Academic Centre
Improving adaptability and resilience of perennial ryegrass for safe and sustainable food systems through CRISPR-Cas9 technology – EditGrass4Food, ID No. EEA-RESEARCH-64, Contract No. EEZ/BPP/VIAA/2021/4 is one of the Baltic Research Programme's projects, which is financially supported by the European Economic Area (EEA) grants. The project is developed in cooperation with University of Latvia as Promoter, Norwegian University of Life Sciences, Lithuanian Research Centre for Agriculture and Forestry, and Tallinn University of Technology.
During the recent project partner meeting in Riga March 30 – 31 it was concluded that a significant progress was made in WP1, WP2 and WP3. In particular, plant in vitro material was established for further transformation applications. Callogenesis has been achieved in all L.perenne target lines of the project and rapidly propagating callus cultures are being maintained. Sucessfull protoplast culture establishment protocol has been developed and protoplast cultures with protoplast density relevant for further transformation applications (> 10^5/ml) are being achieved on a regular basis. Application of rigorous antibiotic treatment for in vitro tiller propagation has allowed for elimination of bacterial contamination however endophytic fungal contaminants are still precluding further application of in vitro tiller cultures for the transformation. Experiments with cycloheximide application aiming at full eradication of microbial contamination from L.perenne tiller cultures are currently under progress.
In WP1 and WP3 several drought and freezing-tolerance genes have been selected for CRISPR/Cas9 knock-out generation in L.perenne based on literature search. Target genes will be sequenced in April from a selection of drought and freezing tolerant genotypes using PacBio HiFi long-read technology which allows to resolve haplotypes. Single gRNA plasmid constructs for three target genes have been developed and multiple gRNA plasmid construct development is in progress. The most efficient gRNAs for each target gene will be selected based on genome editing efficiency in protoplast cultures before application in callus transformation.
In WP2 Freezing tolerance evaluation experiments have been performed on a set of target lines representing the wide spectrum tolerance. RNA has been isolated from the plant material subjected to the freezing conditions and RNA sequencing will be used to quantify changes in the gene expression during the stress. Drought tolerance experiment is sceduled on April 2023 and will also include evaluation of several physiological parameters as well as collection of RNA for further sequencing.
The next project online meeting will take place in June 2023, while the next face-to-face meeting and public event will take place in Lithuania in October 2023.
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