Rapid Assay of Tissue Specific Promoters in Vitis

Grapevine (Vitis vinifera) is one of the world’s most economically valuable and prolific fruit crops with an area of 7.8 million hectares producing 65 million metric tons of fruit. Grapevine yields are negatively affected by a multitude of diseases every year requiring growers to utilize unsustainable methods of disease control such as pesticide and fungicide applications. Effective disease resistance cannot be incorporated into elite cultivars using conventional breeding due to the constraints of a long breeding cycle, self-incompatibility, and inbreeding depression. Precision breeding allows for the insertion of specific genetic elements from any cross fertile species into elite cultivars. The ability to rapidly test the expression of these genetic elements in living tissues by using particle bombardment provides a highly convenient and accurate method to determine the function of putative tissue specific genes, promoters, and other genetic elements. In this study, fruit-specific expression was determined by using grape berries and optimized parameters for particle bombardment. Microcarrier type, DNA concentration, rupture disk pressure, and target distance were determined using a test plasmid containing the enhanced green fluorescent protein (EGFP) gene that was overexpressed by a CaMV 35S promoter. Transient GFP expression was observed within 48 hours post-bombardment. Berries were assessed for both skin and mesocarp expression by determining the number of cells that expressed GFP. Resulting optimized bombardment parameters were subsequently used to evaluate tissue-specific expression of six putative fruit-specific promoters by comparing relative GFP expression in epidermis and mesocarp with somatic embryos, leaf, and stem tissue controls. Promoters evaluated due to their abilities to drive high levels of expression in fruit tissues included the V. vinifera thaumatin-like protein, (VvTL1) as well as orthologous fruit-specific promoters from Pyrus pyrifolia (polygalacturonase PG2), and fruit-ripening promoters from Lycopericum esculentum (phosphoenolpyruvate carboxylase PPC2, P119 and E8). Of these, only PG2 and VvTL1 were determined to exhibit fruit specific activity. By incorporating this rapid screening technique into precision breeding-based cultivar development, numerous genetic elements may be tested for functionality well before the trait is to be incorporated into elite cultivars.