Use of an anthocyanin production phenotype as a visible selection marker system in transgenic tobacco plant

To develop a potentially alternative method for the selection of transgenic plants instead of antibiotic and herbicide resistance, anthocyanin pigmentation phenotype was examined to provide a visible selection marker system. Two regulatory genes of the anthocyanin biosynthetic pathway, the R2R3 MYB mPAP1 gene from Arabidopsis and the basic helix loop helix B-Peru gene from maize, were amplified by RT-PCR and then individually cloned into a plant expression vector. The requirement of these two genes for anthocyanin pigmentation was pre-confirmed via an in vivo assay using tobacco agro-infiltration. The mPAP1 and B-Peru vectors were further stably co-transformed into tobacco plants using Agrobacterium tumefaciens strain LBA4404. Tobacco plants harboring both genes could be readily selected through the manifestation of a red color due to anthocyanin accumulation in the whole plant body. The T1 segregants showed red or green phenotypes depending on the genotype. The need for both the mPAP1 and B-Peru genes for a red color phenotype due to anthocyanin pigmentation was further confirmed by genotyping of the T1 generation by genomic PCR analysis and an in vivo assay using agro-infiltration. From these results, we conclude that co-transformation with two individual vectors harboring a critical anthocyanin transcriptional factor has potential utility as an alternative visible selectable marker system for transgenic progeny selection in plants.