Construction and genetic analysis of anthocyanin-deficient mutants induced by T-DNA insertion in ‘Tsuda’ turnip (Brassica rapa)

We previously showed that anthocyanin biosynthesis is specifically induced either by UV-A or co-irradiation with blue and UV-B, but not by monochromatic blue or UV-B light in the epidermis of the storage root of ‘Tsuda’ turnip (Brassica rapa L. subsp. rapa). To gain further molecular insights into the light signal transduction pathway of anthocyanin accumulation, over 10,000 germinated seeds of Tsuda turnip were sonicated and transformed using an improved Agrobacterium-mediated vacuum infiltration method. We obtained 17 anthocyanin-rich and 60 anthocyanin-deficient mutant lines from the mutant library. PCR identification, GFP assay and GUS staining showed that six anthocyanin-deficient mutants were generated by T-DNA insertion. Real-time qRT-PCR results demonstrated that expression of structural and regulatory genes of anthocyanin synthesis in the anthocyanin-deficient lines significantly decreased compared with wild type, which coincided with their anthocyanin levels in the epidermis of storage roots. In a genetic analysis of the F2 population of the mutants (g56w, g83w, g142w, and g143w) backcrossed with the wild type, the phenotypic proportions between the wild type and mutants were 3:1 following Mendelian segregation. Therefore, we speculated that the mutated trait of each mutant is controlled by a single recessive gene. This study provides a series of stable, homozygous and valuable mutant resources for elucidating the mechanisms of UV-A and blue + UV-B induced anthocyanin biosynthesis in higher plants.