Combinatorial expression of transcription factor genes B-Peru and mPAP1 enhances anthocyanin accumulation in transgenic Petunia hybrid

• We report an improved Agrobacterium-mediated transformation protocol for Petunia.
• PPT used throughout the selection procedure reduced the total number of escapes.
• Clavamox controlled Agrobacterium after co-cultivation and supported regeneration.
• The protocol was utilized to transfer anthocyanin regulatory genes into Petunia.
• Co-expression of B-peru and mPAP1D significantly improved anthocyanin in Petunia.

The present study aimed to determine the role of transcription factors (bHLH and MYB) in enhancing anthocyanin production in Petunia ‘Mirage rose’. Initially, we optimized an Agrobacterium-mediated transformation protocol to over-express transcription factors (B-Peru, mPAP1, and B-Peru + mPAP1) in Petunia. Phosphinothricin (PPT) concentrations of 0.5, 1.0, and 1.5 mg l−1 were found to be ideal for the selection and regeneration of transformed shoots at different developmental stages. Clavamox (250 mg l−1) efficiently eliminated Agrobacterium after co-cultivation (2 days) and favored maximum shoot regeneration. In addition, incubation for 30 min in Agrobacterium suspension increased the number of transformed cells and resulted in improved regeneration in the selection medium. The transformed plants were successfully developed through a direct organogenesis system. The transformed plants were selected using BASTA® and the presence of transgenes was assessed using PCR. Visible anthocyanin accumulation was evident only in plantlets (shoots, stem, leaves, and roots) carrying the gene combination B-Peru + mPAP1. The expression of biosynthetic genes involved in the flavonoid pathway was analyzed using quantitative real-time PCR. Expression levels of PAL, CHS, CHI, F3H, DFR, and ANS were higher in the young and mature leaves of plants transformed with B-Peru + mPAP1 compared to those transformed with B-Peru, mPAP1, and/or non-transformed plants. Furthermore, the highest level of anthocyanin was recorded in the leaves, stem, and roots of plants transformed with B-Peru + mPAP1. These results validate the combinatorial requirement of B-Peru and mPAP1 to enhance anthocyanin content in Petunia hybrida ‘Mirage rose’.