Optimizing Parameters for Precision Breeding of Grapevine

Reporter genes such as the green fluorescence protein (GFP) and β-glucoridinase (GUS) are routinely used to monitor gene insertion and expression in plant transformation studies. However, their use is limited by the need for expensive equipment and/or enzymatic assays for tracking gene expression. Additionally, the occurrence of such sequences in transgenic crops has resulted in unsubstantiated claims of health and environmental hazards. The use of plant derived-reporter gene systems serve as valuable tools for gene insertion and plant regeneration of perennial fruit species such as grapevine. To evaluate the efficiency of a grapevine-derived reporter gene system, the Vitis vinifera MybA1 gene (VvMybA1) was placed along with a nptII gene under the control of a CaMv35S promoter. Embryogenic cultures of grape cultivars ‘Bronx Seedless’, ‘Merlot’ and ‘Thompson Seedless’ were produced from leaves or floral tissues. Somatic embryos at the mid-cotyledonary stage of development were co-cultivated with Agrobacteriumharboring the VvMybA1 gene. Transient and stable gene expression was recorded in co-cultivated embryogenic cultures and independent plant lines were recovered via secondary embryogenesis. Stable gene insertion and expression in independent plant lines was confirmed by PCR and RT-PCR. Scanning electron microscopy was performed to study potential changes in leaf cell structure caused by VvMybA1 gene expression and subsequent anthocyanin accumulation. Transient anthocyanin expression was evidenced by red spots on somatic embryo explants after 3 days of co-cultivation.  No difference in stable gene expression and embryo line production was observed between the VvMybA1reporter gene system and existing reporter genes, GFP and GUS. Although plant lines expressing the VvMybA1 gene exhibited normal growth and development, leaf cells appeared much larger and distorted, apparently due to hyperaccumulation of anthocyanin. We are currently exploring the possibility of using the VvMYbA1 reporter gene specifically in cell culture, with an embryo specific promoter. Gene expression at the cell culture stage would enable convenient identification of gene insertional events, while allowing normal growth and development following plant regeneration.