Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a 'shooter mutant' A. tumefaciens.
In this work, Potato virus Y (PVY) resistant potatoes were generated using an environmentally safe construct. For this purpose, a 'shooter' mutant Agrobacterium-based transformation system was used. The isopentenyl transferase gene (ipt) present on the Ti plasmid of 'shooter' strains enhances shoot regeneration and can be used as a phenotypic selection marker. The introduced marker-free binary vector carried a hairpin construct derived from the coat protein gene of PVY-NTN strain in order to induce gene silencing. Transformation resulted in high regeneration rates (1.4-5.7 shoots per explant). With pre-selection for the ipt (+) phenotype the transformation frequency was 24-53%, while without selection 12-28% of the shoots were PCR positive. The presence of the transgene was verified by Southern hybridization. In 16 of 31 challenged transformant lines PVY could be detected neither by RT-PCR nor by back inoculation. A 62.5% of these resistant lines proved to be also ipt-free. This transformation system was reproducible in four potato cultivars, suggesting that it could easily be adapted for other species.
Bukovinszki
Agnes
A
Agricultural Biotechnology Center, Szent-Györgyi A u 4, 2100, Gödöllo, Hungary. bagnes@abc.hu
Divéki
Zoltán
Z
Csányi
Márta
M
Palkovics
László
L
Balázs
Ervin
E
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D015139:Blotting, Southern; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D009154:Mutation; D010641:Phenotype; D010935:Plant Diseases / Q000235:genetics / Q000821:virology; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D017800:Potyvirus / Q000254:growth & development*; D011198:Solanum tuberosum / Q000235:genetics* / Q000821:virology*; D014170:Transformation, Genetic
D018744:DNA, Plant