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Binding of Bt proteins to soil particles

(2008 – 2011) Institute of Applied Biotechnology in the Tropics (IBT) at the University of Göttingen

Topic

The aim of this project was to investigate the effects of the cultivation of the genetically modified Bt maize cultivar MON89034xMON88017, which produces three different Bt proteins, on the soil of the crop area. The Bt proteins from the maize plants enter the soil via root exudates, harvest residues and pollen and bind to soil particles. The binding (sorption) to soil particles partially inhibits the breakdown of the Bt proteins, so that their insecticidal effect remains intact. This could alter humus formation and the composition of the organic substance in the soil.

The project examined the following questions:

  • What are the chemical-physical properties of the soil fractions on the release site and of the three different Bt proteins produced by the maize variety under investigation?
  • What quantities of Bt proteins are exuded via the roots and how quickly are they broken down?
  • What binding behaviour do the Bt proteins exhibit towards the soil fractions on the release site?
  • Do the three Bt proteins compete for binding sites in the soil?
  • Is it likely that Bt proteins can be transported to lower soil layers over longer periods of time?

Summary

Soil samples were taken from all the plots on the trial field and examined to determine their chemical properties and mineral composition. The soil type was banded parabraunerde. The soil of the release site was generally very homogeneous.

All three Bt proteins were found to enter the soil continuously via the maize roots.

An investigation of the binding behaviour of the Bt proteins in relation to the soil fractions on the release site showed that the Bt proteins are most likely to bind to clay particles in the soil. Of the three proteins, Cry3Bb1 was found to bind the most. No competition for binding sites was observed between the Cry1A.105 and Cry2Ab2 proteins.

Experiment description

Soil characterisation

The preceding projects showed that the binding of Bt proteins to soil particles is significantly affected by the structure and chemical properties of both the clay particles in the soil and the Bt proteins. Since this project investigated a new genetically modified maize variety on a different release site, the soil and the Bt proteins first had to be adequately characterised to serve as a basis for further research.

Taking soil samples on the trial field

Obtaining root exudates in the climate chamber

Soil pits were dug on three plots to a depth of 1.5 metres in order to expose the soil profile.

Soil samples were taken from all the plots on the trial field at different depths (topsoil 0-20 cm, subsoil 40-60 cm) and tested for their chemical properties and mineral composition. To investigate the possible influence of Bt maize on the decomposition of maize litter, the project measured the level of mineral nitrogen, taking soil samples from the topsoil in the spring of 2008 and 2009 before the start of sowing and the first application of fertiliser.

Examination of root exudates

GM plants were grown in a nutrient solution. The concentration of the Bt proteins in this nutrient solution was measured at various times using the ELISA detection method. This determined whether and at what point the Bt proteins enter the soil through the maize roots.

Sorption measurements

The smallest soil particles (grain size < 2 micrometres, clay fraction) have the highest binding capacity. Therefore, exact quantities of clay and fine earth were agitated with a Bt protein solution for 30 minutes. Once the soil had been separated by centrifugation, the Bt protein concentration in the overlaying solution was measured. The experiment was conducted with all three Bt proteins and with different starting concentrations, corresponding to the Bt protein concentrations measured in the root exudates.

To check whether Bt proteins compete for binding sites, other sorption measurements were made using solutions containing the Bt proteins Cry1A.105 and Cry2Ab2.

Production of antibodies

As part of the project, new antibodies for the different Bt proteins were developed and produced. The antibodies were used to develop and optimise a special ELISA test. They were also made available to other teams within the research group.

Production of antibodies

As part of the project, new antibodies for the different Bt proteins are being developed and produced. They are to be used in the ELISA test and made available to the whole of the research group.

Results

Soil characterisation

The soil type was banded parabraunerde. The soil of the release site was generally very homogeneous:

  • The pH levels of the topsoil were within a range typical of farmland sites. The pH levels of the subsoil were higher than in the topsoil, because the soil naturally becomes more acidic from top to bottom.
  • The clay content was 6.4 per cent in the topsoil and 5.8 per cent in the subsoil. Bt proteins bind more easily to particles in the clay fraction. These particles are very small and, taken together (as the clay fraction of the soil), they have a very large surface area. Clay particles have a grain size smaller than 0.002 mm (= 2 micrometres).
  • The organic matter in the clay fraction was 5.4 per cent in the topsoil and 2.4 per cent in the subsoil. The organic matter closes small spaces between the soil particles, preventing Bt proteins from binding to particle surfaces in the spaces.
  • There were no great differences in the negative surface charge of the silt and clay particles within the plots. Nevertheless, the lower the negative charge of the soil particles, the more Bt protein was bound.

The nitrogen levels in the soil were somewhat lower in 2009 than in 2008. The statistical evaluation found that the maize variety had no significant effect on the level of mineral nitrogen in the soil.

Examination of root exudates

The laboratory experiments demonstrated that Bt maize MON88017 x MON89034 produces all three Bt proteins continuously throughout the growth periods observed and releases them into the soil through the plant roots. It is likely that Bt proteins also enter the soil continuously in the field. The exudation rate decreased over the course of the cultivation period.

Binding behaviour of the three Bt proteins in the collective clay sample from the topsoil (OB) and subsoil (UB)

Sorption measurements

Because of the low clay content, samples were combined into three collective topsoil and subsoil samples. The clay fraction is the soil component most actively involved in binding. This is because of the large surface area of the clay fraction.

The Bt protein Cry2Ab2 consistently bound more strongly to the clay fraction of the topsoil than to the corresponding subsoil sample. By contrast, the Cry1A.105 protein bound more strongly in the subsoil. Cry3Bb1 showed no clear preference but, of the three proteins, was found to bind the most.

No competition for binding sites was observed between the Cry1A.105 and Cry2Ab2 proteins.

Production of antibodies

To produce antibodies, goats were immunized against the three Bt proteins and the antibodies were obtained from the serum and purified. The antibodies obtained in this way are polyclonal, i.e. different antibodies that target the same protein. Monoclonal antibodies were obtained for the Bt proteins Cry1A.105 and Cry2Ab2 with the help of antibody-producing cells.