Institut für Züchtungsforschung an Obst
An impact of the advancing climate change can already be observed in the earlier flowering time of apples. The resulting increased risk of damage to the flower buds caused by spring frosts has a negative effect on yield stability and fruit quality. The duration of bud dormancy is crucial for the flowering time. In apple, several candidate genes have already been identified that are thought to play a role in the regulation of dormancy and bud break, including several homologs of the Dormancy Associated MADS-Box (DAM) gene family. Knowledge of the exact function of the individual genes involved in flowering time regulation is still very limited and represents an important basis for the future development of apple cultivars with climate-adapted flowering behavior. For functional gene characterization, the project will use a targeted mutagenesis approach to generate apple lines that are characterized by a defect in the respective gene involved in flowering time regulation. In a first step, the genomic target loci will be sequenced to characterize them more precisely. This will allow the design of specific guide RNAs to induce targeted double-strand breaks in the respective target gene using a CRISPR/Cas nuclease system. Mutagenesis constructs based on binary T-DNA plasmids will be cloned and subsequently used for stable transformation of apple. After multiple regeneration steps of the transformed plant material on selection medium, the obtained in vitro shoots will be screened for the presence of insertion or deletion mutations by fragment length analysis. The potential mutant lines will be characterized in detail using various molecular methods such as PCR analysis, gene expression analysis and, next-generation sequencing methods. These studies will serve to select stable knock-out mutant lines that will subsequently be available for experimental approaches in reverse genetics and provide a basis for studying the gene function.
Federal Ministry of Food and Agriculture
