Yann Devos, Dirk Reheul and Adinda De Schrijver
March, 2006

With the inscription of 17 genetically modified (GM) maize (Zea mays L.) varieties derived from the event MON810 in the common catalogue of varieties of agricultural plant species of the European Union (EU) on 8 September 2004, the acreage of MON810 hybrids increased in Germany, France, and Spain, and their commercial cultivation expanded to the Czech Republic and Portugal in 2005. On 14 December 2005, Germany accepted the listing of 3 GM MON810 hybrids in the national catalogue, and on 30 December 2005, 14 additional Spanish GM MON810 hybrids entered the common EU catalogue. These evolutions may further boost the adoption of transgenic maize by European farmers and illustrate the urgent need for legal and practical frames dealing with coexistence in order to maintain conventional, organic, and genetically modified (GM) crop production, and to guarantee a high degree of consumer choice. In the EU, specific tolerance thresholds have been established or are discussed for the adventitious and technically unavoidable presence of approved GM material in non-GM produce: 0.9% for food and feed, 0.3-0.7% for seeds (crop specific), and 0.1-0.9% for organic produce (country specific). In addition to the mentioned thresholds, the product needs to be labeled as consisting of, containing, or produced from a genetically modified organism (GMO). In the case of maize seeds, a threshold of 0.3% is currently proposed.

Member states will impose strict technical management measures to keep the adventitious presence of GM material in non-GM produce below the labeling thresholds. As maize is a cross-pollinated crop relying on wind for dispersal of its pollen, on-farm measures may rely on spatial isolation.1 The task may be difficult, since various biological, physical, experimental, and analytical parameters with varying levels of importance have been identified to play a role in the study of cross-fertilization in maize. The number of variables and their variability may hamper the comparison between research results and make it difficult to define the appropriate length of isolation distances and/or pollen barriers. How some of the parameters can hamper the comparison between research results is addressed below.1

- Definition of isolation distance and pollen barrier: Although the terms isolation distance and pollen barrier (or buffer zone) are clearly distinct, they are regularly confused in the scientific literature. An isolation distance separates fields by a zone of open ground or a zone with low growing crops, while a pollen barrier consists of plants that are sown or planted around the source or recipient field. If outer parts of fields function as a barrier, the distance between inner parts increases. Barriers may also produce competing pollen (if the barrier is of the same species as the crop) and/or may serve as a physical barrier to air flow and consequently pollen flow. A pollen barrier of maize has been proven to reduce cross-fertilization levels more effectively than an isolation distance of the same length.2 For the future, it might be advisable to match the common vocabulary to similar definitions.

- Measuring cross-fertilization: Cross-fertilization is measured in different ways. Out-crossing may be noted in the following ways: (1) in the hybrid ears by phenotypic markers (e.g., xenia); (2) by detecting off-types in hybrid progeny; (3) by exposing the seedlings to an appropriate selection pressure (e.g., herbicide treatment in case of herbicide-resistant plants); and (4) by the qualitative detection of transgenic DNA and/or proteins in the seeds or seedlings. None of these methods quantifies the share of transgenic DNA. A quantitative DNA analysis expresses the GMO content as a percentage of haploid genomes. However, the latter results differ depending on the genetic constitution of the analyzed tissue (zygotic or maternal), the relative shares of these tissues in the sample, the ploidy levels of the tissue (triploid endosperm vs. diploid maternal tissue), the moment of sampling (early or late stage of kernel development), the copy number of transgenic DNA, and the DNA extractability, which may differ between plant tissues (Fig. 1).3 As a consequence, results based on quantitative DNA analyses are not smoothly convertible to results based on qualitative analyses.

- Hemizygosity: In the production of current GM hybrid varieties, the transgene generally is present in either the seed parent or the pollinator: as a result GM hybrids are hemizygous for the transgenic trait. Hence only half of the pollen produced on the hybrid carries the transgene, and only half of the cross-fertilization is measured compared to a pollen donor that is homozygous for the screened trait (Fig. 2).

- Analyzed plant tissue: The material to be analyzed for the adventitious presence of GM material depends on the use of maize. In grain maize, adventitious mixing is restricted to the grain fraction of the plant: the cross-fertilization level is expressed per grain lot. In corn cob mix and in fodder maize, transgene presence is diluted if expressed as a percentage of genomes since vegetative plant parts (maternal tissue) are included in the harvested material (Fig. 1). In non-processed fresh sweet maize, cross-fertilization is expressed per individual ear.

- Experimental design: The results of field trials will differ according to the implemented design. In different studies, small recipient plots or even individual plants have been planted at various distances from a source in order to measure how far viable maize pollen can successfully fertilize a maize ovule. Such designs do not reflect the real agricultural situation and are not suited to quantify the adventitious GMO content of recipient fields of commercial size. Individual plants or small recipient plots are much more prone to cross-fertilization than large recipient fields, which may result in an overestimation of the out-crossing level when making extrapolations. Recent studies carried out in France5, Germany6, Spain7, and the UK8 mimicked worst-case commercial on-farm situations (e.g., pollen source next to or completely surrounded by a recipient field) with a trend towards out-crossing studies in real agricultural situations.9 As the probability of cross-fertilization diminishes with increasing distances, sampling was performed at different positions within the recipient fields in order to calculate the average percentage of cross-fertilization over the whole field. The recommendations previously made for isolation distances and/or pollen barriers, based on discrete out-crossing levels, may therefore be too conservative and thus larger than the ones actually needed.

Apart from the previously discussed parameters, out-crossing is also affected by the distance between the pollen source and recipient; size, shape, and orientation of the pollen source and recipient; wind characteristics; rain; local environment; pollen viability; water status of pollen; climatic conditions; male fertility; and flowering synchrony.1 When research results are compared in order to define the appropriate isolation distances and/or pollen barriers limiting out-crossing, the various parameters at play should always be considered.


1. Devos Y, Reheul D & De Schrijver A (2005) The co-existence between transgenic and non-transgenic maize in the European Union: a focus on pollen flow and cross-fertilization. Environ. Biosafety Res. 4, 71-87

2. Melé E, Peñas G, Serra J, Salvia J, Ballester J, Bas M, Palaudelmàs M & Messeguer J (2005) Quantification of pollen gene flow in large maize fields by using a kernel colour trait. In Messéan A, ed, Proceedings of the 2nd International Conference on Co-existence between GM and non-GM based agricultural supply chains, Agropolis Productions, pp. 289-291

3. Taverniers I (2005) Development and implementation of strategies for GMO quantification in an evolving European context. Ph.D. thesis, University of Ghent, Ghent, Belgium

4. Trifa Y & Zhang D (2004) DNA content in embryo and endosperm of maize kernel (Zea mays L.): impact on GMO quantification. J. Agric. Food Chem. 52, 1044-1048

5. Bénétrix F, Foueillassar X & Poeydomenge C (2005) Coexistence OGM, non OGM: des outils opérationnels pour gérer les productions. Perspectives agricoles N 317, 8-11

6. Weber WE, Bringezu T, Broer I, Holz F & Eder J (2005) Koexistenz von gentechnisch verändertem und konventionellem Mais. Mais 1+2, 1-6

7. Melé E (2004) Spanish study shows that coexistence is possible. ABIC 3, 2

8. Henry C, Morgan D, Weekes R, Daniels R & Boffey C (2003) Farm scale evaluations of GM crops: monitoring gene flow from GM crops to non-GM equivalent crops in the vicinity: part I: forage maize. DEFRA report EPG 1/5/138

9. Messeguer J, Peñas G, Ballester J, Serra J, Salvia J, Bas M & Melé E (2005) Pollen mediated gene flow in maize in real situations of co-existence. In Messéan A, ed, Proceedings of the 2nd International Conference on Co-existence between GM and non-GM based agricultural supply chains, Agropolis Productions, Montpellier, pp. 83-87

Yann Devos and Dirk Reheul
Dept of Plant Production, Bioscience Engineering Faculty
University of Ghent, Ghent, Belgium

Adinda De Schrijver
Division of Biosafety and Biotechnology
Scientific Institute of Public Health
Brussels, Belgium