Efficient genetic transformation and CRISPR/Cas9-mediated genome editing in Lemna aequinoctialis

The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium-mediated transformation to 5-6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9-mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9-mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.