A robust genome-editing method for wild plant speciesNicotiana attenuata

CRISPR genome-editing techniques theoretically enable us to edit any genes in any plants. However, plant tissue culture is required for generating targeted mutants in plants, except in some model plant species such asArabidopsis thaliana. To modify ecologically or agronomically important traits in plants using the CRISPR system, a robust plant regeneration method and efficient genome-editing tools must be developed. This study shows the entire process of genome editing and the regeneration process for wild tobacco,Nicotiana attenuata. We delivered T-DNA harboringStreptococcus pyogenesCas9 (SpCas9) and guide RNA (gRNA) viaAgrobacterium-mediated transformation into the hypocotyl cells of the wild tobacco and regenerated gene-edited plants. The efficacy of genome editing was measured inN. attenuataprotoplasts in which SpCas9 and gRNA were transiently expressed. Light intensity (476.66-627.00 mu W/cm(2), 20.69-52.21 mu E) was optimized to enhance the emergence of plant shoots during callus induction, and the core step of dealing with plant tissues was recorded. In addition, we found that wounding the bottom part of mature plants was critical for root regeneration. By tracking mutation patterns and efficiency at each regeneration step, we found that the mutation was induced early on in the tissue culture process and maintained throughout the regeneration process. Genome-editing techniques have opened the way to study the function of the genes in all plants, and this study will provide guidelines for editing a gene in the plant of interest.