Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFLA |
Texto Completo: | http://repositorio.ufla.br/jspui/handle/1/11155 |
Resumo: | This study aimed was to establish genetic transformation parameters for the hybrid Eucalyptus grandis X Eucalyptus urophylla, mediated by Agrobacterium tumefaciens as well as perform the cryopreservation of shoot tips for the same hybrid, aiming storage and conservation. The organogenic hybrid calluses were subjected to different transformation process, vacuum infiltration (4 to 8 minutes at 400 mmHg), sonication (15 and 30 seconds at a frequency of 40 kHz), combined and isolation. At the same time, were evaluated three concentrations of acetosyringone (100 μM, 200 μM and 300 μM) and three temperatures of cocultivation (19° C, 22° C and 25° C). Among the studied parameters, the mean value of the transient expression was obtained when using the infection method by vacuum infiltration for 4 minutes with mean transient expression of GUS activity (4,4), the addition of 100μM of acetosyringone mean transient expression (0.76), and co-cultivation at a temperature of 19° C with higher average transient expression (2.6). For the cryopreservation process was pre-determined regeneration medium of shoot tips using different concentrations of BAP (0,14 μM; 1,42 μM) and BAP (0,14 μM) in combination with IAA (5,10 μM), after 30 days it was evaluated the percentage of plant regeneration, calluses formation and vitrified plants. To cryopreservation was performed pre-cultivation of shoot tips on MS medium supplemented with 0.2 M sucrose for 24 hours and 0.5 M sucrose for 24 hours and were evaluated six times of exposure to PVS2 (20, 40, 60, 80, 100 and 120 minutes) and subsequent immersion in liquid nitrogen, after 30 days it was evaluated percent survival and after 60 days it was evaluated the percentage of shoots. Histological and ultrastructural observations of the apices were made in different stages of the cryopreservation process, and after one week of recovery, through light and scanning microscopy, of which measurements were made using plasmolysis image-Pro Plus software. The concentration of BAP 0,14 μM best promoted regeneration rates (80%) without the presence of vitrified plants. Cryopreservation was successful using 0.2 M and 0.5 M sucrose in the precultivation and exposure to a cryoprotectant for 60 minutes, providing no irreversible damage cells, resulting in higher survival rates (66%). The histopathological and ultrastructural analysis of the shoot tips were made during the cryopreservation process for the treatment of exposure for 60 minutes was found to increased cell integrity without the presence of picnotics nucleus with low plasmolysis rates when compared with treatments exposed to longer times (80 and 120 minutes) to the PVS2. |
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Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophyllaAgrobacterium tumefaciensÁpices caulinaresDroplet-vitrificationShoot tipsFisiologia VegetalThis study aimed was to establish genetic transformation parameters for the hybrid Eucalyptus grandis X Eucalyptus urophylla, mediated by Agrobacterium tumefaciens as well as perform the cryopreservation of shoot tips for the same hybrid, aiming storage and conservation. The organogenic hybrid calluses were subjected to different transformation process, vacuum infiltration (4 to 8 minutes at 400 mmHg), sonication (15 and 30 seconds at a frequency of 40 kHz), combined and isolation. At the same time, were evaluated three concentrations of acetosyringone (100 μM, 200 μM and 300 μM) and three temperatures of cocultivation (19° C, 22° C and 25° C). Among the studied parameters, the mean value of the transient expression was obtained when using the infection method by vacuum infiltration for 4 minutes with mean transient expression of GUS activity (4,4), the addition of 100μM of acetosyringone mean transient expression (0.76), and co-cultivation at a temperature of 19° C with higher average transient expression (2.6). For the cryopreservation process was pre-determined regeneration medium of shoot tips using different concentrations of BAP (0,14 μM; 1,42 μM) and BAP (0,14 μM) in combination with IAA (5,10 μM), after 30 days it was evaluated the percentage of plant regeneration, calluses formation and vitrified plants. To cryopreservation was performed pre-cultivation of shoot tips on MS medium supplemented with 0.2 M sucrose for 24 hours and 0.5 M sucrose for 24 hours and were evaluated six times of exposure to PVS2 (20, 40, 60, 80, 100 and 120 minutes) and subsequent immersion in liquid nitrogen, after 30 days it was evaluated percent survival and after 60 days it was evaluated the percentage of shoots. Histological and ultrastructural observations of the apices were made in different stages of the cryopreservation process, and after one week of recovery, through light and scanning microscopy, of which measurements were made using plasmolysis image-Pro Plus software. The concentration of BAP 0,14 μM best promoted regeneration rates (80%) without the presence of vitrified plants. Cryopreservation was successful using 0.2 M and 0.5 M sucrose in the precultivation and exposure to a cryoprotectant for 60 minutes, providing no irreversible damage cells, resulting in higher survival rates (66%). The histopathological and ultrastructural analysis of the shoot tips were made during the cryopreservation process for the treatment of exposure for 60 minutes was found to increased cell integrity without the presence of picnotics nucleus with low plasmolysis rates when compared with treatments exposed to longer times (80 and 120 minutes) to the PVS2.Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)O objetivo do trabalho foi estabelecer parâmetros de transformação genética para o híbrido Eucalyptus grandis X Eucalyptus urophylla, mediado por Agrobacterium tumefaciens, bem como realizar a criopreservação de ápices caulinares para o mesmo híbrido, visando seu armazenamento e sua conservação. Os calos organogênicos deste híbrido foram submetidos a diferentes processos de transformação, infiltração a vácuo (4 e 8 minutos com pressão de 400 mmHg), sonicação (15 e 30 segundos a uma frequência de 40 kHz), os tratamentos foram aplicados de forma combinada e isolada. Paralelamente, foram avaliados três concentrações de acetoseringona (100 µM, 200 µM e 300 µM) e três temperaturas de co-cultivo (19 ºC, 22 ºC e 25 ºC). Dentre os parâmetros estudados, a maior média da expressão transiente foi obtida quando se utilizou o método de infecção por infiltração a vácuo durante 4 minutos com média de expressão transiente da atividade do GUS (4,4), a adição de 100 μM de acetoseringona com média de expressão transiente (0,76), e o co-cultivo sob temperatura de 19 ºC com maior média de expressão transiente (2,6). Para o processo de criopreservação foi determinado o meio de cultura para a regeneração dos ápices, testando diferentes concentrações de BAP (0,14 µM; 1,42 µM) e BAP (0,14 µM) em combinação com AIA (5,10 µM), após 30 dias avaliou-se percentagem de regeneração de plantas, formação de calos e plantas hiper-hídricas. Para a criopreservação realizou-se o pré-cultivo dos ápices em meio MS suplementado com sacarose a 0,2 M por 24 horas e sacarose 0,5 M por mais 24 horas e foram avaliados seis tempos de exposição ao PVS2 (20, 40, 60, 80, 100 e 120 minutos) e posterior imersão em nitrogênio líquido. Após 30 dias avaliou-se percentagem de sobrevivência e após 60 dias percentagem de regeneração dos ápices. As observações histológicas e ultra-estruturais dos ápices foram realizadas em diferentes fases do processo de criopreservação e após uma semana de recuperação. As variáveis analisadas foram plasmólise, danos celulares, núcleos pinóticos e ruptura de membrana. Para avaliação da percentagem de plasmólise foram realizadas medições da área da parede celular e do citoplasma em três camadas dos ápices caulinares (L1; L1 -L3; L7-L9), utilizando o software imagePro Plus®. A concentração de 0,14 µM de BAP promoveu melhores taxas de regeneração (80%) sem presença de plantas hiper-hídricas. A criopreservação foi bem sucedida quando os ápices foram expostos ao crioprotetor por 60 minutos de PVS2, com maiores taxas de regeneração (66%), proporcionando uma maior integridade celular, sem a presença de núcleos pinóticos, com baixas taxas de plasmólise.Universidade Federal de LavrasPrograma de Pós-Graduação em Agronomia/Fisiologia VegetalUFLAbrasilDepartamento de BiologiaPaiva, Luciano VilelaSilva, Diogo Pedrosa Corrêa daMendonça, Evânia GalvãoVaz, Chaiane Fernandes2016-05-16T18:17:46Z2016-05-16T18:17:46Z2016-05-162016-03-11info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfVAZ, C. F. Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla. 2016. 112 p. Dissertação (Mestrado em Fisiologia Vegetal)-Universidade Federal de Lavras, Lavras, 2016.http://repositorio.ufla.br/jspui/handle/1/11155porinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFLAinstname:Universidade Federal de Lavras (UFLA)instacron:UFLA2023-04-27T17:50:36Zoai:localhost:1/11155Repositório InstitucionalPUBhttp://repositorio.ufla.br/oai/requestnivaldo@ufla.br || repositorio.biblioteca@ufla.bropendoar:2023-04-27T17:50:36Repositório Institucional da UFLA - Universidade Federal de Lavras (UFLA)false |
dc.title.none.fl_str_mv |
Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla |
title |
Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla |
spellingShingle |
Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla Vaz, Chaiane Fernandes Agrobacterium tumefaciens Ápices caulinares Droplet-vitrification Shoot tips Fisiologia Vegetal |
title_short |
Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla |
title_full |
Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla |
title_fullStr |
Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla |
title_full_unstemmed |
Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla |
title_sort |
Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla |
author |
Vaz, Chaiane Fernandes |
author_facet |
Vaz, Chaiane Fernandes |
author_role |
author |
dc.contributor.none.fl_str_mv |
Paiva, Luciano Vilela Silva, Diogo Pedrosa Corrêa da Mendonça, Evânia Galvão |
dc.contributor.author.fl_str_mv |
Vaz, Chaiane Fernandes |
dc.subject.por.fl_str_mv |
Agrobacterium tumefaciens Ápices caulinares Droplet-vitrification Shoot tips Fisiologia Vegetal |
topic |
Agrobacterium tumefaciens Ápices caulinares Droplet-vitrification Shoot tips Fisiologia Vegetal |
description |
This study aimed was to establish genetic transformation parameters for the hybrid Eucalyptus grandis X Eucalyptus urophylla, mediated by Agrobacterium tumefaciens as well as perform the cryopreservation of shoot tips for the same hybrid, aiming storage and conservation. The organogenic hybrid calluses were subjected to different transformation process, vacuum infiltration (4 to 8 minutes at 400 mmHg), sonication (15 and 30 seconds at a frequency of 40 kHz), combined and isolation. At the same time, were evaluated three concentrations of acetosyringone (100 μM, 200 μM and 300 μM) and three temperatures of cocultivation (19° C, 22° C and 25° C). Among the studied parameters, the mean value of the transient expression was obtained when using the infection method by vacuum infiltration for 4 minutes with mean transient expression of GUS activity (4,4), the addition of 100μM of acetosyringone mean transient expression (0.76), and co-cultivation at a temperature of 19° C with higher average transient expression (2.6). For the cryopreservation process was pre-determined regeneration medium of shoot tips using different concentrations of BAP (0,14 μM; 1,42 μM) and BAP (0,14 μM) in combination with IAA (5,10 μM), after 30 days it was evaluated the percentage of plant regeneration, calluses formation and vitrified plants. To cryopreservation was performed pre-cultivation of shoot tips on MS medium supplemented with 0.2 M sucrose for 24 hours and 0.5 M sucrose for 24 hours and were evaluated six times of exposure to PVS2 (20, 40, 60, 80, 100 and 120 minutes) and subsequent immersion in liquid nitrogen, after 30 days it was evaluated percent survival and after 60 days it was evaluated the percentage of shoots. Histological and ultrastructural observations of the apices were made in different stages of the cryopreservation process, and after one week of recovery, through light and scanning microscopy, of which measurements were made using plasmolysis image-Pro Plus software. The concentration of BAP 0,14 μM best promoted regeneration rates (80%) without the presence of vitrified plants. Cryopreservation was successful using 0.2 M and 0.5 M sucrose in the precultivation and exposure to a cryoprotectant for 60 minutes, providing no irreversible damage cells, resulting in higher survival rates (66%). The histopathological and ultrastructural analysis of the shoot tips were made during the cryopreservation process for the treatment of exposure for 60 minutes was found to increased cell integrity without the presence of picnotics nucleus with low plasmolysis rates when compared with treatments exposed to longer times (80 and 120 minutes) to the PVS2. |
publishDate |
2016 |
dc.date.none.fl_str_mv |
2016-05-16T18:17:46Z 2016-05-16T18:17:46Z 2016-05-16 2016-03-11 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
VAZ, C. F. Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla. 2016. 112 p. Dissertação (Mestrado em Fisiologia Vegetal)-Universidade Federal de Lavras, Lavras, 2016. http://repositorio.ufla.br/jspui/handle/1/11155 |
identifier_str_mv |
VAZ, C. F. Otimização de parâmetros de transformação genética e criopreservação de Eucalyptus grandis X Eucalyptus urophylla. 2016. 112 p. Dissertação (Mestrado em Fisiologia Vegetal)-Universidade Federal de Lavras, Lavras, 2016. |
url |
http://repositorio.ufla.br/jspui/handle/1/11155 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Lavras Programa de Pós-Graduação em Agronomia/Fisiologia Vegetal UFLA brasil Departamento de Biologia |
publisher.none.fl_str_mv |
Universidade Federal de Lavras Programa de Pós-Graduação em Agronomia/Fisiologia Vegetal UFLA brasil Departamento de Biologia |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFLA instname:Universidade Federal de Lavras (UFLA) instacron:UFLA |
instname_str |
Universidade Federal de Lavras (UFLA) |
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UFLA |
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UFLA |
reponame_str |
Repositório Institucional da UFLA |
collection |
Repositório Institucional da UFLA |
repository.name.fl_str_mv |
Repositório Institucional da UFLA - Universidade Federal de Lavras (UFLA) |
repository.mail.fl_str_mv |
nivaldo@ufla.br || repositorio.biblioteca@ufla.br |
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1807835221482012672 |