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Mediated Transformation (mediated + transformation)
Selected AbstractsTargeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in transgenic silkwormsINSECT MOLECULAR BIOLOGY, Issue 5 2007S. Kanginakudru Abstract RNA interference (RNAi)-mediated viral inhibition has been used in a few organisms for eliciting viral resistance. In the present study, we report the use of RNAi in preventing baculovirus infection in a lepidopteran. We targeted the baculoviral immediate early-1 (ie-1) gene in both a transformed lepidopteran cell line and in the transgenic silkworm Bombyx mori L. Constitutive expression of double-stranded RNA was achieved by piggyBac -mediated transformation of Sf9 cell line with a transgene encoding double-stranded ie-1 RNA (dsie-1). Strong viral repression was seen at early stages of infection but subsequent recovery of viral proliferation was observed. In contrast, the same transgene inserted into the chromosomes of transgenic silkworms induced long-term inhibition of B. mori nucleopolyhedrovirus infection, with nearly 40% protection compared with nontransgenic animals. Protection was efficient at larval stages after oral infection with occlusion bodies or hemocoel injection of budded viruses. Virus injected pupae also displayed resistance. These results show that heritable RNAi can be used to protect silkworm strains from baculovirus infection. [source] Advances in Research on Genetically Engineered Plants for Metal ResistanceJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 11 2006Ri-Qing Zhang Abstract The engineering application of natural hyperaccumulators in removing or inactivating metal pollutants from soil and surface water in field trials mostly presents the insurmountable shortcoming of low efficiency owing to their little biomass and slow growth. Based on further understanding of the molecular mechanism of metal uptake, translocation, and also the separation, identification, and cloning of some related functional genes, this article highlights and summarizes in detail the advances in research on transgenic techniques, such as Agrobacterium tumefaciens -mediated transformation and particle bombardment, in breeding of plants for metal resistance and accumulation, and points out that deepening the development of transgenic plants is one of the efficient approaches to improving phytoremediation efficiency of metal-contaminated environments. From the viewpoint of sustainable development, governments should strengthen support to the development of genetic engineering for metal resistance and accumulation in plants. (Managing editor: Li-Hui Zhao) [source] Colonization process of olive tissues by Verticillium dahliae and its in planta interaction with the biocontrol root endophyte Pseudomonas fluorescens PICF7MICROBIAL BIOTECHNOLOGY, Issue 4 2009Pilar Prieto Summary The colonization process of Olea europaea by the defoliating pathotype of Verticillium dahliae, and the in planta interaction with the endophytic, biocontrol strain Pseudomonas fluorescens PICF7 were determined. Differential fluorescent protein tagging was used for the simultaneous visualization of P. fluorescens PICF7 and V. dahliae in olive tissues. Olive plants were bacterized with PICF7 and then transferred to V. dahliae -infested soil. Monitoring olive colonization events by V. dahliae and its interaction with PICF7 was conducted using a non-gnotobiotic system, confocal laser scanner microscopy and tissue vibratoming sections. A yellow fluorescently tagged V. dahliae derivative (VDAT-36I) was obtained by Agrobacterium tumefaciens -mediated transformation. Isolate VDAT-36I quickly colonized olive root surface, successfully invaded root cortex and vascular tissues via macro- and micro-breakages, and progressed to the aerial parts of the plant through xylem vessel cells. Strain PICF7 used root hairs as preferred penetration site, and once established on/in root tissues, hindered pathogen colonization. For the first time using this approach, the entire colonization process of a woody plant by V. dahliae is reported. Early and localized root surface and root endophytic colonization by P. fluorescens PICF7 is needed to impair full progress of verticillium wilt epidemics in olive. [source] Cladosporium fulvum (syn. Passalora fulva), a highly specialized plant pathogen as a model for functional studies on plant pathogenic MycosphaerellaceaeMOLECULAR PLANT PATHOLOGY, Issue 4 2005BART P. H. J. THOMMA SUMMARY Taxonomy:,Cladosporium fulvum is an asexual fungus for which no sexual stage is currently known. Molecular data, however, support C. fulvum as a member of the Mycosphaerellaceae, clustering with other taxa having Mycosphaerella teleomorphs. C. fulvum has recently been placed in the anamorph genus Passalora as P. fulva. Its taxonomic disposition is supported by its DNA phylogeny, as well as the distinct scars on its conidial hila, which are typical of Passalora, and unlike Cladosporium s.s., which has teleomorphs that reside in Davidiella, and not Mycosphaerella. Host range and disease symptoms:, The presently known sole host of C. fulvum is tomato (members of the genusLycopersicon). C. fulvum is mainly a foliar pathogen. Disease symptoms are most obvious on the abaxial side of the leaf and include patches of white mould that turn brown upon sporulation. Due to stomatal clogging, curling of leaves and wilting can occur, leading to defoliation. C. fulvum as a model pathogen:, The interaction between C. fulvum and tomato is governed by a gene-for-gene relationship. A total of eight Avr and Ecp genes, and for four of these also the corresponding plant Cf genes, have been cloned. Obtaining conclusive evidence for gene-for-gene relationships is complicated by the poor availability of genetic tools for most Mycosphaerellaceae,plant interactions. Newly developed tools, including Agrobacterium -mediated transformation and RNAi, added to the genome sequence of its host tomato, which will be available within a few years, render C. fulvum attractive as a model species for plant pathogenic Mycosphaerellaceae. Useful websites:,http://www.sgn.cornell.edu/help/about/index.html; http://cogeme.ex.ac.uk [source] Development of insect-resistant transgenic rice with Cry1C*-free endospermPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 9 2009Rongjian Ye Abstract BACKGROUND: Yellow stem borer (Tryporyza incertulas Walker), striped stem borer (Chilo suppressalis Walker) and leaf folder (Cnaphalocrocis medinalis Guenec) are three lepidopteran pests that cause severe damage to rice in many areas of the world. In this study, novel insect-resistant transgenic rice was developed in which Bt protein expression was nearly absent in the endosperm. The resistant gene, cry1C*, driven by the rice rbcS promoter (small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase), was introduced into Zhonghua 11 (Oryza sativa L. ssp. japonica) by Agrobacterium -mediated transformation. RESULTS: A total of 83 independent transformants were obtained, 19 of which were characterised as single-copy foreign gene insertion. After preliminary screening of the T1 families of these 19 transformants in the field, six highly insect-resistant homozygous lines were selected. These six homozygous transgenic lines were field tested for resistance to leaf folders and stem borers, and for their agronomic performance. The Cry1C* protein levels in leaves and endosperm were measured by ELISA. Subsequently, the elite transgenic line RJ5 was selected; this line not only possessed high resistance to leaf folders and stem borers, normal agronomic performance, but also Cry1C* expression was only 2.6 ng g,1 in the endosperm. CONCLUSION: These results indicated that RJ5 has the potential for widespread utility in rice production. Copyright © 2009 Society of Chemical Industry [source] Development of a strategy for transgenic studies and monitoring of transgene expression in two closely related Moricandia species possessing a C3 or C3,C4 intermediate photosynthetic phenotypePHYSIOLOGIA PLANTARUM, Issue 1 2003Vera Thole In order to establish a model system for comparative studies of C3 and C3,C4 intermediate photosynthesis, the development of efficient transformation systems and the monitoring of transgene behaviour and stability were carried out in two closely related Moricandia species (Brassicaceae): the C3,C4 photosynthetic intermediate species M. arvensis and the C3 species M. moricandioides. In this study the green fluorescent protein (gfp) reporter gene was used as a vital marker gene while the use of the , -glucuronidase (gusA) gene was based on the highly sensitive detection of its activity. For Agrobacterium -mediated transformation of leaf explants, a cauliflower mosaic virus 35S promoter-driven, modified version of gfp, the mgfp5-ER gene and the gusA gene, respectively, were introduced into the new dual binary transformation vector system pGreen/pSoup (Hellens et al. 2000, Plant Mol Bio 42: 819,832). GFP5 produced bright-green fluorescence in transformed tissues that was distinctly detected 5,12 days following transformation in developing calli of the two species. Visual screening, combined with antibiotic selection, enabled early and easy identification of transformation events and contributed to improvements in the transformation strategies. Transgene integration studies demonstrated that mgfp5-ER was inserted with low copy number in the M. arvensis plant lines and the transgene was transmitted in a Mendelian fashion to T1 and T2 progenies. GFP5 expression levels in a population of 100 independent primary transformed M. arvensis plant lines (T0) showed great variation between transformation events (coefficient of variation of 108%). The mgfp5-ER or gusA reporter genes were expressed in 90,95% of the kanamycin-resistant M. arvensis plant lines and in up to 98% of the independent M. moricandioides plant lines. [source] Agrobacterium -mediated transformation of cereals: a promising approach crossing barriersPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2006Ashok Kumar Shrawat Summary Cereal crops have been the primary targets for improvement by genetic transformation because of their worldwide importance for human consumption. For a long time, many of these important cereals were difficult to genetically engineer, mainly as a result of their inherent limitations associated with the resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. The delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique. However, there are still serious handicaps with Agrobacterium -mediated transformation of other major cereals. In this paper, we review the pioneering efforts, existing problems and future prospects of Agrobacterium -mediated genetic transformation of major cereal crops, such as rice, maize, wheat, barley, sorghum and sugarcane. [source] Disease resistance conferred by the expression of a gene encoding a synthetic peptide in transgenic cotton (Gossypium hirsutum L.) plantsPLANT BIOTECHNOLOGY JOURNAL, Issue 6 2005Kanniah Rajasekaran Summary Fertile, transgenic cotton plants expressing the synthetic antimicrobial peptide, D4E1, were produced through Agrobacterium -mediated transformation. PCR products and Southern blots confirmed integration of the D4E1 gene, while RT-PCR of cotton RNA confirmed the presence of D4E1 transcripts. In vitro assays with crude leaf protein extracts from T0 and T1 plants confirmed that D4E1 was expressed at sufficient levels to inhibit the growth of Fusarium verticillioides and Verticillium dahliae compared to extracts from negative control plants transformed with pBI-d35S,- uidA-nos (CGUS). Although in vitro assays did not show control of pre-germinated spores of Aspergillus flavus, bioassays with cotton seeds in situ or in planta, inoculated with a GFP-expressing A. flavus, indicated that the transgenic cotton seeds inhibited extensive colonization and spread by the fungus in cotyledons and seed coats. In planta assays with the fungal pathogen, Thielaviopsis basicola, which causes black root rot in cotton, showed typical symptoms such as black discoloration and constriction on hypocotyls, reduced branching of roots in CGUS negative control T1 seedlings, while transgenic T1 seedlings showed a significant reduction in disease symptoms and increased seedling fresh weight, demonstrating tolerance to the fungal pathogen. Significant advantages of synthetic peptides in developing transgenic crop plants that are resistant to diseases and mycotoxin-causing fungal pathogens are highlighted in this report. [source] Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) genePLANT BIOTECHNOLOGY JOURNAL, Issue 5 2005Lin Wang Summary Atrazine is one of the most widely used herbicides in the USA. Atrazine chlorohydrolase (AtzA), the first enzyme in a six-step pathway leading to the mineralization of atrazine in Gram-negative soil bacteria, catalyses the hydrolytic dechlorination and detoxification of atrazine to hydroxyatrazine. In this study, we investigated the potential use of transgenic plants expressing atzA to take up, dechlorinate and detoxify atrazine. Alfalfa, Arabidopsis thaliana and tobacco were transformed with a modified bacterial atzA gene, p -atzA, under the control of the cassava vein mosaic virus promoter. All transgenic plant species actively expressed p -atzA and grew over a wide range of atrazine concentrations. Thin layer chromatography analyses indicated that in planta expression of p -atzA resulted in the production of hydroxyatrazine. Hydroponically grown transgenic tobacco and alfalfa dechlorinated atrazine to hydroxyatrazine in leaves, stems and roots. Moreover, p -atzA was found to be useful as a conditional-positive selection system to isolate alfalfa and Arabidopsis transformants following Agrobacterium -mediated transformation. Our work suggests that the in planta expression of p -atzA may be useful for the development of plants for the phytoremediation of atrazine-contaminated soils and soil water, and as a marker gene to select for the integration of exogenous DNA into the plant genome. [source] Overexpression of bacterial catalase in tomato leaf chloroplasts enhances photo-oxidative stress tolerancePLANT CELL & ENVIRONMENT, Issue 12 2003E.-A. MOHAMED ABSTRACT The Escherichia coli gene katE, which is driven by the promoter of the Rubisco small subunit gene of tomato, rbcS3C, was introduced into a tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens -mediated transformation. Catalase activity in progeny from transgenic plants was approximately three-fold higher than that in wild-type plants. Leaf discs from transgenic plants remained green at 24 h after treatment with 1 µm paraquat under moderate light intensity, whereas leaf discs from wild-type plants showed severe bleaching after the same treatment. Moreover, ion leakage from transgenic leaf discs was significantly less than that from wild-type leaf discs at 24 h after treatment with 1 µm paraquat and 10 mm H2O2, respectively, under moderate light intensity. To evaluate the efficiency of the E. coli catalase to protect the whole transgenic plant from the oxidative stress, transgenic and wild-type plants were sprayed with 100 µm paraquat and exposed to high light illumination (800 µmol m,2 s,1). After 24 h, the leaves of the transgenic plants were less damaged than the leaves of the wild-type plants. The catalase activity and the photosynthesis activity (indicated by the Fv/Fm ratio) were less affected by paraquat treatment in leaves of transgenic plants, whereas the activities of the chloroplastic ascorbate peroxidase isoenzymes and the ascorbate content decreased in both lines. In addition, the transgenic plants showed increased tolerance to the oxidative damage (decrease of the CO2 fixation and photosystem II activity and increase of the lipid peroxidation) caused by drought stress or chilling stress (4 °C) under high light intensity (1000 µmol m,2 s,1). These results indicate that the expression of the catalase in chloroplasts has a positive effect on the protection of the transgenic plants from the photo-oxidative stress invoked by paraquat treatment, drought stress and chilling stress. [source] A SELDI-TOF MS procedure for the detection, quantitation, and preliminary characterization of low-molecular-weight recombinant proteins expressed in transgenic plantsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2009M. Amine Badri Abstract We describe a SELDI-TOF MS procedure for the rapid detection and quantitation of low-molecular-weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens -mediated transformation, and then used as test material for the analyses. Real-time RT-PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI-TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2,h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production. [source] Heritable targeted mutagenesis in maize using a designed endonucleaseTHE PLANT JOURNAL, Issue 1 2010Huirong Gao Summary The liguleless locus (liguleless1) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I- CreI homing endonuclease. A single-chain endonuclease, comprising a pair of I- CreI monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 (LG1) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium -mediated transformation of immature embryos, and transgenic T0 plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T0 plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re-design of an endonuclease can produce a functional enzyme capable of introducing double-strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re-designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant. [source] Consistent transcriptional silencing of 35S-driven transgenes in gentianTHE PLANT JOURNAL, Issue 4 2005Kei-ichiro Mishiba Summary In this study, no transgenic gentian (Gentiana triflora × Gentiana scabra) plants produced via Agrobacterium -mediated transformation exhibited transgene (GtMADS, gentian-derived MADS-box genes or sGFP, green fluorescent protein) expression in their leaf tissues, despite the use of constitutive Cauliflower mosaic virus (CaMV) 35S promoter. Strikingly, no expression of the selectable marker gene (bar) used for bialaphos selection was observed. To investigate the possible cause of this drastic transgene silencing, methylation-specific sequences were analysed by bisulfite genomic sequencing using tobacco transformants as a control. Highly methylated cytosine residues of CpG and CpWpG (W contains A or T) sites were distinctively detected in the promoter and 5, coding regions of the transgenes 35S- bar and 35S- GtMADS in all gentian lines analysed. These lines also exhibited various degrees of cytosine methylation in asymmetrical sequences. The methylation frequencies in the other transgene, nopaline synthase (NOS) promoter-driven nptII, and the endogenous GtMADS gene coding region, were much lower and were variable compared with those in the 35S promoter regions. Transgene methylation was observed in the bialaphos-selected transgenic calluses expressing the transgenes, and methylation sequences were distributed preferentially around the as-1 element in the 35S promoter. Calluses derived from leaf tissues of silenced transgenic gentian also exhibited transgene suppression, but expression was recovered by treatment with the methylation inhibitor 5-aza-2,-deoxycytidine (aza-dC). These results indicated that cytosine methylation occurs exclusively in the 35S promoter regions of the expressed transgenes during selection of gentian transformants, causing transcriptional gene silencing. [source] Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutationsTHE PLANT JOURNAL, Issue 1 2003Richard J. Lawrence Summary Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical. We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes. Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A. thaliana and A. arenosa. We demonstrate that A. suecica can be genetically transformed using the floral dip method for Agrobacterium -mediated transformation. Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs. Expressing a double-stranded (ds) RNA corresponding to the A. thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A. thaliana - and A. arenosa -derived centromere repeats. These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs. [source] Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1THE PLANT JOURNAL, Issue 6 2000Zongrang Liu Summary To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium -mediated transformation of the uvh1-1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3, splice site and introducing an in-frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues. [source] Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformationTHE PLANT JOURNAL, Issue 1 2000Kirankumar S. Mysore Summary Germ-line transformation (vacuum infiltration) is frequently used to transform Arabidopsis thaliana using Agrobacterium tumefaciens. We have recently identified several Arabidopsis ecotypes and T-DNA-tagged mutants that are recalcitrant to Agrobacterium -mediated transformation of cut root segments. Some of these ecotypes and mutants are deficient in their ability to bind bacteria. Some are deficient in T-DNA integration. We report here that using a germ-line transformation protocol we transformed these ecotypes and mutants, including attachment- and integration-defective Arabidopsis plants, with a frequency similar to that of highly susceptible wild-type plants. However, we could not transform otherwise highly susceptible Arabidopsis plants by germ-line or root transformation using several vir and attachment-deficient Agrobacterium mutants. These results indicate that certain plant factors important for transformation may exist in germ-line tissue but may be lacking in some somatic cells. [source] | |||||||||||||