Efficient double-flowered gentian plant production using the CRISPR/Cas9 system

Japanese cultivated gentians are highly valued ornamental flowers in Japan, but the flower shape is mostly limited to single-flower type, unlike other such as roses and carnations. To overcome this limitation, we used CRISPR/Cas9 genome editing system increase double-flowered genetic resources gentians. Our approach targeted an AGAMOUS (AG) floral homeotic gene (AG1), which responsible for natural mutation that causes double We designed two targets exon 1 of AG1 found 9 12 herbicide-resistant shoots had biallelic mutations target regions AG1. These nine lines all produced flowers, with stamens converted into petaloid organs, similar mutant. also analyzed off-target effects AG2, homologous AG1, occurred gentian low frequency. Furthermore, successfully transgene-free genome-edited plants (null segregants) by crossing wild-type pollen. F1 seedlings were subjected PCR analysis determine whether foreign DNA sequences, partial CaMV35S promoter Cas9 gene, present genome. As a result, genes segregated at : ratio, indicating successful null segregant production. Using analysis, confirmed four representative segregants did not contain transfer DNA. In summary, our study demonstrates can efficiently produce gentians, be obtained. valuable future breeding programs.