Modeling gene flow distribution within conventional fields and development of a simplified sampling method to quantify adventitious GM contents in maize.

Genetically modified (GM) crops have been commercially grown for two decades. GM maize is one of 3 species with the highest acreage and specific events. Many countries established a mandatory labeling of products containing GM material, with thresholds for adventitious presence, to support consumers' freedom of choice. In consequence, coexistence systems need to be introduced to facilitate commercial culture of GM and non-GM crops in the same agricultural area. On modeling adventitious GM cross-pollination distribution within maize fields, we deduced a simple equation to estimate overall GM contents (%GM) of conventional fields, irrespective of its shape and size, and with no previous information on possible GM pollen donor fields. A sampling strategy was designed and experimentally validated in 19 agricultural fields. With 9 samples, %GM quantification requires just one analytical GM determination while identification of the pollen source needs 9 additional analyses. A decision support tool is provided.

Gene expression profiles of MON810 and comparable non-GM maize varieties cultured in the field are more similar than are those of conventional lines.

Maize is a major food crop and genetically modified (GM) varieties represented 24% of the global production in 2007. Authorized GM organisms have been tested for human and environmental safety. We previously used microarrays to compare the transcriptome profiles of widely used commercial MON810 versus near-isogenic varieties and reported differential expression of a small set of sequences in leaves of in vitro cultured plants of AristisBt/Aristis and PR33P67/PR33P66 (Coll et al. 2008). Here we further assessed the significance of these differential expression patterns in plants grown in a real context, i.e. in the field. Most sequences that were differentially expressed in plants cultured in vitro had the same expression values in MON810 and comparable varieties when grown in the field; and no sequence was found to be differentially regulated in the two variety pairs grown in the field. The differential expression patterns observed between in vitro and field culture were similar between MON810 and comparable varieties, with higher divergence between the two conventional varieties. This further indicates that MON810 and comparable non-GM varieties are equivalent except for the introduced character.

Effect of volunteers on maize gene flow.

Regulatory approvals for deliberate release of GM maize events into the environment have lead to real situations of coexistence between GM and non-GM, with some fields being cultivated with GM and conventional varieties in successive seasons. Given the common presence of volunteer plants in maize fields in temperate areas, we investigated the real impact of GM volunteers on the yield of 12 non-GM agricultural fields. Volunteer density varied from residual to around 10% of plants in the field and was largely reduced using certain cultural practices. Plant vigour was low, they rarely had cobs and produced pollen that cross-fertilized neighbour plants only at low--but variable--levels. In the worst-case scenario, the estimated content of GMO was 0.16%. The influence of GM volunteers was not enough to reach the 0.9% adventitious GM threshold but it could potentially contribute to adventitious GM levels, especially at high initial densities (i.e. above 1,000 volunteers/ha).

Lack of repeatable differential expression patterns between MON810 and comparable commercial varieties of maize.

The introduction of genetically modified organisms (GMO) in many countries follows strict regulations to assure that only products that have been safety tested in relation to human health and the environment are marketed. Thus, GMOs must be authorized before use. By complementing more targeted approaches, profiling methods can assess possible unintended effects of transformation. We used microarrays to compare the transcriptome profiles of widely commercialized maize MON810 varieties and their non-GM near-isogenic counterparts. The expression profiles of MON810 seedlings are more similar to those of their corresponding near-isogenic varieties than are the profiles of other lines produced by conventional breeding. However, differential expression of approximately 1.7 and approximately 0.1% of transcripts was identified in two variety pairs (AristisBt/Aristis and PR33P67/PR33P66) that had similar cryIA(b) mRNA levels, demonstrating that commercial varieties of the same event have different similarity levels to their near-isogenic counterparts without the transgene (note that these two pairs also show phenotypic differences). In the tissues, developmental stage and varieties analyzed, we could not identify any gene differentially expressed in all variety-pairs. However, a small set of sequences were differentially expressed in various pairs. Their relation to the transgenesis was not proven, although this is likely to be modulated by the genetic background of each variety.

Assessment of real-time PCR based methods for quantification of pollen-mediated gene flow from GM to conventional maize in a field study.

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1-10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.

Pollen-mediated gene flow in maize in real situations of coexistence.

We present the first study on cross-fertilization between Bt and conventional maize in real situations of coexistence in two regions in which Bt and conventional maize were cultivated. A map was designed and the different crops were identified, as were the sowing and flowering dates, in Bt and conventional maize fields. These data were used to choose the non-transgenic fields for sampling and analysis by the real-time quantification system-polymerase chain reaction (RTQ-PCR) technique. In general, the rate of cross-fertilization was higher in the borders and, in most of the fields, decreased towards the centre of the field. Nine fields had values of genetically modified organism DNA to total DNA of much lower than 0.9%, whereas in three the rate was higher. Some differences were found when comparing our results with those of common field trials. In real conditions of coexistence and in cropping areas with smaller fields, the main factors that determined cross-pollination were the synchronicity of flowering and the distances between the donor and receptor fields. By establishing an index based on these two variables, the rate of the adventitious presence of genetically modified maize could be predicted, as well as the influence of other factors. By applying this index, and in the case of a fully synchronous flowering time, a security distance between transgenic and conventional fields of about 20 m should be sufficient to maintain the adventitious presence of genetically modified organisms as a result of pollen flow below the 0.9% threshold in the total yield of the field.