SiFo project: Outcrossing from transgenic maize and quantifying outcrossing rates

Bt maize and outcrossing: The flight of the pollen cloud

Squelch, squelch, squelch: a small group of researchers and their visitors, trudge across the sodden trial field near Braunschweig. Less than 24 hours ago an unusually fierce storm tore through the fields of the Federal Biological Research Centre for Agriculture and Forestry (BBA) for the second time this year. Despite this, Sara Meier-Bethke is enthusiastic about the summer. The weather has been ideal for her investigations into the outcrossing of genetically modified maize.

Male inflorescence at the tip of the maize plant. It supplies the pollen.

The male flowers at the tip of the maize plants produce the pollen.

The female flowers are on the sides of the plant stems. They form thread-like sticky silks to which the pollen sticks.

The female inflorescences produce thread-like sticky silks, which the pollen adheres to.

Pollen.  During the flowering phase the male flowers produce huge quantities of fine yellow pollen.

During the flowering period the maize plants produce large quantities of yellow pollen.

The trial set-up in 2000: Outcrossing rates based on a 1% threshold. Even at a distance of 25 m (Row 5) isolated values were above 1%.

Red: Values above 1%, Blue: Values below 1%.
Z: Central plot with transgenic maize

Row 1 3 m (from the central plot)
Row 2: 4,5 m
Row 3: 7,5 m
Row 4: 12,5 m
Row 5: 25,5 m
Row 6: 49,5 m

Experiment design in 2002: The transgenic maize is the dark green strip on the left-hand edge of the picture. Top right are 28 small maize control plots with a clover/grass mix and bottom right are 28 more with cereal stubble. The surrounding crops

The trial set-up in 2002: The narrow, dark green strip on the left-hand side of the picture is the transgenic maize. Top right are 28 small maize control plots in a clover/grass mixture, bottom right 28 further plots with corn stubble.

Plots with conventional maize at varying distances from a transgenic maize field.

Control plots with conventional maize are located at varying distances from the transgenic trial field.

Sara Meier-Bethke assesses a germination test. Only the green maize plants are herbicide-resistant, all other seedlings have withered after treatment with glufosinate.

Pollen trap for measuring the intensity of the pollen drift. The traps automatically turn to face the wind, the pollen is sucked in through a small opening and sticks to a sticky strip.

The intensity of the pollen drift is determined using pollen traps like this.

Wind speedometer

The wind speed at different heights is measured in the transgenic maize field.

Air humidity meter
The film of moisture on the leaf surfaces is measured.

Instruments for measuring air humidity and moisture on the surface of the leaves

When it really mattered, just as the maize was flowering, it was very sunny at the trial site with temperatures above 30° Celsius. This was just what the young scientist wanted. Together with meteorologists and mathematicians, she is studying the effect of weather and thermals on pollen dispersal and the outcrossing of genetically modified maize. Warm air streams evidently play an important role in transporting the relatively heavy maize pollen over distances of more than 20 metres. Sara Meier-Bethke is therefore delighted with the sunny weather during the flowering period as it creates ideal conditions for producing measurable thermals. Once the maize has flowered, a cloud burst is of no importance.

One trial, many questions

In muddy shoes (after all, not everyone routinely puts a pair of Wellingtons in their car boot when setting out for a walk in August) the group reaches its first destination: one of 56 control plots with conventional maize. The plots, each measuring 36 square metres, are situated in the prevailing wind direction beyond the 1.2-hectare transgenic maize field. They are arranged at seven different distances from the transgenic maize up to a maximum of 200 metres. The graduated distribution enables the researchers to make statements about outcrossing rates at different distances. In this way the trial serves partly to repeat and verify previous field experiments with herbicide-tolerant maize.

In 2000 and 2001 BBA scientists Joachim Schiemann and Sara Meier-Bethke were able to gather a large amount of data, which touches the very heart of current discussion on transgenic contamination of harvested crops and GM thresholds. Evaluation of the crop trial conducted in 2000 showed, among other things, that outcrossing to conventional maize varieties at a distance of ten metres from the transgenic maize field lay on average below the 1% threshold under discussion in the European Union. However, isolated samples downwind attained or even exceeded the 1% mark even at a distance of 25 meters. Outcrossing rates fell sharply even over the first few metres outside the transgenic plot, but isolated outcrossings were nevertheless detected over greater distances.

Up and away

Having carefully analysed the data, Joachim Schiemann and Sara Meier-Bethke speculate that different laws come into play for incrossings at close range than for those at distances of more than 20 meters. So this year they plan to take into account thermal effects and their influence on pollen dispersal as well. Over each field a thermal develops that is typical for the crop grown there.

“The air over a ripe cornfield warms up more than the air over a green meadow”, explains Sara Meier-Bethke. “The rising warm air carries some of the pollen to higher layers of the atmosphere. The average wind speed increases as altitude increases, carrying the pollen over greater distances.” To observe this effect, the control plots with the conventional maize were surrounded by two different main crops – winter barley and a clover/grass mixture. The winter barley promotes strong thermal transport, whereas weak thermals prevail above the clover/grass field.

Manual work

How do the researchers determine for certain whether the genetically modified maize has transferred the trait of herbicide resistance to the adjacent control plants? Sara Meier-Bethke points out the small, coloured, plastic streamers attached to many of the maize stems: “We use these labels to mark the plants which we intend to take samples from.” The researchers record the exact location of the plants and the start and finish of their flowering period on the streamers. Then in October, 60 maize cobs from each sampling point - or more precisely from each sampling area - each containing 300 to 400 grains, are harvested by hand. The grains are removed from the cobs, dried and then germinated in the greenhouse in special containers. About 2500 grains are collected from each sampling point. For the scientists and their assistants this involves a lot of manual work. But they need the large number of grains in order to make statistically reliable statements even with low outcrossing frequencies. When the seedlings are between one and two weeks old they are sprayed with the herbicide to which the transgenic maize is resistant. If they survive this treatment, it is considered to be proof of outcrossing, since glufosinate, the active substance in the herbicide, kills conventional maize plants.

Pollen trap

It is not just the maize plants decorated with streamers that stand out on the trial field. The field is littered with measuring instruments. Franz Josef Löpmeier from the German Meteorological Office (DWD) knows what they are for: “This is a pollen trap; we use it to measure the intensity of the pollen drift. The traps automatically align themselves with the wind direction. The pollen is sucked through a narrow opening and sticks fast to an adhesive strip. Together with data on wind speed and wind direction, we are able to accurately determine when and how far how much pollen has drifted.” Löpmeier also measures the air and soil temperature, air humidity, solar radiation and precipitation. The warming of the air directly on the soil surface is measured with infrared thermometers, which look like pistols. Even the moisture on the surface of the leaves is recorded. All this provides the researchers with a comprehensive overview of the climatic conditions on the trial site.

Maths on film

The two data streams from the meteorological records and the germination test are brought together by the mathematicians under the aegis of Professor Otto Richter from the Institute of Geoecology at TU Braunschweig. Their task is to model, for the very first time, a mathematical formula for the dispersal of genetically modified maize under local climatic conditions on the basis of the measurements. The idea is that with this model it will be possible to describe the pollen drift so accurately that scientists will be able to make prognoses about outcrossing behaviour in maize even under different growing conditions. If this is successful, the model will become extremely important as the basis for safety concepts and cultivation recommendations for avoiding transgenic contamination. Ralf Seppelt, a colleague of Otto Richter, outlines the procedure. The geo-ecologists adopt two different approaches, one based on the physical meteorological data and the other on probability calculations, which draw on the observed outcrossings. From the point of view of a mathematician, both methods have advantages and disadvantages. However, both of the models that have been developed enable very good predictions of outcrossing frequency to be made. A computer simulation illustrates the “flight of the pollen cloud” which until now has only been available in the form of calculations. The scientists are now eagerly awaiting this year’s maize harvest, to compare their prognoses with the results of the new germination tests so they can further refine their model.