TESTBIOTECH Background 10- 4 - 2015
Stress test for genetically engineered plants
shows major gaps in risk assessment
Christoph Then for Testbiotech
Content
Summary...............................................................................................................................................1
1. What reactions might stressors trigger in genetically engineered plants?........................................2
2. Which effects are known?................................................................................................................3
3. Pilot project: trials in a climate chamber..........................................................................................6
Aims and methods...........................................................................................................................6
Results..............................................................................................................................................7
Discussion........................................................................................................................................8
4. Conclusions......................................................................................................................................9
Summary
Currently, European Food Safety Authority (EFSA) risk assessment does not include any in-depth
investigation of interactions between the transgenic plant genome and the environment. Neither
does it require data to show how the plant metabolism reacts to changing or extreme environmental
conditions. There are, in fact, hardly any data at all on how genetically engineered plants could react
to ongoing climate change.
Driven by additional promotors, the DNA inserted into the plants could escape the plant’s gene
regulation. Further, the transgenic plants are not adapted to the newly inherited metabolism. In
result environmental stressors such as heat, drought, saline soils or diseases might trigger genetic
instabilities or unpredictable changes in the metabolism of the plants. Several studies with
genetically engineered plants such as petunia, cotton, potato, soybean and wheat have reported
unexpected reactions to environmental stress conditions.
In 2015, scientists from Switzerland and Norway published results (Trtikova et al., 2015) from an
investigation into genetically engineered maize MON810, which produces an insecticidal protein, a
so-called Bt toxin. This pilot project was realised with the support of Testbiotech and with funding
1
from the Manfred Hermsen Umweltstiftung. Further funding was made available by AltnerCombecher-Foundation, GEKKO Foundation and Foundation on Future Farming. Two varieties of
maize MON810 were grown in climate chambers and subjected to defined stress conditions i.e.
cold/wet and hot/dry.
According to the authors, this is the first study to report on whether there is any relationship
between transgene expression and protein production in Bt maize under changing environmental
conditions. The study also investigated how transgene expression and protein content in two Bt
maize varieties containing the same transgene cassette (MON810) responded to stressful
environmental conditions.
The results were surprising: In general, the Bt content was on average higher in one variety
compared to the other. Under cold/wet conditions the content of Bt increased in one of the varieties,
but not in the other. The activity of the DNA construct inserted into the plants was lowered
significantly under hot/dry conditions in one variety, but this had no influence on the Bt content.
In conclusion, these results show that the stress reactions of maize containing the DNA for the
MON810 event are not predictable. Further, the results indicate major gaps in the current
understanding of transgene regulation in genetically engineered crops. These findings are highly
relevant for the risk assessment of MON810 or 1507 maize and other genetically engineered plants
expressing single or several Bt proteins. Reliable data on the Bt content are needed to assess
potential toxicity in non-target organisms. For example, risks for non-target organisms such as soil
organisms or the larvae of protected butterflies can be much higher than assumed if the Bt content
shows a high range of variation. It should also be taken into account that immune reactions due to
the consumption of food and feed derived from transgenic plants have been observed in several
feeding studies. It is likely that these effects are dose-dependent and therefore the content of the Bt
toxins also plays a decisive role in the risk assessment for food and feed.
There are also some general implications: If there is no correlation between transgene expression
and content of the transgene protein in the plant, a basic paradigm applied in current risk assessment
of genetically plants has to be reviewed. This can have major implications for any granted or
pending authorisation process for transgenic plants.
2
Testbiotech recommends:

The reassessment of authorisations issued for transgenic plants in the EU in regard to
transgene expression and content of transgene protein under stressful conditions.

Stress tests are conducted to investigate interactions between genetically engineered plants
and various environmental conditions before any new transgenic plants are authorised for
release, import or cultivation.

The investigations should be conducted systematically and in contained systems that enable
assessment of specific stressors under defined conditions.

For each authorisation, adequate methods must be made available that allow independent
monitoring of the concentration of the content of transgene proteins in the plants.
1. What reactions might stressors trigger in genetically
engineered plants?
The production of genetically engineered plants is different to conventional plant breeding. While
conventional breeding is based on the whole genome and natural cell regulation, genetic
engineering works with DNA isolated from its cellular context. In many cases, these DNA
sequences are randomly inserted into the genome. Its biological activity is enforced by technical
means so its activity is (partially) independent of normal cell regulation: The DNA is restructured
and combined with additional promotors to avoid gene silencing or down regulation in the cells
(Diehn et al., 1996). This approach is contrary to mechanisms in conventional plant breeding.
While genetic engineering tries to enforce new biological functions in the transgenic plants that
have not arisen through evolutionary processes, conventional plant breeding uses existing biological
diversity and natural mechanisms, such as mutagenesis.
Driven by additional promotors, the DNA inserted into the plants could escape the plant’s gene
regulation. Further, the transgenic plants are not adapted to the newly inherited metabolism. In
result environmental stressors such as heat, drought, saline soils or diseases might trigger genetic
instabilities or unpredictable changes in the metabolism of the plants. This could lead to a much
3
higher content of the new additional proteins being produced in the plants than intended.
Disturbance or interruption of gene regulation might also cause a higher incidence of plant diseases,
lower yields, reduced tolerance to stressors (such as climate changes), a rise in unintended plant
compounds such as anti-nutritional, allergenic or toxic compounds. Higher fitness (due to a higher
amount of pollen or seeds) might be a further consequence. Some of these effects might become
evident only after several generations or under certain environmental conditions.
The producers of genetically engineered plants have taken some notice of these problems in gene
regulation but it appears to be a problem that they hardly ever mention or acknowledge. One
example in a patent application made by Monsanto (WO 2004/053055) says that:
“Nonetheless, the frequency of success of enhancing the transgenic plant is low due to a
number of factors including the low predictability of the effects of a specific gene on the
plant's growth, development and environmental response, the low frequency of maize
transformation, the lack of highly predictable control of the gene once introduced into the
genome, and other undesirable effects of the transformation event and tissue culture
process.”
Genetically engineered plants should be systematically subjected to suitable stress tests in order to
generate reliable risk assessment data. Such testing can provide a broad range of data relevant for
risk assessment before genetically engineered plants are released or cultivated on large scale. The
stress tests should be conducted in contained systems, which allow a detailed assessment of the
impact of defined stressors. This data can be used to derive hypotheses on how plants might react to
various environmental conditions (such as climate change) and how this might impact their
associated risks. Currently no such tests are required before plants are authorised.
2. Which effects are known?
The environmental impact on genetically engineered plants first attracted attention during
experimental field trials growing petunias in Germany. Most of the reddish coloured transgenic
petunias lost their colour after a period of warm weather with temperatures of up to 36°C. These
changes were explained by heat stress silencing the transgenes (Meyer et al., 1992).
4
As Zeller et al (2010) showed, the ecological behaviour of plants can render effects that are highly
relevant for the risk assessment of genetically engineered plants. Genetically engineered wheat
showed a complex reaction of reduced fitness, higher incidence of fungal disease and higher burden
of toxic residues from the fungal disease. These effects only occurred in the field conditions, but not
in the greenhouse. The effects were explained by environmental differences between greenhouses
and field conditions. Zeller et al (2010) also pointed out that so far there has been hardly any
investigation of these interactions:
“(...) a careful search in the literature for replicated and randomized studies about the
ecological behaviour of GM and control plants in glasshouse versus field environments did
not return any published references.”
Findings in genetically engineered maize producing the Bt toxin show that its associated risks are
dynamic and subject to environmental conditions. Interference from light, fertiliser and climate can
affect the Bt content in maize MON810 substantially (see Table 1, from Then&Lorch, 2008).
Tab 1: Factors influencing or correlating with the Bt content in transgenic plants MON810.
Author
Factor
Abel & Adamczyk (2004) photosynthesis
Impact
Bt
content
and
photosynthesis
are
positively correlated
Bruns & Abel (2007)
nitrogen fertiliser
Bt content and nitrogen fertiliser are
positively correlated
Griffiths et al. (2006)
soil quality
can increase or decrease Bt content
Griffiths et al. (2006)
pesticide use
spraying of insecticide increases Bt
content in leaves and roots
Griffiths et al. (2006)
growing process
Bt content increases towards flowering
Nguyen & Jehle (2007)
different climatic regions
significant difference between field sites
Nguyen & Jehle (2007)
growing process
Bt content in leaves increases during
growing season
It is also known from genetically engineered cotton that its Bt content is influenced by
environmental conditions. Chen et al. (2005) showed, that high temperatures caused a lower level of
Bt toxins in the plants. Dong & Li (2006) published a review showing that Bt content depends on
the age of plants as well as on environmental factors. Huang et al (2014) show that the quality and
5
quantity of fertiliser applied impacts the Bt content in genetically engineered cotton. Luo et al.
(2008) reported that waterlogging alone and in combination with salinity reduced Bt content in
transgenic cotton. Control of pest insects was also reduced by these stressors.
Investigations from Brazil show that genetically engineered maize responds differently to regional
environmental conditions compared to conventionally bred maize: Agapito-Tenfen et al. (2013)
identified 32 differences in protein content of genetically engineered maize compared to isogenic
plants if grown under various regional environmental conditions.
Gertz et al. (1999) reported that stalks of genetically engineered soybean plants are more likely to
split in hot conditions than conventionally grown soybean plants. According to an Athenix patent
application (WO2007/103768), the enzyme produced in genetically engineered soybeans to make
them resistant to herbicide applications with glyphosate is deactivated at higher temperatures.
Furthermore, investigations into genetically engineered potatoes show that they can have
unexpected reactions in stressful conditions that are not seen in ‘normal’ conditions. Matthews et al.
(2005) subjected genetically engineered potato plants to several biotic (e.g. infections) and abiotic
stress factors. They found several differences between the potatoes in the formation of defending
compounds.
In conclusion, several findings show that genetically engineered plants can indeed react differently
to environmental factors compared to plants derived from conventional breeding. Not only is the
effect of the content of new proteins of concern, but also more generally plant metabolism and
related biological functions. Some of the effects such as a higher content in insecticidal toxins,
alkaloids, fungal toxins, allergenic compounds or higher potential to spread into the environment
are highly relevant for risk assessment. Currently, none of these issues are taken into account during
risk assessment.
6
3. Pilot project: trials in a climate chamber
Maize MON810, in all of its parts and throughout the whole period of vegetation, produces an
insecticidal toxin that naturally occurs in soil bacteria (Bacillus thuringiensis). This toxin is
distributed in the environment via pollen and parts of the plants, and also exuded via roots into the
soil. While it is known that the Bt content in genetically engineered maize is influenced by
environmental conditions, there have been no systematic investigations into its true variations and
impact. From 2009, Testbiotech therefore actively sought to raise funds for a project titled “stress
test for genetically engineered maize”. In 2010, the Manfred Hermsen Umweltstiftung provided
funding which was used to support a pilot project at the ETH in Zurich (Switzerland). The results of
the project were published in 2015 in the peer reviewed journal PLOS one (Trtikova et al., 2015).
Aims and methods
The aim of the project was to establish how genetically engineered maize MON810 responds to
various defined environmental stressors.
Two Bt maize varieties, so-called white and yellow maize, containing the same transgene cassette
(MON810), were exposed in the climate chambers to cold/wet or hot/dry conditions. Temperatures
applied were from 13 °C to 45 °C. In addition to the Bt content in the leaves, the level of RNA was
determined as an indication for transgene expression or in other words, the biological activity of the
inserted DNA. It was expected that the level of RNA and Bt toxin would be correlated.
Since there was no existing method evaluated and agreed by several laboratories to measure the Bt
content in the plants, another study was performed in advance (also with support of Testbiotech): A
ring test was conducted involving four laboratories to find out which protocols were the most
reliable and reproducible. The results were published in 2011 (Székács et al., 2011) and taken into
account in the current study.
7
Figure 1: Maize was grown in climate chambers
Figure 2: Samples from leaves collected for Bt protein content analysis and transgene expression
8
Results
The investigations brought some surprising results:
(1) The Bt content in the leaves of the two varieties were different, despite both expressing the
same gene construct in their genome: On average, the Bt content in yellow maize was higher
than in the white maize.
(2) When exposed to cold/wet conditions, the Bt content in white maize was raised
significantly, while in the yellow maize this was not the case.
(3) Hot/dry conditions did not have a significant impact on the Bt content.
These results show that different maize varieties which contain the same transgene can react very
differently to environmental stressors in terms of Bt toxin concentration.
Figure 3 (with kind permission of the authors): Bt content in white and yellow maize under optimal, cold/wet and
hot/dry conditions. Means labelled with different letters are significantly different.
9
The results from measuring the transgene expression rate in the maize show a different and
surprising outcome:
(1) Transgene expression was reduced under hot/dry conditions in both varieties compared to
optimal conditions, being significant reduced in white Bt maize (but not the Bt content).
(2) Cold/wet conditions did not show a significant impact.
Figure 4 (with kind permission of the authors): Transgene expression in white and yellow maize under optimal,
cold/wet and hot/dry conditions. Means labelled with different letters are significantly different.
According to these results, the Bt protein content was correlated with the transgene expression in
only one of the varieties and only under optimal growth conditions. There are similar results from
an investigation with Bt cotton (Li et al., 2013). The authors suggest that transgene expression is not
the only factor determining and regulating Bt protein content in the investigated maize varieties.
Discussion
According to the authors, this is the first study to report on whether there is a relationship between
transgene expression and protein production in Bt maize under changing environmental conditions.
It further investigated how transgene expression and protein content in two Bt maize varieties
10
containing the same transgene cassette (MON810) responded to stressful environmental conditions
i.e. cold/wet and hot/dry.
In most cases, data provided by companies for the authorisation process only show a limited
variation in the Bt content of the transgenic plants. However, there is evidence from investigations
carried out in Germany that in the case of MON810 the data provided by industry do not represent
the true range of variations (Nguyen & Jehle, 2007, Lorch & Then, 2007). The investigations
described above were performed independently of industry and showed a much higher variation in
the Bt content than is suggested by Monsanto, the company that produces maize MON810.
Results reported by Trtikova et al. (2015) support the evidence that the data from industry is
insufficient to establish the genetic stability of these plants. The reaction of maize MON810 to
environmental stressors does not seem to be predictable. Furthermore and surprisingly, the Bt
content cannot be concluded from the transgene expression rate.
According to the scientists, this suggests that the transgene is not the only factor in determining and
regulating Bt protein. If there is no correlation between transgene expression and content of the
transgene protein in the plant, a basic paradigm currently used in the risk assessment of genetically
plants has to be reviewed. This can have major implications for any granted or pending
authorisation process.
In general, a lot more investigation is needed in order to fully understand how genetic modifications
alter the plant’s response to environmental changes such as climate change. The plant’s reactions are
also highly relevant for risk assessment. Reliable data on the Bt content are needed to assess
potential toxicity in non-target organisms. For example, risks for non-target organisms such as soil
organisms or the larvae of protected butterflies can be much higher than assumed if the Bt content
shows a high range of variation. On the other hand, an unexpectedly low level in the Bt content can
accelerate the rise of resistance in pest insects.
It should also be taken into account that immune reactions due to the consumption of food and feed
derived from transgenic plants have been observed in several feeding studies. The investigations
examined fish (Sagstad et al., 2007, Gu et a., 2012), pigs (Walsh et al., 2011, Carman et al., 2013),
mice (Finamore et al., 2008, Adel-Patient et al., 2011), and rats (Kroghsbo et al., 2008, Gallagher
2010). In addition, Andreassen et al (2014) also found IGE mediated immune (allergic) reactions to
11
Cry 1Ab if mice were exposed to the protein via inhalation. In summary, one can conclude there is
evidence of the immunogenic properties of Bt proteins such as Cry1Ab. It is likely that these effects
are dose-dependent and therefore the content of the Bt toxins also plays a decisive role in the risk
assessment for food and feed.
These findings are not only relevant for maize MON810 but even more so for stacked events such
as SmartStax, which produce up to six different Bt toxins in parallel so that the overall
concentration of Bt toxins is much higher in these plants. In this case it would be even more
relevant for risk assessment to find out how much Bt is actually produced in the plants. But reliable
and systematic results are missing in the case of SmartStax as well as for MON810 and maize
1507, which is assumed to have an extremely high concentration of Bt protein Cry1F in its pollen.
4. Conclusions
There is evidence that
(1) data from industry are generally not sufficient to assess technical qualities such as the Bt
content in the plant, which is necessary to perform reliable risk assessment;
(2) many more investigations are needed to gain a sufficient understanding of the interaction
between the transgene and the overall cell regulation in the plants.
(3) systematic stress tests need to be conducted to provide a sufficient range of data which can
be used as a starting point for further risk assessment. This is not only relevant for the
cultivation of genetically engineered plants but also for its usage in food and feed.
In the light of these findings, Testbiotech recommends:

The reassessment of authorisations issued for transgenic plants in the EU in regard to
transgene expression and content of transgene protein under stressful conditions.

Stress tests are conducted to investigate interactions between genetically engineered plants
and various environmental conditions before any new transgenic plants are authorised for
release, import or cultivation.

The investigations should be conducted systematically and in contained systems that enable
assessment of specific stressors under defined conditions.
12

The stress tests should also take into account impact factors such as salinity of soils,
pesticide applications and plant diseases, which were not integrated in the current pilot
project (for relevant factors see for example Bhatnagar-Mathur et al., 2007).

After authorisation, adequate methods must be made available that allow the monitoring of
the true composition of the plants and content of transgene proteins.

The investigations should be performed by institutions that are independent of industry.
References
Abel, C.A., & Adamczyk J.J. (2004) Expression of Cry1A in maize leaves and cotton bolls with diverse
chlorophyll content and corresponding larval development of fall armyworm (Lepidoptera: Noctuldae) and
southwestern corn borer (Lepidoptera: Crambidae) on maize whorle leaf profiles. J. Econ. Entomol., 97:
1737-1744.
Adel-Patient, K. Guimaraes, VD., Paris, A., Drumare, M_F., Ah-Leung, S., Lamourette, P., Nevers, M.,
Canlet, C., Molina, J., Bernard, H., Creminon, C., Wal, J. (2011) Immunological and metabolomic impacts of
administration of Cry1Ab protein and MON 810 maize in mouse, Plos ONE 6(1): e16346.
Agapito-Tenfen, S.Z., Guerra, M.P., Wikmark, O.G., Nodari R.O. (2013) Comparative proteomic analysis of
genetically modified maize grown under different agroecosystems conditions in Brazil. Proteome Science,
2013, 11(1): 46
Andreassen, M., Rocca, E., Bøhn T., Wikmark O-G., van den Berg J., Løvik, M., Traavik T., Nygaard U.C.
(2014) Humoral and cellular immune responses in mice after airway administration of Bacillus thuringiensis
Cry1Ab and MON810 cry1Ab-transgenic maize , Food and Agricultural Immunology
Bhatnagar-Mathur, P., Vadez, V., Sharma, K.K. (2007) Transgenic approaches for abiotic stress tolerance in
plants: retrospect and prospects. Plant Cell Reports, 27(3): 411-424.
Bruns, H.A., & Abel, C.A. (2007) Effects of nitrogen fertility on Bt endotoxin levels in maize, Journal of
Entomological Science, 42: 35-43.
Carman, J.A., Vlieger, H.R., Ver Steeg, L.J., Sneller, V.E., Robinson, G.W., Clinch-Jones C.A., Haynes J.I.,
Edwards J.W. (2013) A long-term toxicology study on pigs fed a combined genetically modified (GM) soy
and GM maize diet. Journal of Organic Systems 8 (1): 38-54.
Chen, D., Ye, G., Yang, C., Chen Y., Wu, Y. (2005) The effect of high temperature on the insecticidal
properties of Bt Cotton. Environmental and Experimental Botany, 53: 333–342.
Diehn, S.H., De Rocher, E.J., Green, P.J. (1996) Problems that can limit the expression of foreign genes in
plants: Lessons to be learned from B.t. toxin genes. Genetic Engineering, Principles and Methods, 18: 83-99.
Dong, H.Z., & Li W.J. (2006) Variability of endotoxin expression in Bt transgenic cotton. J. Agronomy &
Crop Science, 193: 21-29.
Finamore, A., Roselli, M., Britti, S., Monastra, G., Ambra, R., Turrini, A. & Mengheri, E. (2008) Intestinal
and peripheral immune response to MON810 maize ingestion in weaning and old mice. Journal of
13
Agricultural and Food Chemistry, 56: 11533–11539.
Forlani, G., Kafarski, P., Lejczak, B., Wieczorek, P. (1997) Mode of action of herbicidal derivatives of
aminomethylenebisphosphonic acid. J Plant Growth Regul, 16(3): 147-152.
Gallagher, L. (2010) Bt Brinjal Event EE1The Scope and Adequacy of the GEAC Toxicological Risk
Assessment, Review of Oral Toxicity Studies in Rats. http://www.testbiotech.de/node/444
Gertz, J.M., Vencill, W.K., Hill, N.S. (1999) Tolerance of transgenic soybean (Glycine max) to heat stress.
British Crop Protection Conference – Weeds, 15-19 Nov 1999,. Brighton: 835-840.
Griffiths, B.S., Caul, S., Thompson, J., Birch, A.N., Scrimgeour, C., Cortet, J., Foggo, A., Hacket, C.A.,
Krogh, P.H. (2006) Soil microbial and faunal community responses to Bt maize and insecticide in two soils.
J.Environ. Qual., 35: 734-741.
Gu J., Krogdahl A., Sissener N.H., Kortner T.M., Gelencser E., Hemre G.-I., Bakke A.M. (2012) Effects of
oral Bt-maize (MON810) exposure on growth and health parameters in normal and sensitised Atlantic
salmon, Salmo salar L. , British Journal of Nutrition
Huang, J., Mi, J., Chen, R., Su, H., Wu, K., Qiao, F., & Hu, R. (2014). Effect of farm management practices
in the Bt toxin production by Bt cotton: evidence from farm fields in China. Transgenic research, 1-10.
http://link.springer.com/article/10.1007/s11248-013-9775-7
Kroghsbo, S., Madsen, C., Poulsen, M., Schrøder, M., Kvist, P.H., Taylor, M., Gatehouse, A., Shu, Q.,
Knudsen, I. (2008) Immunotoxicological studies of genetically modified rice expressing PHA-E lectin or Bt
toxin in Wistar rats. Toxicology, 245: 24-34.
Li, M.Y., Li, F.J., Yue, Y.S., Tian, X.L., Li, Z.H., Duan, L.S. (2013) NaCl-Induced Changes of Ion Fluxes in
Roots of Transgenic Bacillus thuringiensis (Bt) Cotton (Gossypium hirsutum L.). Journal of Integrative
Agriculture, 12: 436-444.
Lorch, A. & Then, C. (2007), How much Bt toxin do GE MON810 maize plants actually produce?
Greenpeace Report, www.testbiotech.org/node/1028
Luo, Z., Dong, H., Li, W., Ming, Z., & Zhu, Y. (2008). Individual and combined effects of salinity and
waterlogging on Cry1Ac expression and insecticidal efficacy of Bt cotton. Crop protection, 27(12), 14851490.
Matthews, D., Jones, H., Gans, P., Coates, S., Smith, L.M.J. (2005) Toxic secondary metabolite production in
genetically modified potatoes in response to stress. Journal of Agricultural and Food Chemistry, 53(20):
7766-7776.
Meyer, P., Linn, F., Heidmann, I., Meyer, H., Niedenhof, I., Saedler, H. (1992) Endogenous and
environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic
petunia and its colour phenotype. Molecular and General Genetics MGG, 231(3): 345-352.
Nguyen, H.T. & Jehle, J.A. (2007) Quantitative analysis of the seasonal and tissue-specific expression of
Cry1Ab in transgenic maize Mon810. Journal of Plant Diseases and Protection, 114(2), 82-87.
Sagstad, A., Sanden, M., Haugland, Ø., Hansen, A.C., Olsvik, P.A., Hemre, G.I. (2007) Evaluation of stressand immune-response biomarkers in Atlantic salmon, Salmo salar L., fed different levels of genetically
modified maize (Bt maize), compared with ist near-isogenic parental line and a commercial suprex maize.
Journal of Fish Diseases, 30: 201–212.
Székács, A., Weiss, G., Quist, D., Takács, E., Darvas, B., Meier, M., Swain, T., Hilbeck, A., (2011)
14
Interlaboratory comparison of Cry1Ab toxin quantification in MON 810 maize by ezyme-immunoassay.
Food and Agricultural Immunology, 23(2): 99-121.
Then, C. & Lorch A. (2008), A simple question in a complex environment: How much Bt toxin do
genetically engineered MON810 maize plants actually produce? In: Breckling, B., Reuter, H. & Verhoeven,
R. (2008) Implications of GM-Crop Cultivation at Large Spatial Scales. Theorie in der Ökologie 14.
Frankfurt, Peter Lang: 17-21.
Online: http://www.mapserver.uni-vechta.de/generisk/gmls2008/beitraege/Then.pdf
Trtikova, M., Wikmark, O.G., Zemp, N., Widmer, A., Hilbeck, A. (2015) Transgene expression and Bt
protein content in transgenic Bt maize (MON810) under optimal and stressful environmental conditions,
PLOS one, April 2015 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123011
Walsh, M.C., Buzoianu, S.G., Gardiner G.E., Rea M.C., Gelencser, E., Janosi, A., Epstein, M.M., Ross, R.P.,
Lawlor, P.G. (2011) Fate of transgenic DNA from orally administered Bt MON810 maize and effects on
immune response and growth in pigs.PLoS One 6(11): e271
Zeller, S.L., Kalininal, O., Brunner, S., Keller, B., Schmid, B. (2010) Transgene x Environment Interactions
in Genetically Modified Wheat. 5(7), e11405.
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011405
15