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Transgene Expression and Bt Protein Content in Transgenic Bt Maize (MON810) under Optimal and Stressful Environmental Conditions

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Bt protein content in transgenic insect resistant (Bt) maize may vary between tissues within plants and between plants growing under different environmental conditions. However, it is unknown whether and how Bt protein content correlates with transgene expression, and whether this relationship is influenced by stressful environmental conditions. Two Bt maize varieties containing the same transgene cassette (MON 810) were grown under optimal and stressful conditions. Before and during stress exposure, the upper leaves were analysed for transgene expression using quantitative RT-PCR and for Bt content using ELISA. Under optimal conditions there was no significant difference in the transgene expression between the two investigated Bt maize varieties whereas Bt protein content differed significantly. Transgene expression was correlated with Bt protein content in only one of the varieties. Under stressful environmental conditions we found similar transgene expressions as under optimal conditions but Bt content responded differently. These results suggest that Bt content is not only controlled by the transgene expression but is also dependent on the genetic background of the maize variety. Under stressful conditions the concentration of Bt protein is even more difficult to predict.
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RESEARCH ARTICLE
Transgene Expression and Bt Protein Content
in Transgenic Bt Maize (MON810) under
Optimal and Stressful Environmental
Conditions
Miluse Trtikova
1
*, Odd Gunnar Wikmark
2
, Niklaus Zemp
1
, Alex Widmer
1
,
Angelika Hilbeck
1,2
1ETH Zurich, IBZ, Plant Ecological Genetics, Universitaetstrasse 16, 8092 Zurich, Switzerland, 2Genok
Centre for Biosafety, 9294 Tromso, Norway
*miluse.trtikova@env.ethz.ch
Abstract
Bt protein content in transgenic insect resistant (Bt) maize may vary between tissues within
plants and between plants growing under different environmental conditions. However, it is
unknown whether and how Bt protein content correlates with transgene expression, and
whether this relationship is influenced by stressful environmental conditions. Two Bt maize
varieties containing the same transgene cassette (MON 810) were grown under optimal
and stressful conditions. Before and during stress exposure, the upper leaves were ana-
lysed for transgene expression using quantitative RT-PCR and for Bt content using ELISA.
Under optimal conditions there was no significant difference in the transgene expression
between the two investigated Bt maize varieties whereas Bt protein content differed signifi-
cantly. Transgene expression was correlated with Bt protein content in only one of the varie-
ties. Under stressful environmental conditions we found similar transgene expressions as
under optimal conditions but Bt content responded differently. These results suggest that Bt
content is not only controlled by the transgene expression but is also dependent on the ge-
netic background of the maize variety. Under stressful conditions the concentration of Bt
protein is even more difficult to predict.
Introduction
Genetic modification of crop plants often has the goal to engineer lines that express novel traits
that cannot be introduced into the crop by conventional breeding. Such bioengineering efforts
build on the expectation that target gene(s) conferring the desired trait, in association with suit-
able regulatory elements that are also part of the transgene construct, express the desired trait
in a stable and reliable manner. This expectation remains to be evaluated, for example when
the transgene is introduced into different genetic background (i.e. varieties) or when genetically
modified (GM) plants are exposed to diverse environmental conditions.
PLOS ONE | DOI:10.1371/journal.pone.0123011 April 8, 2015 1/9
OPEN ACCESS
Citation: Trtikova M, Wikmark OG, 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 10(4): e0123011. doi:10.1371/
journal.pone.0123011
Academic Editor: M. Lucrecia Alvarez, Mayo Clinic
Arizona, UNITED STATES
Received: February 12, 2014
Accepted: February 26, 2015
Published: April 8, 2015
Copyright: © 2015 Trtikova et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Funding: This research was funded by ETH Zurich,
Genok, Manfred-Hermsen-Stiftung für Natur und
Umwelt and Testbiotech. The funders had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
One of the two most widely marketed GM traits worldwide is insect resistance, which is
conferred by insecticidal toxins from Bacillus thuringiensis (Bt). This trait has been engineered
into a number of crop plants, including maize and cotton. Maize containing insect resistance
trait is currently grown on more than 47 million hectares worldwide [1], which represents
about 27% of the global area planted with maize [2]. Bt maize cultivars derived from the
MON810 event specifically contain a transgene cassette consisting of the cauliflower mosaic
virus 35S promoter, thet e hsp70 intron and the cry1Ab gene endowing the resulting MON810
Bt maize plants with a resistance to lepidopteran pest species, particularly, the European corn
borer, Ostrinia nubilalis.
It is generally expected that in commercial GM plants, transgenes are constitutively express-
ed at high levels and in all plant tissues and phenological phases [3]. Tight control over trans-
gene expression and Bt protein content is important in light of concerns over the evolution of
Bt toxin resistance in target insects [4]. In recent years, several studies have reported that the Bt
protein concentration may vary within Bt plants (i.e. across tissues) and over growing seasons
[57], while other authors reported that abiotic factors, such as nitrogen fertilization [8], soil
quality and pesticide use [9] can affect Bt protein content. However, to the best of our knowl-
edge, no study has been published to date that jointly investigated the relationship between Bt
transgene expression and Bt protein content in transgenic Bt maize. In Bt cotton, this relation-
ship has been investigated in a limited number of studies [1012]. Olsen et al. [12] and Adamc-
zyk et al. [10] found correlations between mRNA transcript levels of insect resistance
transgene cry1Ac and Bt protein content, whereas Li et al. [11] observed no such relationship
under salt stress. It therefore currently remains open what the relationship between Bt trans-
gene expression and Bt protein content in GM crops is.
Establishing whether such a relationship exists in Bt maize and how it is affected by environ-
mental conditions is an important question. In most countries where cultivation of Bt maize
has been approved, this was done on the condition of installing an insect resistance manage-
ment (IRM) program. One of the pillars of IRM is that plants contain high and stable levels of
the Bt protein that are lethal not only to susceptible target insects but also to heterozygotes that
carry one resistance allele (RS genotypes) [13]. The aim of IRM is to delay the evolution of re-
sistance to Bt toxins in target pests which has been identified as a prime threat to the sustain-
ability of Bt crops [4].
The aims of this study were to explore the relationship between Bt transgene expression and
Bt protein content in two Bt maize varieties, and to experimentally test whether abiotic envi-
ronmental stress conditions influence the relationship between transgene expression and
protein content.
Materials and Methods
Plant material and treatments
Seeds of two Bt maize (MON 810) varieties (white BtPAN 6Q-321B and yellow BtPAN 6Q-
308B) were sown into two litre plastic pots filled with the potting soil (Oekohum Topferde mit
Kokos) and covered with a layer of gravel. Fifteen plants of each variety were first grown in the
climate chambers under optimal conditions (16/8 L/D, 25/20°C, 50/65% rh, watered regularly).
After six weeks, the plants were either kept further under optimal conditions or exposed to stress-
ful environmental conditions for one week. The stressful conditions included a hot/dry treatment
in a greenhouse: 16/8 L/D, temperature varied in a shade between 2130°C and in a full sun
reached up to 45°C, relative humidity varied between 3967%, the plants were watered sparsely,
100 ml per pot on a daily basis. Or a cold/wet treatment was applied: 16/8 L/D, 16/13°C,
65/80% rh, waterlogged for 24 h, afterwards soil kept saturated with water. Before application of
Transgene Expression and Bt Protein Content in Transgenic Bt Maize
PLOS ONE | DOI:10.1371/journal.pone.0123011 April 8, 2015 2/9
the stress treatment, the plants had on average 11 leaves and 7 collars. After one week of cold/wet
and hot/dry stress, the plants had 11 leaves and 8 collars, whereas under optimal conditions they
had on average 12 leaves and 8 collars.
Plant sampling
The second upper fully developed leaves were sampled before the stressful conditions were ap-
plied. The tips of the leaves were cut off and seven about 4 cm
2
pieces were cut next to each
other avoiding the main leaf vein. Following this approach we could reduce the variation in Bt
content within the single leaf [7]. The five leaf pieces assigned to be analysed for transgene ex-
pression were immediately frozen in liquid nitrogen and later stored at -80°C. The two leaf
pieces collected to measure Bt content were kept on ice and later stored at -20°C. After one
week of stressful conditions, the same plants were resampled. When possible, the samples were
not collected from the same previously cut leaves. Instead, seven leaf pieces were cut from the
leaves above or from newly formed leaves.
qRT-PCR
RNA was extracted from 60 leaf samples (15 white Bt and 15 yellow Bt maize plants sampled
before and during stress) using RNeasy Plant Mini Kit (Qiagen). RNA quality was checked on
Bioanalyzer Agilent 2100 using Plant RNA Nano chips. Only samples with RIN higher than 5
were used for further analysis. RNA was treated with RDD Buffer (Qiagen) and DNAse (Qia-
gen) and inactivated with EDTA (Invitrogen). QuantiTect Reverse Transcription Kit (Qiagen),
including the Wipeout Buffer, was used to produce cDNA. The samples were run in triplicates
in a 11 μl reaction volume using TaqMan Gene Expression Master Mix (Applied Biosystems).
The primers and probe sequences for cry1Ab transgene were kindly provided by A. Coll (Insti-
tut de Tecnologia ària (INTEA), Universitat de Girona). Additionally, four reference genes
(mep,ubcp,cul,lug) as recommended by Manoli et al. 2012 [14] were chosen to normalize the
qRT-PCR data. TaqMan primers and probes for reference genes were designed based on the se-
quences obtained from Maize Genetics and Genomics Database (http://www.maizegdb.org/)
using Primer Express 3.0 software (Applied Biosystems). Amplification efficiencies were esti-
mated using LinRegPCR software version 2012.3 [15](S1 Table). The stability of the reference
genes was assessed using geNorm and cul was excluded because it was not stable enough. The
remaining three reference genes were used for normalization (M <0.5 and pair-wise coeffi-
cient variance <0.15) of the expression data using the qbase+ software (Biogazelle).
ELISA
The leaf samples originated from the same plants analysed for transgene expression. Approxi-
mately 10 mg of freeze-dried leaf material was ground using a FastPrep-24 Instrument (MP
Biomedicals, Inc.) and homogenized in 1.5 ml of PBST-buffer (pH 7.4). After centrifugation
the supernatants were diluted 1:50 with PBST-buffer. Standards were prepared using freeze-
dried Cry1Ab toxin (M. Pusztai-Carey, Case Western Reserve University) identical with the
Bt-protein expressed in the Bt maize plants. Thirteen Cry1Ab concentrations were used for the
calibration curve ranging from 0 to 6.8 ng/ml dissolved in PBST-buffer. In addition 3 standards
were prepared with control leaf extracts from conventional maize. The level of Bt protein in
maize leaves was determined using the commercial double antibody sandwich (DAS) ELISA
kit (Agdia). Standards and controls were added to a 96-well ELISA microplate in duplicates,
samples were added in triplicates. The colour development was measured in a kinetic mode at
650 nm using a Bio-Tek Synergy HT multi detection microplate reader. The colour reaction
Transgene Expression and Bt Protein Content in Transgenic Bt Maize
PLOS ONE | DOI:10.1371/journal.pone.0123011 April 8, 2015 3/9
was stopped after 16 minutes by adding 3 M sulphuric acid and colour intensity was read at
450 nm.
Statistical analysis
Due to non-normal distribution, the transgene expression data (5 plants per variety and treat-
ment) and the Bt content data (45 plants per variety and treatment) were log10 transformed.
Three-way ANOVA was used to test for the effects of the variety, stress treatment and the tim-
ing of the sampling (before and during stress) on the transgene expression and Bt content. Fold
change in the transgene expression and the Bt content between the first (before stress) and the
second (during stress) sampling was calculated as the ratio of the during stressvalue to the
before stressvalue. Due to non-normal distribution the fold change data were log10 trans-
formed. The fold change means obtained for different stress treatments and plant varieties
were compared using Tukeys HSD method. Correlation between the transgene expression and
the Bt protein content was analysed using Spearman rank correlation coefficient (Rs), as rec-
ommended by Ponnala et al. [16]. All statistical analyses were performed in JMP 10.0.0 (SAS
Institute Inc. 2012).
Results
Transgene expression
There was no significant difference in cry1Ab expression in the upper leaves of the two Bt
maize varieties (Table 1). Also, the transgene expression did not differ between the treatments
(Table 1). We also compared how transgene expression changed during the stress relative to
the level before stress (i.e. fold change) in the same plants (Fig 1). In the white Bt maize, the
transgene expression under cold/wet stress was similar to the expression under optimal condi-
tions, but was significantly reduced under hot/dry stress. In the yellow Bt maize, the transgene
expression under cold/wet and hot/dry stress was not significantly different from the expres-
sion under optimal conditions (Fig 1).
Bt protein content
There were significant differences in the Bt content in the upper leaves of the two Bt maize vari-
eties (Table 1). The Bt content in the leaves of the yellow Bt maize plants was on average higher
than in the leaves of the white Bt maize plants. There were also significant differences in the Bt
Table 1. Effects of variety (white Bt, yellow Bt), stress treatment (optimal, cold/wet, hot/dry), sampling time (before and during stress) and their in-
teractions on cry1Ab transgene expression and Cry1Ab protein content.
ANOVA cry1Ab transgene
expression
Cry1Ab protein content
df F ratio P F ratio P
Variety 1 0.02 0.889 5.33 0.025
Treatment 2 0.58 0.561 5.70 0.006
Sampling time 1 3.17 0.081 1.36 0.249
Treatment*Variety 2 0.11 0.893 0.14 0.866
Treatment*Sampling time 2 7.41 0.002 1.70 0.195
Sampling time*Variety 1 0.02 0.899 3.66 0.062
Treatment*Sampling time*Variety 2 1.10 0.341 3.10 0.054
Signicant effects (P<0.05) are shown in bold.
doi:10.1371/journal.pone.0123011.t001
Transgene Expression and Bt Protein Content in Transgenic Bt Maize
PLOS ONE | DOI:10.1371/journal.pone.0123011 April 8, 2015 4/9
content between the treatments (Table 1). The Bt content was similar in the plants grown
under optimal and hot/dry conditions. However, the leaves of Bt maize plants exposed to cold/
wet stress had significantly higher Bt content than the leaves of the plants grown under optimal
conditions. When comparing how Bt content changed during the stress relative to the level be-
fore stress (i.e. fold change), the Bt content in the white Bt maize plants exposed to the cold/
wet conditions increased 4-times compared to the plants grown under optimal conditions, but
this was not the case for the same treatment with the yellow Bt maize (Fig 2).
Correlation between transgene expression and Bt protein content
The relationship between transgene expression and Bt protein content differed between the
two Bt maize varieties. The Bt protein content was correlated with cry1Ab transgene expression
in the white Bt maize plants (Rs = 0.536, P= 0.040) but not in the yellow Bt maize plants
(Rs = 0.407, P= 0.133) (Fig 3). Furthermore, the correlation was only found in the white Bt
maize grown under optimal conditions before any stress treatment was applied. No correlation
between cry1Ab transgene expression and Cry1Ab protein content was found in the plants ex-
posed to cold/wet or hot/dry stress (S1 Fig).
Discussion
In this study we investigated whether there is a correlation between transgene expression and
Bt protein content in two Bt maize varieties containing the same transgene cassette (MON
810) and how this relationship is influenced by stressful environmental conditions. Overall, we
found no differences in transgene expression between the two different Bt maize varieties
Fig 1. Transgene expression. Fold change in the transgene expression in the upper leaves ofthe white and
yellow Bt maize between the first (before stress) and the second (during stress) sampling. Calculated as the
ratio of the during stressvalue to the before stressvalue (i.e. 1 = no change). Plants grown under optimal
growth conditions were exposed to no stress. The means labelled with the different letters are significantly
different at P<0.05, Tukeys HSD. Means ±SE, n = 5.
doi:10.1371/journal.pone.0123011.g001
Transgene Expression and Bt Protein Content in Transgenic Bt Maize
PLOS ONE | DOI:10.1371/journal.pone.0123011 April 8, 2015 5/9
Fig 2. Bt protein content. Fold change in the Bt protein content in the upper leaves of the white and yellow
Bt maize between the first (before stress) and the second (during stress) sampling. Calculated as the ratio of
the during stressvalue to the before stressvalue (i.e. 1 = no change). Plants grown under optimal growth
conditions were exposed to no stress. The means labelled with the different letters are significantly different
at P<0.05, Tukeys HSD. Means ±SE, n = 45.
doi:10.1371/journal.pone.0123011.g002
Fig 3. Correlation under optimal conditions. Spearman rank correlation between relative transgene expression and Bt protein content in the plants grown
under optimal conditions: A) white Bt maize and B) yellow Bt maize.
doi:10.1371/journal.pone.0123011.g003
Transgene Expression and Bt Protein Content in Transgenic Bt Maize
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which is in accordance with other studies measuring cry1Ab transgene expression. La Paz et al.
[17] found no significant differences in cry1Ab mRNA levels in the leaves of 28 commercial Bt
maize varieties. Also, Coll et al. [1820] reported that mRNA levels of cry1Ab were similar be-
tween different Bt maize varieties. Thus, our results add to the emerging picture that expression
of the transgene cry1Ab is not influenced by the genetic background in a significant way.
Simultaneously to quantifying transgene expression, we also measured Bt protein levels in
the same plant tissues of the two Bt maize varieties. Despite their similar transgene expression,
Bt protein levels differed significantly in the tissue samples of the two Bt maize varieties. The
yellow Bt maize leaves contained on average 40% more Bt protein than the white Bt maize
leaves. This suggests that mRNA levels of the cry1Ab transgene do not necessarily predict its
protein, i.e. Bt toxin, level. The relationship between mRNA and protein abundances has been
reported to be weak also for native genes [21,22]. Various regulatory processes, including post-
transcriptional, translational and protein degradation regulation, occurring after mRNA is
made, control native protein abundances [23]. Inhibited protein synthesis, degradation and/or
remobilization (or transportation) to developing plant parts have been suggested to cause ob-
served reductions in the amount of Bt protein in transgenic Bt cotton [24]. Similar processes
are presumably involved in the regulation of Bt protein content in Bt maize. However, to mea-
sure mRNA and protein degradation rates is technically challenging and has not yet been fully
explored in plants [16].
Adamczyk et al. [10] reported that cry1Ac mRNA transcript levels correlated with Cry1Ac
protein levels in Bt cotton. We also found a correlation between cry1Ab transgene expression
and Cry1Ab protein content, but only in white Bt maize plants. The fact that the correlation be-
tween transgene expression and protein content was found in one Bt maize variety and not in
the other suggests that other factors also influence Bt protein content. It also shows that Bt pro-
tein content cannot be reliably predicted by measuring only mRNA transcript levels. Our re-
sults therefore do not support the conclusion of Adamczyk et al. [10].
To investigate how stressful environmental conditions influence the relationship between
transgene expression and Bt content, we exposed plants of both Bt maize varieties to cold/wet
and hot/dry treatments. Transgene expression under cold/wet stress was similar to the expres-
sion under optimal conditions, but the expression of the transgene was reduced under hot/dry
stress, though this was only significant in white Bt maize. Also Meyer et al. [25] observed re-
duction in transgene expression (i.e. a reduction in flower coloration) in transgenic petunia
after the plants were exposed to high temperatures. The white flowering plants showed hyper-
methylation of the 35S promoter directing the transgene expression, in contrast to the fully red
flowering plants showing no methylation of the 35S promoter. As cry1Ab transgene expression
in Bt maize is also driven by the CaMV 35S promoter, it is possible that methylation of the pro-
moter might play a role in the reduced transgene expression under hot/dry conditions.
The reduction in transgene expression under hot/dry conditions did not result in a corre-
sponding, systematic effect on the Bt protein concentration. Also, in the white Bt maize plants
the transgene expression under cold/wet stress was similar to optimal conditions, but the Bt
content under cold/wet stress increased 4-times compared to optimal treatment. Thus, while
transgene expression was correlated to Bt protein content in the white Bt maize under optimal
conditions, this correlation was disrupted under stressful conditions. Indeed, during acute
stress and developmental changes involving significant proteome remodelling, mRNA-protein
correlations are often weaker, with either mRNA or protein lagging in abundance response
[16]. Similarly, Li et al. [11] showed that cry1Ac mRNA transcript levels in Bt cotton increased
in plants exposed to NaCl stress but NaCl treatment did not affect the corresponding Bt protein
content in the leaves or roots. In essence, this suggests that under stressful environmental con-
ditions, transgene expression is only a proxy for determining whether the transgene product
Transgene Expression and Bt Protein Content in Transgenic Bt Maize
PLOS ONE | DOI:10.1371/journal.pone.0123011 April 8, 2015 7/9
here Bt proteinis present or absent and that the Bt protein content is affected by the plants
own regulatory system and by outside environmental conditions.
Our findings challenge the general presumption that transgenes in commercially approved
genetically modified plants are almost invariably expressed at high levels in all plant tissues and
phenological phases [3]. We found large variation in the transgene expression and Bt protein
content caused by plant genetic background and environmental conditions. Field-grown Bt
maize plants might therefore not always produce high enough dose of Bt protein to kill the in-
termediate (heterozygous) resistant insect pests. Survival of such intermediate resistant pest
species on individual Bt plants could increase the probability of resistance development to Bt
protein [26]. Moreover, changes in Bt plant efficacy might be mediated through modification
of the plant physiological background without any changes in Bt transgene expression and/or
Bt protein content [27]. Thus, any assessment of transgenic Bt plants will be incomplete with-
out measuring transgene expression in conjunction with Bt protein content and efficacy.
Supporting Information
S1 Fig. Correlation under stressful conditions. Correlation between relative transgene ex-
pression and Bt protein content during cold/wet or hot/dry stress: A) in the white Bt maize and
B) in the yellow Bt maize plants. Plants grown under optimal growth conditions were exposed
to no stress.
(TIF)
S1 Table. Sequences and amplification efficiencies of the TaqMan primers and probes for
the reference genes.
(PDF)
Acknowledgments
We thank Claudia Michel and Matthias Meier for their help in the laboratory. We acknowledge
the services of the Genetic Diversity Centre at the ETH Zurich. We also thank Anna Coll for
providing the primer and probe sequences.
Author Contributions
Conceived and designed the experiments: AH MT AW OW. Performed the experiments: MT.
Analyzed the data: MT NZ OW. Contributed reagents/materials/analysis tools: AH AW OW.
Wrote the paper: MT AH OW AW NZ.
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sponse to two environmental factors: temperature and insect damage. Journal of Economic Entomolo-
gy 98: 13821390. PMID: 16156594
Transgene Expression and Bt Protein Content in Transgenic Bt Maize
PLOS ONE | DOI:10.1371/journal.pone.0123011 April 8, 2015 9/9
... Studies across different Bt crop plants have found that there are not only temporal and spatial variations in the concentration of the Bt insecticidal protein [6][7][8][9][10], but also that environmental stress factors such as temperature, salinity, waterlogging, drought and CO 2 , can induce Bt plants to produce lower Bt toxin concentrations [11][12][13][14][15][16][17][18][19][20][21][22][23][24]. Stress-dependent changes in Bt toxin levels have also been shown not to always correlate with changes in transgene expression [25], and the efficacy of Bt crops cannot always be explained by variations in Bt protein content [15,23,24,26]. The introduction of an actively transcribing DNA sequence from B. thuringiensis into the plant genome and the resulting production of insecticidal Cry1Ab protein, has been useful in exploring transgene expression not only because this is the second most important GM trait, but also because it depends on the simple direct relationship outlined above: transgene-mRNAprotein-bioactivity. ...
... Abiotic stress factors can influence transgene transcription level of Bt maize plants. Trtikova et al. [25] reported that the cry1Ab transgene expression in Bt maize plants under cold/wet stress was similar to the expression under optimal conditions, but that it was significantly reduced under hot/dry stress. However, in another Bt maize variety, the cry1Ab transgene transcription levels measured as mRNA in leaves under cold/wet and hot/dry stresses were similar to levels in the control treatments. ...
... Luo et al. [24] found that waterlogging and the combination of waterlogging and salinity reduced of the Cry1Ac toxin concentration in GM cotton plants. In a study with maize plants, Trtikova et al. [25] showed that Cry1Ab protein concentrations were similar in plants grown under optimal and hot/dry growing conditions, but that Bt maize plants exposed to cold/wet stress conditions had significantly higher Cry1Ab toxin concentrations than plants grown under optimal conditions. Based on such prior reports and our own results, we conclude that biotic and abiotic stresses can affect Cry1Ab protein production but not always, and if it does, the effect is unpredictable, and dependent of many factors, such as crop species and the type of stress factors. ...
Article
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Background Decades after their first commercial release, many theoretical assumptions are still taken for granted in the deployment of genetically modified (GM) crops. Theoretically, in the case of maize, active transcription of the cry1Ab transgene would result in dose-dependent production of the insecticidal Cry1Ab protein, which would in turn induce dose-dependent mortality on lepidopteran pests. We produced data to realistically approach this question by using a model that includes two genetic background contexts from two geographical provenances in Brazil and South Africa, and two lepidopteran pests (Helicoverpa armigera and Spodoptera littoralis). However, in this study, the effect of insect herbivory was superimposed to investigate possible stress-induced effects in transgene expression at three levels: mRNA, protein and bioactivity. Results Overall, we found that herbivore damage by H. armigera was reflected only at the translational level, with a higher level of Cry1Ab protein measured in the Brazilian crosses under herbivore stress. On the other hand, compared to non-stress growing conditions, the herbivore damage by S. littoralis was not directly reflected in mRNA, protein or bioactivity in the South African crosses. Conclusions The differences between South African and Brazilian genetic backgrounds, and between the stressor effect of the two herbivores used, highlight the complexity of transgene expression at the agroecological level.
... As genetic engineering application progresses from sessile microorganisms and plants (e.g. Doron (Trtikova et al. 2015), and the fruit y (Horn and Wimmer 2003), thus suggesting that proper development of safe genetic engineering applications requires further study of transgene e cacy across diverse environmental conditions. ...
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Genetic biocontrol approaches to insect pest management offer high species specificity and reduce insecticidal chemical pollution, but these approaches require the release of genetically modified organisms that must perform across a variety of environmental conditions. Because organisms often display phenotypic and gene expression variability when exposed to different environments, it is reasonable to expect that transgenic systems may be prone to environmentally mediated variation in expression and function. In this study, we examined the influence of two abiotic variables, temperature and nutrition, on the penetrance of an early embryonic Tet-off conditional lethality system in the vinegar fly, Drosophila melanogaster . We manipulated parental and offspring environments independently to determine the impact of life stage of exposure on transgene performance by estimating the probability of larval hatching and measuring transcript abundance of the transgenic system components. Our findings revealed that: 1) transgene performance has distinct norms of reaction to temperature and nutrition, 2) the effects of abiotic challenge are greatest when exposure occurs in the embryos expressing the transgene compared to parental exposure, and 3) stress exposures at the extreme limits of permissible conditions can dramatically decrease the penetrance of transgenic lethality. While variation in transcript abundance of the transgenic system was observed in some environments, these changes were not fully congruent with patterns of phenotypic penetrance, suggesting that the observed variation in lethality is likely driven by processes downstream of transcription.
... The concentration of Bt proteins in Bt maize is influenced by various factors, including environmental conditions (such as temperature and humidity), geographic location, and crop self-regulation [26]. For example, under cold/wet stimulation, the expression of Cry1Ab toxic protein in MON810 maize increased by four times [45]. Previous studies have measured the concentration of Vip3Aa protein in leaf tissues of DBN3601T maize grown indoors at 6.78 µg/g, which is 4.3 times lower than the concentration found in our field-grown leaves [23]. ...
Article
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The beet armyworm, Spodoptera exigua (Hübner), is a major pest of maize, cotton, soybean, and many other crops globally. Despite the widespread deployment of Bt transgenic maize for pest control worldwide, the efficacy of Bt lepidopteran-resistant transgenic maize in managing S. exigua remains rarely studied. In this study, we quantified the expression level of pyramided Cry1Ab and Vip3Aa toxins in Bt maize (event DBN3601T) and evaluated their control efficiency against S. exigua under both laboratory and field conditions. The enzyme-linked immunosorbent assay (ELISA) results showed that the expression levels of Cry1Ab and Vip3Aa proteins in DBN3601T maize tissues followed a decreasing order as follows: V5-leaf > V8-leaf > VT-tassel > R2-kernel > R1-silk. Diet-overlay assay results showed that the LC50 values of Cry1Ab and Vip3Aa proteins against S. exigua larvae were 11.66 ng/cm2 and 27.74 ng/cm2, respectively, with corresponding GIC50 values at 1.59 ng/cm2 and 7.93 ng/cm2. Bioassay using various tissues of the DBN3601T maize indicated that after 7 days of infestation, mortality rates of neonates and third-instar larvae ranged from 86% to 100% and 58% to 100%, respectively. Mortality was highest on V5 and V8 leaves, followed by R2-kernel, VT-tassel, and R1-silk. Field trials demonstrated that DBN3601T maize exhibited significantly lower larval density, damage rate, and leaf damage score compared to non-Bt maize. Field cage trial showed that the control efficacy of DBN3601T maize at the vegetative stage could reach 98%. These findings provide a theoretical basis for utilizing Bt transgenic maize to enhance the sustainable management of S. exigua in Asia.
... If necessary, spraying pesticides and using other methods to carry out integrated prevention and control. The protein expression levels in Bt crops are influenced by internal regulatory factors, as well as external factors such as environmental conditions (temperature, altitude, etc.) and human factors (fertilization levels, weed management, etc.) [32][33][34]. Therefore, the field management and planting environment of Bt maize need to ensure the normal growth and development of maize. ...
Article
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The oriental armyworm, Mythimna separata (Walker), an important migratory pest of maize and wheat, is posing a severe threat to maize production in Asian countries. As source areas of spring–summer emigratory populations, the control of M. separata in southwestern China is of great significance for East Asian maize production. To assess the toxicity of Bt maize against the pest, bioassays of Bt-(Cry1Ab+Vip3Aa) maize (event DBN3601T), Bt-Cry1Ab maize (event DBN9936), and Bt-Vip3Aa maize (event DBN9501) were conducted in Yunnan province of southwest China. There were significant differences in insecticidal activity between the three Bt maize events, and DBN3601T presented the highest insecticidal role. The results also indicated that the insecticidal effect of various Bt maize tissues took an order in leaf > kernel > silk, which is highly consistent with the expression amounts of Bt insecticidal protein in leaf (69.69 ± 1.18 μg/g), kernel (11.69 ± 0.75 μg/g), and silk (7.32 ± 0.31 μg/g). In field trials, all larval population densities, plant damage rates, and leaf damage levels of DBN3601T maize were significantly lower than the conventional maize. This research indicated that the DBN3601T event had a high control efficiency against M. separata and could be deployed in southwest China for the management of M. separata.
... 46 However, this degree of dominance in S. frugiperda calculated by Jin et al. 46 was based on a Vip3Aa concentration of 0.4 μg cm −2 , which is lower than the concentration typically produced by Bt corn and cotton plants. [47][48][49][50] Previous studies show that the degree of dominance varies based on Bt concentrations, with nonrecessive resistance at low concentration and more recessive resistance at high concentrations. 23,51 Resistance to Vip3Aa in strains of Helicoverpa punctigera and Helicoverpa armigera from Australia also was monogenic and essentially recessive. ...
Article
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BACKGROUND Practical resistance of Helicoverpa zea to Cry proteins has become widespread in the US, making Vip3Aa the only effective Bacillus thuringiensis (Bt) protein for controlling this pest. Understanding the genetic basis of Vip3Aa resistance in H. zea is essential in sustaining the long‐term efficacy of Vip3Aa. The objectives of this study were to characterize the inheritance of Vip3Aa resistance in four distinct field‐derived H. zea strains (M1‐RR, AC4‐RR, R2‐RR and R15‐RR), and to test for shared genetic basis among these strains and a previously characterized Texas resistant strain (LT#70‐RR). RESULTS Maternal effects and sex linkage were absent, and the effective dominance level (DML) was 0.0 across Vip3Aa39 concentrations ranging from 1.0 to 31.6 μg cm⁻², in all H. zea resistant strains. Mendelian monogenic model tests indicated that Vip3Aa resistance in each of the four strains was controlled by a single gene. However, interstrain complementation tests indicated that three distinct genetic loci are involved in Vip3Aa resistance in the five resistant H. zea strains: one shared by M1‐RR and LT#70‐RR; another shared by R2‐RR and R15‐RR; and a distinct one for AC4‐RR. CONCLUSION Results of this study indicate that Vip3Aa resistance in all H. zea strains was controlled by a single, recessive and autosomal gene. However, there were three distinct genetic loci associated with Vip3Aa resistance in the five resistant H. zea strains. The information generated from this study is valuable for exploring mechanisms of Vip3Aa resistance, monitoring the evolution of Vip3Aa resistance, and devising effective strategies for managing Vip3Aa resistance in H. zea. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
... For this purpose, guidance on how to achieve targeted drought stress conditions and the recording of relevant phenotypic traits is available in field manuals issued by the International Maize and Wheat Improvement Centre [105]. In combination with an assessment of plant survival (see above), gene expression data as well as proteomic and metabolomic profiling [106][107][108], such assessments could provide useful information on the GMP's ability to survive and perform under drought stress conditions. ...
Article
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Background For market approval of genetically modified plants (GMPs), the evaluation of agronomic and phenotypic plant traits is a standard requirement and part of the comparative assessment of the GMP and its conventional counterpart. This comparative assessment is a starting point for environmental risk assessment (ERA) and should inform all areas of risk. We scrutinize frequently used approaches to characterize GMPs in EU market applications and discuss their usefulness for drawing conclusions on risks related to the plant’s ability to survive, persist or become invasive. Results Our analysis shows that the agronomic and phenotypic characterization of GMPs, although based on guidelines, is confined to plant traits and test designs that are relevant for the quality control and agronomic performance of genetically modified (GM) crops. We provide evidence of how methodological approaches frequently applied during the agronomic and phenotypic characterization of the GMP could be improved and complemented to better inform on potential phenotypic changes relevant to assessing environmental risks. These approaches refer to (i) the assessment of the survival of GM seeds and plants (e.g., volunteers); (ii) the consideration of environmental exposure and (iii) improved methodological approaches for the assessment of biotic and abiotic stress responses for GMPs. Conclusions The comparative assessment of agronomic and phenotypic plant traits currently does not provide suitable data to draw conclusions on environmental risks relating to the persistence and invasiveness of the GMP. Ecologically more realistic assessments should be part of the phenotypic characterization of GMPs and need guidance and decision criteria to be implemented in ERA. This is of considerable importance, as new genomic techniques are expected to increase the diversity and complexity of GM plants and traits, particularly stress tolerance, which may affect the survival of GMPs in the environment.
... The level of transgene expression is affected by many factors related both to the plant and the environment [30][31][32]. Quantitative analysis showed a stable tendency of uidA gene expression in leaves of 19 transgenic pear lines during 12 years of field tests. Silencing of expression was not observed even during the extremely hot and dry season of 2010 [33] and after it. ...
Article
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Stable and high expression of introduced genes is a prerequisite for using transgenic trees. Transgene stacking enables combining several valuable traits, but repeated transformation increases the risk of unintended effects. This work studied the stability and intron-mediated enhancement of uidA gene expression in leaves and different anatomical parts of pear fruits during field trials over 14 years. The stability of reporter and herbicide resistance transgenes in retransformed pear plants, as well as possible unintended effects using high-throughput phenotyping tools, were also investigated. The activity of β-glucuronidase (GUS) varied depending on the year, but silencing did not occur. The uidA gene was expressed to a maximum in seeds, slightly less in the peel and peduncles, and much less in the pulp of pear fruits. The intron in the uidA gene stably increased expression in leaves and fruits by approximately twofold. Retransformants with the bar gene showed long-term herbicide resistance and exhibited no consistent changes in leaf size and shape. The transgenic pear was used as rootstock and scion, but grafted plants showed no transport of the GUS protein through the graft in the greenhouse and field. This longest field trial of transgenic fruit trees demonstrates stable expression under varying environmental conditions, the expression-enhancing effect of intron and the absence of unintended effects in single- and double-transformed woody plants.
... In addition, several studies have shown that the expression of Bt transgenes in plants is affected by environmental factors, especially under some stress conditions affecting the primary and secondary metabolism of Bt plants [29]. For example, Trtikova et al. [30] reported that the expression of the cry1Ab transgene in MON 810 maize could be reduced under hot/dry stressed environments compared to optimal conditions. Recently, Liu et al. [31] also observed that Cry1Ab/c expression in transgenic cotton plants varied in different planting environments. ...
Article
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The wide occurrence of resistance to Cry1A and Cry2A insecticidal toxins from Bacillus thuringiensis (Bt) in the corn earworm/bollworm Helicoverpa zea (Boddie) leaves the Vip3A toxin produced during the vegetative stage of Bt as the only fully active toxin expressed in transgenic crops to control H. zea in the U.S.A. During 2021, the first unexpected survival of H. zea and injury (UXI) on a maize hybrid expressing Cry1A.105, Cry2Ab2, and Vip3Aa in Louisiana, U.S.A. were observed in two sentinel plots used for resistance monitoring. A follow-up intensive investigation was conducted with two H. zea populations established from larvae collected from the two UXI plots. The main goal of this study was to reveal if the unexpected damage was due to resistance development in the insect to the Bt toxins expressed in the maize hybrid. Diet-overlay bioassays showed that the two populations were highly resistant to Cry1A.105, moderately resistant to Cry2Ab2, but still highly susceptible to Vip3Aa when compared to a reference susceptible strain. In 10 d assays with detached ears, the larvae of the two UXI populations exhibited survival on ears expressing only Cry toxins but presented near 100% mortality on maize hybrids containing both cry and vip3A transgenes. Multiple field trials over three years demonstrated that natural H. zea populations in Louisiana were highly resistant to maize expressing only Cry toxins but remained susceptible to all tested hybrids containing cry and vip3A genes. Altogether, the results of this study suggest that the observed UXIs in Louisiana were associated with a resistance to Cry toxins but were not due to a resistance to Vip3A. The possible causes of the UXIs are discussed. The results generated and procedures adopted in this study help in determining thresholds for defining UXIs, assessing resistance risks, and documenting field resistance.
... The insecticidal effect of insect-resistant transgenic maize exogenous Bt depends on its insecticidal protein expression [29,30]. Previous studies have confirmed that Bt protein expression in different regions, different transgenic crops, and different growth stages of the same transgenic crop has significant spatiotemporal variation [21,[31][32][33][34]. For example, the expression level of Cry1Ab in DBN9936 was significantly lower in Xinxiang, Langfang, and Harbin than in Wuhan and Shenyang [21]. ...
Article
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Lepidopteran pests present a key problem for maize production in China. In order to develop a new strategy for the pest control, the Chinese government has issued safety certificates for insect-resistant transgenic maize, but whether these transformation events can achieve high dose levels to major target pests is still unclear. In this paper, the transformation events of DBN9936 (Bt-Cry1Ab), DNB9936 × DBN9501 (Bt-Cry1Ab + Vip3A), Ruifeng 125 (Bt-Cry1Ab/Cry2Aj), and MIR162 (Bt-Vip3A) were planted in the Huang-huai-hai summer corn region of China to evaluate the lethal effects on major lepidopteran pests, Spodoptera frugiperda, Helicoverpa armigera, Ostrinia furnacalis, Conogethes punctiferalis, Mythimna separata, Leucania loreyi, and Athetis lepigone, using an artificial diet containing lyophilized Bt maize tissue at a concentration representing a 25-fold dilution of tissue. The results showed that the corrected mortalities of DBN9936 (Bt-Cry1Ab), DNB9936 × DBN9501 (Bt-Cry1Ab + Vip3A), Ruifeng 125 (Bt-Cry1Ab/Cry2Aj), and MIR162 (Bt-Vip3A) to the seven pests were in the ranges 53.80~100%, 62.98~100%, 57.09~100%, and 41.02~100%, respectively. In summary, the events of DBN9936, DNB9936 × DBN9501, and MIR162 reached high dose levels to S. frugiperda. DNB9936 × DBN9501 only at the R1 stage reached a high dose level to H. armigera. DBN9936, DNB9936 × DBN9501, and Ruifeng 125, at most growth stages, reached high dose levels to O. furnacalis, and these three events at some stages also reached high dose levels to A. lepigone. Ruifeng 125 presented a high dose level only to C. punctiferalis. However, no transformations reached high dose levels to either M. separata or L. loreyi. This study provides a support for the breeding of high-dose varieties to different target pests, the combined application of multiple genes and the commercial regional planting of insect-resistant transgenic maize in China.
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Following the submission of dossier GMFF‐2022‐9450 under Regulation (EC) No 1829/2003 from Bayer Agriculture BV, the Panel on Genetically Modified Organisms of the European Food Safety Authority was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the insect protected genetically modified maize MON 810, for food and feed uses (including pollen), excluding cultivation within the European Union. The data received in the context of this renewal application contained post‐market environmental monitoring reports, an evaluation of the literature retrieved by a scoping review, additional studies performed by or on behalf of the applicant and updated bioinformatics analyses. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. Under the assumption that the DNA sequence of the event in maize MON 810 considered for renewal is identical to the sequence of the originally assessed event, the GMO Panel concludes that there is no evidence in dossier GMFF‐2022‐9450 for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on maize MON 810.
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Bacillus thuringiensis (Bt) cotton is grown worldwide, including in saline soils, but the effect of salinity on ion fluxes of Bt cotton remains unknown. Responses of two transgenic Bt cotton genotypes (SGK321 and 29317) and their corresponding receptors, Shiyuan 321 (SY321) and Jihe 321 (J321), to 150 mmol L−1 NaCl stress were studied in a growth chamber. The root dry weight of SGK321 and 29317 under NaCl treatment was decreased by 30 and 31%, respectively. However, their corresponding receptor cultivars SY321 and J321 were less affected (19 and 24%, respectively). The root length and surface area of the Bt cultivars were significantly decreased relative to their receptors under salt stress. NaCl treatment significantly increased Cry1Ac mRNA transcript levels in SGK321 and 29317 but did not affect Bt protein content in leaves or roots of either cultivar at 1 and 7 d after NaCl treatment. Fluxes of Na+, K+, and H+ in roots were investigated using the scanning ion-selective electrode technique. Both mean K+ efflux rate and transient K+ efflux of the Bt cultivars increased four-fold compared to their corresponding receptors when exposed to salinity stress. There were no significant differences in Na+ efflux between Bt and non-Bt cottons. Furthermore, the Na+ contents in roots and leaves of all genotypes dramatically increased under salt stress, whereas K+ contents decreased. Our results suggested that Bt cotton cultivars are more sensitive to salt stress than their receptor genotypes.
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To help understand regulation of maize leaf blade development, including sink-source transitions and induction of C4 photosynthesis, we compared large scale quantitative proteome and transcriptomes collected at specific stages along the developmental maize leaf blade gradient. Proteome data were based on label-free shotgun proteomics (spectral counting) and transcript data were based on RNA-seq using the same source materials, and were previously published (Li et al. 2010, Majeran et al. 2010).Transcript and protein abundance followed near normal distributions, in contrast to several studies with other organisms. Protein observability correlated to transcript abundance following a 'lazy step function' similar as in bacteria and yeast. mRNA and protein abundance showed significant positive correlations (upto 0.8) for log-transformed length-weighted (NSAF and RPKM) and non-weighted abundances (NadjSPC and COV) in dependence of function and development. Correlations were much weaker in the leaf 'sink-source' transition zone', i.e. the zone with massive investments in leaf chloroplast biogenesis and build-up of photosynthetic capacity. Clustering analyses of gene-specific protein-mRNA ratios revealed coordinated shifts in control points in gene expression along the leaf blade developmental gradient. The highest protein/mRNA ratio for each gene generally corresponded to leaf developmental stages where the protein function was most important, with the exception of the 80S ribosome. Specific examples are discussed in the context of C4 photosynthesis, leaf development and sink-source transitions. This large scale mRNA-protein correlation analysis in plants (maize) using label-free spectral counting for protein quantification and RNA-seq for mRNA abundance will provide a template for future mRNA-protein correlation studies. This article is protected by copyright. All rights reserved.
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Nitrogen deficiencies interfere with protein synthesis and growth in general in maize (Zea mays L.). Transgenic maize hybrids with the Bacillus thuringiensis Berliner (Bt) gene produce proteins that are solubilized and become insecticidal in the highly alkaline midgut of certain lepidopteran insects. The effect N fertility has on Bt δ-endotoxin and how whole-plant N concentrations relate to Bt δ-endotoxin levels has yet to be documented in early-growth maize. Two Bt maize hybrids ('Pioneer 33V08'1 with Bt event MON-810 and 'Dekalb 626Bty'1 with Bt event DBT 418) were grown in pots in duplicate greenhouse experiments with N fertility rates of 0, 112, 224, and 336 kg ha-1 N as NH4NO3. Fertilizer was blended into a potting mixture of a 2:1:1 ratio of peat moss/sand/soil at planting. Plants were harvested at growth stage V5 and assayed for Bt δ-endotoxin using a commercial quantification plate kit. Whole-plant N concentrations were determined by semimicro-Kjeldahl. Whole-plant N concentrations were 25.8, 33.1, 35.1, and 37.7 mg g-1, and Bt δ-endotoxin concentrations were 350, 367, 486, and 534 μg kg-1 at N fertility levels of 0, 112, 224, and 336 kg ha-1, respectively. Increased available N likely increases Bt δ-endotoxin-synthesizing proteins and thus increases the Bt δ-endotoxin concentration. The response of the two Bt hybrids to increased N fertility was similar. Adequate levels of N fertility during early growth appear essential for Bt δ-endotoxin production by the plant and may affect the ability of maize plants to resist insect predation.
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Transgenic cotton expressing Bt (Bacillus thuringiensis) toxins is currently cultivated on a large commercial scale in many countries, but observations have shown that it behaves variably in toxin efficacy against target insects under field conditions. Understanding of the temporal and spatial variation in efficacy and the resulting mechanisms is essential for cotton protection and production. In this review, we summarize current knowledge on variability in Bt cotton efficacy, in particular on the induced variability by environmental stresses. We also discuss the resulting mechanisms and the countermeasures for the inconsistence in efficacy in Bt cotton. It is indicated that insecticidal protein content in Bt cotton is variable with plant age, plant structure or under certain environmental stresses. Variability in Bt cotton efficacy against target insect pests is mainly attributed to the changes in Bt protein content, but physiological changes associated with the production of secondary compounds in plant tissues may also play an important role. Reduction of Bt protein content in late-season cotton could be due to the overexpression of Bt gene at earlier stages, which leads to gene regulation at post-transcription levels and consequently results in gene silencing at a later stage. Methylation of the promotor may be also involved in the declined expression of endotoxin proteins. As a part of total protein, the insecticidal protein in plant tissues changes its level through inhibited synthesis, degradation or translocation to developing plant parts, particularly under environmental stresses, thus being closely correlated to N metabolism. It can be concluded that developing new cotton varieties with more powerful resistance, applying certain plant growth regulators, enhancing intra-plant defensive capability, and maintenance of general health of the transgenic crop are important in realizing the full transgenic potential in Bt cotton.