Saint Louis, United States

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    ABSTRACT: Agrobacterium tumefaciens mediates high frequency of germline transformation of cotton meristem explants. The meristem transformation system we developed is rapid, high throughput and genotype-flexible. We have developed a high throughput cotton transformation system based on direct Agrobacterium inoculation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.). The explants were inoculated with a disarmed A. tumefaciens strain, AB33 harboring a 2 T-DNA binary vector pMON114908. This vector contained a gene of interest, an intron-disrupted β-glucuronidase gene in one T-DNA, and a selectable marker gene, aadA in the other T-DNA. Critical factors, such as method of co-culture, culture temperature during selection, composition of selection medium, and selection scheme were found to influence transformation frequency. The cycle time from initial inoculation to the transplanting of transgenic plants to soil was 7-8 weeks. Stable integration of transgenes and their transmission to progeny were confirmed by molecular and genetic analyses. Transgenes segregated in the expected Mendelian fashion in the T1 generation for most of the transgenic events. It was possible to recover marker-free events in the T1 generation when utilizing a binary vector that contained the selectable marker and gene of interest expression cassettes on independent T-DNAs. The procedure presented here has been used to regenerate thousands of independent transgenic events from multiple varieties with numerous constructs, and we believe it represents a major step forward in cotton transformation technology.
    Plant Cell Reports 10/2013; 33(1). DOI:10.1007/s00299-013-1519-x
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    ABSTRACT: "Field-evolved resistance" is defined as a "genetically based decrease in susceptibility of a population to a toxin caused by exposure to the toxin in the field." The key component of "field-evolved" resistance is that it does confer decreased susceptibility to an insecticide in the field. Another key component is that the decrease in susceptibility to the insecticide is because of previous exposure of the target insect to the toxin in the field. Several studies have reported field-evolved resistance to crops engineered to express proteins from the bacterium, Bacillus thuringiensis (Bt). However, there has not been a consistent standard in the application of the definition of field-evolved resistance for Bt crops. The inconsistency in applying the definition arises from differences in the methods used to detect resistance, the ecology of the interaction between the pest and the Bt crop, and the effective dose the pest encounters while feeding on the Bt crop. Using case studies of reported resistance to Bt crops, it is demonstrated resistance does not come in a single form, and that in most cases, resistance can still be managed.
    Journal of Economic Entomology 08/2013; 106(4):1525-34. DOI:10.1603/EC13103
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    ABSTRACT: The sequence specificity of the endogenous RNA interference pathway allows targeted suppression of genes essential for insect survival and enables the development of durable and efficacious insecticidal products having a low likelihood to adversely impact non-target organisms. The spectrum of insecticidal activity of a 240 nucleotide (nt) dsRNA targeting the Snf7 ortholog in Western Corn Rootworm (WCR; Diabrotica virgifera virgifera) was characterized by selecting and testing insects based upon their phylogenetic relatedness to WCR. Insect species, representing 10 families and 4 Orders, were evaluated in subchronic or chronic diet bioassays that measured potential lethal and sublethal effects. When a specific species could not be tested in diet bioassays, the ortholog to the WCR Snf7 gene (DvSnf7) was cloned and corresponding dsRNAs were tested against WCR and Colorado potato beetle (Leptinotarsa decemlineata); model systems known to be sensitive to ingested dsRNA. Bioassay results demonstrate that the spectrum of activity for DvSnf7 is narrow and activity is only evident in a subset of beetles within the Galerucinae subfamily of Chrysomelidae (>90 % identity with WCR Snf7 240 nt). This approach allowed for evaluating the relationship between minimum shared nt sequence length and activity. A shared sequence length of ≥21 nt was required for efficacy against WCR (containing 221 potential 21-nt matches) and all active orthologs contained at least three 21 nt matches. These results also suggest that WCR resistance to DvSnf7 dsRNA due to single nucleotide polymorphisms in the target sequence of 240 nt is highly unlikely. Electronic supplementary material The online version of this article (doi:10.1007/s11248-013-9716-5) contains supplementary material, which is available to authorized users.
    Transgenic Research 06/2013; 22(6). DOI:10.1007/s11248-013-9716-5


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    Saint Louis, United States
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