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

Area of Science:

• Plant genetics
• Molecular biology
• Horticulture

Background:

• Japanese cultivated gentians are prized ornamental flowers but lack diverse flower shapes, primarily exhibiting single-flower types.
• Existing gentian varieties have limited floral diversity compared to other ornamental flowers like roses and carnations.

Purpose of the Study:

• To utilize CRISPR/Cas9 genome editing to develop double-flowered gentian varieties.
• To increase genetic resources for double-flowered gentians by targeting the AGAMOUS (AG) floral homeotic gene.

Main Methods:

• CRISPR/Cas9 genome editing was applied to target the AGAMOUS1 (AG1) gene in gentians.
• Two target sites in exon 1 of AG1 were designed for precise genome modification.
• Off-target effects on the homologous AGAMOUS2 (AG2) gene were analyzed.
• Transgene-free null segregants were produced through crossing and PCR analysis.

Main Results:

• Biallelic mutations in AG1 were achieved in 9 out of 12 herbicide-resistant shoots.
• These mutated lines consistently produced double flowers, with stamens transformed into petaloid organs.
• Low-frequency off-target mutations were observed in the AG2 gene.
• Transgene-free null segregants were successfully generated, with foreign DNA segregating at a 1:1 ratio.

Conclusions:

• The CRISPR/Cas9 system is an efficient tool for producing double-flowered gentians.
• The generation of transgene-free genome-edited gentians provides valuable genetic resources for future breeding programs.
• This study expands the floral diversity of gentians, offering new possibilities for ornamental horticulture.