Fig. 1
Reproduction of carotenoid-accumulating phenotype of cauliflower Or mutation in rice callus. a Target sequence in the Osor gene of each sgRNA for mimicking aberrant or transcripts expressed in the cauliflower Or mutation. Black and white boxes indicate exons and untranslated regions of the Osor gene, respectively. Sequences of exons are highlighted in gray; 20-nt target sequences are bold and underlined; 3-nt sequence indicating the protospacer adjacent motif (PAM) are boxed in red. b Photographs of hygromycin-resistant calli after 1-month of propagation. Upper and lower panels: calli from GFP and Osor_t3 lines, respectively. White bar = 5 cm. c Mutation patterns of Osor_t3 sgRNA loci. Sequences of 3rd exon and 3rd intron are presented in black and blue, respectively. Target DNA sequence of Osor_t3 sgRNA is shown in WT and underlined at the top of the aligned sequences. A black arrow indicates the expected digestion site by Cas9 with Osor_t3 sgRNA. The PAM sequence is indicated in green and bold. Deletions are indicated as dashes; insertions are in red. Indel size and number of clones are shown on the right (+, insertion; −, deletion; x, number of clones). d RT-PCR analysis of OsOr transcript in callus lines possessing mutations at Osor_t3 loci. Primers were designed to amplify the region from the 3rd exon to the 5th exon in the Osor gene. Expected lengths of PCR products amplified from cDNA or genomic DNA of Osor are 233 and 457 bp, respectively. N, non-transgenic plant. e Composition of in-frame or out-of-frame transcripts in each orange or regular-colored callus line, indicating the percentage of in-frame or out-of-frame transcripts in total sequenced clones shown in Additional file 2: Figure S10 and Additional file 3: Table S1. f Measurement of β-carotene content in orange callus. Left side: HPLC chromatograms of callus extracts. Right side: absorption spectra of the major peak in each chromatogram. V and L in each chromatogram indicate the expected retention times of violaxanthin and lutein, respectively. Materials and methods are listed in Additional files 1 and 3.