Evaluation of plant-based reporter systems for improvement of cold-hardy grape cultivars

Plant-based reporter gene systems enable convenient detection of gene insertion events and recovery of cisgenic/intragenic plants following transformation. For instance, anthocyanin biosynthesis genes isolated from plant species can be used as potential reporter genes for non-destructive analysis of gene expression. Anthocyanin biosynthesis genes from Arabidopsis (MYB75), Citrus sinensis (RUBY), Vitis vinifera (VvMybAl) and Zea mays (LC) were isolated and placed along with a NPTII gene under the control of a CaMV35S-derived bidirectional duplex promoter complex. Embryogenic cultures of Vitis interspecific hybrids 'Bronx Seedless, 'Himrod', 'Interlaken' and 'Seyval Blanc' were initiated from leaf explants for use in expression studies. Somatic embryos were co-cultivated with Agrobacterium in the dark for 3 days. Co-cultivated embryos were then transferred to DM medium containing antibiotics for callus proliferation and secondary embryogenesis. Transient anthocyanin expression, as evidenced by bright red color spots, was observed in somatic embryos after co-cultivation with Agrobacterium harboring anthocyanin-containing vectors. Transient gene expression levels varied from 58-100% depending on the anthocyanin vector. Among the various cultivars, "Interlaken" exhibited the highest expression levels followed by 'Seyval Blanc', 'Himrod' and 'Bronx Seedless'. Anthocyanin-expressing somatic embryos were selected from treated embryogenic cultures by growth on DM medium containing antibiotics and screened for production of stable anthocyanin-expressing embryo lines following secondary embryogenesis. The performance of various anthocyanin-derived reporter genes was compared. Such reporter genes could potentially aid in large-scale testing of functional genes for grapevine improvement.