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Establishing a transient technology for CRISPR/Cas9-based genome editing in apple
Project
Project code: JKI-ZGO-08-4203
Contract period: 01.07.2018
- 30.06.2021
Purpose of research: Project preparation activity
Apple is one of the most economically important fruit crops worldwide. Climate change and the occurrence of new pathogens require that breeding of new and better adapted cultivars is inevitable. Classical breeding is time-consuming and existing problems cannot be solved within a manageable amount of time. However, with the cutting-edge CRISPR/Cas9 genome editing (GE) technology, it has become feasible to improve selected traits in well established cultivars either by mutagenesis or by insertion or replacement of selected genes. First CRISPR/Cas applications were recently established in apple. These GE applications were successful but useless for practical breeding, because transgenic CRISPR/Cas sequences remained in the apple genome. Apple is cross-pollinated and heterozygous. A removal of CRISPR/Cas sequences by sexual recombination will lead to numerous recombination events resulting in the loss of the cultivar specific characteristics. To avoid this problem, the project will focus on the establishment of a method for transient expression of CRISPR/Cas sequences in apple. Therefore different methods will be tested which are either based on the use of cell penetrating peptides or on transient transformation using Agrobacterium or particle bombardment. The most efficient method will then be used in two proof-of-concept experiments. The first experiment focuses on a direct knock-out mutation of the MdPDS gene leading to a visible chlorophyll defect. The second experiment focuses on the exchange of a promoter element in the MdMYB10 promoter which induces red coloration of all vegetative tissues. Resulting GE events will be evaluated for the occurrence of OFF target effects. As a result, a method for GE applications will be established which can be used for directed mutagenesis as well as for the exchange and integration of selected DNA sequences.
Section overview
Subjects
- Plant Breeding
- Process engineering
