CRISPR/Cas9 technology is a versatile tool for targeted mutagenesis in many organisms, including plants. However, this technique has not been applied to the Japanese morning glory (Ipomoea [Pharbitis] nil), a traditional garden plant chosen for the National BioResource Project in Japan. We selected dihydroflavonol-4-reductase-B (DFR-B) of I. nil, encoding an anthocyanin biosynthesis enzyme, as the target gene, and changes in the stem colour were observed during the early stages of plant tissue culture by Rhizobium [Agrobacterium]-mediated transformation. Twenty-four of the 32 (75%) transgenic plants bore anthocyanin-less white flowers with bi-allelic mutations at the Cas9 cleavage site in DFR-B, exhibiting a single base insertion or deletions of more than two bases. Thus, these results demonstrate that CRISPR/Cas9 technology enables the exploration of gene functions in this model horticultural plant. To our knowledge, this report is the first concerning flower colour changes in higher plants using CRISPR/Cas9 technology.

Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.

No abstract

Japanese morning glory, Ipomoea nil, has several coloured flowers except yellow, because it can accumulate only trace amounts of carotenoids in the petal. To make the petal yellow with carotenoids, we introduced five carotenogenic genes (geranylgeranyl pyrophosphate synthase, phytoene synthase, lycopene β-cyclase and β-ring hydroxylase from Ipomoea obscura var. lutea and bacterial phytoene desaturase from Pantoea ananatis) to white-flowered I. nil cv. AK77 with a petal-specific promoter by Rhizobium (Agrobacterium)-mediated transformation method. We succeeded to produce transgenic plants overexpressing carotenogenic genes. In the petal of the transgenic plants, mRNA levels of the carotenogenic genes were 10 to 1,000 times higher than those of non-transgenic control. The petal colour did not change visually; however, carotenoid concentration in the petal was increased up to about ten-fold relative to non-transgenic control. Moreover, the components of carotenoids in the petal were diversified, in particular, several β-carotene derivatives, such as zeaxanthin and neoxanthin, were newly synthesized. This is the first report, to our knowledge, of changing the component and increasing the amount of carotenoid in petals that lack ability to biosynthesize carotenoids.