Application of the CRISPR/Cas9 system for modification of flower color in Torenia fournieri

Abstract

Background: CRISPR/Cas9 technology is one of the most powerful and useful tools for genome editing in various living organisms. In higher plants, the system has been widely exploited not only for basic research, such as gene functional analysis, but also for applied research such as crop breeding. Although the CRISPR/Cas9 system has been used to induce mutations in genes involved in various plant developmental processes, few studies have been performed to modify the color of ornamental flowers. We therefore attempted to use this system to modify flower color in the model plant torenia (Torenia fournieri L.).

Results: We attempted to induce mutations in the torenia flavanone 3-hydroxylase (F3H) gene, which encodes a key enzyme involved in flavonoid biosynthesis. Application of the CRISPR/Cas9 system successfully generated pale blue (almost white) flowers at a high frequency (ca. 80% of regenerated lines) in transgenic torenia T₀ plants. Sequence analysis of PCR amplicons by Sanger and next-generation sequencing revealed the occurrence of mutations such as base substitutions and insertions/deletions in the F3H target sequence, thus indicating that the obtained phenotype was induced by the targeted mutagenesis of the endogenous F3H gene.

Conclusions: These results clearly demonstrate that flower color modification by genome editing with the CRISPR/Cas9 system is easily and efficiently achievable. Our findings further indicate that this system may be useful for future research on flower pigmentation and/or functional analyses of additional genes in torenia.

Keywords: CRISPR/Cas9; Flavanone 3-hydroxylase; Flower color; Genome editing; Torenia fournieri.

Fig. 1

Schematic diagram of the binary vector and the torenia target sequence in the torenia F3H gene. a Schematic diagram of the T-DNA region of pSKAN-pcoCas9-TfF3H used in this study. NPTII, expression cassette of NOSp-nptII-AtrbcsTer; 35Sp, CaMV35S promoter; pcoCas9, plant codon-optimized Cas9 [35]; HSPter, Arabidopsis heat shock protein 18.2 terminator [36]; RB, right border; LB, left border; AtU6p, Arabidopsis small RNA U6–26 promoter; TfF3HsgRNA, torenia F3H targeted single-guide RNA. pcoCas9 contains an intron derived from the intervening sequence 2 (IV2) of the potato St-LS1 gene [35]. b Genomic structure of the torenia F3H gene and exon 1 sequence. Boxes indicate exons, and lines between boxes indicate introns. Framed ATG indicates the start codon, and the gray box indicates the target site F3H sequence. The protospacer-adjacent motif (PAM) is underlined. Primers used for PCR amplification are also shown

Fig. 2

In vitro flowering phenotypes of transgenic torenia plants. Photographs were taken 3 to 8 months after inoculation with Agrobacterium. Numbers indicate transgenic plant lines. Line no. 15 had different-colored flowers and was divided into 15A and 15B

Fig. 3

Flowers of transgenic genome-edited torenia plants cultivated in a closed greenhouse and results of pigment analysis. a Four biallelic transgenic genome-edited torenia plants were grown in a closed greenhouse. Pictures of typical flowers are shown. CrV and CrW indicate cultivars Crown Violet and Crown White, respectively. CrV was the host plant for transformation, and CrW was a white cultivar for comparison. b Pigment analysis by spectrophotometry. Left panel, absorbance spectra of 0.1% HCl–methanol extracts of flower petals (line no. 5, CrV and CrW) are shown in the left panel. Right panel, relative anthocyanin contents of petals of transgenic genome-edited torenia plants and an untransformed control plant (CrV) were determined. Values indicate averages of five flower petals ± standard deviation