Purple Citrus? Utilization of Myb-related Transcription Factor Genes for Anthocyanin Production

Three myb-related transcription factor genes of the anthocyanin biosynthetic pathway, VvmybA1 from Vitis vinifera, Ruby from Citrus sinensis and PAP1 from Arabidopsis thaliana were introduced into Citrus aurantifolia 'Mexican Lime' under the control of the cauliflower mosaic virus 35S promoter. The 35S: VvmybA1 construct had the highest expression levels with some regenerated citrus plants being completely purple. The 35S: Ruby plants expressed anthocyanin at a much lower level than that observed in the 35S: VvmybA1 plants. We did not observe any anthocyanin production in any 35S:PAP1 derived transgenic line. Transgenic citrus plants expressing the VvmybA1 or the Ruby gene gave a range of phenotypes, from green to slightly or intensely purple plants. Expression levels could not be correlated to the copy number in selected transgenic lines. The intensely purple plants lacked vigor and grew slowly. Anthocyanin biosynthetic pathway gene transcripts were observed to be up regulated when analyzed by qRT–PCR.  These myb-related transcription factor genes hold potential as a simple and non-destructive visual marker for citrus transformation when coupled with a tissue specific or inducible promoter. These genes can also be used as a component in a marker free transformation system.