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

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.