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Improving oilseed rape by Cas9-mediated genome editing (OilCas)
Project
Project code: 031B0533
Contract period: 01.07.2018
- 30.06.2020
Budget: 304,177 Euro
Purpose of research: Experimental development
Keywords: Cas9-mediated mutagenesis, TILLING, rapeseed, glucosinolate metabolism, risk assessment
Genome editing by Cas9-mediated mutagenesis has been successfully demonstrated in a number of crop plants including oilseed rape. Commonly, the constructs are stably integrated into the nuclear genome after transformation. Alternatively, innovative genome editing techniques without transgene insertions can be used. Furthermore, many transformation protocols include a tissue culture phase, which is often a time-consuming bottleneck and which needs a lot of expertise, thus limiting high-throughput applications. Consequently, we are aiming at the establishment of a transient sonication-assisted Agrobacterium tumefaciens-mediated transformation technique which could be applied to rapeseed seedlings. Resulting plants will not carry transgenes and regeneration from tissue culture can be omitted. It is to be expected that the protocol can be applied to a broad range of genotypes and will thus strongly contribute to future research and breeding programs. Following up a previous project, we are in the unique position of having mutant plants from both a random EMS mutagenesis experiment (TILLING) and from targeted Cas9 mutagenesis at hand. Using Cas9-mediated gene replacement, we will generate plants with identical mutations to our TILLING mutants. This allows us to assess the general plant fitness of both mutant types. The findings will highlight the advantages of genome editing and contribute to risk assessment of mutant plants. Rapeseed (Brassica napus, 2n = 38, AACC) is a polyploid crop which resulted from the natural hybridization of the progenitors Brassica rapa (2n = 20, AA) and Brassica oleracea (2n = 18, CC). This implies the presence of at least two homoeologous genes for each gene of the model plant Arabidopsis thaliana. In practice, six rapeseed homoeologs with potentially redundant functions are common due to ancient genome duplication events in the evolution of Brassica species. While this drastically complicates rapeseed breeding, multiplexed genome editing can facilitate the introduction of multiple mutations within a single generation. We have successfully applied multi-paralog targeting in rapeseed (Braatz et al. 2017). Now, we will further develop a multiplexing strategy for targeted mutagenesis of rapeseed based on a polycistronic tRNA-gRNA technique. By knocking out redundant genes in parallel, we will produce rapeseed with an increased number of seed chambers, which has a potential to increase seed yield. At the same time, we will target the glucosinolate biosynthesis pathway to reduce anti-nutritive compounds in rapeseed products. By combining innovative genome editing techniques with aiming at phenotypes that are valuable for breeding, we provide promising perspectives for future applications.
Section overview
Subjects
- Plant Breeding
- Process engineering
