Figure 6.

Flow chart of CRISPR‐based Targeted Mutagenesis in F. hindsii by Agrobacterium‐mediated Transformation. (a) The episperm of the collected seeds was removed, and the seeds were sterilized. (b) The sterilized seeds were cultured on MT medium. (c) The germinated seeds were cultured under darkness for 4 weeks. (d) The seedlings were cultured under a 16 : 8 photoperiod for regreening (10 days). (e) The epicotyl segments were co‐cultured with Agrobacterium tumefaciens under darkness (3 days). The red arrow indicates the cut surfaces. (f) The infected segments were cultured on shoot‐inducing medium under darkness (1 week). The red arrow indicates the cut surfaces that would produce white callus. (g) The shoots were induced from white callus under a 16 : 8 photoperiod (4 weeks). The red arrow indicates the regeneration of shoots from the white callus. (h) The regenerated shoots were cultured on root‐inducing medium under a 16 : 8 photoperiod. The red arrow indicates the cut surface and white callus. (i) The regenerated roots were induced from white callus (8 weeks). The red arrow indicates the regeneration of roots from the white callus. (j) to (k) Seedlings with vigorous roots were cultured in water for acclimatization (1 week) by using paper wick method. (l, m) The transgenic plants were genotyped via HRM analysis and Sanger sequencing. (n) Mature fruits and seed (T₁) of T₀ (about 9 months). Bars = 1 cm.