084 Response of Transgenic `Royal Gala' Apple (Malus ×domestica Borkh.) Shoots, Containing the Modified Cecropin MB39 Gene, to Erwinia amylovora

Transgenic `Royal Gala' apple (Malus × domestica Borkh.) shoots were obtained by Agrobacterium-mediated gene transfer using the plasmid binary vector pGV-osm-AC with a T-DNA encoding a chimeric gene consisting of a secretory sequence from barley-amylase joined to the modified cecropin MB39 coding sequence. Shoots were placed under the control of a wound-inducible, osmotin promoter from tobacco. The integration of the cecropin MB39 gene into apple was confirmed by Southern blot analysis. The transformation efficiency was 1.5% when internodes from etiolated shoots were used as explants and 2% when leaf explants were used. Both non- and transgenic tetraploid plants were produced by treatment of leaf explants with colchicine at 25 mg·L-1, and polyploidy was confirmed by flow cytometry. Of the diploid transgenics, three of seven were significantly more resistant to Erwinia amylovora than the non-transgenic `Royal Gala' control. Also, in one instance, a tetraploid transgenic was significantly more resistant than the diploid shoot from which it was derived.

Genetic Modification of `Royal Gala' Apple by Agrobacterium-mediated Transformation and Colchicine-induced Mutation

As part of a program to develop transgenic Malus × domestica Borkh. cv. Royal Gala with improved disease resistance, transgenic diploid and tetraploid plants with cecropin MB39 gene were regenerated. Transgenic diploid plants were obtained from etiolated internodal explants by Agrobacterium tumefaciens-mediated transformation using the plasmid binary vector pGV containing a chimeric gene consisting of a secretory sequence from barley-amylase joined to a modified cecropin MB39 coding sequence and placed under control of wound-inducible osmotin promoter from tobacco. The integration of the cecropin gene into apple genome was confirmed by Southern blot analysis. The transformation efficiency was 1.5%. Both non- and transgenic tetraploid plants were produced by cocultivating leaf explants from wild type and transgenic diploid shoots with colchicine at 25 mg/L in apple regeneration medium containing 10 μM TDZ. Twenty-two tetraploid lines were obtained from 90 explants. Flow cytometry was used for ploidy determination. The tetraploid plants were distinguishable from the diploid on morphological as well as cytogenetic grounds. Both the transgenic diploid and tetraploid plants are now being evaluated for resistance to fireblight.