# Mutagenesis and production of double-flowered gentians via regeneration from ion beam-irradiated leaves

## TL;DR

Researchers developed a new method to create double-flowered gentians using ion beam mutagenesis and plant regeneration.

## Contribution

A novel regeneration-mediated ion beam mutagenesis method was developed for gentian breeding.

## Key findings

- Two double-flowered gentian lines were obtained from neon ion beam irradiation.
- The G. scabra AG1 gene was deleted in the double-flowered line Ne9Gy#34.
- Ne9Gy#34 also showed increased flower size, suggesting additional mutations.

## Abstract

Gentians are important ornamental plants, and gentian cultivars have been actively bred for decades. However, limited genetic resources are currently available for breeding; therefore, artificial mutagenesis has been applied to generate mutants. In this study, we developed a simple and efficient regeneration-mediated method for ion beam mutagenesis in the Japanese gentian hybrid cultivar ‘Albireo’ (Gentiana scabra × G. triflora). Carbon and neon ion species were tested. Effect of ion beam irradiation on callus formation from leaves was initially evaluated. Tissue culture was then continued, adventitious shoots were induced from calli, and many regenerated plants were obtained. These plants were cultivated until flowering, and two cultivated lines exhibiting a double-flowered phenotype were identified from leaves exposed to 9 and 12 Gy of neon ion beam irradiation among approximately 200 individuals. We analyzed one line derived from irradiation with 9 Gy of neon ions, named Ne9Gy#34, in detail. The agamous gene (AG1), previously identified as the gene responsible for the double-flower phenotype in gentians, was not amplified in the G. scabra allele by genomic polymerase chain reaction. Moreover, next-generation sequencing also indicated that the reads were mapped to the genomic region of the G. triflora AG1 but not to that of G. scabra, suggesting that the deletion of G. scabra AG1 led to the double-flowered phenotype. Ne9Gy#34 also exhibited increased flower size, suggesting additional mutations in genes other than AG1. In summary, the developed regeneration-mediated method represents a promising approach for inducing gentian mutagenesis and efficiently producing novel traits in this plant.