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. SLU, Dept. Of Plant Breeding, Alnarp. Alnarp: SLU, Dept. Of Plant Breeding
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Abstract
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.
Main 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 |
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Authors: | Larsson, Sara J. |
Supervisor: | Zhu, Li-Hua |
Examiner: | UNSPECIFIED |
Series: | Bachelor project in the Horticultural Science programme |
Volume/Sequential designation: | 2007:6 |
Year of Publication: | 2007 |
Level and depth descriptor: | Other |
Student's programme affiliation: | YTRIN Horticultural Management Programme (admitted 2005-2006) 120 HEC |
Supervising department: | (NL, NJ) > Dept. Of Plant Breeding |
Keywords: | dao1, M26, Agrobacterium tumefaciens, transformation, selection, selective marker, antibiotics |
URN:NBN: | urn:nbn:se:slu:epsilon-s-8258 |
Permanent URL: | http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-s-8258 |
Subject. Use of subject categories until 2023-04-30.: | SLU > (LTJ, LTV) > Plant Breeding and Biotechnology (until 121231) Plant genetics and breeding |
Language: | English |
Deposited On: | 25 Oct 2017 07:42 |
Metadata Last Modified: | 25 Oct 2017 07:42 |
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