Fil: Noguera, Aldo Sergio. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cie... more Fil: Noguera, Aldo Sergio. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Tucuman. Instituto de Tecnologia Agroindustrial del Noroeste Argentino. Provincia de Tucuman. Ministerio de Desarrollo Productivo. Estacion Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnologia Agroindustrial del Noroeste Argentino; Argentina
Fil: Rigano, Luciano Ariel. Fundacion Pablo Cassara; Argentina. Consejo Nacional de Investigacion... more Fil: Rigano, Luciano Ariel. Fundacion Pablo Cassara; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Parque Centenario. Instituto de Ciencias y Tecnologia "Dr. Cesar Milstein"; Argentina
Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. ves... more Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.
RESUMEN La variedad comercial de caña de azúcar RA 87-3 se transformó mediante biobalística con u... more RESUMEN La variedad comercial de caña de azúcar RA 87-3 se transformó mediante biobalística con una construcción genética que porta el gen epsps de la cepa CP4 de Agrobacterium tumefaciens, y el gen nptII que confieren resistencia a glifosato y resistencia a kanamicina/geneticina, respectivamente. Las líneas transformadas fueron multiplicadas en invernadero y se evaluó la resistencia al herbicida utilizando diferentes concentraciones de glifosato (3, 4 y 8 l/ha). Las líneas resistentes al herbicida (RH) se evaluaron en campo para confirmar la resistencia a glifosato (3 l/ha) y realizar un análisis preliminar del comportamiento de estas con respecto al cultivar parental. Todas las líneas transformadas mantuvieron la resistencia al herbicida pero muchas mostraron cambios fenotípicos. Las diez líneas RH que resultaron muy parecidas a la variedad RA 87-3 se analizaron fenotípica y genéticamente utilizando los nueve descriptores morfológicos obligatorios propuestos por la "International Union for the Protection of New Varieties of Plants" (UPOV) y con 339 marcadores moleculares, respectivamente. Seis líneas de las anteriores presentaron cambios morfológicos y genéticos menores y fueron seleccionadas para ensayos en campo durante dos ciclos vegetativos (caña planta y soca 1), en dos áreas de producción de Argentina. Las seis líneas RH presentaron características agronómicas, industriales y de composición química indistinguibles respecto del cultivar parental. La herencia estable del gen CP4 epsps fue confirmada mediante ensayos de RT-qPCR y Southern blot en diferentes generaciones clonales (caña planta y soca 1). Los estudios confirmaron la utilidad de la transformación genética como una herramienta complementaria a la mejora clásica, y destaca la ventaja del uso de los descriptores de UPOV junto con los marcadores moleculares para una selección temprana de eventos transgénicos que tengan un alto parecido con el genotipo parental. Tomando en cuenta los resultados, una de las seis líneas estudiadas se seleccionó para una posible liberación comercial, la cual debe ser sometida a evaluación por parte de los entes regulatorios (CONABIA, SENASA y DNMA). Palabras clave: Saccharum spp., Glifosato, gen CP4 epsps, resistencia a herbicida. ABSTRACT Development of the transgenic sugarcane event TUC 87-3RG resistant to glyphosate The commercial variety of sugarcane RA 87-3 was transformed by biobalistic with a genetic construct that carries the epsps gene of the CP4 strain of Agrobacterium tumefaciens, and with the nptII gene that confer resistance to glyphosate and resistance to kanamycin / geneticin, respectively. Transformed lines were multiplied in greenhouse and resistance to the herbicide was evaluated using different concentrations of glyphosate (3, 4 and 8 l / ha). Resistant lines to the herbicide (RH) were evaluated in the field to confirm resistance to glyphosate and to carry out a preliminary analysis of their behavior with respect to the parental cultivar. All transformed lines maintained resistance to the herbicide but many showed phenotypic changes. Ten RH lines that were very similar to the RA 87-3 variety were analyzed phenotypically and genetically using the 9 compulsory morphological markers proposed by The International Union for the Protection of New Varieties of Plants (UPOV) and with 339 molecular markers, respectively. Six lines of the previous ones presented minor morphological and genetic changes and were selected for field trials during two vegetative cycles (cane plant and ratoon 1), in two production areas of Argentina. The six RH lines presented agronomic, industrial and chemical composition characteristics indistinguishable from the parental cultivar. The stable inheritance of the CP4 epsps gene was confirmed by RT-qPCR and Southern blot assays in different clonal generations (growth cycles). Our studies confirmed the usefulness of genetic transformation as a complementary tool to classical improvement and highlight the advantage of the use of UPOV descriptors together with mole
Sugarcane commercial variety RA 87-3 was transformed with a genetic construct harboring the epsps... more Sugarcane commercial variety RA 87-3 was transformed with a genetic construct harboring the epsps gene from Agrobacterium strain CP4 conferring tolerance to glyphosate and nptII gene for kanamycin selection. Transformed lines were multiplied in greenhouse, and herbicide tolerance was evaluated using different concentrations (3, 4, 8 and 16 l/ha) of glyphosate (Helm 48 % p/v). All herbicide-tolerant (HT) lines were field tested to confirm glyphosate tolerance and perform preliminary evaluations of phenotypic resemblance to parental cultivar. All transformed lines maintained herbicide tolerance, but many showed phenotypic changes and/or growth aberrations. Ten HT lines, showing close growth resemblance to RA 87-3, were analyzed using nine compulsory morphologic markers proposed by the International Union for the Protection of New Varieties of Plants (UPOV) and 339 molecular markers. Out of the ten HT lines tested, six showed minor morphologic and genetic variations and were selected for field testing over two vegetative crop cycles (plant cane and first ratoon) at two production areas in Argentina. The six field-tested HT lines were found to be almost indistinguishable when comparing agro-nomic and industrial characteristics and chemical composition. Stable heritance of the CP4 epsps gene and glyphosate tolerance throughout different clonal generations were confirmed by RT-qPCR and Southern blot. Taking into account all results, two out of the six lines tested were selected for a possible commercial release. Our study confirms the utility of genetic transformation as a complementary tool to classical breeding procedures and highlights the usefulness of UPOV traits together with molecular markers for early selections of transgenic events that closely resemble their parental genotype.
Fil: Noguera, Aldo Sergio. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cie... more Fil: Noguera, Aldo Sergio. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Tucuman. Instituto de Tecnologia Agroindustrial del Noroeste Argentino. Provincia de Tucuman. Ministerio de Desarrollo Productivo. Estacion Experimental Agroindustrial "Obispo Colombres" (p). Instituto de Tecnologia Agroindustrial del Noroeste Argentino; Argentina
Fil: Rigano, Luciano Ariel. Fundacion Pablo Cassara; Argentina. Consejo Nacional de Investigacion... more Fil: Rigano, Luciano Ariel. Fundacion Pablo Cassara; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Parque Centenario. Instituto de Ciencias y Tecnologia "Dr. Cesar Milstein"; Argentina
Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. ves... more Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.
RESUMEN La variedad comercial de caña de azúcar RA 87-3 se transformó mediante biobalística con u... more RESUMEN La variedad comercial de caña de azúcar RA 87-3 se transformó mediante biobalística con una construcción genética que porta el gen epsps de la cepa CP4 de Agrobacterium tumefaciens, y el gen nptII que confieren resistencia a glifosato y resistencia a kanamicina/geneticina, respectivamente. Las líneas transformadas fueron multiplicadas en invernadero y se evaluó la resistencia al herbicida utilizando diferentes concentraciones de glifosato (3, 4 y 8 l/ha). Las líneas resistentes al herbicida (RH) se evaluaron en campo para confirmar la resistencia a glifosato (3 l/ha) y realizar un análisis preliminar del comportamiento de estas con respecto al cultivar parental. Todas las líneas transformadas mantuvieron la resistencia al herbicida pero muchas mostraron cambios fenotípicos. Las diez líneas RH que resultaron muy parecidas a la variedad RA 87-3 se analizaron fenotípica y genéticamente utilizando los nueve descriptores morfológicos obligatorios propuestos por la "International Union for the Protection of New Varieties of Plants" (UPOV) y con 339 marcadores moleculares, respectivamente. Seis líneas de las anteriores presentaron cambios morfológicos y genéticos menores y fueron seleccionadas para ensayos en campo durante dos ciclos vegetativos (caña planta y soca 1), en dos áreas de producción de Argentina. Las seis líneas RH presentaron características agronómicas, industriales y de composición química indistinguibles respecto del cultivar parental. La herencia estable del gen CP4 epsps fue confirmada mediante ensayos de RT-qPCR y Southern blot en diferentes generaciones clonales (caña planta y soca 1). Los estudios confirmaron la utilidad de la transformación genética como una herramienta complementaria a la mejora clásica, y destaca la ventaja del uso de los descriptores de UPOV junto con los marcadores moleculares para una selección temprana de eventos transgénicos que tengan un alto parecido con el genotipo parental. Tomando en cuenta los resultados, una de las seis líneas estudiadas se seleccionó para una posible liberación comercial, la cual debe ser sometida a evaluación por parte de los entes regulatorios (CONABIA, SENASA y DNMA). Palabras clave: Saccharum spp., Glifosato, gen CP4 epsps, resistencia a herbicida. ABSTRACT Development of the transgenic sugarcane event TUC 87-3RG resistant to glyphosate The commercial variety of sugarcane RA 87-3 was transformed by biobalistic with a genetic construct that carries the epsps gene of the CP4 strain of Agrobacterium tumefaciens, and with the nptII gene that confer resistance to glyphosate and resistance to kanamycin / geneticin, respectively. Transformed lines were multiplied in greenhouse and resistance to the herbicide was evaluated using different concentrations of glyphosate (3, 4 and 8 l / ha). Resistant lines to the herbicide (RH) were evaluated in the field to confirm resistance to glyphosate and to carry out a preliminary analysis of their behavior with respect to the parental cultivar. All transformed lines maintained resistance to the herbicide but many showed phenotypic changes. Ten RH lines that were very similar to the RA 87-3 variety were analyzed phenotypically and genetically using the 9 compulsory morphological markers proposed by The International Union for the Protection of New Varieties of Plants (UPOV) and with 339 molecular markers, respectively. Six lines of the previous ones presented minor morphological and genetic changes and were selected for field trials during two vegetative cycles (cane plant and ratoon 1), in two production areas of Argentina. The six RH lines presented agronomic, industrial and chemical composition characteristics indistinguishable from the parental cultivar. The stable inheritance of the CP4 epsps gene was confirmed by RT-qPCR and Southern blot assays in different clonal generations (growth cycles). Our studies confirmed the usefulness of genetic transformation as a complementary tool to classical improvement and highlight the advantage of the use of UPOV descriptors together with mole
Sugarcane commercial variety RA 87-3 was transformed with a genetic construct harboring the epsps... more Sugarcane commercial variety RA 87-3 was transformed with a genetic construct harboring the epsps gene from Agrobacterium strain CP4 conferring tolerance to glyphosate and nptII gene for kanamycin selection. Transformed lines were multiplied in greenhouse, and herbicide tolerance was evaluated using different concentrations (3, 4, 8 and 16 l/ha) of glyphosate (Helm 48 % p/v). All herbicide-tolerant (HT) lines were field tested to confirm glyphosate tolerance and perform preliminary evaluations of phenotypic resemblance to parental cultivar. All transformed lines maintained herbicide tolerance, but many showed phenotypic changes and/or growth aberrations. Ten HT lines, showing close growth resemblance to RA 87-3, were analyzed using nine compulsory morphologic markers proposed by the International Union for the Protection of New Varieties of Plants (UPOV) and 339 molecular markers. Out of the ten HT lines tested, six showed minor morphologic and genetic variations and were selected for field testing over two vegetative crop cycles (plant cane and first ratoon) at two production areas in Argentina. The six field-tested HT lines were found to be almost indistinguishable when comparing agro-nomic and industrial characteristics and chemical composition. Stable heritance of the CP4 epsps gene and glyphosate tolerance throughout different clonal generations were confirmed by RT-qPCR and Southern blot. Taking into account all results, two out of the six lines tested were selected for a possible commercial release. Our study confirms the utility of genetic transformation as a complementary tool to classical breeding procedures and highlights the usefulness of UPOV traits together with molecular markers for early selections of transgenic events that closely resemble their parental genotype.
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