Inserci?n de genes cry3Ca1 y cry7Aa1 en Ipomoea batatas (L.) Lam. cv. Huachano para conferir resistencia a Cylas puncticollis y C. brunneus ?gorgojos del camote? (Coleoptera: Curculionidae)

Abstract

Con la finalidad de insertar los genes cry3Ca1 y cry7Aa1 en Ipomoea batatas (L.) Lam. cv. ?Huachano? para conferir resistencia a Cylas puncticollis y C. brunneus ?gorgojos del camote?, se desarroll? la transformaci?n gen?tica mediada por Agrobacterium tumefaciens, siguiendo dos protocolos de regeneraci?n y transformaci?n. Partiendo de 46 meristemos y 282 hojas con peciolo, se obtuvieron 8 eventos transg?nicos de inserci?n completa del ADN - T (4 por cada protocolo de transformaci?n) y un evento de inserci?n incompleta. Con una eficiencia de transformaci?n de 8.70%, el protocolo a partir de meristemos demostr? ser m?s eficaz en la obtenci?n de eventos transg?nicos de inserciones completas que el protocolo a partir de hojas con peciolo, con el cual se obtuvo una eficiencia de transformaci?n de 1.42%. A su vez, los regenerantes fueron evaluados mediante pruebas in vitro (resistencia a kanamicina) y moleculares (PCR), con los an?lisis de PCR se confirm? que los 8 regenerantes callo positivos (resistentes a la prueba de kanamicina) tambi?n presentaron la inserci?n de los transgenes de inter?s ?cry3Ca1 y cry7Aa1? y del gen marcador selector ?nptII?. Asimismo, se determin? que 4 de los eventos transg?nicos integraron secuencias externas al ADN - T ?backbone? en el genoma de la planta, uno de los cuales, present? la inserci?n del gen bacteriano ?virD2?.With the purpose of inserting the genes cry3Ca1 and cry7Aa1 in Ipomoea batatas (L.) Lam. cv. "Huachano" to develop resistance to Cylas puncticollis and C. brunneus ?sweet potato weevils?, the genetic transformation was developed using Agrobacterium tumefaciens, following two protocols of regeneration and transformation. Starting from 46 meristems and 282 leaves with petiole, 8 transgenic events were obtained with complete insertion of T - DNA (4 for each transformation protocol) and one event with incomplete insertion. With an efficiency of transformation of 8.70 %, the protocol of meristems proved to be more effective in obtaining transgenic events of complete inserts than the protocol of leaves with petiole, which obtained a efficiency of transformation of 1.42 %. The regenerants were evaluated by testing in vitro (resistance to kanamycin) and molecular (PCR), the PCR analysis confirmed that the 8 regenerants positive callus (resistant to kanamycin test) also presented the insertion of transgenes of interest ?cry3Ca1 and cry7Aa1? and selection marker gene ?nptII?. Likewise, 4 of the transgenic events integrated sequences outside the T - DNA ?backbone? into the plant genome, one of whom, showed the insertion of the bacterial gene ?virD2?.