Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca

Detalhes bibliográficos
Autor(a) principal: Vaez, Juliana Rocha
Data de Publicação: 2007
Tipo de documento: Tese
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/286
Resumo: Cassava (Manihot esculenta Crantz) is one of the most important crops for human consumption. It is considered the fourth most important source of carbohydrates in the tropics. The increasing importance of the culture achieved during the last years, raised the interest in the improvement and development of new cassava varieties. Therefore, the aims of the present work were to evaluate factors associated to the in vitro regeneration and genetic transformation of five cassava cultivars. Explants from different genotypes were evaluated for their embryogenic response to two growth regulators, picloram and 2,4 D. The growth regulator 2,4 D led to the best embryogenic response and the concentrations of this regulator for cultivars Mantiqueira, Parazinho, Vassourinha and Urubu were two, one, two and two mg/L, respectively. Somatic embryogenesis is a process by which haploid cells develop through different embryogenic states, originating a plant. Embryos in the cotyledonary stage may have their development induced by several combinations of growth regulators. Aiming to evaluate which combination promotes the best stretching frequency, the following combinations were tested: 0.4 mg/L BAP; 0.25 mg/L BAP + 0.25 mg/L AIB; 0.4 mg/L GA3 and 0.25 mg/L GA3 + 0.25 mg/L AIB. Among the evaluated media, the most adequate was the one which contained 0.4 mg/L GA3. In order to induce organogenesis, the following combinations of growth regulators were tested: 1 mg/L BAP + 0.5 mg/L AIB and 1.6 mg/L BA + 1.6 mg/L AIB were evaluated. The highest organogenic frequency was obtained with the use of the first combination mentioned. The organogenic process may be influenced by many factors, such as genotype, type of explants and developmental stage of the explants. Different cotyledonal regions and maturation periods of somatic embryos at the cotyledonal stage which produced the greatest amount of shoots were evaluated. In the case of cultivar Rosinha, the central cotyledonal region was the most adequate for organogenesis induction and the 15-day maturation period was the one which produced the highest amount of in cotyledons. Various protocols for manihot transformation were developed, however, none of them have universal applicability and the selection systems used still either generate many escapes or are deleterious. There are more than 1,500 manihot cultivars reported, however, only a few was successfully used for transformation. Among the transformation systems used, there is a preference for the transformation via Agrobacterium tumefaciens for the production of transgenic plants. To increase the transformation efficiency by A. tumefaciens, parameters which could decrease histodifferentiation were evaluated in cultivar Rosinha. The parameters that interfered in the transformation were paramomycin at a concentration of 500 mg/L and the bacterial strain EHA 105. Os fatores que interferem no processo foram a paramomicina na concentração de 500mg/L e a estirpe bacteriana EHA105. A major factor to be defined during the transformation process is the antibiotic dosage. The concentration defined should prevent false positives without interfering with the regeneration potential of the explants used in the transformation process. The effects of different concentration of kanamycin and paramomycin on somatic embryos were evaluated in globular and cotyledonal stages. The concentrations of 10mg/L (kanamycin) and 20 mg/L (paramomycin) were chosen for embryo selection in globular stage of cultivar Rosinha. For embryo selection in cotyledonal stage of cultivar Rosinha the concentration of 10mg/L paramomycin was chosen. The transformation via SAAT (Sonication-assisted Agrobacterium-mediated transformation) uses ultrasound pulses to wound the tissue and this factor can affect transformation efficiency. In order to evaluate the efficiency of this transformation system, experiments were conducted which evaluated the regenerative response of explants that had undergone the SAAT procedure as well as the frequency of the GUS gene expression. It was verified that SAAT caused an increase on the transient expression of the GUS gene and the regeneration of explants were slightly affected by the process.
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spelling Vaez, Juliana Rochahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4768327D5Guimarães, Valéria Montezehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3Campos, Francisco de Assis Paivahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788290A6Barros, Everaldo Gonçalves dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781285J6Otoni, Wagner Camposhttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786133Y6Oliveira, Luiz Orlando dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781626T2Lima, Andréia Barcelos Passoshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4766019H42015-03-26T12:15:12Z2008-07-222015-03-26T12:15:12Z2007-12-10VAEZ, Juliana Rocha. Factor evaluations associated to in vitro regeneration of five cultivars of cassava. 2007. 138 f. Tese (Doutorado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2007.http://locus.ufv.br/handle/123456789/286Cassava (Manihot esculenta Crantz) is one of the most important crops for human consumption. It is considered the fourth most important source of carbohydrates in the tropics. The increasing importance of the culture achieved during the last years, raised the interest in the improvement and development of new cassava varieties. Therefore, the aims of the present work were to evaluate factors associated to the in vitro regeneration and genetic transformation of five cassava cultivars. Explants from different genotypes were evaluated for their embryogenic response to two growth regulators, picloram and 2,4 D. The growth regulator 2,4 D led to the best embryogenic response and the concentrations of this regulator for cultivars Mantiqueira, Parazinho, Vassourinha and Urubu were two, one, two and two mg/L, respectively. Somatic embryogenesis is a process by which haploid cells develop through different embryogenic states, originating a plant. Embryos in the cotyledonary stage may have their development induced by several combinations of growth regulators. Aiming to evaluate which combination promotes the best stretching frequency, the following combinations were tested: 0.4 mg/L BAP; 0.25 mg/L BAP + 0.25 mg/L AIB; 0.4 mg/L GA3 and 0.25 mg/L GA3 + 0.25 mg/L AIB. Among the evaluated media, the most adequate was the one which contained 0.4 mg/L GA3. In order to induce organogenesis, the following combinations of growth regulators were tested: 1 mg/L BAP + 0.5 mg/L AIB and 1.6 mg/L BA + 1.6 mg/L AIB were evaluated. The highest organogenic frequency was obtained with the use of the first combination mentioned. The organogenic process may be influenced by many factors, such as genotype, type of explants and developmental stage of the explants. Different cotyledonal regions and maturation periods of somatic embryos at the cotyledonal stage which produced the greatest amount of shoots were evaluated. In the case of cultivar Rosinha, the central cotyledonal region was the most adequate for organogenesis induction and the 15-day maturation period was the one which produced the highest amount of in cotyledons. Various protocols for manihot transformation were developed, however, none of them have universal applicability and the selection systems used still either generate many escapes or are deleterious. There are more than 1,500 manihot cultivars reported, however, only a few was successfully used for transformation. Among the transformation systems used, there is a preference for the transformation via Agrobacterium tumefaciens for the production of transgenic plants. To increase the transformation efficiency by A. tumefaciens, parameters which could decrease histodifferentiation were evaluated in cultivar Rosinha. The parameters that interfered in the transformation were paramomycin at a concentration of 500 mg/L and the bacterial strain EHA 105. Os fatores que interferem no processo foram a paramomicina na concentração de 500mg/L e a estirpe bacteriana EHA105. A major factor to be defined during the transformation process is the antibiotic dosage. The concentration defined should prevent false positives without interfering with the regeneration potential of the explants used in the transformation process. The effects of different concentration of kanamycin and paramomycin on somatic embryos were evaluated in globular and cotyledonal stages. The concentrations of 10mg/L (kanamycin) and 20 mg/L (paramomycin) were chosen for embryo selection in globular stage of cultivar Rosinha. For embryo selection in cotyledonal stage of cultivar Rosinha the concentration of 10mg/L paramomycin was chosen. The transformation via SAAT (Sonication-assisted Agrobacterium-mediated transformation) uses ultrasound pulses to wound the tissue and this factor can affect transformation efficiency. In order to evaluate the efficiency of this transformation system, experiments were conducted which evaluated the regenerative response of explants that had undergone the SAAT procedure as well as the frequency of the GUS gene expression. It was verified that SAAT caused an increase on the transient expression of the GUS gene and the regeneration of explants were slightly affected by the process.A mandioca (Manihot esculenta Crantz) é uma das culturas fundamentais para a alimentação humana, visto que é a quarta fonte mais importante de carboidratos nos trópicos. Devido à importância que a cultura atingiu nos últimos anos, o interesse no melhoramento e desenvolvimento de novas cultivares vem crescendo. Assim, os objetivos deste trabalho foram avaliar fatores associados à regeneração in vitro e transformação genética de cinco cultivares de mandioca. Para tanto, explantes de mandioca de diferentes cultivares tiveram sua resposta embriogênica a dois reguladores de crescimento, picloram e 2,4 - D, avaliada em diversas concentrações. O regulador de crescimento 2,4 - D foi o que apresentou maior freqüência embriogênica, e as suas concentrações mais indicadas para as cultivares Mantiqueira, Parazinho, Vassourinha e Urubu foram, respectivamente, 2, 1, 2 e 2 mg/L. A embriogênese somática é um processo pelo qual células haplóides se desenvolvem através de diferentes estádios embriogênicos, dando origem a uma planta. Embriões em estádio cotiledonar podem ter seu alongamento induzido por diversas combinações de reguladores de crescimento. Com o intuito de avaliar o meio que promoveria maior freqüência de alongamento, foram testadas as seguintes combinações: 0,4 mg/L de BAP, 0,25 mg/L de BAP + 0,25 mg/L AIB, 0,4 mg/L de GA3 e 0,25 mg/L de GA3 + 0,25 mg/L AIB. Entre os meios avaliados, o mais adequado foi o que continha 0,4 mg/L de GA3. Para induzir organogênese, faz-se uso de combinações entre reguladores de crescimento. As combinações hormonais de 1 mg/L BAP + 0,5 mg/L AIB e 1,6 mg/L BA + 1,6 mg/L AIB foram avaliadas e a maior freqüência organogênica, obtida com o uso da primeira combinação citada. O processo de organogênese pode ser influenciado por vários fatores, por exemplo genótipo, tipo de explante e estádio de desenvolvimento do explante. Foram avaliadas diferentes regiões cotiledonares e período de maturação de embriões somáticos no estádio cotiledonar que produziriam maior quantidade de brotos. Na cultivar Rosinha, a região cotiledonar central foi a mais adequada para indução de organogênese e o período de 15 dias de maturação, o que produziu a maior quantidade de brotos em cotilédones. Diversos protocolos de transformação de mandioca foram desenvolvidos, mas nenhum deles tem aplicabilidade universal e os sistemas de seleção utilizados ainda geram muitos escapes ou são deletérios. Há mais de 1.500 cultivares de mandioca, contudo apenas um pequeno número foi utilizado com sucesso em processos de transformação. Entre os sistemas de transformação utilizados, na cultura da mandioca há preferência pela transformação via Agrobacterium tumefaciens para a produção de plantas transgênicas. Com o intuito de aumentar a eficiência de transformação, foram avaliados os parâmetros que afetariam a transformação via A. tumefaciens da cultivar Rosinha, por diminuírem a freqüência de histodiferenciação. Os fatores que interferem no processo foram a paramomicina na concentração de 500 mg/L e a estirpe bacteriana EHA105. Um fator de grande importância a ser determinado no processo de transformação é a dose do antibiótico, de maneira a impedir o aparecimento de falsos positivos sem interferir, ao mesmo tempo, no potencial regenerativo dos explantes usados na transformação. Foram avaliados os efeitos de diferentes concentrações dos antibióticos canamicina e paramomicina em embriões somáticos nos estádios globular e cotiledonar. Das concentrações avaliadas, as concentrações de 10 mg/L de canamicina e 20 mg/L de paramomicina foram as escolhidas para a seleção de embriões em estádio globular da cultivar Rosinha. Já das concentrações avaliadas a concentração de 10 mg/L de paramomicina foi a escolhida para a seleção de embriões em estádio cotiledonar da cultivar Rosinha. A transformação via SAAT (Sonication-assisted Agrobacterium- mediated transformation) utiliza pulsos de ultra-som para ferir o tecido-alvo da transformação. O ferimento é um fator que pode ser manipulado e ter como resposta direta o aumento na eficiência do processo de transformação. Para avaliar a eficiência desse sistema de transformação, foram realizados experimentos que analisassem a resposta regenerativa dos explantes submetidos a SAAT e a freqüência da expressão do gene GUS. Foi verificado que SAAT aumentou de forma sensível a expressão transiente do gene GUS e a regeneração dos explantes foi pouco afetada pelo processo.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de ViçosaDoutorado em Bioquímica AgrícolaUFVBRBioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animalManihot esculentaCultura de tecidosManihot esculentaTissue cultureCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARAvaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandiocaFactor evaluations associated to in vitro regeneration of five cultivars of cassavainfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf1792013https://locus.ufv.br//bitstream/123456789/286/1/texto%20completo.pdf11be69400a74e69ebfc6e7b1f59b102cMD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain263626https://locus.ufv.br//bitstream/123456789/286/2/texto%20completo.pdf.txtbd1ae24308cc78b9b587ae3765be9ea2MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3535https://locus.ufv.br//bitstream/123456789/286/3/texto%20completo.pdf.jpg5435e6457eccfdcdc9ec7ef2d681a86aMD53123456789/2862016-04-06 23:01:50.207oai:locus.ufv.br:123456789/286Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-07T02:01:50LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca
dc.title.alternative.eng.fl_str_mv Factor evaluations associated to in vitro regeneration of five cultivars of cassava
title Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca
spellingShingle Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca
Vaez, Juliana Rocha
Manihot esculenta
Cultura de tecidos
Manihot esculenta
Tissue culture
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
title_short Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca
title_full Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca
title_fullStr Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca
title_full_unstemmed Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca
title_sort Avaliação de fatores associados à regeneração in vitro e transformação genético de cinco cultivares de mandioca
author Vaez, Juliana Rocha
author_facet Vaez, Juliana Rocha
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4768327D5
dc.contributor.author.fl_str_mv Vaez, Juliana Rocha
dc.contributor.advisor-co1.fl_str_mv Guimarães, Valéria Monteze
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3
dc.contributor.advisor-co2.fl_str_mv Campos, Francisco de Assis Paiva
dc.contributor.advisor-co2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788290A6
dc.contributor.advisor1.fl_str_mv Barros, Everaldo Gonçalves de
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781285J6
dc.contributor.referee1.fl_str_mv Otoni, Wagner Campos
dc.contributor.referee1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4786133Y6
dc.contributor.referee2.fl_str_mv Oliveira, Luiz Orlando de
dc.contributor.referee2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781626T2
dc.contributor.referee3.fl_str_mv Lima, Andréia Barcelos Passos
dc.contributor.referee3Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4766019H4
contributor_str_mv Guimarães, Valéria Monteze
Campos, Francisco de Assis Paiva
Barros, Everaldo Gonçalves de
Otoni, Wagner Campos
Oliveira, Luiz Orlando de
Lima, Andréia Barcelos Passos
dc.subject.por.fl_str_mv Manihot esculenta
Cultura de tecidos
topic Manihot esculenta
Cultura de tecidos
Manihot esculenta
Tissue culture
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
dc.subject.eng.fl_str_mv Manihot esculenta
Tissue culture
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
description Cassava (Manihot esculenta Crantz) is one of the most important crops for human consumption. It is considered the fourth most important source of carbohydrates in the tropics. The increasing importance of the culture achieved during the last years, raised the interest in the improvement and development of new cassava varieties. Therefore, the aims of the present work were to evaluate factors associated to the in vitro regeneration and genetic transformation of five cassava cultivars. Explants from different genotypes were evaluated for their embryogenic response to two growth regulators, picloram and 2,4 D. The growth regulator 2,4 D led to the best embryogenic response and the concentrations of this regulator for cultivars Mantiqueira, Parazinho, Vassourinha and Urubu were two, one, two and two mg/L, respectively. Somatic embryogenesis is a process by which haploid cells develop through different embryogenic states, originating a plant. Embryos in the cotyledonary stage may have their development induced by several combinations of growth regulators. Aiming to evaluate which combination promotes the best stretching frequency, the following combinations were tested: 0.4 mg/L BAP; 0.25 mg/L BAP + 0.25 mg/L AIB; 0.4 mg/L GA3 and 0.25 mg/L GA3 + 0.25 mg/L AIB. Among the evaluated media, the most adequate was the one which contained 0.4 mg/L GA3. In order to induce organogenesis, the following combinations of growth regulators were tested: 1 mg/L BAP + 0.5 mg/L AIB and 1.6 mg/L BA + 1.6 mg/L AIB were evaluated. The highest organogenic frequency was obtained with the use of the first combination mentioned. The organogenic process may be influenced by many factors, such as genotype, type of explants and developmental stage of the explants. Different cotyledonal regions and maturation periods of somatic embryos at the cotyledonal stage which produced the greatest amount of shoots were evaluated. In the case of cultivar Rosinha, the central cotyledonal region was the most adequate for organogenesis induction and the 15-day maturation period was the one which produced the highest amount of in cotyledons. Various protocols for manihot transformation were developed, however, none of them have universal applicability and the selection systems used still either generate many escapes or are deleterious. There are more than 1,500 manihot cultivars reported, however, only a few was successfully used for transformation. Among the transformation systems used, there is a preference for the transformation via Agrobacterium tumefaciens for the production of transgenic plants. To increase the transformation efficiency by A. tumefaciens, parameters which could decrease histodifferentiation were evaluated in cultivar Rosinha. The parameters that interfered in the transformation were paramomycin at a concentration of 500 mg/L and the bacterial strain EHA 105. Os fatores que interferem no processo foram a paramomicina na concentração de 500mg/L e a estirpe bacteriana EHA105. A major factor to be defined during the transformation process is the antibiotic dosage. The concentration defined should prevent false positives without interfering with the regeneration potential of the explants used in the transformation process. The effects of different concentration of kanamycin and paramomycin on somatic embryos were evaluated in globular and cotyledonal stages. The concentrations of 10mg/L (kanamycin) and 20 mg/L (paramomycin) were chosen for embryo selection in globular stage of cultivar Rosinha. For embryo selection in cotyledonal stage of cultivar Rosinha the concentration of 10mg/L paramomycin was chosen. The transformation via SAAT (Sonication-assisted Agrobacterium-mediated transformation) uses ultrasound pulses to wound the tissue and this factor can affect transformation efficiency. In order to evaluate the efficiency of this transformation system, experiments were conducted which evaluated the regenerative response of explants that had undergone the SAAT procedure as well as the frequency of the GUS gene expression. It was verified that SAAT caused an increase on the transient expression of the GUS gene and the regeneration of explants were slightly affected by the process.
publishDate 2007
dc.date.issued.fl_str_mv 2007-12-10
dc.date.available.fl_str_mv 2008-07-22
2015-03-26T12:15:12Z
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dc.identifier.citation.fl_str_mv VAEZ, Juliana Rocha. Factor evaluations associated to in vitro regeneration of five cultivars of cassava. 2007. 138 f. Tese (Doutorado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2007.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/286
identifier_str_mv VAEZ, Juliana Rocha. Factor evaluations associated to in vitro regeneration of five cultivars of cassava. 2007. 138 f. Tese (Doutorado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2007.
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