ISHS Acta Horticulturae 590: IX International Workshop on Fire Blight
TRANSFORMATION OF SR1 TOBACCO AND JTE-H APPLE ROOTSTOCK WITH THE EPS-DEPOLYMERASE GENE FROM AN ERWINIA AMYLOVORA PHAGE |
| Authors: | S. Sule, W.-S. Kim, K. Geider, E. Kiss |
| DOI: | 10.17660/ActaHortic.2002.590.63 |
| Abstract:
The gene of an Erwinia amylovora bacteriophage encoding an extracellular polysaccharide depolymerase (Dpo), which specifically degrades the surface polysaccharides of the fire blight pathogen, was cloned into pUC-vectors including His-tagged fusions. These insertions were transferred into plasmid pBinAR and named pBinAR-dpo. pBinAR-dpo was transformed into Agrobacterium tumefaciens strain LBA 4404 and were used to transform SR1 tobacco and JTE-H apple rootstock leaves. The putative transformant shoots appearing were analysed by PCR for the presence of nptII gene and the 35S promoter. Plants which were positive in PCR analysis, were further analysed by Western blots. Antiserum against recombinant EPS depolymerase was prepared in order to detect expression of the dpo gene. Several transformed tobacco and apple plants were positive for the presence of EPS-depolymerase. The tobacco clone EPS-dpo 6 that expressed the protein at a high level was chosen for the production of seeds and the establishment of a transgenic Nicotiana tabacum line. Seeds of the transgenic plants were germinated on 1/2 MS medium containing 100 mg kanamycin per litre. Out of 100 seeds 62 exhibited normal roots and leaf phenotypes. Several problems were encountered in transformation of the JTE-H apple rootstock. Out of 432 infected leaves, 24 transformed cell lines were obtained. Incorporation of the 35S promoter, the nptII and dpo genes were confirmed by PCR analysis. These clones are currently under investigation for expression of the EPS-depolymerase and their resistance to E. amylovora. Preliminary data with five transformed shoots showed that four out five plants were resistant to the E.amylovora infection. All of the five control plants were sensitive. | |
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