Negative Selection Marker

Comparable with screenable marker genes, so-called negative selection markers are used to optimize transformation efficiency. Thereby negative selection systems kill the transformed cells. This allows new strategies to limit the production of vector-backbone-containing plants by flanking the T-DNA with negative selection marker genes. The most used negative selection marker gene is the codA gene from E. coli encoding cytosine deaminase. The usefulness of codA as a conditional toxic gene was explored in different Agrobacterium-mediated transformation protocols (Koprek et al. 1995; Schlaman 1997). Plant cells which are transgenic for codA show sensitivity to 5-fluorocytosine (5-FC) at different developmental stages. The negative selection marker confers a lethal phenotype on the transformant and is therefore often part of co-transformation systems. Co-transformation with codA is a viable method for the production of easily distinguished, selectable marker genefree transgenic plants (Park et al. 2004). As described by Verweire et al. (2007) the cytosine deaminase gene can be used as a counter-selectable marker. In this system CodA is a component of a germline-specific auto-excision vector, in which codA is present in tandem with the recombinase and the positive selection marker between lox sites. After auto-excision of the whole Lox-cassette, marker-free regenerates can be identified by growing on medium containing 5-fluorocytosine.

Often mentioned in literature concerning marker genes are the tms2 and the dao1 gene as negative selection marker genes (Upadhyaya et al. 2000; Erikson et al. 2004). The marker gene, dao1, encoding D-amino acid oxidase (DAAO) can be used for either positive or negative selection, depending on the substrate. D-Alanine and D-serine are toxic to plants, but are metabolized by DAAO into non-toxic products, whereas D-isoleucine and D-valine have low toxicity but are metabolized by DAAO into the toxic products respectively. Hence, both positive and negative selection is possible with the same marker gene.

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