RESUMO
The anatomical research of the vegetative organs of the cytoplasmatic hybrid in vitro plants containing the Brassica napus nucleus and the Lesquerella fendleri chloroplasts had been conducted in comparison to the parental forms. It was found, that the anatomical structure of the cybrid had been similar to rapeseed. Anomalous changes in the epithelial, parenchymal and connective tissue of the leaf, stalk, stem, and root of the cybrid were detected. The appearance of the anatomical defects can be explained by nuclear-cytoplasmatic incompatibility which is the cause of low adaptability of the cybrid to in vivo conditions and takes place due to alien chloroplast gene expression in the remote species.
Assuntos
Brassica napus/anatomia & histologia , Brassicaceae/anatomia & histologia , Núcleo Celular/ultraestrutura , Quimera , Cloroplastos/ultraestrutura , Citoplasma/ultraestrutura , Brassica napus/genética , Brassicaceae/genética , Núcleo Celular/genética , Cloroplastos/genética , Cruzamentos Genéticos , Citoplasma/genética , Regulação da Expressão Gênica de Plantas , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/genética , Caules de Planta/anatomia & histologia , Caules de Planta/genéticaRESUMO
This work is the first to our knowledge to describe the successful attempt of Agrobacterium rhizogenes-mediated transformation of topinambour in order to obtain the transgenic H. tuberosus plants, callus and "hairy" root cultures. The plasmid vectors contained the sequence of interferon gene fused with Nicotiana plumbagenifolia L. calreticulin apoplast targeting signal driven by 35S CaMV promoter or root-specific Mll promoter. Nearly 75% isolated Ri-root lines and callus cultures were proved (by PCR analysis) to contain HuINFa-2b transgene. We also managed to obtain H. tuberosus transgenic plants through somatic embryogenesis on the transgenic "hairy" root culture. The obtained transgenic H. tuberosus cultures exhibited high-level antiviral activity that ranged from 2000 to 54500 IU/g FW that makes this crop considered a promising source of recombinant interferon alpha 2b protein.
Assuntos
Agrobacterium tumefaciens/genética , Antivirais/isolamento & purificação , Helianthus/genética , Fatores Imunológicos/biossíntese , Interferon-alfa/biossíntese , Plantas Geneticamente Modificadas , Animais , Antivirais/farmacologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/virologia , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Helianthus/metabolismo , Helianthus/microbiologia , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/farmacologia , Interferon alfa-2 , Interferon-alfa/genética , Interferon-alfa/isolamento & purificação , Interferon-alfa/farmacologia , Masculino , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Técnicas de Embriogênese Somática de Plantas/métodos , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Suínos , Testículo/efeitos dos fármacos , Testículo/imunologia , Testículo/virologia , Técnicas de Cultura de Tecidos , Nicotiana/química , Nicotiana/genética , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimentoRESUMO
In order to investigate the high temperature tolerance of spring canola plants (Brassica napus L.) constitutively expressing cyp11A1 gene which encodes bovine cytochrome P450(scc) the growth features were analyzed under short time heat stress (42 degrees C) in growth chamber. Earlier it was documented that results of the heat tolerance test positively correlated with improvement of high temperature resistance in field trial. Higher relative water content (by 13%) and superoxide dismutase (SOD) activity, lower electrolyte leakage (up 1.4-fold) and smaller increase in chlorophyll a and carotenoid contents in cyp11A1 canola leaves in comparison with wild-type plants under stress allowed to conclude cyp11A1 plants are more tolerant to high temperature than the control ones. We suppose that SOD activity increase which revealed in our transgenic canola in normal condition plays the defining role in the biochemical alterations in plant metabolism for the thermotolerance improvement. SOD activity increment could be caused by heterologous cytochrome P450(scc) activity which resulted in the superoxide radical formation. Cyp11A1 canola plants might be resistant to the other stress conditions of different origin.
Assuntos
Brassica napus/enzimologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Temperatura Alta , Plantas Geneticamente Modificadas/enzimologia , Estresse Fisiológico , Animais , Antioxidantes/metabolismo , Brassica napus/genética , Brassica napus/crescimento & desenvolvimento , Bovinos , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Fotossíntese/fisiologia , Pigmentos Biológicos/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Estresse Fisiológico/genéticaRESUMO
Spring rapeseed transgenic lines expressing human interferon alpha 2b were created by Agrobacterium-mediated transformation of aseptic plant leaf explants. The maximum antiviral activity of the leaf extracts reached 4500 IU/g fresh weight. It was determined that the antioxidant activity and the activity of an enzyme of plant antioxidant system--superoxide dismutase (SOD)--in the leaf tissues of transgenic plants increased compared to controls. There were no correlations between the interferon and antioxidant activities, as well as between SOD and interferon activities. Using the obtained transgenic rapeseed plants with high interferon and antioxidant activities as a feed additive for animals might have preventive effect on their body, increasing resistance to infections of various origins.
Assuntos
Brassica napus/genética , Interferon-alfa/genética , Plantas Geneticamente Modificadas/genética , Animais , Antioxidantes/metabolismo , Brassica napus/química , Brassica napus/enzimologia , Células Cultivadas , Efeito Citopatogênico Viral/efeitos dos fármacos , Humanos , Interferon alfa-2 , Extratos Vegetais/farmacologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/enzimologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Superóxido Dismutase/metabolismo , Suínos , Transformação Genética , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacosRESUMO
Phosphinothricin (PPT) resistant hybrid plants between Brassica napus L. cv. Kalinovsky and Orychophragmus violaceus (L.) O.E. Shulz. were obtained as a result of somatic hybridization experiments. The hybrids inherited PPT resistance from O. violaceus plants which were previously transformed by the vector containing Spm/dSpm Zea mays transposon system with bar gene located within the nonautonomous transposon. The obtained plants had intermediate morphology. Their hybrid nature has been confirmed by isozyme (esterase and amilase activity) and PCR (bar, gus, Spm/dSpm integration) analyses. The hybrids combined B. napus plastom and O. violaceus mithochondrion that was revealed by PCR-RFLP. The hybrid plants might be included to rapeseed breeding programme after examination of their oil quality as well as to chloroplast transformation experiments that is still urgent for B. napus.
Assuntos
Aminobutiratos/farmacologia , Brassica napus/genética , Brassicaceae/genética , Herbicidas/farmacologia , Hibridização Genética , Plantas Geneticamente Modificadas , Brassica napus/efeitos dos fármacos , Brassica napus/crescimento & desenvolvimento , Brassicaceae/efeitos dos fármacos , Brassicaceae/crescimento & desenvolvimento , Cloroplastos , Elementos de DNA Transponíveis/genética , DNA de Plantas/genética , Resistência a Herbicidas/genética , Zea mays/genéticaRESUMO
Transferring of Lesquerella fendleri genetically transformed plastids to Brassica napus plants has been performed with the somatic hybridization method. The plastome of the previously engineered transplastomic L. fendleri plants contained the aadA16gfp selective marker gene conferring spectinomycin/streptomycin resistance and green fluorescence under UV light. The protoplasts of B. napus chlorophyll-deficient plants were fused with gamma-irradiated protoplasts of L. fendleri transplastomic plants. A total of 59 green hybrid colonies have been isolated followed by spectinomycin/streptomycin selection. Shoot regeneration has been observed for two cell lines. Morphologically normal plants have been regenerated for one of them. PCR and isozyme analyses showed that the plants were transplastomic cybrids containing B. napus nuclei and L. fendleri transformed chloroplasts.
Assuntos
Brassica napus/crescimento & desenvolvimento , Cloroplastos/genética , Plantas Geneticamente Modificadas , Transformação Genética , Brassica napus/genética , Brassicaceae/genética , Brassicaceae/crescimento & desenvolvimento , DNA de Plantas/genética , Genes de Plantas , Engenharia Genética , Proteínas de Fluorescência Verde , Reação em Cadeia da Polimerase , Espectinomicina/farmacologia , Estreptomicina/farmacologia , Raios UltravioletaRESUMO
Data concerning plant transposable elements and their contribution to plant genome evolution are reviewed. Much attention is focused on utilization of transgenic plants as heterologous hosts of transposons for investigation of transposition mechanisms and gene cloning. Probable ways of the use of plant transposons as genetic tools in biotechnology are discussed.
Assuntos
Biotecnologia , Elementos de DNA Transponíveis , Evolução Molecular , Genética , Plantas/genéticaRESUMO
Plastid genetic transformation has been performed using both the PEG-treatment of protoplasts of somatic hybrids of B. oleracea carrying A. thaliana chloroplasts and the particle bombardment of regenerable calluses of B. napus sv. Westar. The chloroplast transformation vector pCB040 carried resistance (aadA) gene flanked by rapeseed plastid DNA sequences to target its insertion between the trnV-rps7 fragments. Selection of transplastomic cell lines has been performed according to their ability to grow on the medium supplied with spectinomycin and streptomycin in high concentrations. Antibiotic resistant cell lines have been obtained using the both transformation methods. The presence of the aadA gene in the A. thaliana and B. napus plastomes was confirmed by PCR analysis for two cell lines of B. oleracea (+ A. thaliana) and three lines of B. napus.
Assuntos
Arabidopsis/genética , Brassica/genética , Plantas Geneticamente Modificadas , Espectinomicina/farmacologia , Estreptomicina/farmacologia , Transformação Genética , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Brassica/citologia , Brassica/efeitos dos fármacos , Brassica napus/citologia , Brassica napus/efeitos dos fármacos , Brassica napus/genética , Cloroplastos/efeitos dos fármacos , DNA de Plantas/genética , Genes de Plantas , Engenharia Genética , Plasmídeos , Reação em Cadeia da Polimerase , Protoplastos/efeitos dos fármacos , Seleção GenéticaRESUMO
Functionally asymmetric somatic hybrids possessing heterologous transposable element Spm/dSpm were obtained following intertribal somatic hybridization between Brassica napus and transgenic Arabidopsis thaliana. Mobile genetic elements actively transposed in the hybrid genomes. Complete elimination of A. thaliana genome was not observed.
Assuntos
Arabidopsis/genética , Brassica napus/genética , Elementos de DNA Transponíveis/genética , DNA de Plantas/genética , Hibridização Genética/genética , Zea mays/genética , Genes de Plantas , Plantas Geneticamente Modificadas , Transcrição GênicaRESUMO
Intertribal somatic hybridization between wild type Brassica juncea (L.) Czern. & Coss. and transgenic A. thaliana L. has been carried out. Genome of A. thaliana plants contained heterologous transposable element Spm/dSpm, reporter GUS gene, selective genes for kanamycin- (npt II) and phosphinothricin (bar) resistance. Hybrid nature of obtained plants was confirmed with their morphology, GUS hystochemical assay, PCR-RFLP, RAPD and isozyme analyses. It was determined that heterologous transposable element Spm/dSpm is able to function in hybrid plants. There was no complete elimination of A. thaliana genetic material in the hybrids and the transgenes were stably maintained.
Assuntos
Arabidopsis/genética , Elementos de DNA Transponíveis/genética , DNA de Plantas/genética , Hibridização Genética/genética , Mostardeira/genética , Plantas Geneticamente Modificadas , Amilases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Primers do DNA , Eletroforese em Gel de Ágar , Esterases/metabolismo , Genes de Plantas/genética , Genes Reporter/genética , Vetores Genéticos , Mostardeira/enzimologia , Mostardeira/crescimento & desenvolvimento , Brotos de Planta/enzimologia , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Protoplastos/enzimologia , Seleção GenéticaRESUMO
A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) by vacuum infiltration has been developed. Transgenic sugarbeet plants of Ukraine breeding were selected for their resistance either to the antibiotic kanamycin or to the herbicide glufosinate ammonium. Integration of transgenes was confirmed by PCR and GUS-assay.
