Abstract
Development and commercialization of a transgenic crop depends upon the availability of a reliable quantitative assay sensitive enough to detect low level presence of transgenic material in mixed seed or in a commercial product. An appropriate species-specific endogenous control also is necessary for accurate quantification. A novel mode of transgenic tolerance to glyphosate, whereupon an engineered acetylase detoxifies glyphosate, was recently developed and incorporated into transgenic canola (Brassica napus). Here, we describe two highly specific and sensitive PCR-based assays for the transgenic event, DP-O73496-4. We also developed and evaluated an appropriate endogenous control to be used in conjunction with the event-specific assays. This endogenous control is specific to the A genome of cultivated canola-quality oilseed rape based on our analysis of the diversity of FatA gene sequences.
Highlights
Modified (GM) crops providing herbicide tolerance and insect resistance, as well as other desirable agronomic and nutritionally enhanced traits, are being rapidly adopted in agricultural practice
We describe a gelbased PCR assay for the same transgenic event, which has the advantage of simplicity and sensitivity
Genomic DNA isolated from 53 varieties of B. napus, B. juncea, B. rapa, B. nigra, B. carinata, and B. oleracea from various geographical regions (Belgium, Canada, China, France, Germany, Hong Kong, Hungary, India, Italy, Japan, South Korea, Netherlands, New Zealand, Poland, Portugal, Spain, Sweden, USA, UK) (Supplementary Table 1) was used as template for PCR amplification of the region of interest
Summary
Modified (GM) crops providing herbicide tolerance and insect resistance, as well as other desirable agronomic and nutritionally enhanced traits, are being rapidly adopted in agricultural practice. The glyphosate resistance trait, mediated by transgenic EPSP synthase, has been rapidly adopted in maize, soybean, and other crops (James 2010). As part of the development of such crops, analytical methods are being developed that enable quantitative analysis of the presence of specific transgenic events in any genetic material of interest. Such assays are needed to determine seed purity, obtain regulatory approval, and provide a means to monitor low level presence (LLP) (König et al 2004; McHughen and Smyth 2008).
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