January 12-16, 2008
Town & Country Convention Center
San Diego, CA
Ewa E. Borejsza-Wysocka1 , John L. Norelli2 , Angela M. Baldo3 , Robert E. Farrell, Jr.4 , Carole L. Bassett2 , Herb S. Aldwinckle1
A high-efficiency transformation and selection system was used to create apple RNAi mutants for determination of function of candidate genes in resistance of apple to Erwinia amylovora (fire blight). The M.26 apple genotype was transformed with a mixture of five RNAi EST-silencing vectors in each transformation experiment to allow selection of multiple types of mutants from a single experiment. Bioinformatics analysis was used to identify ESTs associated with response to E. amylovora. The silencing constructs were transferred to Agrobacterium tumefaciens strain EHA105pCH32, which was used to transform leaf-slice explants. Regenerants were selected with kanamycin using nptII and then screened by PCR using universal primers to assess integration of a silencing construct. In most of the lines screened, PCR showed that only single genes had been inserted. Thus far ESTs from genes in six functional categories, general metabolism (1), photosynthesis (2), nucleic acid metabolism (1), protein metabolism (3), signaling (4), and defense/stress (4), have been subjected to this protocol. The regenerants will be assayed for phenotype of reaction to E. amylovora by inoculation of young plantlets. This project is supported by a National Research Initiative Competitive Grant 2005-35300-15462 from the USDA Cooperative State Research, Education, and Extension Service.