Transformação genética e rejuvenescimento in vitro de híbridos naturais de Eucalyptus urophylla

• Ano de defesa: 2011
• Autor(a) principal: Mendonça, Evânia Galvão
• Orientador(a): Paiva, Luciano Vilela
• Banca de defesa: Andrade, Helder Bolognani, José, Anderson Cleiton, Carneiro, Andrea Almeida
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Lavras
• Programa de Pós-Graduação: Programa de Pós-graduação em Agronomia/Fisiologia Vegetal
• Departamento: Departamento de Biologia
• País: BRASIL
• Palavras-chave em Português: Biotecnologia; Cultivo in vitro; Enraizamento; Gene repórter; Biotechnology; Cultivation in vitro; Rooting; Reporter gene
• Área do conhecimento CNPq: Fisiologia Vegetal
• Link de acesso: https://repositorio.ufla.br/handle/1/1962

Eucalyptus is a species native to Australia and it belongs to the Family Myrtaceae. Due to the species´ intrinsic genetic variability and good adaptability, Eucalyptus can be used for plantations in critical areas and its wood, destined to several purposes. In order to search for a methodology that associates characteristics of economic interest, this work has been carried out in two steps. The first step aimed to promote the rejuvenation in vitro and increase the rooting rate of two commercial clones (VM 02 and VM 04) of natural hybrids of Eucalyptus urophylla from the company V&M Florestal. The cultivation in vitro started with meristematic apexes and the plants originated from these meristems were subcultured, rooted and acclimatized in order to be rooted ex vitro. From this rooting, it was possible to observe that, for clone VM 02, three subcultures were enough to provide the rooting of 100% of the mini-cuttings, rooted ex vitro, while for VM 04, only one subculture in vitro was necessary to provide plant rejuvenation. This result has also been observed with the rooting of approximately 100% of the cuttings ex vitro. By means of histochemical analysis with lugol and ferric chloride, it could be seen that the number of amyloplasts and the amount of phenols are higher in the generations in which the mini-cuttings do not root. From these results, it can be concluded that rejuvenation was an important factor for the rooting of mini-cuttings of clones VM 02 and VM 04. The aim of the second experiment was to optimize a protocol of genetic transformation for a hybrid of Eucalyptus urophylla via Agrobacterium rhizogenes. For this study, sprouts of this hybrid were used as vegetal material, cultivated in vitro, in multiplication media. The sprouts were subjected to two methods of infection with the agrobacteria: injury by cutting the base of the bud and vacuum infiltration. The co-culture lasted for 5 days and the analysis of the potentially transgenic roots was carried out by visual analysis of GFP expression and by PCR with specific primers for gfp gene. It was possible to observe the plants´ growth from the tenth day after the infection, being that some of them already had the phenotype of hairy root. It could be verified that the studied hybrid of eucalyptus is susceptible to genetic transformation via Agrobacterium rhizogenes and that the infection by vacuum infiltration provided a lower mortality rate of the explants and a transformation efficiency of 44%. With these results, we conclude that it is possible to transform roots of the hybrid of E. urophylla using A. rhizogenes.