Development of a simple multiple mutation detection system using seed-coat flavonoid pigments in irradiated Arabidopsis M(1) plants

Ionizing radiation induces genetic variations in plants, which makes it useful for plant breeding. A theory that the induced mutations occur randomly in the genome has long been accepted, but is now controversial. Nevertheless, a comparative analysis of the mutations at multiple loci has not been conducted using irradiated M(1) genomes that contain all types of mutations. In this study, we identified Arabidopsis mutants (pab2 and pab3) in a mutagenized population of an anthocyanin-positive seed mutant (ban). Both pab2 and pab3 were revealed to be double mutants (tt4 ban and tt8 ban, respectively) that produced similar anthocyanin-less immature seeds, but differentially colored mature seeds. These features enabled the seed color-based detection of de novo M(1) mutations in TT4 or TT8 following the irradiation of double heterozygous plants (TT4/tt4 TT8/tt8 ban/ban). Most of the irradiated double heterozygous plants produced anthocyanin-positive immature seeds, but 19 plants produced anthocyanin-less immature seeds. Of these 19 mutants, 2 and 17 exhibited tt4- and tt8-type mature seed coloration, respectively. The molecular analysis of the seed coat DNA from randomly selected anthocyanin-less seeds detected mutations at the locus predicted on the basis of the phenotype. Thus, the simple system developed in this study can reliably detect radiation-induced mutations at multiple loci in irradiated Arabidopsis M(1) plants.