Etablierung einer Flp/<em>FRT</em>-vermittelten Rekombination des Selektionsmarkers und Untersuchungen zur RNAi-induzierten Resistenzerhöhung gegenüber pilzlichen Schaderregern beim Apfel (<em>Malus x domestica</em> BORKH.)

Abstract

One of the most important goals in genetic engineering is the generation of marker-free transgenic crops. Subsequent to an Agrobacterium tumefaciens-mediated gene transfer, the nptII/kanamycin system is usually applied for the selection of transgenic apple cells (Malus x domestica BORKH.). In October 2002 the European government enacted a Council Directive (2001/18/EG) which prohibits the release of genetically modified (gm) organisms carrying selective markers such as the nptII gene. The establishment of alternative selection strategies is therefor compulsory. Still, until now no comparable system is available to replace the nptII/kanamycin selection system for apple transformation experiments. In the present study, the operability and applicability of the Flp/FRT recombination system in order to remove the nptII gene after the selection of transgenic apple cells was analyzed. The overall aim was the generation of gm apple plants which do not carry the selective marker. A monitoring vector was constructed and transfered into the Agrobacterium strain GV3101pMP90RK. The transgenic strain was then used to inoculate 4,080 in-vitro apple leaves in 17 different apple transformation experiments and nine gm apple lines were finally selected. The functional T-DNA of the vector contains the CaMV 35S promoter and a gusA reporter gene which were spatial separated by a FRT-flanked box. This FRT-flanked box contained the nptII gene and the flp recombinase gene which was regulated by the heat-shock-inducible promoter Gmhsp17.5E from the soybean Glycine max. Consequently, expression of gusA was only expected after elimination of the FRT-flanked box. Each recombination event could be detected by using histochemical GUS Assays. The first obtained transgenic apple line, T670, was used to investigate different time and temperature regimes for the Gmhsp17.5E promoter induction. An effective promoter induction and Flp-mediated recombination was observed after treatment of apple shoots at 42°C. However, the heat-shock-induced recombination did not occur concurrently in all cells, because shoots with chimeric tissues were often detected. To generate fully nptII-free gm apple plants, the leaves of the lines T670, T781, T782 and T793 were sampled after heat-shock-treatment and the explants placed on regeneration medium without the selective agent kanamycin for 16 weeks. Up to forty shoot regenerates were randomly chosen and analyzed using the GUS Assay after regeneration process in order to detect putative nptII-free apple shoots up. Using this method, up to 38 percent of the studied regenerates were identified as GUS positive and putatively nptII-free. In addition, the molecular analysis on genomic DNA and mRNA of selected shoot regenerates of the lines T781 and T782 showed that the regeneration of heat-shock-treated leaves generate fully nptII-free gm apple shoots. To increase the efficiency of Flp/FRT recombination system another monitoring vector was created which contained a second selection system. Using this second selection system, nptII-free apple plants could potentially be identified without the use of gusA. For the long therm the transfer of target genes and the generation of gm apple plants which do not carry antibiotic resistance genes will be possible. Another important aim in genetic engineering of apple breeding programs is the increase of resistance against biotic pathogens. Especially an improved resistance against pathogenic fungi like the apple powdery mildew Podosphaera leucotricha and apple scab Venturia inaequalis is highly requested. Althougt molecular markers exist for different apple resistant loci, nucleotide sequences of involved genes are yet unknown. Hence the use of apple resistance genes is not possible in genetic modification experiments of apple plants. A promising method is the RNAi-mediated gene silencing in pathogenic fungi. In the second part of the present study, experiments were thus conducted, performed in which a hostinduced defense mechanism against fungal pathogens was analyzed. First, we showed the transport of synthetic, Fluorescein-labeled siRNA molecules from plant leaves into the hyphae of P. leucotricha. The treatment of the fungus with a respiration blocker additionally illustrated that absorption of herbal siRNAs into fungal infection structures is probably an ATP-depended process. For an efficient application of RNAi-induced resistance mechanisms in gm apple plants, the silencing of an essential fungal gene is required. We chose the chitinsynthase class V gene as a potential target and started with the gene isolation from P. leucotricha and V. inaequalis. The expression of ChsV genes is essential for cell wall stability and fungal development (AMNUAYKANJANASIN et al. 2003; MADRID et al. 2003, WERNER et al. 2007). Due to these facts the silencing of ChsV may trigger a reduced or inhibited disease development of the apple powdery mildew disease or apple scab.