?Inserci?n del gen RB en las variedades Revoluci?n y D?sir?e de Solanum tuberosum para conferir resistencia a Phytophthora infestans.?

Abstract

Con el fin de introducir el gen RB que confiere resistencia al tiz?n tard?o se desarrollaron dos construcciones gen?ticas, la primera s?lo con el gen de inter?s (RB) y la segunda con el gen de inter?s y un gen de resistencia a kanamicina, que sirvi? como marcador de selecci?n. Ambas construcciones fueron usadas para transformar mediante Agrobacterium tumefaciens, explantes de Solanum tuberosum de las variedades Revoluci?n y D?sir?e Se desarrollaron medios para propagaci?n y regeneraci?n de las pl?ntulas de Revoluci?n, y se determin? la concentraci?n de antibi?tico necesaria para la selecci?n de los explantes transformados y la eliminaci?n de Agrobacterium. Se realizaron los eventos de transformaci?n con la primera construcci?n carente del gen marcador de selecci?n; por lo cual, la mayor?a de los explantes regeneraron. Los regenerantes fueron analizados por PCR y se obtuvieron 7 l?neas posiblemente transg?nicas, pero en los resultados con Southern blot resultaron negativas. De la transformaci?n con la segunda construcci?n, en la variedad D?sir?e se obtuvieron 12 plantas putativamente transg?nicas que fueron luego analizadas por Southern blot confirmando que 10 de ellas eran transg?nicas y conten?an el gen de inter?s. In order to insert the RB gene conferring resistance to late blight, two genetic constructs were developed. The first construct only contains the gene of interest (RB) and the second construct, the gene of interest plus a kanamycin resistance gene, which served as a selection marker. Both constructs were used to transform explants of Solanum tuberosum, D?sir?e and Revolution varieties, by Agrobacterium tumefaciens. Culture medium were developed for propagation and regeneration of Revolution?s seedling. Concentration of required antibiotic to select transformed explants and eliminate Agrobacterium were also determined. Transformation events were conducted with the first construct lacking the selection marker gene. As a result, most of the explants were regenerated. PCR was used to analyzed the regenerants and 7 lines were candidates to be transgenics. However, Southern blot analisys ruled out this posibility. Transformation events with the second construct, in the D?sir?e variety, resulted in 12 putative transgenic plants. PCR and Southern blot analisys confirmed that 10 of them were transgenic and contained the gene of interest.