Genetic Engineering of Grapevine (Vitis sp.) for Enhancement of Disease Resistance
Kikkert, J.R., Gul S. Ali, Michael J. Striem, Mary-Howell Martens, Patricia G. Wallace, Linda Molino, and Bruce I. Reisch. 1997. Genetic Engineering of Grapevine (Vitis sp.) for Enhancement of Disease Resistance. Proc. Third International Symposium on In Vitro Culture and Horticultural Breeding. ISHS. 16-21 June 1996. Jerusalem, Israel (A. Altman and M. Ziv, eds.) Acta Horticulturae 447:273-279.
Department of Horticultural Sciences, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456-0462 USA.
Additional index words: biolistics, chitinase, gene gun, gene transfer, microprojectile bombardment, somatic embryogenesis, transgenic plants.
Abstract
Chitinolytic enzymes exhibit antifungal activity against several grapevine pathogens in in vitro assays, including Uncinula necator (powdery mildew) and Botrytis. The genes that encode such enzymes have the potential to confer tolerance to fungal pathogens when engineered into disease susceptible cultivars. Our laboratory has previously regenerated transgenic 'Chancellor' grapevine plants that were transformed with marker genes using the biolistic (gene gun) process (Kikkert et al., 1996, Plant Cell Reports, 15:311-316). More recently, embryogenic cultures of 'Chancellor' were bombarded with a chitinase gene. Following selection on kanamycin, the culture medium adjacent to the putative transformants was assayed for chitinase activity using a fluorescent assay. Out of 470 culture lines tested, 11 (2.3%) and 66 (14%) exhibited 4 and 3 times, respectively, more intense fluorescence than non-bombarded control lines. The recovered embryos are being further analyzed by PCR and Southern blotting.
To extend the biolistic transformation technology to commercially important grapevine cultivars, embryogenic cultures of V. vinifera L. 'Chardonnay', 'Merlot', and 'Pinot noir', and V. Labruscana 'Concord', and 'Niagara' were initiated as a source of tissue for bombardment. Anthers and ovaries were extracted 10-14 days pre-bloom, and cultured on MS- or NN-based media with different combinations of growth regulators. Somatic embryos were observed from all cultivars at frequencies ranging from 0.1 to 20.0% depending on the explant and growth medium. An embryogenic suspension of 'Merlot' was bombarded with tungsten particles coated with a plasmid containing the b-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII) genes or the chitinase and NPTII genes. Strong transient GUS expression was observed 2 days post-bombardment. Selection for kanamycin-resistant embryos, which should also contain the chitinase gene, is currently underway. Preliminary tests of chitinase expression on a small number of regenerated 'Merlot' embryos were positive. Similar bombardments are being conducted with 'Chardonnay' and 'Concord' embryogenic calli grown on semi-solid medium. Our goal is to test the chitinase gene, as well as other antifungal genes, in the five grapevine cultivars listed above for their effectiveness in controlling powdery mildew and other fungal diseases.
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