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Registro Completo |
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Biblioteca(s): |
Embrapa Café. |
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Data corrente: |
06/05/2019 |
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Data da última atualização: |
06/05/2019 |
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Tipo da produção científica: |
Artigo em Periódico Indexado |
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Autoria: |
VARGAS-GUEVARA, C.; VARGAS-SEGURA, C.; VILLALTA-VILLALOBOS, J.; PEREIRA, L. F. P.; GATICA-ARIAS, A. |
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Afiliação: |
Cristian Vargas-Guevara, Laboratorio Biotecnología de Plantas/Escuela de Biología/Universidad de Costa Rica; César Vargas-Segura, Laboratorio Biotecnología de Plantas/Escuela de Biología/Universidad de Costa Rica; Jimmy Villalta-Villalobos, Laboratorio Biotecnología de Plantas/Escuela de Biología/Universidad de Costa Rica; LUIZ FILIPE PROTASIO PEREIRA, CNPCa; Andrés Gatica-Arias, Laboratorio Biotecnología de Plantas/Escuela de Biología/Universidad de Costa Rica. |
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Título: |
A simple and efficient agroinfiltration method in coffee leaves (Coffea arabica L.): assessment of factors affecting transgene expression. |
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Ano de publicação: |
2018 |
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Fonte/Imprenta: |
3 Biotech, v. 8, n. 11, 2018 |
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Idioma: |
Português |
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Conteúdo: |
The establishment of a simple, rapid and efficient transient expression system is a necessary tool for the functional validation of candidate genes in coffee biotechnology. The effects of Agrobacterium strain, age of the donor plant, infiltration method, and infiltration medium on transgene expression in detached coffee leaves were evaluated. Regarding the effect of Agrobacterium strain, the expression of uidA was higher in GV3101-treated coffee disks than in LBA4404 and ATHV-treated samples. On the other hand, transient expression of uidA was significantly higher in leaf disks from young plants (6-weeks-old) (13.1 ± 1.4%) than in mature tissue (12-weeks-old) (1.6 ± 1.2%). Transient uidA expression was higher in detached coffee leaf disks from young plants infiltrated with one injection of 15 μL of Agrobacterium strain GV3101::1303 suspended in MS salts supplemented with 30 g/L sucrose, 1.9 g/L MES and 200 uM AS with subsequent sanding of the abaxial epidermis. Using the optimized protocol, expression of the uidA gene was observed 6, 24 and 48 h and 5 weeks after bacterial injection. DNA was extracted from coffee disks with positive GUS expression and specific mgfp5 and uidA fragments were amplified 5 weeks post-agroinfiltration. On the other hand, using the optimized protocol, a specific cry10Aa (500 bp) fragment was amplified in the agro-infiltrated coffee leaf disks 5 weeks post-agroinfiltration with the plasmid pB427-35S-cry10Aa. Moreover, the expression of the gene cry10Aa in two infiltrated coffee leaf disks was verified by RT-PCR and an expected 500 bp fragment was amplified. MenosThe establishment of a simple, rapid and efficient transient expression system is a necessary tool for the functional validation of candidate genes in coffee biotechnology. The effects of Agrobacterium strain, age of the donor plant, infiltration method, and infiltration medium on transgene expression in detached coffee leaves were evaluated. Regarding the effect of Agrobacterium strain, the expression of uidA was higher in GV3101-treated coffee disks than in LBA4404 and ATHV-treated samples. On the other hand, transient expression of uidA was significantly higher in leaf disks from young plants (6-weeks-old) (13.1 ± 1.4%) than in mature tissue (12-weeks-old) (1.6 ± 1.2%). Transient uidA expression was higher in detached coffee leaf disks from young plants infiltrated with one injection of 15 μL of Agrobacterium strain GV3101::1303 suspended in MS salts supplemented with 30 g/L sucrose, 1.9 g/L MES and 200 uM AS with subsequent sanding of the abaxial epidermis. Using the optimized protocol, expression of the uidA gene was observed 6, 24 and 48 h and 5 weeks after bacterial injection. DNA was extracted from coffee disks with positive GUS expression and specific mgfp5 and uidA fragments were amplified 5 weeks post-agroinfiltration. On the other hand, using the optimized protocol, a specific cry10Aa (500 bp) fragment was amplified in the agro-infiltrated coffee leaf disks 5 weeks post-agroinfiltration with the plasmid pB427-35S-cry10Aa. Moreover, the expression of the gene... Mostrar Tudo |
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Palavras-Chave: |
In planta genetic transformation; Leaf agroinfection. |
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Thesagro: |
Agrobacterium Tumefaciens; Bacillus Thuringiensis. |
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Thesaurus Nal: |
Genetic transformation. |
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Categoria do assunto: |
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Marc: |
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Registro original: |
Embrapa Café (CNPCa) |
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