Resistencia aumentada a Rhizoctonia solani por la expresión combinada de quitinasa y proteínas inactivantes de los ribosomas en patatas transgénicas

Potato (Solanum tuberosum L.) is susceptible to many fungal pathogens including Rhizoctonia solani. In the present study, the potato cultivar Desirée was transformed via Agrobacterium tumefaciens strain GV3101 containing the binary plasmid pGJ132 harboring both the chitinase (chiA) and rip30 genes. The potato leaf disc was used as an explant for transformation. PCR, Southern blot and Western blot were used for characterization of the transgenic plants. In this study it was shown that not all the plants developed in selective medium were positive for the corresponding gene using the PCR technique. Southern blot analysis confirmed that transgenic plants integrated 2-3 copies of chiA and rip30 genes respectively into their genome. The expression of the CHIA and RIP30 proteins was confirmed in the leaf extracts of the transgenic clones by Western blot analysis. Transgenic potato plants expressing rip30 and chiA genes showed enhanced resistance to R. solani in a greenhouse assay.La patata (Solanum tuberosum L.) es susceptible a muchos hongos fitopatógenos, incluyendo Rhizoctonia solani. En el presente estudio, se transformó el cultivar de patata ‘Desirée’ mediante Agrobacterium tumefaciens, cepa GV3101, que contiene el plásmido binario pGJ132 que alberga los genes quitinasa (chiA) y rip30. Se utilizaron discos de hojas como explante para la transformación de plantas. Se utilizaron las técnicas de PCR, Southern y Western blot para la caracterización de las plantas transgénicas. En este estudio se demostró, mediante PCR, que no todas las plantas que se desarrollaron en medio selectivo fueron positivas para el gen correspondiente. El análisis de Southern blot confirmó que las plantas transgénicas integraron en su genoma 2-3 copias de los genes chiA y rip30. Se llevó a cabo un ensayo de invernadero para evaluar la resistencia a R. solani de los clones transgénicos que expresan los transgenes. Las plantas transgénicas que expresan los genes rip30 y chiA mostraron una resistencia completa a R. solani

Ribosome Inactivating Protein of barley enhanced resistance to Rhizoctonia solani in transgenic potato cultivar 'Desirée' in greenhouse conditions

In the present study, the potato cultivar 'Desirée' was transformed via Agrobacterium tumefaciens strain LBA4404 containing the plasmid pBIN19 which harbors the Ribosome Inactivating Protein (rip30). The potato leaf discs were used as an explant for transformation. The in vitro regeneration parameters (percentage of callus regenerated, number of shoots per callus, percentage of regenerated roots and percentage of the transgenic plants) were evaluated. The PCR technique was used for identification of transformed plants. Southern and Western blot analyses were applied for molecular characterization of the transgenic clones. A greenhouse assay was carried out to evaluate the resistance to Rhizoctonia solani pathogen of transgenic clones expressing the rip30 gene. The results revealed that not all the plants developed in selective medium were positive for the corresponding gene using the PCR technique. Southern blot analysis demonstrated that the tested transgenic plants integrated three copies of rip30 gene into their genome. The expression of the RIP30 protein was confirmed in the leaf extracts of the transgenic clones by Western blot analysis. Resistance evaluation of the transgenic plants in greenhouse conditions showed that disease incidence and severity were reduced for R. solani