HEAT-SHOCK REGULATED EXCISION OF THE NPTII MARKER GENE IN TRANSGENIC APPLE (MALUS × DOMESTICA BORKH.)
Apple (Malus × domestica Borkh.) breeding is time consuming and expensive. Genetic engineering seems to be useful to overcome the limitations of classical breeding. Selected genes/traits can be introduced into the genome of existing cultivars via Agrobacterium-mediated plant transformation. The final product will be a cultivar with improved traits which is already established on the market. The transfer of genes from a crossable species into existing cultivars followed by the elimination of all unwanted DNA sequences (e.g., selectable marker gene) from the final product is expected to be more acceptable for consumers than classical transgenic plants. In the present study, a method to develop marker-free genetically- modified plants in apple was established. The method is based on a site-specific excision of the nptII marker gene which is mediated by heat-shock inducible expression of the FLP recombinase driven by the soybean GmHsp17.5-E promoter. A monitoring vector containing the nptII and the flp genes in a box flanked by two direct repeated FRT recognition sites was developed. The FRT-flanked box separates the gusA reporter gene from the CaMV 35S promoter. GUS expression is only expected after excision of the FRT-flanked box. Using this vector, transgenic lines were produced and investigated by PCR, RT-PCR and Southern hybridization. Different temperature regimes were tested and after exposure of transgenic shoots to 42°C for 4 hours a high number of recombination events were detectable. Subsequent shoot regeneration from leaf explants was necessary to obtain transgenic apple plants completely free of nptII. To avoid the additional shoot regeneration cycle, we tested three different induction methods on leaves or leaf explants, respectively. Furthermore, first results of additional experiments aimed on improving the system will be presented.
Würdig, J., Herzog, K., Flachowsky, H. and Hanke, M.-V. (2013). HEAT-SHOCK REGULATED EXCISION OF THE NPTII MARKER GENE IN TRANSGENIC APPLE (MALUS × DOMESTICA BORKH.) . Acta Hortic. 976, 439-444
transgene technology, marker-free, site-specific recombination