Publicação
Molecular pharming in Lactuca sativa L. (Lettuce): development of protocols
| Resumo: | Agrobacterium tumefaciens- mediated genetic transformation is an indispensable tool used for the production of genetically modified plants. In our laboratory, we have established a transformation system for lettuce (Lactuca sativa L.) cultivars Batávia Blonde, Butterhead and Queen of May, using Agrobacterium strain EHA 105. Five-day-old mature cotyledon explants was used for transformation study. The selection of transformed shoots was carried out in MS medium fortified with BA (0.1 mg L), NAA (0.1 mg/L) and under the selection of Kanamycin sulphate (50 to BB and 100mg/L to BH and QM) and Ticarcillin (250 mg/L). The transient GUS expression assay was carried out in order to find transformed shoots and to asses the influence of the genotype in the infection process. No differences were found between varieties in the T-DNA transfer rate (100%), but it was observed different behaviours during the regeneration process. Thus, it was shown that the genotype influences the response of different varieties during regeneration but not influences the predisposition to infection by A. tumefaciens. The molecular confirmation by PCR and Southern blot of transformed shoots revealed the foreign gene integration into lettuce genome. The reporter gene show a segregation pattern of Mendel in lettuce, since the generations T1 and T2 had a segregation ration of 3:1. It was proven that the gene gusA has no influence in the phenolic profile of the plants, since no differences were found in the type of compounds produced between transgenic plants and non-transgenic. Having established the protocol for infection of L. sativa L., in an attempt to demonstrate the applicability of this method, it was constructed an expression vector for an animal protein, leukaemia inhibitor factor (LIF), wich has not yet been expressed in plants. The construction of this vector will allow the expression of LIF in plants like lettuce and future studies on LIF yield production and purification in systems like plants. In this way will be possible to compare the different methods to produce LIF and select the ideal. |
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| Autores principais: | Oliveira, Ana Lúcia da Silva |
| Ano: | 2009 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Agrobacterium tumefaciens- mediated genetic transformation is an indispensable tool used for the production of genetically modified plants. In our laboratory, we have established a transformation system for lettuce (Lactuca sativa L.) cultivars Batávia Blonde, Butterhead and Queen of May, using Agrobacterium strain EHA 105. Five-day-old mature cotyledon explants was used for transformation study. The selection of transformed shoots was carried out in MS medium fortified with BA (0.1 mg L), NAA (0.1 mg/L) and under the selection of Kanamycin sulphate (50 to BB and 100mg/L to BH and QM) and Ticarcillin (250 mg/L). The transient GUS expression assay was carried out in order to find transformed shoots and to asses the influence of the genotype in the infection process. No differences were found between varieties in the T-DNA transfer rate (100%), but it was observed different behaviours during the regeneration process. Thus, it was shown that the genotype influences the response of different varieties during regeneration but not influences the predisposition to infection by A. tumefaciens. The molecular confirmation by PCR and Southern blot of transformed shoots revealed the foreign gene integration into lettuce genome. The reporter gene show a segregation pattern of Mendel in lettuce, since the generations T1 and T2 had a segregation ration of 3:1. It was proven that the gene gusA has no influence in the phenolic profile of the plants, since no differences were found in the type of compounds produced between transgenic plants and non-transgenic. Having established the protocol for infection of L. sativa L., in an attempt to demonstrate the applicability of this method, it was constructed an expression vector for an animal protein, leukaemia inhibitor factor (LIF), wich has not yet been expressed in plants. The construction of this vector will allow the expression of LIF in plants like lettuce and future studies on LIF yield production and purification in systems like plants. In this way will be possible to compare the different methods to produce LIF and select the ideal. |
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