated charcoal in both light and dark, but no somatic embryos were induced. Additionally, partial dehydration did not induce improved peach palm multiplication nor butiá SE. However, 40% and 27.5% placed on MS media containing 2.5g/L activated charcoal and 300µM or 450µM, respectively, picloram responded with tissue growth similar to that found in peach palm highly embryogenic tissue. Açaí multiplication was greatly improved through optimizing multiplication media with addition 2.5g/L activated charcoal and increased picloram up to 50µM from 10µM. The effects of the activated charcoal led to 400% reduction in number of oxidized explants, an increase in number of explants producing embryos, embryos per explant, number of embryos producing polyembryogenic masses, and number of polyembryogenic masses per explant. However, instances of organogenic root growth and increased callogenesis were observed on açaí explants placed on media with activated charcoal, which suggests that the decreased effect of auxin caused by activated charcoal can reduce embryogenic potential. Together, these works contribute to better understanding the trends found in multiple palms, factors involved in the valuable technology of Protoplast Culture, the biochemical characteristics of several types of peach palm tissue with different embryogenic potential, and cultural optimization, especially for the economically valuable açaí palm
Yutaka Hirata - One of the best experts on this subject based on the ideXlab platform.
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plant regeneration from hypocotyl Protoplasts of red cabbage brassica oleracea by using nurse Cultures
Plant Cell Tissue and Organ Culture, 2004Co-Authors: Liping Chen, Qiubing Xiao, Jianguo Wu, Mingfang Zhang, Yutaka HirataAbstract:A protocol for rapid and efficient plant regeneration from Protoplasts of red cabbage was developed by a novel nurse Culture method. When the Protoplasts of red cabbage were Cultured in modified MS medium containing various combinations of BA, NAA and 2,4-D, they did not continue dividing due to browning. However, they successfully divided and formed micro-calli at a high efficiency when they were mixed and co-Cultured with those of tuber mustard at a 1:1 ratio. The presence of tuber mustard Protoplasts used as nurse cells was essential for sustainable divisions and colony formation of red cabbage Protoplasts. Red cabbage-like plantlets were regenerated from these Protoplast-derived calli at a frequency ranging from 33 to 56% in all the experiments where three cultivars of red cabbage were tested. Over 120 Protoplast-derived cabbage plants were transferred to the greenhouse, and they showed no noticeable abnormalities in morphological features. Chromosome observation revealed that all of the plants examined had the normal chromosome number of cabbage (2n = 18), suggesting that no spontaneous fusion between the two species had occurred during Protoplast Culture.
Dennis T Thomas - One of the best experts on this subject based on the ideXlab platform.
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isolation callus formation and plantlet regeneration from mesophyll Protoplasts of tylophora indica burm f merrill an important medicinal plant
In Vitro Cellular & Developmental Biology – Plant, 2009Co-Authors: Dennis T ThomasAbstract:Protoplast Culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and Cultured at a density of 5 × 105 Protoplasts per gram fresh weight, which is required for the highest frequency of Protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of Culture at 25°C in the dark, Protoplasts formed colonies consisting of about 100 cells. The Protoplast-derived microcalli were visible to the naked eye within 60 d of Culture and reached a size of 0.2–0.4 mm in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 µM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 µmol m−2 S−1 of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM) and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ and 0.4 μM NAA. On this medium, 44% Cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid.
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the role of activated charcoal in plant tissue Culture
Biotechnology Advances, 2008Co-Authors: Dennis T ThomasAbstract:Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue Culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther Culture, synthetic seed production, Protoplast Culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the Culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of Culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue Culture and the recent developments in this area.
Adela Adamus - One of the best experts on this subject based on the ideXlab platform.
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peptide growth factor phytosulfokine α stimulates cell divisions and enhances regeneration from b oleracea var capitata l Protoplast Culture
Journal of Plant Growth Regulation, 2019Co-Authors: Agnieszka Kielkowska, Adela AdamusAbstract:Protoplasts of six cabbage accessions were isolated from leaf mesophyll and Cultured in the presence of 0.01, 0.1 and 1.0 µM phytosulfokine–α (PSK-α) and in a PSK-free control medium. PSK-α was applied for 10 days and later, Protoplast-derived cells were Cultured in the PSK-free medium. Supplementation of the Culture medium with PSK-α showed a dose-dependent effect on the mitotic activity of Cultured cells. On the 15th day of Culture, the highest mitotic activity of Protoplast-derived cells was observed in Cultures treated with 0.1 µM of PSK-α, and ranged from 14 to 60% dependent on the accession. The number of multi-cell structures was also higher (90–93%) on this medium compared to the control (77–80%). Analysis of cellulose regeneration in Cultured Protoplasts after Calcofluor White staining showed that this process was not synchronous, but depended instead on the presence of PSK-α in the Culture medium, and was more pronounced in the low-responding accession. Sustained cell divisions led to formation of microcallus colonies, subjected to regeneration on solid media. Supplementation of the regeneration media with 0.1 µM of PSK significantly increased shoot regeneration compared to the control media. Moreover, enhanced regeneration was observed from calluses developed from cells treated with PSK-α at the early stages of development and later transferred for regeneration onto the media supplemented with 0.1 µM of this peptide.
Liping Chen - One of the best experts on this subject based on the ideXlab platform.
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plant regeneration from hypocotyl Protoplasts of red cabbage brassica oleracea by using nurse Cultures
Plant Cell Tissue and Organ Culture, 2004Co-Authors: Liping Chen, Qiubing Xiao, Jianguo Wu, Mingfang Zhang, Yutaka HirataAbstract:A protocol for rapid and efficient plant regeneration from Protoplasts of red cabbage was developed by a novel nurse Culture method. When the Protoplasts of red cabbage were Cultured in modified MS medium containing various combinations of BA, NAA and 2,4-D, they did not continue dividing due to browning. However, they successfully divided and formed micro-calli at a high efficiency when they were mixed and co-Cultured with those of tuber mustard at a 1:1 ratio. The presence of tuber mustard Protoplasts used as nurse cells was essential for sustainable divisions and colony formation of red cabbage Protoplasts. Red cabbage-like plantlets were regenerated from these Protoplast-derived calli at a frequency ranging from 33 to 56% in all the experiments where three cultivars of red cabbage were tested. Over 120 Protoplast-derived cabbage plants were transferred to the greenhouse, and they showed no noticeable abnormalities in morphological features. Chromosome observation revealed that all of the plants examined had the normal chromosome number of cabbage (2n = 18), suggesting that no spontaneous fusion between the two species had occurred during Protoplast Culture.
