Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium
The aim of this project was to evaluate the possibility to use D-alanine as a selective marker instead of commonly used antibiotics and herbicides in transformation of apple rootstock M26. The transformation was carried out using Agrobacterium tumefaciens strain C58C1(pGV3101) containing the vec...
| Autor principal: | |
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| Formato: | Otro |
| Lenguaje: | sueco Inglés |
| Publicado: |
2008
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| Materias: | |
| Acceso en línea: | https://stud.epsilon.slu.se/12562/ |
| _version_ | 1855572089193365504 |
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| author | Larsson, Sara J. |
| author_browse | Larsson, Sara J. |
| author_facet | Larsson, Sara J. |
| author_sort | Larsson, Sara J. |
| collection | Epsilon Archive for Student Projects |
| description | The aim of this project was to evaluate the possibility to use D-alanine as a selective marker instead of commonly used antibiotics and herbicides in transformation of apple rootstock M26.
The transformation was carried out using Agrobacterium tumefaciens strain C58C1(pGV3101) containing the vector pPCV70:dao1. The vector contained the dao1 gene resistant to D-amino acids and the nptII gene resistant to antibiotic, kanamycin. The dao1 encodes for the enzyme D-amino-acid oxidase (DAAO). The enzyme detoxifies D-amino acids, such as, D-alanine, which are harmful to plants.
The transformed explants were grown on medium containing increased concentrations of D-alanine, with a starting concentration of 1.2 mM. Eight putative transgenic clones were obtained from the selection medium. However, further molecular analyses have to be carried out to confirm the result.
A second experiment was performed to transform the GA20 oxidase gene into A. tumefaciens to get a ready-to-use transformation vector. The vector containing the GA20 oxidase gene was transformed into the A. tumefaciens strain C58C1(pGV3850). DNA from the strain was isolated and digested. The result was visualized on an agarose gel under UV light. The conclusion of the experiment is that the vector containing GA20 oxidase gene has been transformed into the strain C58C1. |
| format | Otro |
| id | RepoSLU12562 |
| institution | Swedish University of Agricultural Sciences |
| language | Swedish Inglés |
| publishDate | 2008 |
| publishDateSort | 2008 |
| record_format | eprints |
| spelling | RepoSLU125622017-10-25T07:42:47Z https://stud.epsilon.slu.se/12562/ Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium Larsson, Sara J. Plant Breeding and Biotechnology (until 121231) Plant genetics and breeding The aim of this project was to evaluate the possibility to use D-alanine as a selective marker instead of commonly used antibiotics and herbicides in transformation of apple rootstock M26. The transformation was carried out using Agrobacterium tumefaciens strain C58C1(pGV3101) containing the vector pPCV70:dao1. The vector contained the dao1 gene resistant to D-amino acids and the nptII gene resistant to antibiotic, kanamycin. The dao1 encodes for the enzyme D-amino-acid oxidase (DAAO). The enzyme detoxifies D-amino acids, such as, D-alanine, which are harmful to plants. The transformed explants were grown on medium containing increased concentrations of D-alanine, with a starting concentration of 1.2 mM. Eight putative transgenic clones were obtained from the selection medium. However, further molecular analyses have to be carried out to confirm the result. A second experiment was performed to transform the GA20 oxidase gene into A. tumefaciens to get a ready-to-use transformation vector. The vector containing the GA20 oxidase gene was transformed into the A. tumefaciens strain C58C1(pGV3850). DNA from the strain was isolated and digested. The result was visualized on an agarose gel under UV light. The conclusion of the experiment is that the vector containing GA20 oxidase gene has been transformed into the strain C58C1. Syftet med detta projekt var att undersöka möjligheten att vid transformering av äpplegrundstammen M26 genomföra selektion med hjälp av D-aminosyra (D-alanin) istället för andra vanligt förekommande selektionsämnen, så som antibiotika och herbicider. Transformeringar med D-alanin som selektionsämne genomfördes, med Agrobacterium tumefaciens stammen C58C1(pGV3101) innehållande vektorn pPCV70:dao1. Vektorn innehöll även genen nptII, som ger de transformerade explantaten resistans mot antibiotika, kanamycin. dao1 genen kodar för enzymet D-amino-acid oxidase (DAAO). Enzymet bryter ner det giftiga ämnet D-alanin till ofarliga ämnen för växten. De transformerade explantaten odlades på medium innehållande ökande koncentration av D-alanin, med utgångskoncentrationen 1.2 mM. Åtta putativa transformerade kloner selekterades på selektionsmediet. Ytterligare molekylära analyser måste dock genomföras för att bekräfta resultaten. Försök genomfördes även för att transformera en vektor innehållande GA20 oxidas genen i A. tumefaciens, för att få fram en vektor färdig för användning vid transformering av växter. Vektorn innehållande GA20 oxidas genen och nptII genen transformerades in i A. tumefaciens stammen C58C1(pGV3850). DNA från A. tumefaciens var sedan isolerad och fragmenterad. Resultatet visualiserades på en agarose gel under UV ljus. Slutsatsen av försöket är att vektorn transformerats in i A. tumefaciens stammen C58C1. 2008-03-06 Other NonPeerReviewed application/pdf sv https://stud.epsilon.slu.se/12562/1/larsson_s_171025.pdf Larsson, Sara J., 2007. Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium. UNSPECIFIED, Alnarp. Alnarp: (NL, NJ) > Dept. Of Plant Breeding <https://stud.epsilon.slu.se/view/divisions/LMNR=3A642.html> urn:nbn:se:slu:epsilon-s-8258 eng |
| spellingShingle | Plant Breeding and Biotechnology (until 121231) Plant genetics and breeding Larsson, Sara J. Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium |
| title | Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium |
| title_full | Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium |
| title_fullStr | Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium |
| title_full_unstemmed | Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium |
| title_short | Evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock M26 as well as transformation of a vector containing GA20 oxidase gene into Agrobacterium |
| title_sort | evaluation of the dao1 gene as a selectable marker in transformation of apple rootstock m26 as well as transformation of a vector containing ga20 oxidase gene into agrobacterium |
| topic | Plant Breeding and Biotechnology (until 121231) Plant genetics and breeding |
| url | https://stud.epsilon.slu.se/12562/ https://stud.epsilon.slu.se/12562/ |