Production of a Bt toxin standard and development of a measuring procedure to assess the amount of the toxin in Bt maize

Topic

Summary

Between 2001 and 2003, a total of 635 mg of Bt toxin and 122 mg of Bt protoxin were made available to the project partners. The toxin was capable of being stably stored and was monitored for biological activity.

In Mon810, Bt toxin expression in the roots remained almost constant during the entire vegetation period. The toxin content in the stalks and leaves increased over the course of the vegetation period. The toxin content was highest in the leaves and lowest in the ears.

In Bt176 maize, the expression of Cry1Ab was very low in roots, stalks and ears. In the leaves, the toxin was more strongly expressed in the later development stages than in the earlier stages.

The toxin content fluctuates both seasonally and between different plant parts. In some cases, the measured toxin content values differ considerably from those familiar from similar experiments in the United States, but the general trends were confirmed. This finding highlights the importance of carrying out such experiments under local climatic conditions and with local varieties.

Experiment description

Production of Bt toxin

Bt toxin was produced in E. coli using an internationally accepted method. The toxin is comparable with the toxins produced in the transgenic plants, although the precursors are not identical. Following activation in the insects’ midgut, what remains of the toxin produced here and the toxin in the Bt plants is the same insect-active protein core, which is insensitive to the digestive enzyme trypsin.

The produced Bt toxin precursor, called protoxin, was treated with trypsin and purified further to produce usable Bt toxin. The necessary process steps were optimised for use on a larger scale.

Several tests were used to check that consistent quality standards were respected for the Bt toxin:

• Biochemical proof of equivalence between Bt toxin produced in E. coli and in the transgenic plants (e.g. comparison of molecular weights and tests for antibody reactions).

• Biological effect test: Measuring the acute toxicity of the standardised Bt toxin compared with other variants of the toxin with regard to the European corn borer. The biological activity of the protoxin and toxin was checked in bioassays using the European corn borer larvae.

Measuring standard for Bt toxin

During the vegetation period, plant samples were taken from the roots, leaf, stalk, pollen and ears on both sites on four occasions. The Bt toxin content of the samples was measured using an immunological method (ELISA).

Investigating the stability and keeping quality of Cry1Ab
Stability: After completion of the project, the stability and keeping ability of the toxin continued to be examined at minus 20 and minus 80 degrees Celsius. Parts of the stored toxins were examined biochemically for biological activity every six months.

Bioassay: As a parameter for the biological activity, bioassays (determining the dose at which fifty per cent of the trial animals die (LD50)) are being carried out in collaboration with the Institute for Biological Control of the Federal Biological Research Centre for Agriculture and Forestry (BBA Darmstadt). Different toxin concentrations were applied to the surface of the food source of newly emerged corn borer larvae. The bioassays were evaluated after seven/fourteen days and repeated at intervals of approximately three to ten months in order to establish whether the storage conditions had any effect on the stability of the toxin.
 

Results

After about 48 hours formation of the protein inclusion body was clearly visible in the E. coli cells under the light microscope (phase contrast).

Production of Bt toxin The production of Bt toxin was carried out with a transformed E. coli strain. The Bt toxin was isolated and purified. The size and purity of the produced toxin was checked using SDS polyacrylamide gel electrophoresis. The protoxin and toxin were capable of being stored stably at minus twenty degrees Celsius for at least eight months. A total of 635 mg of Bt toxin and 122 mg of Bt protoxin were made available to the project partners between 2001 and 2003.

Biological effect test: the LC50 value (toxin concentration at which 50 per cent of the test animals die) was 50 or 72 ng/cm2 for surface application. These two different concentrations were the result of different purity levels of the batches. The activity of the protoxin remained stable over four to six weeks of storage at four degrees Celsius.

Biotests using dried transgenic maize powder (MON810) revealed total growth inhibition of the European corn borers at 0.45 µg/ml medium, despite a low mortality rate among the larvae.

Measuring standard for Bt toxin

From 2001 to 2003, the toxin content in different plant parts of the transgenic lines Mon810 and Bt176 was measured over the course of the vegetation period. A total of 2023 tissue samples were taken and their Bt toxin content measured.

The toxin levels of the transgenic maize samples were measured using an ELISA method (DAS ELISA, QuantiplateTM kit). No toxin was found in any tissue in around nine per cent of all plant samples. These plants were not taken into account in the statistical evaluation.

Bt toxin content in Mon810

Examination of the level of expression of the Bt toxin in the four development stages over the course of the vegetation period revealed the following picture: expression in the roots was almost constant. By contrast, the toxin content in the stalks and leaves increased over the course of the vegetation period. The toxin content was highest in the leaves and lowest in the ears. A comparison of the experiment years showed that the toxin content was generally lower in 2002 than in 2001 and 2003.

The Bt toxin values showed similar patterns for the different plant parts on the two sites. However, the toxin volumes at one of the sites were about six to 49 per cent higher than those of the other site in almost all development stages during the three experiment years.

Bt toxin content in Bt176

In the Bt176 maize line, Cry1Ab toxin was not found in 32 per cent of root samples. The toxin content in the upper leaves was much higher in the later development stages than in the earlier stages. As with Mon810, there was a clear difference between the Bt toxin content measured in 2001 and 2003, compared with the measurements from 2002.

Investigating the stability and keeping quality of Cry1Ab
Stability: The toxin remained biochemically stable over a period of 18 to 22 months at minus 20 degrees and minus 80 degrees Celsius.

Bioassay: It was found that the activity of the Cry1Ab toxin stored at minus 20 degrees fell one- to 200-fold over a period of one to thirty months. The activity of the toxin in one batch fell so dramatically that no LD50 was obtained. The steepest decline was observed after ten or 18 months’ storage.

At minus 80 degrees, the bioactivity of the toxin remained much more stable than at minus 20 degrees.

Freeze-dried Cry1AB showed no significant differences in LC50 values after one and twelve months.