RESUMO
Wheat (Triticum aestivum L. var. Shiranekomugi) seeds were soaked in water at 22 degrees C for 1 d. Thereafter, the embryo of the soaked seeds was inoculated with Agrobacterium tumefaciens by piercing a region of the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were incubated at 22 degrees C for 2 d and sterilized by cefotaxime (Claforan) (1000 ppm water solution) treatment and then vernalized at 5 degrees C for 25 d. Finally, the seedlings were grown to maturation (T(0) plants) and allowed to pollinate naturally for seed setting (T(1) plants) in pots under nonsterile condition. To examine the transformation by various means, four different strains of A. tumefaciens were used for transformation. The following five lines of evidence proved the transformation: altered phenotype and its transmittance to the next generation, resistance of T(1) seed germination to geneticin or hygromycin B, the detection of a transgene in T(1) plants by PCR analysis and Southern hybridization and the rescue of the plasmid consisting of the integrated T-DNA and flanking wheat genome DNA from T(1) plants. The transformation efficiency of T(1) plants, which were transformed using different A. tumefaciens strains, was estimated to be 33% by PCR analysis, 75% by Southern hybridization and 40% by plasmid rescue.
Assuntos
Agrobacterium tumefaciens/genética , Sementes/genética , Transformação Genética , Triticum/genética , Vetores Genéticos/genética , Plantas Geneticamente Modificadas , Plasmídeos/genética , Sementes/microbiologiaRESUMO
Seeds of rice (Oryza sativa L. var. Koshihikari) were soaked in water for 2 d. Thereafter, the embryo containing an apical meristem was inoculated with Agrobacterium tumefaciens by piercing a site of the husk overlying the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were then grown to maturation (T0 plants) and allowed to pollinate naturally to set seeds (T1 plants) in pots under nonsterile conditions. To examine the transformation by various means, three different strains of A. tumefaciens were used for transformation: an M-21 mutant, which is an avirulent mutant with a Tn5 insertion in the iaaM gene, and two LBA4404 strains each with a different binary vector. Five different lines of evidence were demonstrated the transformation: the altered phenotype and its inheritance by the next generation, histochemical detection of beta-glucuronidase, resistance to hygromycin B, detection of the transgene by PCR and rescue of a plasmid consisting of the integrated T-DNA and the flanking rice genome DNA. Transformation efficiency of T1 plants was estimated to be 40% and 43% by PCR and a histochemical assay of beta-glucuronidase, respectively.
Assuntos
Agrobacterium tumefaciens/genética , Técnicas de Transferência de Genes , Oryza/genética , Transformação Genética , Proteínas de Bactérias/genética , Sequência de Bases , Elementos de DNA Transponíveis/genética , Vetores Genéticos/genética , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos/genética , Sementes/genética , TransgenesRESUMO
Kenaf was transformed by inoculation of Agrobacterium tumefaciens onto the meristems of young plants in pots. The transformation was demonstrated by three lines of evidence: a phenotypic inheritance from T(0) to T(1) plants, detection of the transgene in both T(0) and T(1) plants, and rescue of plasmids composed of T-DNA of the binary vector and flanking plant genomic DNA from T(1) plants.
RESUMO
Chromosomal virulence genes acvB, abvA, chvA of Agrobacterium tumefaciens were cloned with the technique of transposon 5 insertion. The chromosome genes are necessary for Agrobacterium tumfaciens absorbing to cell ular surface of plant, the adherence reaction can't be executed and result in losing the toxicity if mutations are occurred in some chromosome genes. The chromosome toxicity gene is inactivated due to transposon Tn5 be inserted and the accept ant cell infected with Agrobacterium tumefaciens can't cause tumor ultimately. This article briefly introduces the research way of thinking and strategy of this technique and the important roles of every gene, which are taken of in the process of T-DNA's form, transfer, integration, and expression etc. This article also gives a presumption to T-DNA's transport: The plant cell wall's porin may be T-DNA's natural channel.