CRISPR/Cas9-mediated Targeted Mutagenesis of Inulin Biosynthesis in Rubber Dandelion

Rubber dandelion (Taraxacum kok-saghyz) is a natural rubber-producing dandelion that has the potential to become an industrial crop. Inulin is a storage carbohydrate in rubber dandelion, and its synthesis competes with rubber production for assimilated carbon. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system to simultaneously target two sites in the gene that encodes 1-fructan: fructan-1-fructosyl transferase gene (1-FFT), a key enzyme in inulin biosynthesis. Agrobacterium rhizogenes and Agrobac-terium tumefaciens-mediated plant transformation methods were used to generate transgenic plants with CRISPR/Cas9 elements. Transformation rates were 71% and 64% via A. rhizogenes and A. tumefaciens-mediated transformations, re-spectively. Mutagenesis was confirmed by the loss of restriction site method and Sanger sequencing. Of 13 transgenic plants obtained via A. rhizogenes, six showed editing of both target sites within the 1-FFT gene. Transgenic rubber dan-delion plants were obtained within 10 weeks using A. rhizogenes-mediated transformation, which was much faster than the 6 months required for A. tumafaciens transformants. Of 11 transgenic plants obtained via A. tumefaciens, five showed mutations in both target sites. Reverse-transcription polymerase chain reaction confirmed Cas9 expression in all edited transformants. Both A. rhizogenes-mediated double-mutant transformants and A. tumefaciens-mediated double mutant transformants had lower levels of inulin than wild-type plants. Moreover, A. rhizogenes-mediated transformants had a higher rubber content than wild-type plants. Therefore, the present study validates the use of CRISPR/Cas9 gene editing as an efficient tool for the generation of useful mutations in rubber dandelion and could be implemented in future crop improvement approaches.