Environmental risks of Bt-maize and
transgenic drought tolerant maize.
A. Nijmeijer
3253430
Utrecht University
June 2013
Supervised by:
Dr. P. A. H. M. Bakker
Utrecht University
Table of Content
Abstract ..................................................................................................................................... 3
1. Introduction ............................................................................................................................. 4
2. The cultivation of maize ...................................................................................................... 5
2.1 Plant physiology ............................................................................................................................. 5
2.2 Plant origin ....................................................................................................................................... 5
2.3 Spatial growth limitations .......................................................................................................... 6
2.4 Production area .............................................................................................................................. 7
3. History of breeding and genetical modification in maize ....................................... 9
3.1 Transgenic Technologies ............................................................................................................ 9
4. Bt-maize and drought tolerant maize events .............................................................12
4.1 Bt-Maize .......................................................................................................................................... 12
4.2 Drought tolerant maize............................................................................................................. 13
5. Environmental risks of Bt-maize ....................................................................................15
5.1 Biodiversity risks ........................................................................................................................ 15
5.2 Effects on non–target species................................................................................................. 17
5.3 Interaction with target organism and development of resistance ......................... 18
6. Discussion ...............................................................................................................................20
References ..............................................................................................................................22
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
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Abstract
The use of genetically modified crops could offer a solution for the food problem that
probably will arise due to the increasing world population and a climate change induced
decrease in agricultural productivity (Whitford et al., 2010). However, the
commercialisation of transgenic crop events encounters heavy resistance in many
countries. Especially the environmental effects of transgenic crops lead to concerns.
These concerns include risks of biodiversity loss and health and survival risks for nontarget species.
This study discusses the available data on the impact that Bt-maize has had on the
environment since its commercialization in 1996. Additionally, the putative
environmental risks of drought-tolerant maize events are assessed using the data
available for Bt-maize. For a better perception on the impact of transgenic maize
cultivation a general introduction in maize cultivation and genetical modification is
given. Subsequently, the Bt-maize and drought tolerant maize events are described.
Overall, laboratory and field studies showed that the risks of Bt-maize for the
environment are negligible. Also the expected impact of drought tolerant maize appears
to be low. Furthermore, the introduction of transgenic events, especially in the tropical
regions, can result in increased yields per square meter. This could be beneficial for
overall biodiversity by decreased conversion of natural ecosystems to agricultural land.
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
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1. Introduction
Maize (Zea Mays) originates from Mexico but has been domesticated for tens of
thousands of years (James 2003). By selection for traits such as ear size, number of ears
per plant and the grain size during the last century only, the yield has increased by
eightfold (Tian et al., 2011). Today, maize is worlds most widely grown crop and after
wheat and rice it is the third most important food crop, and especially in developing
countries it is of high importance (Naqvi et al., 2011; Ribaut et al., 2009). In many
African countries maize accounts for more than 30% of the total calories consumed
daily. However, the majority of maize is produced as livestock feed. Besides this, maize
is used for the production of bioethanol and biodegradable plastics as well.
Furthermore, it is used for industrial purposes such as the production of high value
pharmaceutical products and specialty chemicals (Naqvi et al., 2011). The production of
large amounts of one plant species, attracts all kinds of pests and diseases. Maize suffers
not only from these biotic stresses, but also abiotic stresses influence the yield. Drought
is the most important constraint for the production of maize and many other crops
(Westgate and Hatfield 2011; Ribaut et al., 2009). In 1999 it was estimated that a
quarter of all the produced maize was affected by drought (Cairns et al., 2012). Since
weather conditions became less predictable over the last decades and will become even
less liable in the near future, with more severe droughts and increased temperatures,
more and more pressure is set on the plasticity of maize (Westgate and Hatfield 2011).
In addition, the world population is growing, and it is expected that by 2050 the world
population will pass 9 billion people (Cairns et al., 2012). The current levels of
productivity will fall short for the future demands. Therefore, better and faster methods
are needed for the introduction of better adapted varieties of crops.
At the moment, besides traditional breeding methods, molecular breeding and
genetic modification techniques are used for the improvement of crops. Genetic
modification of organisms and the offset of transgenic seeds has grown fast over the
past decades. Between 1996, when the first GMO crops were introduced, and 2012 the
total area of biotech crops have increased every single year. At the moment in 30
countries more than 170 thousand hectares of land are cultivated with a transgenic crop
by in total more than 17 million farmers (ISAAA, International service for the acquisition
of agri-biotech application, 2012). Along with the fast adoption of the new techniques
the amount of criticism has been rising as well. Besides a lot of media attention, many
scientific papers on the use of transgenic food crops have been published. The main
concerns focus on contamination of wild lines and the effect on human health and the
environment. The threat that transgenic strains pose on the environment compared to
wild-type varieties depends on the specific transgenic trait and the type of plant. For
example, in the case of the wind pollinator maize the chance for cross contamination is
higher compared to self-pollinators such as potatoes and wheat (Gewin 2003).
Previous to the growing season of 2013 Monsanto will commercialize a transgenic
drought tolerant maize variety. However, before the commercialisation of the maize
event numerous environmental risk assessments need to be performed. These
assessments contribute to the official acceptance of an event. To predict the
environmental risks of a transgenic maize variety such as drought tolerance, I have
chosen to focus on the environmental effects of one of the first commercialized
transgenic maize events, Bt-maize. Bt-maize belongs, along with herbicide resistant
events, to the most prevalent transgenic crops (Gewin 2003). Over the last two decades
extensive research has been done on the environmental effects of the various Bt-maize
events. In this thesis an overview of the effects of Bt-maize is given and this is used to
give some insight in what can be expected of the risks of the drought-tolerant maize
events.
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
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2. The cultivation of maize
Next to the cultivated maize varieties, in South- and Central-America wild-type
varieties can be found. After its domestication, around 10.000 years ago, maize spread
out to the South and Northern regions of America. In Europe maize was first introduced
by Columbus, who brought it back from the West Indies to Spain in 1493 (Rebourg et al.,
2003). Not long after the introduction of these tropical species other varieties were
introduced from more northern areas of America. These lines were very important for
the establishment of maize in northern Europe (Rebourg et al. 2003).
2.1 Plant physiology
Maize is an angiosperm that belongs to the family of the poaceae, a family of
monocotyledonous, C4 grasses. The maize grown today can be recognized as a singular
stem that is comparable to bamboo, with on top of it the tassel, the male inflorescence.
On average maize plants reach a height of 2.5 m with leaves growing from each node in
the stem. These leaves can reach a length upto 1.2 m with a width of 9 cm. Female
inflorescence appear above some of the leaves in the midsection of the plant. Each
flower is enveloped in several layers of husks (layers of ear leaves) from which
elongated stigmas, silks, emerge. After pollination kernels are formed on an ear. On
average one ear contains around 600 kernels, the colour of these kernels can vary from
nearly black to yellow and red.
Maize is a wind pollinator, when it is warm and dry, the tassel starts releasing ± 104
to 2×106 pollen grains per day for a period of 5-8 days (Naqvi et al., 2011; Jarosz et al.,
2003). These pollen play an important role in the gene-flow of maize, however, Luna et
al. (2001) found a maximum possible crosspollination distance of 200 meter. Beyond
this range crosspollination is highly unlikely, due to the high settling velocity and the
size of the pollen.
2.2 Plant origin
By human selection maize is derived from the wild-type progenitor grass, called
teosinte (Figure: 1). Taxonomically both plants are termed Zea mays, however, their
subspecies names are respectively mays for maize and parviglumis for teosinte. Despite
their close relationship and their morphological similarities such as their tassels, stems,
leafs, and root structures, several differences can be distinguished as well. In contrast to
maize, teosinte is branched with a tassel on top of every single branch. Furthermore, the
female inflorescences (ears) of teosinte consist of a small ear with kernels projecting
from two sides only, where maize has a cob covered with kernels. Additionally, kernels
of maize are naked attached to the cob, only covered by a thin layer of chaff where at
teosinte every single kernel is covered in a hard glume. During maturing the seeds of the
teosinte will disperse where the kernels of maize will stay on the plant (Meyerowitz
1994).
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
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Figure 1: Left maize, plant and ear with kernels, right teosinte, the ancestor of maize
with ear and seeds (Meyerowitz 1994).
The differences in plant agriculture between teosinte and maize are the result of only
a few differences in quantitative trade loci (QTL; Doebley et al., 1997). Much of the traits
of maize are based on the retrotransposon of tb 1 (Doebley et al., 1997). However, for
the original transition of the wild-type plant to the now known maize took several
centuries.
A maize plant is a 20 chromosome counting diploid (n = 10). In 2009, after a project
of four years, for the first time the entire genome of maize has been sequenced
(Schnable et al., 2009). The inbred line B73 genome sequence is 2.3 billion base pairs
long and includes more than 32000 protein-coding genes. When comparing maize to
teosinte the genome of teosinte seems to be nearly 400 million nucleotide smaller and
contains nearly 20% less retrotransposons (Schnable et al., 2009). In maize the amount
of transposable elements consisted of nearly 85% of the genome.
2.3 Spatial growth limitations
Maize is produced in more countries than any other crop because maize is able to
grow in climates as divers as arid desert plains up to high mountain plains. Geographic
growth is mainly limited by its disability to grow in cold areas. Optimally, maize grows
in a well-drained, moist loam soil with a pH ranging between 5.8 and 6.5. This pH
improves maize ability to take up soil nutrients. On hectare base the soil nutrient need
for maize seem to lay higher compared to average fertility needs. Particularly the soil
nutrients potassium, phosphate and nitrogen are needed for maize growth and
development. In commercial maize production, often tillage is used for improvement of
the seedbed and soil incorporation of fertilizers (James 2003).
As any other crop, maize production suffers from pests and diseases, but also low soil
fertility and drought cause yearly significant yield and economical losses (Cairns et al.,
2012). Major pests that threaten maize include rootworms, earworms, borers and
weevils. The western corn rootworm Diabrotica virgifera virgivera is one of the most
serious pests. Other pests include the corn earworm Helicoverpa armigera that feeds on
the ears of maize in its larval stage and ground weevils protostrophus spp. that feeds on
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
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the leaves of young maize. Finally there are the caterpillars of the european corn borer,
Ostrinia nubilalis, that eat through the maize stems and damages the ears of maize
causing annual production losses between 7 and 10% (James 2003).
Major diseases of maize include leaf blights, stalk rots, ear rots and viral diseases.
Most common diseases are stalk rots, including gibberella stalk rot (Gibberella zeaeI)
and anthracnose stalk rot (Collentotrichum graminicola), causing annual losses between
5 to 10% of total production. Within viral diseases maize dwarf mosaic and Maize
chlorotic dwarf are potentially damaging. Leafhoppers and aphids mainly transmit these
viruses (James 2003).
2.4 Production area
Maize is grown in more countries than any other crop due to the wide climatically
range where it can be produced. However, maize that performs well in the temperate
zone can generally not directly be introduced in tropical regions. Many of the open
pollinated varieties and the hybrids are special bred for countries in Europe, the United
States or China that fall within the temperate regions. The differences in yield are
around 5 t/ha-1 (tonnes per hectare) where the average maize yield in industrialized
countries is more than 8 t/ha-1, while in the developing countries maize yields are
slightly less than 3 t/ha-1 (Edmeades 2008). In contrary to people in these temperate,
well developed regions, a large proportion of the people within the tropical region
depend on the production of maize, as it is their primary staple food. To decrease the
differences in yield several breeding programs focussed on tropical maize in African
countries have been set up (CIMMYT, International Maize and Wheat Improvement
centre, 2012), since the production of maize in Africa is still much less compared to
other parts of the world (Figure: 2).
Figure 2: Maize production per country for 2010, ranging from 10 – 2.195.200 metric
tonnes (green) to 56.060.400 – 314.165.000 metric tonnes (red) (Targetmap, 2013).
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
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In 2010 the global production of maize was around 850445143.2 tonnes (FAOSTAT,
Food and Agricultural organization of the United Nations, 2011). The USA is globally the
largest producer of maize > 316165000 tonnes, even though the total production in
2010 was around 5% less than the previous year due to a severe drought in the east and
extensive rainfall in the west of the United States (Figure: 3; FAOSTAT, 2011). The
second biggest producer is China and there after is Brazil the largest producer.
Figure 3: Fluctuations in maize production over three years (tonnes per country), of the
ten leading maize producing countries (FAOSTAT, 2011)
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
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3. History of breeding and genetic modification in maize
Modifying food is as old as people started cultivating crops and started selecting and
breeding for increasing taste, growth or colour. Last century the transformation
efficiency improved due to the introduction of several techniques for inducing
mutations, including the use of chemicals or increased irradiation (Gewin 2003). The
introgression of traits by backcrossing with the cultivated parent is a slow and complex
process. The introduction of biotechnology during the 70’s, including molecular markers
in breeding has accelerated the process (Whitford et al., 2010; Songstad 2010). Within
maize genetic modification started with the regeneration of maize plants from callus
(Green and Phillips 1975) and the isolation of protoplasts (Chourey and Zurawski
1981). A difference can be made between the use of molecular techniques for speeding
up conventional breeding processes and genetically modification by altering the genome
of a crop.
Within the first option conventional breeding makes use of marker-assisted selection
(MAS) by identification of QTL’s and variation in DNA sequences. During backcrossing
activities MAS can be very useful for the maintenance of recessive alleles and for the
introgression of QTL’s. There are several options in which MAS is used in conventional
breeding, however, overall MAS is combined with phenotypic selection even when
relationships between phenotypical variations and genes are well defined (Whitford et
al., 2010). Other possibilities in which MAS is used are marker assisted pyramiding or
early generation MAS. During this process of pyramiding several QTL’s or monogenetic
traits together provide one target trait. In early generation MAS a screening is done
mainly using genotyping traits within germplasm rather than selecting for phenotypical
traits after plant growth (Whitford et al., 2010).
3.1 Transgenic Technologies
In the second option, genetical modification, specific alternation in an organisms DNA
is used to provide plants with certain traits (Whitford et al., 2010). This can include
transferring genes between organisms, moving, deleting, modifying or multiplying genes
within an organism or the incorporation of newly constructed genes into an organism
(Whitford et al., 2010).
Transformation of organisms got its breakthrough in the 80’s when the first gene
transfers were accomplished using hypovirulent Agrobacterium tumefaciens. Originally
Agrobacterium is a pathogen of dicotyledonous plants that causes symptoms that are
located on the border of the root system and the stem, forming opine producing tumour
cells through the transfer of a piece of DNA into its host DNA (Songstad 2010).
Other methods for transferring foreign DNA in a host cell include electroporation,
micro-particle bombardments of plant cells or tissue, cross hybridization and selection
involving transgenic donors. Electroporation is a method where foreign DNA can be
transferred into a host cell after making the membrane more permeable by causing
pores with electrical pulses. Micro-particle bombardment has as disadvantage that it
includes more DNA rearrangements and higher copy numbers of unstable transgenes
compared to Agrobacterium mediated transformation. Micro-particle bombardment is a
technique where micron sized metal particles covered in DNA get emitted into target
cells at velocities fast enough to penetrate cell walls but not too fast to prevent lethal
damage. Once into the cells interior the DNA will detach from the micro particles and
integrate into the genome of the host (Taylor and Fauquet 2002).
A transgene inserted in an organism, often includes a gene of interest, promoters and
a marker gene. Markers are used to identify if the gene of interest is incorporated in the
target organism. Within marker genes two different types can be distinguished,
selectable marker genes and reporter genes. Selectable marker genes are used to
identify successful incorporation of the trait gene by conferring resistance to a selective
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
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agent like an antibiotic or an herbicide. Reporter genes produce a product that can be
detected biochemically or visually. Furthermore, promoters are part of the incorporated
gene. Promoters are pieces of DNA that mediate the transcription of the marker- and
trait genes. The best-known promoters are Ubiquitin, Actin and 35S promoter. Ubiquitin
promoters (Ubi) are natural plant promoters that are continuously active but which are
further induced by heat shock (Streatfield et al., 2004). Actin (Act 1) is, like Ubiquitin, a
constitutively active plant promoter, derived from rice. The most frequent used 35S
promoter is a promoter derived from the cauliflower mosaic virus. Finally, also the gene
of interest is incorporated in the target organism genome. These traits genes can be
divided in two subclasses. In the first class, the transgene encoded proteins are the
desired product. In the second class the transgenes produce a molecule that alters the
metabolic system of the plant, where the alternation in a plants behavior is the wanted
result (Naqvi et al., 2011; Whitford et al., 2010). Bt-maize is an example of the first class
where the cry-protein, a toxin for Lepidoptera, is the desired trait. Transgenic drought
tolerant maize belongs to the second class, since the cold shock protein produced by the
transgene ensures the maintenance of normal cellular functions under water limited
circumstances. The total of commercialized traits in maize can be divided in five groups,
abiotic traits, herbicide tolerance, insect resistance, modified production qualities and
pollination control system (Table: 1).
Table 1: Commercialized transgene maize events whereof 4 drought tolerant events and 99 insect
resistant events (ISAAA 2012)
Commercial Trait
trait
Gene
name
Gene source
Product
Function
Abiotic (4)
CspB (4)
Bacillus subtilis
Cold shock protein B
Maintains normal cellular
functions under water stress
conditions by preserving RNA
stability and translation
Glufosinate
Pat (69)
herbicide
tolerance (80)
Bar (16)
Streptomyces
viridochromogenes strain Tu
494
Streptomyces hygroscopicus
Phosphinothricin Nacetyltransferase (PAT)
enzyme
Eliminates herbicidal activity
of glufosinate
(phosphinothricin) herbicides
by acetylation
Pat (syn)
(4)
Synthetic form of pat gene
derived from Streptomyces
viridochromogenes strain Tu
494
Zea maize
Modified 5enolpyruvylshikimate-3phosphate synthase (EPSPS)
enzyme
Herbicide tolerant form of 5enolpyruvulshikimate-3phosphate synthase (EPSPS)
enzyme
Glyphosate Nacetyltransferase enzyme
Confers tolerance to
applications of glyphosateammonium based herbicides
Drought
tolerance (4)
Disease
resistant (0)
Herbicide
tolerant
(103)
Glyphosate
Mepsps
herbicide
(28)
tolerance (66)
Cp4 epsps Agrobacterium tumefaciens
(aroA:CP4) strain CP4
(34)
Gat4621
(4)
Insect
resistance
(99)
Lepidopteran Cry1Ab
insect
(47)
resistance (83)
Bacillus licheniformis
Bacillus thuringiensis subsp.
kurstaki
Cry1Ab delta-endotoxin
Modified Cry1F protein
Vip3Aa20
(14)
Synthetic form of cry1F gene
derived from Bacillus
thuringiensis var. aizawai
Bacillus thuringiensis strain
AB88
Cry9C
(1)
Bacillus thuringiensis subsp.
tolworthi strain BTS02618A
Cry9C delta endotoxin
Cry1Fa2
(37)
Decreases binding affinity for
glyphosate and confers
increased tolerance to
glyphosate herbicide
Detoxifies glyphosate and
confers tolerance to
glyphosate herbicides
Confers resistance to feeding
damage caused by
lepidopteron insects by
selectively damaging their
midgut
Vegetative insecticidal protein
(vip3Aa variant)
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
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Cry1A.105 Bacillus thuringiensis subsp.
(12)
kumamotoensis
Cry2Ab2
(12)
Bacillus thuringiensis subsp.
kumamotoensis
Cry1F
(1)
Bacillus thuringiensis var.
aizawai
Cry1F delta-endotoxin
Mocry1F
(1)
Synthetic form of cry1F gene
from Bacillus thuringiensis
var. aizawai
Solanum tuberosum
Modified Cry1F protein
Synthetic form of cry3A gene
from Bacillus thuringiensis
subsp. tenebrionis
Bacillus thuringiensis strain
PS149B1
Modified Cry3A deltaendotoxin
PinII
(1)
Coleopteran
Mcry3A
insect
(28)
resistance (60)
Cry34Ab1
(31)
Modified
production
quality (11)
Pollination
control
system (6)
Protease inhibitor protein
Cry34Ab1 delta-endotoxin
Cry35Ab1 Bacillus thuringiensis strain
(31)
PS149B1
Cry35Ab1 delta-endotoxin
Cry3Bb1
(14)
Cry3Bb1 delta endotoxin
Modified alpha Amy797E
amylase
(8)
dihydrodipicoli
nate synthase
enzyme
Male sterility
(6)
Cry1A.105 protein which
comprises the Cry1Ab, Cry1F
and Cry1Ac proteins
Cry2Ab delta-endotoxin
CordapA
(2)
Bacillus thuringiensis subsp.
kumamotoensis
Synthetic gene derived from
Thermococcales spp.
Thermostable alpha-amylase Enhances bioethanol
enzyme
production by increasing the
thermo stability of amylase
used in degrading starch
Corynebacterium glutamicum Dihydrodipicolinate synthase Increases the production of
enzyme
amino acid lysine
Zm-aa1
(1)
Zea maize
Alpha amylase enzyme
Dam
(3)
Escherichia coli
DNA adenine methylase
enzyme
Barnase
(2)
Bacillus amyloliquefaciens
Barnase ribonuclease
(RNAse) enzyme
Zea maize
Ms45 protein
Fertility
Ms45
restoration (1) (1)
Enhances defence against
insect predators by reducing
the digestibility and
nutritional quality of the
leaves
Confers resistance to
coleopteran insects
particularly corn rootworm
pests by selectively damaging
their mid-gut lining
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
Hydrolyses starch and makes
pollen sterile when expressed
in immature pollen
Confers male sterility by
interfering with the
production of functional
anthers and pollen
Causes male sterility by
interfering with RNA
production in the tapetum
cells of the anther
Restores fertility by restoring
the development of the
microspore cell wall that gives
rise to pollen
11
4. Bt-maize and drought tolerant maize events
4.1 Bt-Maize
In 1996 Bt-maize was commercialized as one of the first transgenic crops. Currently,
17 years later, 99 commercialized events have been described (ISAAA, 2012). Bt-maize
contains one or multiple Cry genes derived from the gram-positive endospore forming
bacterium Bacillus thuringiensis. This bacterium can be found in many environments
such as the soil, plant surfaces and cereal storage dust (Schnepf et al., 1998). B.
thuringiensis can be distinguished from other bacillus species by the production of
parasporal crystals during sporulation. One group of proteins present in these crystals
are the Cry proteins, which are toxic to insects. Due to the production of these Cry
proteins, B. thuringiensis was characterized as an insect pathogen (Schnepf et al., 1998).
However, there is also evidence suggesting that the bacterium can accomplish an insect
independent lifecycle (Marco and Porcar 2012).
After ingestion of crystals by insects, the alkaline environment of the midgut causes
the crystals to dissolve resulting in the release of protoxins. After trimming of the N- and
C-termini by gut proteases of the insect, the activated toxins bind to specific receptors
on the cell membrane of the midgut epithelial cells (Schnepf et al., 1998). Once the
activated proteins are bound to the membrane, the toxin forms pores in the epithelial
cell membranes, killing the cell due to colloid osmotic lysis (de Maagd et al., 2001). The
activated toxin consists of three domains (Figure: 4). Domain I (blue) is important for
insertion in the membrane and the forming of pores. Domain II (green) and III (red) are
important for recognition of, and binding to the receptor (de Maagd et al., 1999).
The toxins of B. thuringiensis strains are highly
specific to binding receptor proteins on the
epithelial cells. Since these receptor proteins differ
between insect species the Cry proteins differ
among different strains of B. thuringiensis. More
than 290 different Cry proteins have been
identified and are classified according to their
amino acid sequence. Specific Cry proteins are toxic
against specific insect families, such as Lepidoptera
(catapilars), Diptera (flies and mosquitos) and
Coleoptera (beetles) (Crickmore et al., 2013; de
Maagd et al., 2001). In addition, some activity of
Cry-proteins against mites, nematodes, flatworms,
protozoa and some other insects have been
reported as well (de Maagd et al., 2001). This
species specificity is mainly dependent on the
structure of domain II and domain III.
Since 1930 spraying of B. thuringiensis has been
Figure 4: Cry-protein, adapted from
used to control insect damage. However, in the (de Maagd et al., 2001)
1950s the introduction of commercial Thuricide, a
mixture of B. thuringiensis spores and crystals, led to larger-scale production and usage
of B. thuringiensis as an insecticide (de Maagd et al., 2001). Although the usage of B.
thuringiensis as a natural insecticide increased, it never was a very important product.
Nowadays, B. thuringiensis sprays are mainly used by organic farmers. This lack of
success was caused by a number of disadvantages of directly applying spores and
crystals. The most important drawbacks are: 1) the lack of stability of the cry protein
(El-Sharkawey et al., 2009), 2) a high percentage of the product does not penetrate the
plant tissue, and therefore will never reach insects inside the plant tissue, 3) Cry
proteins act very specific and therefore you need to apply the right toxins for each
insect, and 4) Cry proteins need to enter the digestive system of the insect to be
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
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effective. The introduction of Cry genes in plants can overcome some of these
drawbacks that come along with directly applying B. thuringiensis as an insecticide.
Transgenic plants expressing Cry genes of B. thuringiensis continuously produce cry
proteins. This continuous production of Cry protein in plants gets around the problem of
fast protein degradation. Furthermore, sap sucking and piercing insects come in direct
contact with the toxins when feeding on a plant. Additionally, insects that do not feed on
the Bt crops will not be exposed to the toxin. Finally, the specificity of the Cry proteins
can be an advantage, since non-targeted organisms will not be affected by the proteins
(de Maagd et al., 1999).
In the first transgenic plants the expression of unmodified cry-genes was not high
enough. Probably the high A/T content in bacteria compared to plants decreased the Cry
gene expression levels, since the high A/T regions may contain plant transcription
termination sites, mRNA splice sites and/or mRNA instability motifs. Therefore,
extensive modifications to the gene, including removal of several polyadenylation sites,
removal of mRNA instability motifs and the introduction of tissue specific promoters,
were necessary for sufficient Cry protein production level (Gatehouse 2008). In maize
Cry protein levels can reach up to 0.4% of the total amount of soluble protein (de Maagd
et al., 1999) . In case of introduction of cry-genes in the genome of chloroplasts, even
higher levels of protein expression can be reached (up to 3–5% of the total soluble
protein). An additional advantage of expressing Cry genes in the chloroplast is that the
proteins are not produced in pollen, since plastid-encoded characteristics are
predominantly maternally inherited (Gatehouse 2008). However, good techniques for
stable transformation of chloroplast genomes have not been established yet. The
introduction of single Cry genes in maize was followed by the introduction of multiple
gene constructs for the expression of multiple cry proteins against a variety of insect
pest (Gatehouse 2008; de Maagd et al., 1999).
4.2 Drought tolerant maize
Worldwide, drought is the biggest threat for the agricultural production (Westgate
and Hatfield 2011; Jewell et al., 2010). Also maize can be severely affected by drought,
and especially during the flowering period it is vulnerable (Ribaut et al., 2009).
Therefore, research on drought tolerance has a high priority for breeding companies.
However, the response of a plant to drought is complex which makes research to
identify genes involved in drought responses difficult. In addition, vary the responses
due to variation in timing and duration of drought stress. Roughly, the responses of
plants to drought stress can be divided into avoidance of drought and tolerating of
drought. Drought avoidance mechanisms in maize lead to a diversity of changes in
phenotypic characteristics. Deeper rooting with less lateral branching that leads to more
water uptake. Shorter and thicker stems result in a more efficient transportation of
sugars. A reduction in tassel size saves energy. Smaller leaves above the ears and more
erected positioned leaves result in improved light penetration into the canopy. This
leads to a more homogenous light uptake, which increases the light use efficiency by
avoiding light saturation and decreases the leaf temperature. Both are important to
improve the plants water use efficiency (Jewell et al., 2010).
Genetic improvements in drought tolerance mechanisms are difficult since drought
tolerance mechanisms are complex and play a role within the entire plant (Jewell et al.,
2010). Drought tolerance mechanisms primary involve turgor maintenance,
protoplasmic resistance and dormancy. In maize numerous genomic regions (QTL’s)
have been found that are involved in morphological traits important for drought
tolerance. Genes involved in drought tolerance can roughly be grouped into three
groups: 1) involvement in signal transduction pathways and transcriptional control, 2)
genes that haver membrane and protein protection functions, and 3) genes involved in
water and ion transport and uptake. Today, several successful improvements have been
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
13
made involving manipulation of a single or a few genes. However, due to all interactions,
transformations in one or a few genes have often undesirable phenotypic or pleiotropic
side effects (Jewell et al., 2010).
Nevertheless, the first transgenic drought tolerant maize species will be
commercially available in 2013 in the Western Great Plains area within the USA
(Monsanto, 2013). The increase of drought tolerance in this maize event has been
accomplished by transferring a modified cold shock protein B (modified CspB),
originating from the soil bacterium Bacillus subtilis. During drought or other kind of
stresses, in B. subtilis this protein stabilizes the RNA molecules and facilitates the
translation of mRNA by disassociating the secondary structures formed of the RNA
molecules (Monsanto Japan Limited 2012; Westgate and Hatfield 2011). It has been
suggested that this protein functions in a similar way in maize and thus helps
maintenance of cell functions during drought stress. Under water-limited conditions,
mainly moderate drought events, CspB-maize events gave 7.5% more yield compared to
conventional varieties (p < 0.01). However, this was still only half of the production level
compared to yield levels that can be reached under well watered conditions (Westgate
and Hatfield 2011). Under well-watered conditions no significant differences in yields
were detected between the transgenic CspB maize and the non-transgenic control group.
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
14
5. Environmental risks of Bt-maize
The commercialization of transgenic maize in 1996 was surrounded by many
concerns around the environmental fate of the transgenic trades. The concerns varied
from place to place, however, the differences are probably best visible when comparing
the USA and Europe. The USA chose for fast adoption of transgenic crops, in 2009 85%
of the planted maize was transgenic. Worldwide nearly half of the share of transgenic
grown crops comes from the USA. In contrast, Europe takes a more conservative
approach, there it is assumed that transgenic maize has hazardous site effects and
therefore tests need to be done to prove its safety (Naqvi et al., 2011). These differences
resulted in the fast adoption of Bt-maize in some countries and the exclusion of the
production of Bt-maize in many other countries. Now 17 years later still a lot of research
money is invested in studies research on environmental effects of Bt-maize. A risk
assessment includes several steps for evaluating the potential adverse effects of a
transgenic crop. First the potential hazards are identified, which is followed by assessing
the hazards characteristics and the exposure characteristics. This results in
determination of the risk characteristics and subsequently risk management strategies
are formulated and an overall evaluation of the risks is made. For these risk assessments
of the environmental effects of transgenic crops it is important that the effects of these
crops are investigated in todays intensified agricultural practices, because these
practices already influence the environment by conversion of natural ecosystems into
agricultural land and the input of fertilizers and pesticides (Sanvido et al., 2007).
Since transgenic drought tolerant maize is a rather new transgene event no data on
the environmental impact of these crops is available yet. However, many studies on the
environmental impact of Bt-maize have been performed and reported. Therefore, in this
chapter I will focus on environmental effects of Bt-maize. First, the impact of Bt-maize
on biodiversity will be described. Next, the environmental risks are subdivided in direct
and indirect effects of Bt-maize on non-target organisms, such as beneficial insects, and
on the soil microbial community.
5.1 Biodiversity risks
Crop diversity
Crop diversity refers to the genetic variation within a crop species. This diversity
ensures that different traits are included in a crop species. In this way it ensures the
production of a crop in a wider range of conditions (Carpenter 2011). Especially since
the climate is changing, maintaining and enhancing the genetic diversity is of high
importance for the resilience of food crop production. One of the concerns regarding the
introduction of transgenic crops is that this would endanger the rich genetic diversity of
several crop species (Jacobsen et al., 2013). However, only a small number of studies
have investigated the effect of genetically modified crops on the within crop diversity,
and no studies have been performed on the influence of the introduction of transgenic
maize on maize diversity (Carpenter 2011; Ammann 2005). A study by Bowman et al.
(2003) on the uniformity of cotton in the USA revealed that the genetic diversity within
the crop increased over the period 1995-2000. During this study, uniformity data from
1995, when no transgenic cotton was grown, were compared with data from 2000,
when 72% of planted cotton was transgenic. They found that the field genetic
uniformity decreased with 28%. This was probably due to increased availability of
transgenic and conventional cotton varieties (Bowman et al., 2003). However, a similar
study on the uniformity of Bt-cotton in India found a decrease in crop diversity. Though,
this decrease in diversity could be diminished, according to the researchers, by the
release of more transgenic Bt-maize varieties (Krishna et al., 2009).
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
15
Farm scale diversity
Besides the concerns around genetic crop diversity also concerns were raised around
the effects of Bt-maize on organisms that naturally come in contact with the transgenic
crops. The farm scale diversity describes the diversity of organisms that primarily live
within the boundaries of a farm. In general it is thought that the introduction of Btmaize has a positive impact on on-farm biodiversity, since the in planta production of
cry-proteins manages the insect pests in a very specific way compared to chemical
insecticides (Ammann 2005). When comparing the biodiversity on fields of Bt-maize
with the biodiversity of conventional crops that were not sprayed with insecticides, no
difference in biodiversity were observed when looking at the community structure or
individual species abundance. The only species that were negatively influenced by the
Bt-maize were the target insects and their natural enemies (Ammann 2005). However,
when comparing diversity between fields of conventional maize varieties that were
sprayed with insecticides and non-sprayed Bt-maize fields, the conventional sprayed
fields showed a lower biodiversity compared to the non-sprayed Bt-maize fields (Rose
and Dively 2007). This was due to the strong effects that the spraying had on many nontarget species within the sprayed fields, leading to lower non-target population within
these fields compared to the Bt-fields (Sanvido et al., 2007; Ammann 2005). These
results show that the use of Bt-maize indeed results in increased farm biodiversity
levels compared to conventional farming methods.
Landscape scale diversity
Besides the local effects of Bt-maize, the effects of transgenic crops on the broader
environment have been investigated. Of special concern were the effects of gene flow on
the environment, the effects of transgenic crops on vertebrates consuming the maize
products and the landscape wide diversity loss. Still, the most important reason for
biodiversity loss is habitat loss caused by conversion of natural ecosystems into
agricultural land. Increases in yield per m2 could result in reduced conversion of natural
ecosystems to agricultural land. Analyzing 49 studies, Carpenter et al. (2010) found that
overall yields increased for farmers who adopted transgenic crops. Meaning that the
adoption of transgenic crops could reduce the conversion of natural areas in agricultural
land resulting in better conservation of biodiversity levels.
The biggest concerns around the introduction of Bt-maize on landscape level include
the transfer of genes to nearby non-Bt-maize crops causing unintended spreading of the
trait. Further concerns were the transfer of the Bt-gene to non-cultivated, wild relatives
of maize and the potential establishment of Bt-maize as a weed. Maize is a wind
pollinator and releases its pollen grains in high quantities. However, since the pollen
grains of maize are relatively large, 90-125 micrometer, they tend to settle relatively
close to the source, 90 percent has been found at a distance of 2 – 10 meter (Naqvi et al.,
2011). However, to minimize the risks of gene flow predetermined standards for
isolation distances between fields have been set on 1.6 km within the USA, even though
201 meter distance between fields already guarantees 99.5 percent grain purity (Naqvi
et al., 2011). The risk of gene flow to natural related species would only be of a concern
if the insect species that the Bt-gene provides protection against are important in
regulating the population of the non-crop relatives (Storer et al., 2010). In addition,
could traits from other conventional grown maize varieties influence the natural
relatives as much as the transgenic incorporated traits. The potential establishment of
Bt-maize as a weed is not likely, since agricultural crops do not do well when competing
with native vegetation.
Overall it is believed that the use of Bt-maize leads to an area-wide decline of the
target species populations. This will not only be beneficial for the adopters of Bt-maize
but will also positively influence the amount of pest species in areas where conventional
maize is grown. An investigation on moth populations, Ostrinia nubilalis and Helicoverpa
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
16
zea, in Maryland, USA, revealed a correlation between the introduction of Bt-maize in
1996 and a decline in abundance of the target moth species (Storer et al. 2008).
5.2 Effects on non–target species
As mentioned above, growing Bt-maize appears to have less effect on non-target
organisms compared to conventional maize growing in combination with spraying of
insecticides. Research on the effects of Bt-maize on non-target organisms includes
monitoring the parasitoids and predators that are important for natural pest regulation
on Bt-maize but also the level of pollinators and butterflies. Additionally, the effects that
Bt-maize has on the soil ecosystem and on the health of vertebrates, including humans,
have been reviewed.
Non-target insects
In general insects are polyphagous and are therefore able to switch to other preys
when one specific food source is scarce. The growth of Bt-maize does have an effect on
certain insect species. Greenhouse experiments revealed that not the cry-protein
directly affects the beneficial insects and predators present on maize fields. Rather, the
effects of Bt-maize on non-target insects are the result of the decreased level of the
targeted insects that serve as prey to the beneficial insect. However, the most used
technique for assessing the effect of Bt-maize on beneficial insects includes field-testing
the abundance of these insects. A few studies have compared biological control
functions of the natural enemies in a conventional field and a Bt-maize field (Sanvido et
al., 2007). During these experiments, where Bt-maize was compared with normal
insecticide use (such as pyrethroids and organophosphates), reduced abundances of
insects in conventional fields were found, despite the studies were limited in their
spatial scale and thus lack statistical power (Sanvido et al., 2007).
Even though maize is a wind pollinated crop, the effects of cry proteins on pollinators
such as bees and bumblebees have been investigated. No effects were found when
feeding cry proteins to these pollinators. In addition, it was long thought that the pollen
of maize would have an adverse effect on other Lepidoptera species feeding on plants
that are contaminated with the pollen of Bt-maize. In a study by Jesse and Obrycki
(2000), significant increased mortality levels of Danaus plexippus larvae feeding on
contaminated Asclepias syriaca (common milkweed) growing in a Bt-cornfield were
found. Within the first 48 hour of exposure to accumulating Bt-maize pollen of event
176, the mortality rate of the D. plexippus larvae was 20% compared to 0% mortality in
the non-Bt pollen treatment and 3% mortality in larvae feeding on washed A. syriaca
leaves (P=0.0415; Jesse and Obrycki 2000). During later studies it turned out that only
the pollen of Bt-maize event 176 had these adverse effect on D. plexippus larvae.
Therefore, this variety has been withdrawn from the market (Sanvido et al., 2007). Since
other Bt-events produce lower amount of Cry-proteins (80 times less), risks for the
larvae will be lower as well (Sanvido et al., 2007). A model from Dively et al. (2004)
showed that Bt-maize caused an additional mortality risk for the monarch larvae of
0.6%. Additionally, other studies found that the impact of Bt-maize pollen on D.
plexippus populations was negligible (Oberhauser and Rivers 2003; Sears et al., 2001).
Overall, there is no clear evidence that Cry-proteins affect insects other than the
target organisms and its predator populations.
Soil ecosystem
Besides concerns about the influence of Bt-maize on non-target insects also concerns
have been raised about the effect of Cry-proteins on soil ecosystems. Bt-proteins can
enter the soil ecosystem via root exudates or biomass degradation. The amount of toxins
entering the soil depends on the type Bt-gene, the expression level of the toxins and the
amount of decaying plant material. The persistence of the toxins depends on numerous
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
17
biotic and abiotic factors. In general, degradation of the Cry-protein will take between a
few hours to several months (Sanvido et al., 2007). Several studies have investigated the
effects of the Bt-toxins on the soil ecosystem. However, due to the great diversity of
biotic factors, such as bacterial and fungal communities and the presence of certain
macro soil fauna, and abiotic factors, such as soil type, pH and availability of nutrients
and water, research on this topic has proven to be difficult.
Mulder et al. (2006) found an increase in soil bacterial activity within the first 72
hours after application of Cry1Ab Bt-maize litter compared to conventional maize litter.
However, after three weeks no significant differences were found between Bt-maize and
conventional maize. In two other studies, where the relative abundance of rhizosphere
bacteria and endophyte communities in conventional and several Bt-maize lines,
producing Cry1Ab2, Cry2Ab2, Cry3Bb1, Cry1A105 were compared, no significant
differences were found between the control- and the Bt-maize treated groups
(Dohrmann et al., 2012; Prischl et al., 2012). In a different experiment in a tropical
agrosystem not only alterations in the bacterial community but also changes in the
fungal community were investigated. Also in this study no differences were found
between the Bt-maize and their parental lines (Cotta et al., 2013).
Besides soil microorganisms also the effect of Bt-maize on the soil macro fauna have
been studied. This macro fauna include, nematodes earthworms, soil mites etc. Studies
on nematodes and woodlice that were exposed to Bt-maize during different lengths of
time did not show differences in animal behavior (Sanvido et al., 2007). From all studies
that investigated the effects of Cry1Ab of Bt-maize on earthworms (Lubricus terrestris),
only 1 study found significant differences in weight of the earthworms feeding on Btmaize (Sanvido et al., 2007). In this study of 200 days the weight loss of earthworms
exposed to Bt-maize was 18%. In contrast, earthworms feeding on conventional maize
gained 4% in weight (Zwahlen et al., 2003).
Overall, the effect of Bt-maize on soil ecosystem cannot be eliminated entirely, since
an effect has been found in earthworms. However, overall no differences have been
found between conventional and Bt-maize growing.
Non-target vertebrates
Another great concern is that transgenic plants will affect animal and human health.
The exposure risks of transgenic plants compared to conventional parent lines include
allergenicity and adverse reactions to the transgenic plants or its products. The possible
allergenicity is of special concern to maize since so far maize is one of the few crops that
has been eaten for millennia by billions of people without causing illness (Naqvi et al.,
2011). In a study by Buzoianu et al. (2012) on the effect of Bt-maize on inflammations or
allergy effects in sows no indications for increased risks were found. However, in
studies performed over periods of 90 days to long-term health effects in rats feeding on
Bt-maize, some adverse effects on the liver and kidneys were found. In four of the eleven
studies statistical differences were found in one or both organs. Therefore, potential
chronic toxicity as a result of Bt-maize based diets cannot be excluded and longer study
periods, of two years or more, are recommended (Séralini et al., 2011).
So far chronic toxicity risks of Bt-maize based diet cannot be excluded. However,
based on the available data the risks of negative side effects appear to be small.
5.3 Interaction with target organism and development of resistance
Besides the uncertainties around the unknown side effects of Bt-maize, target
organisms could develop resistance against the Bt toxins. At this moment there are
several field cases known where different target species showed increased resistance
levels against specific Cry-proteins from Bt-maize events (Gassmann et al. 2011, Sorer et
al. 2010, Rensburg et al 2007). In 2007, farmers in Puerto Rico reported reduced
performances of a Cry-1F protein producing maize event against the Lepidoptera
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
18
species Spodoptera frugiperda. In a laboratory bioassay to the resistance of the S.
frugiperda colonies from Puerto Rico, Storer et al. (2010), found that the resistance was
recessive and that it was autosomally inherited. Besides increased resistance against the
Cry-1F protein the research showed a minor decrease in sensitivity levels against the
Cry1Ab and Cry1Ac proteins as well (Storer et al. 2010). The resistance against the Cry1F event was probably initiated by a lack of different host species causing increased
selection pressure for S. frugiperda. In another survey increased resistance of a western
corn rootworm population against Cry-3Bb1 protein producing maize was found.
Gassmann et al. (2011), argued that the consecutive amount of years that the same Btmaize event was grown on the same field could be the reason for the increased
resistance of the rootworm population. The presence of several resistance occasions
against Bt-maize events needs to be taken very serious. However, this problem is not
only restricted to the genetic modification technology since it is also a present problem
in case of using insecticides for controlling maize related pests.
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
19
6. Discussion
Maize has been cultivated for many centuries. Because of this cultivation and
targeted breeding the maize teosinte diverged into the maize we grow and eat today
(James 2003). Now maize is one of the most important food sources in the world and is
produced in more countries than any other crop. Especially in the tropical regions maize
is one of the most important staple crops. However, spatial differences show that in the
temperate zone yields are much higher compared to the tropical zone (Edmeades 2008).
These differences in yields are mainly the result of different maize hybrids, differences
in agricultural practises, differences in access to fertilizers and pesticides and different
abiotic factors. Other factors that influence yield are pest and plague invasions or
extreme climate events. These yield losses could potentially be reduced by the
introduction of transgenic maize varieties. Bt-maize reduces yield losses due to insect
herbivory by the production of insecticidal Cry-proteins, and drought tolerant maize
could decrease losses that are the result of drought events. However, prior to the
commercialisation of a transgenic crop event, comprehensive environmental risk
assessments are required (EFSA, European Food and Safety Authority, 2010). These risk
assessments are done to exclude potential unintended side-effects of the use of
transgenic crops.
Since the first field introduction of transgenic maize in 1996, 30 countries have
adopted the production of transgenic crops. In the USA even 85% of the maize grown is
transgenic (Jacobsen et al., 2013; Naqvi et al., 2009). However, in other countries, like in
Europe, the introduction of transgenic crops encounters severe resistance. In these
countries many concerns have been raised around the environmental risks of the
introduction of transgenic crops. These concerns focus mainly on the effects of
transgenic crops on human health, effects on non-target organisms and the possibility of
gene-flow between transgenic and non-transgenic plants.
After more than a decade of commercialized growth of Bt-maize, there is a better
view of the potential long-term environmental side effects. The use of Bt-maize seems
not to have increased pressure on crop- and biodiversity compared to conventional
agricultural practices (Carpenter 2011; Sanvido et al., 2007). When looking at insect
diversity, the use of Bt-maize even appears to result in an increased level of diversity
and abundance compared to conventional insecticide sprayed fields (Ammann 2005). In
addition, decreases crop uniformity have been found after the introduction of Bt-maize
events (Bowman et al., 2003). However, these investigations to the crop uniformity
were done in highly developed areas, where maize is mainly grown in monocultures. In
the research done by Krishna et al. (2009), performed in a non-industrialized area, a
decrease in cotton diversity was found. Thus, the introduction of transgenic maize
varieties, such as drought tolerant maize, could lead to a decrease in the number of
available genetic traits and diversity in tropical regions. A decrease in the number of
traits might result in the loss of traits important for the plasticity of the crop, which
could have adverse effects on the production, particularly now climatic changes are
expected (Rusch et al., 2010; Soleri and Cleveland 2006). However, as seen in Mexico,
this is not necessarily due to the introduction of a transgenic crop, but rather is caused
by the introduction of intensive cultivation of monoculture maize from hybrids, and the
use of increasing amounts of agro- chemicals (Wise, 2007). Besides the negative effects
of more industrialized agricultural practices, intensified cropping also leads to increased
yields. Currently, industrialized countries reach grain yields up to 8 t/ha-1 compared to 3
t/ha-1 in developing countries (CIMMYT, 2013).
Next to the risks of a decrease in maize diversity, there is the risk of unintended
spreading of the transgenic traits by gene flow. This risk is unrelated to the trait and is
therefore not limited to Bt-maize but is a concern for every transgenic event. Because
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
20
nearly no pollen settles at distances of more than 200 meter from the source, the risk of
gene flow between maize varieties is relative low when there is a buffer zone of more
than 200 meters between fields of transgenic and non-transgenic maize (Naqvi et al.,
2011; Luna et al., 2001). In industrialized countries field regulations are strict and
chances on gene flow are limited. For example, in the USA the distance between fields
with transgenic maize and conventional varieties needs to be 1.6 km. In Mexico most of
the genetic variation originates from high levels of gene-flow between small scale farms
(Soleri and Cleveland 2006). Also in other developing countries agricultural practices
are based on small scale farming. Therefore, implementing such regulations as in the
USA is much more difficult in developing countries.
If a maize variety is a threat for the landscape diversity depends on the trait and the
ability of a crop plant to become a weed (Sanvido et al., 2007). In maize the ability for
seed dispersal is for instance rather low, since the kernel sticks to the ear and the ear
sticks to the plant (Meyerowitz 1994). However, when by gene flow a certain trait gets
incorporated in a natural variety of maize this could lead to enhanced fitness. This could
be a concern for Bt- or drought tolerant maize events. For the drought-tolerance trait
this could lead to the invasion of the crop into new habitats, since the niche in which a
maize plant with this trait could grow is larger compared to conventional maize
varieties. However, even though there are significant fitness advantages for the
transgenic species, drought tolerant maize gives 7.5% more yield during drought events
than conventional varieties, the differences are still small and therefore invasion risks
might be negligible. Still, there are a few cases known where hybridization between a
transgenic crop species and their wild relatives caused ecological problems (Sanvido et
al., 2007). In Europe the F1 of Bt-Oilseed rape (Brassica rapa) that entered natural
patches, had an enhanced fitness under high herbivore pressure by producing 1.4 times
more seeds compared to their wild type relatives. However, without herbivore pressure
wild type plants produced more seeds (6.2 times more; Vacher et al., 2004).
Nevertheless, for maize so far no evidence has been found that hybridization between
transgenic maize and teosinte occurred.
For insects no substantial evidence has been found that Cry-proteins have negative
side effects on non-target insect species, but do affect the target species and their
predators populations. Similar results were found in studies on soil macro-fauna like
worms and nematodes. However, there is still a small chance that insects will feed on
transgenic maize pollen spread by wind and that lands on other plants. This risk can
mostly be eliminated when the Bt-gene is incorporated in plastid DNA of maize, since
plastid DNA is mainly maternally inherited and therefore will not be present in pollen
(Gatehouse 2008). It is not very likely that transgenic drought tolerant maize events
have a strong effect on insect diversity and abundance. However, research data on this
subject is still missing.
Also the effects of the Cry-proteins on soil microbial communities have been studied
extensively. However, these studies are difficult due to the biotic and abiotic variations
of the soil ecosystems. So far tests on the impact of transgenic maize on the soil
microbial community did not reveal any effects compared to the control groups. In
addition, there are concerns for horizontal gene transfer of recombinant transgenic
maize DNA to soil bacteria (EFSA, 2010). Events of horizontal gene transfer from plants
to bacteria are rare and in experiments with Bt-maize no horizontal gene transfer could
be demonstrated. For drought tolerant maize this has not been tested yet. Besides this,
the traits for drought tolerant maize and Bt-maize originate both from soil bacteria
(Mendelsohn et al., 2003).
Potential risks for human and animal health need to be taken very seriously, since
maize is such an important food and feed crop. So far no adverse effects have been found
on sows, or humans due to feeding on Bt-maize (Naqvi et al., 2011; Hammond et al.,
2006). However, some adverse effects have been found in liver and kidneys of rat fed on
Bt-maize for 90 days. To exclude potential chronic toxicity effects experiments of 2 years
Environmental risk of Bt-maize and drought tolerant maize – A.Nijmeijer – June 2013
21
were recommended (Séralini et al., 2011). For the drought tolerant maize events the
amino acid sequence of the CspB-protein has been compared with allergen amino acid
sequences this test did not reveal any similarities between the CspB-protein and
allergen sequences (Monsanto Japan Limited 2012; Castiglioni et al., 2008). This is an
indication that drought tolerant maize may not have acute adverse effects. However, in
vivo experiments on animals should be done to exclude acute and chronic toxicity.
Overall, potential environmental drawbacks of transgenic crops are tested over and
over without substantial evidence for negative effects that are not present already in
conventional agricultural practices. There are large differences between regulations
regarding the admittance of transgenic crops in different countries, which has a lot to do
with the local public opinion. In addition, scientific data on the environmental effects of
transgenic crops tend to remain controversial. Therefore, clear scientific criteria to
evaluate the environmental effects of transgenic crops should be set, as has been
proposed by Smit et al. (2011) for surveillances on the effect of transgenic crops on soil
ecosystems.
Bt-maize and drought tolerant maize events together could help increase or stabilise
yields and could therefore be very beneficial for the food security and the risks involved
in growing these crops appears to be low. However, adoption of these events in tropical
regions still needs more in-depth research.
Environmental risk of Bt-maize and drought tolerant maize – A. Nijmeijer – June 2013
22
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