Improved fire blight resistance in transgenic apple lines by constitutive overexpression of the mbr4 gene of Malus baccata

Abstract

The improvement of resistance to fire blight caused by Erwinia amylovora is one of the most important goals of conventional apple breeding. The breeding of highly resistant cultivars with marketable fruits is limited by the fact that most commercially used cultivars are susceptible to fire blight and resistance genes are mostly present in small-fruited wild species. That's why we used the Agrobacterium tumefaciens mediated gene transfer to introduce the apple resistance gene mbr4 driven by the constitutive CaMV 35S promoter into the genome of 'Pinova', an apple cultivar that is established on the market. We produced fourteen putative transgenics and tested them for the presence of the mbr4 and the nptII marker gene via PCR. All plants amplified PCR products of the expected size for each transgene. The plants were propagated in vitro and fourteen transgenic clones were established. These clones were evaluated for the number of integrated T-DNA's by southern hybridization. The expression of the nptll and mbr4 genes was studied by reverse transcriptase PCR (RT-PCR) and quantitative RT-PCR (qRT-PCR). Twenty in vitro leaves of each line were infected with a gfp gene expressing E. amylovora strain and evaluated after five days of incubation using a fluorescence microscope. Most clones were less susceptible than the non-transgenic genotype 'Pinova'. Three clones showed significantly increased resistance compared to the parental line. It is interesting to note that one clone was significantly more resistant than the wild species M. ×robusta persicifolia used as the fire blight resistant control. Twenty shoots of each line were rooted and transferred to a greenhouse. These shoots were evaluated for their resistance to fire blight via artificial shoot inoculation. Three out of fourteen clones showed significantly increased resistance compared to 'Pinova'.