Genetic transformation of Vitis vinifera to improve disease resistance

Resistance to several major diseases is seriously deficient in most cultivars of Vitis vinifera. We sought to develop disease-resistant forms of important cultivars by transformation with an endochitinase-producing gene (ThEn42) from Trichoderma, as well as with two magainin-type antimicrobial peptide genes (MagII and MSI-99). Embryogenic cultures of Vitis vinifera L. `Chardonnay' were transformed using the biolistic method. After bombardment with pBin19ESR (harboring both the nptII and ThEn42 genes), 93 `Chardonnay' lines were regenerated on media containing 25 or 50 mg/L kanamycin. Quantitative fluorescent chitinase assays were performed to measure the level of enzyme activity from leaves of in vitro-grown plants. Forty to fifty percent of `Chardonnay' lines expressed chitinase at levels 10- to 100-fold higher than non-bombarded controls. Integration of the ThEn42 and nptII genes in the chitinase positive lines was confirmed by PCR and Southern blot analysis. Western blotting confirmed gene expression in both in vitro-grown and field-grown plants. In a preliminary detached leaf assay, the lesion size after Botrytis infection was 65% less in a high chitinase expressing line as compared to a non-bombarded line. Six of 25 `Chardonnay' lines were more tolerant of powdery mildew after natural inoculation in the field. Co-transformation was used to obtain antimicrobial peptide-transgenic vines of 'Chardonnay'. One plasmid harbored the nptII selectable marker and another carried the peptide gene. Both genes were driven by Arabidopsis ubiquitin promoters. From 165 embryos selected on media with 15 mg/L kanamycin, 94 lines regenerated into plants. Results from PCR and dot blot hybridization screening of 72 plants showed a frequency of co-transformation of approximately 50%. Evidence showed that an Arabidopsis ubiquitin promoter functioned in Vitis, and that biolistics can be used to co-transform genes on separate plasmids. These vines are undergoing further greenhouse and field analyses for disease resistance.