www.checkbiotech.org ; www.raupp.info ; www.czu.cz

Commercial cultivation of genetically engineered (GE) maize in Europe has
been well legislated since 2003 (Directive, 2001/18/CE; Regulation (EC),
1829/2003, 1830/2003). December 2006 by Joaquima Messeguer and Enric Melé.

In these regulations, the concept of coexistence has been established as
'the principle that farmers should be able to cultivate freely the
agricultural crops they choose, be it GE crops, conventional, or organic
crops'.

All European countries need to develop national strategies to ensure
coexistence (Commission Recommendation, 2003), taking into account that the
threshold value of 0.9% for labeling GE maize food and feed has been
established.

Coexistence can be affected by the adventitious presence of one crop within
another, which can arise for a variety of reasons. These include seed
impurities, cross-pollination, volunteer presence, and harvesting and
storage practices. The adventitious presence of genetically engineered
organisms (GEOs) as a result of cross-pollination is one of the factors that
needs to be evaluated in different cropping areas, as local climatic
conditions may influence the extent of pollen-mediated gene flow. Maize
pollen is relatively large and heavy, but it can travel long distances on
airflow, when suitable meteorological conditions occur, and therefore
cross-pollination will take place to some extent. The rate of
cross-pollination between fields depends on pollen viability,
synchronization of flowering, and relative concentrations of pollen in donor
and receptor plots (for reviews see Treu and Emberlin 2000; Brookes et al.,
2004).

Several field trials have been performed to evaluate gene flow from GE to
non-GE maize (reviewed in Devos et al., 2005; Brookes et al., 2006). In
these trials it has been demonstrated that when a neighboring non-GE field
is at least 1 ha in size, an isolation distance of 20 ? 25 m is sufficient
to ensure purity levels in harvest material below the 0.9% threshold. It has
also been demonstrated that a buffer zone (some maize rows) is more
effective than an empty gap (Pla et al., 2006).

In general, field trials were designed by planting a nucleus of maize (GE or
a cultivar with a special phenotypic trait) and then studying the occurrence
of cross-fertilization in an adjacent field. In most trials, both genotypes
had been sown at the same time to increase synchronicity of flowering, in
order to detect cross-fertilization in the worst situation that could be
found in an area in which GE and non-GE maize coexist. However, could these
results be applied to real situations of coexistence? To answer this
question a study has been published recently (Messeguer et al., 2006) to
elucidate to what extent the results encountered in these field trials can
be applied to real situations of coexistence in which GE and non-GE maize
fields are sown with different cultivars, with different sowing dates, mixed
with other crops, and with different barriers that may influence pollen
dissemination.

To perform this study, two crop regions located in Catalunya (Spain) were
chosen during the 2004 growing season in which irrigated transgenic Bt
(resistant to the corn borer attack) and conventional maize fields coexisted
with other crops. Both regions are characterized by small size of the fields
(0.5?5 ha, with a mean of about 2 ha). In both regions, two areas were
defined: the central area in which conventional fields were selected for
sampling; and the surrounding area that may influence rate of
pollen-mediated gene flow. The total surface area studied in Térmens was 300
ha, with a central area of 43 ha; whereas in Pla de Foix? the area was 400
ha, with a central area of 100 ha. The different crops (cereals, fruit
trees, maize, etc.) were identified during the cropping season, and data on
maize cultivar, sowing and flowering dates, and wind speed and orientation
were recorded. All of these data were used to choose the non-transgenic
fields for sampling. At harvest, samples were collected and analyzed by
real-time quantification system-polymerase chain reaction (RTQ-PCR) as
described in Pla et al., 2006.

Five conventional fields in the Térmens area and seven in the Foix? area
were chosen to detect and quantify the rate of cross-fertilization. In the
Térmens area, fields were analyzed for both Mon810 and Bt176 events;
whereas, in the Foix? area, analysis was performed for the Mon810 event
only, because neither the fields of the selected zone nor the fields of the
surrounding zone had been sown with Bt176 maize. A stratified sampling
system was applied by dividing fields into different zones according to the
distance from the borders. The number of sampling points for each border
depended on the size and particular shape of the field. In total 488
analyses were performed.

Values obtained from RTQ-PCR analysis were used to estimate GEO content of
the studied fields. All fields were divided into zones by the transects used
for sampling and by two virtual lines that follow the field perimeter at 3
or 10 m inside. The partial GEO content of each zone was calculated by
averaging four samples that delimited the surface. These values were used to
make a graphical representation of the local distribution of adventitious
flow in the field. The average of these local values, weighted by their
corresponding area, was used to obtain a representative estimation of the
global field value. In general, the distribution of the GEO content was not
uniform in the field, showing some affected areas and providing useful
information about the putative pollen donors. In general, the rate of
cross-fertilization was higher in borders and decreased towards the center
of the field. Nine of the 12 analyzed fields gave values much lower than
0.9%; whereas, in the other three, the values were higher than 0.9%.

To identify which transgenic fields influence the adventitious presence of
Bt maize in a conventional field and to what extent, we used an empirical
ECP (estimated cross-pollination) index for each pair of transgenic and
non-transgenic fields. This index takes into account only two scalar
factors?synchronicity of flowering and distance between the fields?that are
independent of orientation. In the ECP index, it is assumed that
adventitious pollination is directly proportional to the number of days of
synchronous flowering and inversely proportional to the square of the
distance. High values of this index indicate a significant contribution to
adventitious presence of transgenic maize detected in the non-transgenic
field. So, with this criterion, we were easily able to visualize and
classify the fields most responsible for the adventitious flow detected.
Moreover, for each individual field studied, a global index (GI) was
calculated by adding ECP indices of surrounding transgenic fields, giving an
estimation of global gene flow produced, considering only distance and
synchronicity of flowering.

A good correlation (R2 = 0.9491, n = 15) was found between the average
percentage of GEO obtained by RTQ-PCR analysis of samples and the GI of the
ECP indices, showing that GI may be useful for estimating the percentage of
GEO in fields before harvesting.

Data obtained in several field trials especially conducted to quantify the
adventitious presence of GEOs have been used to predict the separation
distance between transgenic and non-transgenic fields that needs to be
established to ensure coexistence. However, almost none of the Bt fields
studied in the Foix? and Térmens areas had a separation distance or a buffer
zone (recommended by seed producers only for fields larger than 5 ha).
Several different situations were encountered, depending on agronomical and
physical factors and on the fact that, in the real situation of coexistence,
there was competition between the pollen produced by the analyzed field and
pollen coming not from one field only, but from several fields both close to
and at different distances away from the analyzed field.

Although the reliability of this approach must be confirmed by accurate
calculations using mathematical models, we can obtain some idea of the size
of the security distance needed to maintain adventitious presence of GEO
under the 0.9% threshold required by European Union regulations. The slope
of the regression line of GEO content vs. GI is 0.0689, and on the basis of
a field surrounded by four transgenic crops with total synchronicity of
flowering, the expected value of the GEO content is 2.76% if the distance is
within the first 10 m, 0.69% within the second, and 0.30% within the third.
This means that a security distance between 10 and 20 m should be sufficient
to maintain the GEO content below the 0.9% threshold under the worst
circumstances. The studied areas must be considered ?difficult zones? for
controlling pollen flow, because they are flat and windy, with small fields
(2 ha average), and with a high percentage of transgenic content.

Results obtained in this study perfectly match those obtained in field
trials specially designed to study pollen mediated gene flow in maize. We
have shown that coexistence between GE and conventional fields can be
achieved by establishing simple rules that take into account the
synchronicity of flowering and distance between fields. Moreover, data
collected in this study will be very useful for validation of models to
predict pollen flow at landscape level with different spatial distributions
(smaller and larger fields).

References


Brookes G, et al. (2004) Genetically Modified Maize: Pollen Movement and
Crop Coexistence. Dorchester, UK: PG Economics Ltd.
[www.pgeconomics.co.uk]
Brookes G, et al. (2006) Coexistence of genetically modified and
non-genetically modified maize: Making the point on scientific evidence and
commercial experience. PG Economics Ltd. [www.pgeconomics.co.uk]
Devos Y, et al. (2005) The coexistence between transgenic and non-transgenic
maize in the European Union: a focus on pollen flow and cross-fertilisation.
Environmental Biosafety Research 4, 71-87
Messeguer J, et al. (2006) Pollen-mediated gene flow in maize in real
situations of coexistence. Plant Biotechnology Journal 4, 633-645
Pl? M, et al. (2006). Assessment of real-time PCR based methods for
quantification of pollen-mediated gene flow from GM to conventional maize.
Transgenic Research 15,219-228
Treu R, and Emberlin J. (2000) Pollen Dispersal in the Crops Maize (Zea
mays), Oil Seed Rape (Brassica napus ssp. oleifera), Potatoes (Solanum
tuberosum), Sugar Beet (Beta vulgaris ssp. vulgaris) and Wheat (Triticum
aestivum). Evidence from Publications. A Report Commissioned by the Soil
Association. Worcester: National Pollen Research Unit, University College
Worcester.

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