Application of CRISPR/Cas9 System for Modification of Flower Colors in Tobacco and Torenia Plants

Genome modification technologies such as ZFN (Zinc Finger Nuclease), TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR/Cas9 technology have been successfully applied to improve various crop traits. Among them, CRISPR/Cas9 system is now conveniently available as a precise and efficient genome engineering tool. Targeted mutagenesis using CRISPR/Cas9 system is especially useful for plant breeding and gene functional analysis. 

In this study, we applied the CRISPR/Cas9 system to modify flower colors in higher plants using tobacco (Nicotiana tabacum cv. SR1) and torenia (Torenia fournieri) as models. Binary vectors harboring expression cassettes of Cas9 nuclease, single guide RNA (sgRNA) targeting the flavanone 3-hydroxylase (F3H) and nptII as a selectable marker gene were constructed and used for production of transgenic tobacco and torenia plants via Agrobacterium-mediated transformation. Targeted mutations were analyzed in kanamycin-resistant shoots by sequencing of the F3H amplicons (PCR products). Transgenic tobacco and torenia plants clearly contained DNA mutations such as nucleotide substitutions, insertions and deletions at the target site, which varied depending on the transgenic lines. Flower colors of some transgenic lines turned pale or white. Interestingly, some lines displayed variegated flowers. Data describing relationship between flower-color phenotypes and mutations in these transgenic plants will be presented.