RESUMEN
Sugar beet is highly sensitive to imidazolinone herbicides thus rotational restrictions exist. In order to develop imidazolinone tolerant sugar beets als gene from Arabidopsis thaliana encoding acetolactate synthase with S653N mutation was used for genetic transformation. Transgenic sugar beet plants were obtained by Agrobacterium-mediated transformation of aseptic seedlings using vacuum-infiltration. The efficiency of genetic transformation was 5.8%. RT-PCR analysis of obtained plants revealed accumulation of specific als transcript. The resistance to imidazolinone was proved for developed transgenic sugar beet plants in vitro and in greenhouse conditions after spraying with imazethapyr (Pursuit, BASF).
Asunto(s)
Beta vulgaris/efectos de los fármacos , Herbicidas/farmacología , Imidazolinas/farmacología , Ácidos Nicotínicos/farmacología , Plantas Modificadas Genéticamente/efectos de los fármacos , Rhizobium/genética , Transformación Genética , Arabidopsis/genética , Beta vulgaris/genética , Beta vulgaris/crecimiento & desarrollo , Resistencia a los Herbicidas , Herbicidas/química , Imidazolinas/química , Ácidos Nicotínicos/química , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , Semillas/efectos de los fármacos , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
Transgenic sugar beet plants carrying maize Spmn/dSpm transposable elements system have been constructed. Heterologous system of maize transposable elements Spm/dSpm was active in transgenic sugar beets that permits transposon-based gene tagging and obtaining of marker-free transgenic sugar beet.
Asunto(s)
Beta vulgaris/genética , Elementos Transponibles de ADN/genética , Genes de Plantas , Plantas Modificadas Genéticamente , Zea mays/genética , Agrobacterium tumefaciens/genética , Beta vulgaris/enzimología , Cartilla de ADN , Vectores Genéticos , Resistencia a los Herbicidas/genética , Plantas Modificadas Genéticamente/enzimología , Reacción en Cadena de la Polimerasa , Transformación Genética , Transgenes , Zea mays/enzimologíaRESUMEN
Phosphinothricin resistant plants of two rapeseed (Brassica napus L. var. oleifera DC.) spring industrial cultivars were obtained by Agrobacterium tumefaciens leaf disk transformation. Vector constructions contained the promoterless coding sequence of phosphinothricin acetyltransferase (bar) gene located between two inverted lox-sites (elements of Cre/lox recombination system of P1 phage) and selective neomycinphosphotransferase II gene (nptII). Integration of the alien genes was confirmed by the PCR analyses. Stable and linked inheritance of foreign genes in T1 and T2 progeny was shown.
Asunto(s)
Acetiltransferasas/genética , Brassica napus , Expresión Génica , Genes de Plantas , Plantas Modificadas Genéticamente , Recombinación Genética , Agrobacterium tumefaciens/patogenicidad , Aminobutiratos/farmacología , Brassica napus/genética , Brassica napus/crecimiento & desarrollo , Brassica napus/microbiología , Vectores Genéticos , Resistencia a los Herbicidas/genética , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/microbiología , Reacción en Cadena de la Polimerasa , Regiones Promotoras GenéticasRESUMEN
We demonstrate that localization of lox site between the right border of T-DNA and promoterless bar gene (RB-lox-bar-) led to its highly efficient expression in transgenic plants of Nicotiana tabacum and N. africana. Plasmid vectors used in gene integration experiments contained neomycin phosphotransferase II (npt II) gene under nos promoter as well. Transgenic plants were selected according to their capacity to grow on the medium with kanamycin and then they were tested on the selective medium containing phosphinothricin. 80% of transgenic plants expressed bar gene at the level similar to that in plants transformed with the bar gene under widely used constitutive promoter. Transformation of plants with the plasmid vector containing only promoterless bar gene near T-DNA right border (RB-bar-) and with the vector containing lox site and promoterless bar gene in the middle of the construction (-lox-bar-) led to obtaining no more than 4.5% of transgenic plants resistant to phosphinothricin. PCR analyses confirmed both the absence of tandem repeats and of plasmid recombination resulting in transference of bar gene under promoter in plasmid vector. Nos-terminator situated between the lox site and the right border of T-DNA did not decrease bar gene expression.
Asunto(s)
Expresión Génica , Genes de Plantas , Nicotiana/genética , Plantas Modificadas Genéticamente , Recombinación Genética , ADN Bacteriano/genética , ADN de Plantas/genética , Vectores Genéticos/genética , Integrasas/genética , Plásmidos/genética , Regiones Promotoras Genéticas , Rhizobium/genética , Proteínas Virales/genéticaRESUMEN
Transgenic pea (Pisum sativum L.) plants containing mutant ahas/als gene were obtained using Agrobacterium-mediated genetic transformation. Transformation has been carried out using cocultivation of pea explants with Agrobacterium tumefaciens strain lBA4404 carrying genetic vectors pCB004, pCB006 and pCB007 containing ahas/als and nptII genes. The presence of transferred genes in the genomes of transgenic plants has been confirmed by PCR analysis.
Asunto(s)
Ingeniería Genética/métodos , Herbicidas , Ácidos Nicotínicos , Pisum sativum/genética , Plantas Modificadas Genéticamente/genética , Transformación Genética , Resistencia a Medicamentos/genética , Genes de Plantas/genética , Mutación , Pisum sativum/crecimiento & desarrollo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Reacción en Cadena de la PolimerasaRESUMEN
Normal phenotype sugarbeet plants transformed with Agrobacterium rhizogenes were produced using direct regeneration from explants without hairy root phase. Kanamycin resistant plants and Ri-roots carrying the genes of neomycin phosphotransferase II and b-glucuronidase have been obtained. Integration of transgenes into sugarbeet genome was confirmed with GUS-assay and PCR using primers for the introduced genes.
Asunto(s)
Beta vulgaris/crecimiento & desarrollo , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Rhizobium/genética , Transformación Genética , Beta vulgaris/genética , Beta vulgaris/microbiología , ADN de Plantas/análisis , Glucuronidasa/biosíntesis , Inmunohistoquímica , Proteínas de Plantas/biosíntesis , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/microbiología , Reacción en Cadena de la Polimerasa , Rizoma/genética , Rizoma/crecimiento & desarrollo , Rizoma/microbiologíaRESUMEN
A method of Agrobacterium-mediated genetic transformation of sugarbeet (Beta vulgaris L.) with vacuum infiltration has been developed. Aseptic 3-weeks old etiolated seedlings of two diploid O-type sugarbeet lines (KS3 and KS7) have been used for genetic transformation. Transgenic sugarbeet plants carrying the reporter beta-glucuronidase gene have been selected for their resistance to glufosinate ammonium herbicide. Integration of transgenes into sugarbeet genome was confirmed with GUS assay and PCR using primers for bar and gusA genes.
Asunto(s)
Aminobutiratos/farmacología , Beta vulgaris/genética , Plantas Modificadas Genéticamente , Agrobacterium tumefaciens/genética , Beta vulgaris/efectos de los fármacos , Resistencia a Medicamentos/genética , Glucuronidasa/genética , Glucuronidasa/metabolismo , Herbicidas/farmacología , Plantas Modificadas Genéticamente/metabolismo , Regeneración , Transformación GenéticaRESUMEN
An efficient genetic transformation method for african tobacco Nicotiana africana Merxm. has been established. African tobacco is a valuable source for cytoplasmic male sterility (CMS) and nuclear encoded resistance to potato virus Y (PVY). N. africana transgenic plants have been obtained using both Agrobacterium-mediated and direct transformation of leaf explants with gold particle bombardment using particle inflow gun. Plasmid vectors containing phosphinothricin resistance gene (bar gene) coding region without promoter and independent 35S promoter between lox sites (lox-bar-35S-lox) and nptII gene were used. Transgenic plants were selected according to growth capacity on the selective medium containing 50 mg/l kanamycin. PCR analyses of kanamycin-resistant plants confirmed the presence of nptII and bar genes in their genome. Agrobacterium-mediated transformation of root explants has proved to be the most efficient transformation method for N. africana.
Asunto(s)
Genes de Plantas/genética , Nicotiana/genética , Plantas Modificadas Genéticamente , Plásmidos/genética , Recombinación Genética , Transformación Genética/genética , Agrobacterium tumefaciens/genética , Clonación Molecular , Genes Bacterianos/genética , Vectores Genéticos/genética , Raíces de Plantas/genética , Semillas/genéticaRESUMEN
New model system of plastid transformation has been proposed using a wild representative of Solanaceae family--S. sinuata. Earlier obtained cybrid plants N. tabacum (+ S. sinuata) were used for transformation experiments by PEG treatment of protoplasts with aadA gene that confers resistance to spectinomycin. Transformed S. sinuata plastome was transferred from N. tabacum (+ S. sinuata) cybrid to S. sinuata wild type plants by somatic hybridization. Molecular analysis of nuclear and mitochondrial DNA has been performed.
Asunto(s)
ADN de Plantas/genética , Ingeniería Genética , Nicotiana/genética , Plantas Modificadas Genéticamente/genética , Plastidios/genética , Solanaceae/genética , ADN Mitocondrial/genética , ADN Espaciador Ribosómico/genética , Reacción en Cadena de la Polimerasa , Recombinación GenéticaRESUMEN
The Orychophragmus violaceus chlorophylldefective line of "albino" type has been obtained by spectinomycin treatment. Somatic hybridization between Orychophragmus violaceus and Brassica napus was performed by fusion of green mesophyll protoplasts of rape and callus protoplasts of the O. violaceus "albino" line. Near two hundred of regenerant plants were selected according to the regeneration type and ability to become green, and were determined as hybrids. Chloroplast DNA in selected hybrids was identical to rape chlDNA, which was confirmed by the PCR-RFLP analysis of plastid DNA fragments. Fragments of hybrid mitochondrial DNA analyzed by the PCR-RFLP analysis were identical to fragments of O. violaceus. The nuclear genome of the majority of hybrids was represented by the O. violaceus genome, which was demonstrated by analyses of isoenzymes, DNA telomeric sequences, ribosomal and satellite DNAs, and the RAPD analysis. The cytogenetic analysis of a number of lines has shown variability in the number of chromosomes in the obtained lines.
Asunto(s)
Brassica napus/genética , Brassicaceae/genética , Cloroplastos/genética , Hibridación Genética , Brassica napus/enzimología , Brassica napus/ultraestructura , Brassicaceae/enzimología , Brassicaceae/ultraestructura , Cromosomas de las Plantas/ultraestructura , ADN Mitocondrial/genética , Proteínas de Plantas/genética , Protoplastos/metabolismo , Técnica del ADN Polimorfo Amplificado Aleatorio , Selección GenéticaRESUMEN
Transposon mediated insertional mutagenesis is one of the approaches for the unique gene cloning. A wild species of Cruciferae family Orychophragmus violaceus (L.) O.E. Schulz, which is of interest for practical breeding as a donor of improved plant oil, was an object of the investigation. Plasmid construction used in the experiments included selective NPT II gene, reported GUS gene serving as an excision marker, structural BAR gene located within the dSpm element and Spm transposase. The GUS gene of this plasmid had not his own promoter and became functional only after Spm-transposition. Transformed Orychophragmus violaceus (L.) O.E. Schulz. plants were obtained by direct mesophyll protoplast transformation as well as Agrobacterium tumefaciens-mediated root explant transformation. Gene transfer and the transposition event were confirmed by the GUS activity and the PCR analysis. Relative transformation efficiency using protoplasts was 5.8%.