Evaluation of a grapevine-derived reporter gene system for precision breeding of Vitis

Abstract

A grapevine-derived VvMybA1 transcription factor was evaluated for its efficiency as a reporter gene by comparing it with existing reporter genes, the green fluorescent protein (GFP) and β-glucuronidase (GUS). Embryogenic cultures of Vitis cultivars Thompson Seedless and Bronx Seedless were initiated from leaves of in vitro grown micropropagation cultures and somatic embryos were used as explants for Agrobacterium-mediated transformation. Transient and stable expression of the MybA1 gene was characterized by intense red pigmentation in co-cultivated explants, callus tissues and secondary embryos lines compared to GFP and GUS that exhibited green fluorescence and blue coloration following a substrate assay. No differences were observed in transient gene expression frequencies between the MybA1 gene and GUS among the two compared reporter genes. Visual levels of stable gene expression were higher in GFP and GUS expressing cultures compared to MybA1 expressing cultures. The presence of the inserted genes and their expression in regenerated plant lines was confirmed by PCR and RT-PCR. Embryo and plant lines expressing the MybA1 gene accumulated varying levels of anthocyanin pigment in plant tissues and organs, and were characterized by slower growth compared to plant lines expressing GFP and GUS. Scanning electron microscopy analyses revealed a significant change in abaxial and adaxial leaf epidermal cells of MybA1-expressing plant lines compared to those expressing GFP and GUS, and non-transformed control plants. The study demonstrated the utility of the VvMybA1 transcription factor as a reliable reporter gene for identification of gene insertion events in cell culture and regeneration of modified plants.