The coat protein gene (CP) from a highly virulent, necrotic and dominant strain of potato virus Y (PVY) originated from the Hungarian flora has been engineered via Agrobacterium infection into different Hungarian tobacco breeding lines and cultivars. The integration of the CP was confirmed by polymerase chain reaction (PCR) using genomic preparations. The transcription and the expression of the integrated CP gene were detected by Northern and Western analysis. The pathogen-derived resistance was demonstrated by inoculation of the R1 progeny of transformant plants with purified PVY. The efficiency of the protection varied between different transgenic tobaccos ranging from complete to no protection. The challenge infection of the plants was monitored by dot blot hybridisation at different intervals after mechanical inoculation. Western blot analysis showed that there is no correlation between the level of expressed CP and the extent of resistance. From tobacco cultivars namely Virgin D, Stamm C2 and Hevesi 11, 38, 55 and 23 transformants were obtained, respectively. After several years of greenhouse experiments, only the extreme resistant tobaccos were planted field under the special licence, given by the competent authority. In field conditions, transgenic tobacco varieties showed extreme resistance against natural infection of PVY.
Authors:R. Józsa, Z. Stasevski, I. Wolf, S. Horváth and E. Balázsi
The coat protein (CP) gene from a necrotic strain of potato virus Y (PVY) has been engineered into two-old Hungarian cultivars, namely cv. Mindenes and Somogyi kifli. The integration of the coat protein gene was confirmed by polymerase chain reaction (PCR) using genomic DNA preparations. The transcription and the expression of the integrated CP gene was followed by Northern and Western blot. The pathogene derived resistance was demonstrated by mechanical inoculation of the transformant plants after the transfer of the regenerants into soil. The efficiency of the virus protection varied between different potatoes ranging to complete protection to no protection. All plants were tested in field conditions under the special licence, given the competent authority instructed by the Hungarian gene technology act. In two consecutive years three Mindenes independent transformants proved to be highly resistant against two different strains of PVY in provocative experiment.