Figure 4
From: CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)
Serial observation of somatic embryogenesis and regeneration from embryogenic tissue (ET) line #42, exhibiting patchy GFP fluorescence under visible light (a–e) and blue light (f–j), in which pZK_FFCas9 vector containing the gRNA of GFP target #1 driven by CjU6_#2 promoter was introduced. (a,f) Day 0, ET line #42 in the somatic embryo maturation medium. Bars, 5 mm. (b,g) Day 39, induction of cotyledonary somatic embryos in the same medium. GFP fluorescence is partially quenched in the cell mass. Bars, 5 mm. (c,h) Higher magnifications of the framed boxes in (b) and (g), respectively. White arrows indicate GFP-knock-out embryos. Red arrowheads indicate embryos with active GFP. Bars, 1 mm. (d,e,i,j) Germination of cotyledonary somatic embryos at 7 days after culture on germination medium. (d,i) Embryo with active GFP. Bars, 2 mm. (e,j) GFP-knock-out embryo. Bars, 2 mm. (k) Genotype of regenerated somatic cotyledonary embryos from selected ET lines. The target region and PAM sequence are highlighted in blue and green, respectively. A deletion is denoted by “−”. Letters in red and lower-case pink indicate inserted and replaced bases, respectively. d#, i#, and r# denote the number of bp deleted, inserted, and replaced at the target site, respectively. We extracted genomic DNAs from the whole embryos and determined the nucleotide sequences by direct sequencing.
