RESUMEN
Abstract we report A. rhizogenes-induced hairy root formation in S. bryopteris, a medicinally and commercially important plant. A. rhizogenes strain LBA1334 co-cultivated with explants (root, rhizophore, stem portion near the root, and stem with intact fronds) for 24 and 48 h after transformation for induction of hairy roots. The induction of hairy root was observed after 6 days of infection in case of 48 h co-cultivation only. PCR with rolA and virC gene specific primers confirmed the induced hairy roots were due to Ri T-DNA integration and not due to contaminating A. rhizogenes. The root network as explants showed the maximum transformation efficiency. We tested different media like MS, SHFR (Stage Hog Fern Root) and KNOP's during transformation for hairy root induction. The SHFR based media showed good response in transformation as well as propagation. Further, transformation efficiency was enhanced by addition of TDZ (2 mg/L) and Bevistin (0.1%) in SHFR media. The present work would be helpful in hairy roots-based in vitro production of secondary metabolites and on aspect of functional genomics of S. bryopteris.
Asunto(s)
Transformación Genética/genética , Reacción en Cadena de la Polimerasa , Selaginellaceae/microbiología , Agrobacterium/genética , GenómicaRESUMEN
Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have developed a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito haemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Cx. quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Cx. quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions.
Asunto(s)
Animales , Culex/genética , Expresión Génica/genética , Insectos Vectores/genética , Proteínas Luminiscentes/genética , Transformación Genética/genéticaRESUMEN
The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.
A transformação das plântulas de café com o gene cry1ac de Bacillus thuringiensis foi realizada por biobalística, utilizando todo o pUBC plasmídeo ou só o cassete genético UBI-cry1ac-nos. O gene cry1ac foi inserido no cafeeiro a fim de conferir resistência à folha mineiro Leucoptera coffeella, um inseto responsável por perdas consideráveis nas culturas de café. Tendo em conta que ao plasmídeo e ao cassete genético utilizados para este estudo faltam genes repórteres e/ou de seleção, os parâmetros para o processo de transformação por biobalística foram previamente padronizados com um plasmídeo transportando o gene repórter gus. A presença do gene cry1ac em tecidos de jovens plântulas foi determinada por PCR, Southern blot e transcrição reversa-PCR. Nossos resultados mostram que a obtenção de plântulas de café, transformado por bombardeamento com o gene cry1ac sem genes de seleção genética nem repórteres é viável.
Asunto(s)
Animales , Proteínas Bacterianas/genética , Coffea/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Plantas Modificadas Genéticamente/genética , Transformación Genética/genética , Western Blotting , Biolística/métodos , Lepidópteros , Reacción en Cadena de la Polimerasa , Enfermedades de las Plantas/parasitología , Enfermedades de las Plantas/prevención & controlRESUMEN
The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.
Asunto(s)
Animales , Masculino , Femenino , Aedes/genética , Elementos Transponibles de ADN/genética , Técnicas de Transferencia de Gen , Insectos Vectores/genética , Transformación Genética/genética , Genes de Insecto , Células Germinativas , MicroinyeccionesRESUMEN
A system for the genetic transformation of maize was developed for two Costa Rican varieties: CR-7 and Diamantes 8843, that can allow the subsequent transfer of viral-derived genes in order to confer resistance to the disease caused by maize rayado fino virus (MRFV). The method is based on particle bombardment of organogenic calli derived from shoot tips. On the other hand, the molecular construction pRFcp-bar, containing the coat protein gene of MRFV and the marker gene bar, was elaborated. For the visual selection of the transformed material was used also the plasmid pDM803 that contains the reporter gene uidA (GUS). The results indicate that devices evaluated: the PIG ("Particle Inflow Gun") and the Bio-Rad are both enough efficient to transfer foreign genes to the genome of the maize.
Asunto(s)
Plantas Modificadas Genéticamente/virología , Transformación Genética/genética , Zea mays/genética , Biolística , Costa Rica , Virus de Plantas/genética , Virus de Plantas/patogenicidad , Zea mays/virologíaRESUMEN
Penicillium griseoroseum, a deuteromycete fungus producer of pectinolytic enzymes, was transformed with a gene encoding for green fluorescent protein (GFP). The selection of transformants was based on the homologous nitrate reductase gene (niaD). Protoplasts of a P. griseoroseum Nia mutant (PG63) were co-transformed with the plasmids pNPG1 and pAN52-1-GFP. The plasmid pNPG-1 carries the homologous niaD gene and pAN52-1-GFP carries the SGFP-TYG version of GFP. The highest transformation efficiency (102 transformants/µg of pNPG1) resulted from the utilization of equimolar amounts of transforming and co-transforming vectors. Analysis of pAN52-1-GFP insertions into the genomic DNA of the transformants revealed single and multiple copy integrations. The transformants possessing a single copy of the gfp gene showed a low level of fluorescence, whereas multicopy transformants displayed strong fluorescence under visualization with fluorescent light. The transformants showing high expression of the gfp gene had the normal mycelia pigmentation altered, displaying a bright green-yellowish color, visible with the naked eye on the plates, without the aid of any kind of fluorescent light or special filter set.
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ADN de Hongos/genética , Genoma Fúngico , Proteínas Luminiscentes/genética , Mutación , Penicillium/genética , Transformación Genética/genética , Proteínas Luminiscentes/análisis , Microscopía Fluorescente , Penicillium/enzimología , Plásmidos/genética , Poligalacturonasa/genética , Protoplastos/enzimologíaRESUMEN
Este artículo discute algunas de las posibilidades aplicaciones de técnicas de modificación genética para introducir algunos compuestos en plantas o animales. El potencial de estas técnicas de modificación de plantas es muy amplio y va desde mejoras en la producción de alimentos para consumo humano y para el desarrollo pecuario, pasando por la producción de anticuerpos o proteínas para uso terapéutico hasta modificaciones que implican la inclusión de nutrientes para mejorar el valor nutritivo de un alimento y la producción de vacuna. Debe tenerse presente, sin embargo, que el alcance o las consecuencias de estas modificaciones no están totalmente dilucidadas. Hay preocupación por efectos secundarios o la aparición de nuevos virus por recombinación genética que no han sido totalmente descartados, aunque la tendencia es considerar el proceso como seguro, Finalmente se presentan evidencias sobre la posibilidad de introducir en vegetales la capacidad de sintetizar vitamina A o de producir arroz con un alto contenido de hierro como alternativas reales para combatir algunas de las deficiencias nutricionales mas importantes a nivel mundial