Journal of Agricultural Science, Page 1 of 9.

Cambridge University Press 2015

doi:10.1017/S0021859615000325

CROPS AND SOILS RESEARCH PAPER

Assessment of Bt cotton genotypes for the Cry1Ac transgene and

its expression
H. M. N. CHEEMA 1 *, A. A. KHAN 1 , M. I. KHAN 1 , U. ASLAM 1 , I. A. RANA 2
1
2

A ND

I. A. KHAN 2

Plant Genetic Resources Lab, Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan
Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad, Pakistan

(Received 12 August 2014; revised 30 December 2014; accepted 9 March 2015)

SUMMARY
Genetically modified (GM) plants expressing Bt toxin provide protection against lepidopteran pests. The only GM
crop in Pakistan is Bt cotton, which was illegally imported and adopted rapidly by cotton producers. Farmers
gained access to the seed of many unapproved Bt genotypes before the matter was picked up and formal approval
granted by the relevant governmental agencies. The present study was conducted to evaluate the samples of Bt
cotton, collected from farmers and seed dealer, for transgene integration and expression. Seeds of 52 cotton genotypes, labelled as Bt, were collected from various farmers and seed dealers. An immunoblot strip test was carried
out, which showed that only 086 of the samples collected were synthesizing Cry1Ac toxin. According to multiplexed polymerase chain reaction (PCR) results, 086 of the genotypes tested were positive for the Mon531 event
(an event is a specific genetic modification in a specific species) and 014 were negative for any transgene.
Transcript analysis of transgenes in positive genotypes by real-time Rt-PCR confirmed the synthesis of mRNA
in all genotypes but with significant variation. The concentration of Bt toxin revealed by enzyme linked immunosorbent assay (ELISA) showed that only 002 genotypes had the reported optimum level. The real-time PCR and
ELISA results further confirmed the attenuation of transgene expression at transcriptional and translational level
by various internal and external factors. The same type of event was found in all genotypes, with significant variation in toxin level, revealing the impact of genetic background on transgene expression. The findings support the
recommendation to improve the existing quality criteria for transgenic cotton variety approval and certification in
Pakistan, with the inclusion of toxin concentration in the list of parameters to be considered.

INTRODUCTION
Soybean, maize and cotton are three major crops
which have been commercialized on the basis of
different transgenes for traits such as herbicide tolerance and insect resistance, and are also being cultivated all over the world (Gaskell et al. 1999; Clive
2013). The development and commercial-scale plantation of transgenic crops necessitate continuous
monitoring of their performance in the ecosystem, particularly in relation to the target gene expression and
its implications, if any.
Pakistan is among the top cotton producers of the
world, with an area of 288 million ha, producing
209 million tons per annum (Wasti 2013). One of
* To whom all correspondence should be addressed. Email:
masooma@uaf.edu.pk

the major threats to this economic crop is bollworm,

causing yield losses of 3040% (Cororaton et al.
2008; Masood et al. 2011; Khan et al. 2012).
Massive pesticide applications had been used to
control these pests, leading to increases in pesticide
imports and in the costs of production (Tariq et al.
2007). Environmental pollution and the hazardous
impact of pesticides on farmers and livestock are
additional side effects (Ali et al. 2010). After the introduction of Bt technology in the developed world,
unapproved and unregulated Bt cotton genotypes
were widely imported and adopted as bollwormresistant crops in Pakistan. Some Bt cotton varieties
were formally approved in Pakistan for the first time
in 2010. The time lag between the illegal introduction
and the formal approval of Bt cotton seeds led to the
spread and adoption of sub-standard seeds among

H. M. N. Cheema et al.

cotton growers. Therefore, this technology has not

enhanced yield as much as expected.
The efficacy and performance of Bt cotton depend
mainly upon expression of the Cry1Ac gene
(Greenplate et al. 2000; Holt et al. 2002; Gutierrez
et al. 2006), which fluctuates throughout the
growing season (Kranthi et al. 2005; Olsen et al.
2005; Adamczyk et al. 2009; Bakhsh et al. 2010).
Expression of the transgene hinges on genotype, age,
tissue type and site of insertion in the host plant
genome (Adamczyk & Sumerford 2001; Gore et al.
2001; Abel & Adamczyk 2004; Jackson et al. 2004;
Wan et al. 2005). Therefore, it is critical to include a
standard level of Bt gene expression in the list of
variety approval and seed certification criteria, along
with monitoring the performance of cultivated Bt
cotton for toxin concentration. It should be practiced
in a developing country such as Pakistan, where the
success story of this technology in the developed
world prompted seed companies to sell whatever
was available with the label of Bt cotton. Hence, the
present study was planned to collect seed randomly
from farmers and seed dealers to evaluate the presence and expression of transgenes. Although only
first generation Bt technology (Cry1Ac) was supposed
to be present in Pakistan, the primers for all transgenic
events reported in cotton were used to evaluate the
genotypes collected. Among the various diagnostic
tools used for transgene detection in crop plants, polymerase chain reaction (PCR) is the most sensitive and
widely used analytical method (Singh et al. 2008;
Randhawa et al. 2009). Real time PCR and enzyme
linked immunosorbent assay (ELISA) have been used
effectively for quantitative detection of several genetically modified (GM) crops such as maize, cotton,
rapeseed and cassava (Li et al. 2004; Lee et al.
2006a, b; Wu et al. 2007; Aguilera et al. 2009;
Beltrn et al. 2009).Therefore, these qualitative and
quantitative methods were employed to test the transgenicity and expression of Cry1Ac in the collected
genotypes.

districts of the Punjab province in Pakistan. The collected genotypes were grown at the research area of
the Department of Plant Breeding and Genetics,
University of Agriculture, Faisalabad, Pakistan (3115
N, 733E, 1844 m asl), during the 2012 crop season.
The row to row and plant to plant distances were 75
and 30 cm, respectively. Standard agronomic practices
were adopted for the cultivation of cotton crop.
Oligonucleotide primers
The reported primers, targeting various regions of transgene construct and the events (Table 1), were synthesized by Gene Link, USA. The designed primers
were reconstituted in double distilled and de-ionized
water for further use at a concentration of 50 ng/l.
Genomic DNA extraction
The genomic DNA from fresh leaf tissue of 10-weeksold Bt and non-Bt plants was extracted using the cetyl
trimethyl ammonium bromide (CTAB) method (Doyle
& Doyle 1990). The DNA samples were quantified
using a Nano Drop spectrophotometer (Beckman,
USA) at 260 nm/280 nm. The concentrated DNA
was finally diluted to 50 ng/l.
Qualitative polymerase chain reaction
Multiplex PCR assay was performed in a single reaction, using the combination of primer sets for Sad1
gene, Cry1Ac and MON 531, to make the Bt detection
technique more economical, cost effective and
reliable. Each reaction mixture contained 400 ng of
template DNA, 1X Taq PCR buffer, 2 mM magnesium
chloride (MgCl2), 02 mM deoxynucleotide triphosphates dNTPs mix, 05 U of Taq DNA Polymerase
and 04 M of each primer. The amplification conditions were: One cycle of initial denaturation for 5
min at 95 C, 35 cycles of denaturation at 94 C for
1 min, annealing at 48 C for 1 min, and elongation
at 72 C for 1 min. Thirty five cycles were followed
by a final extension of 5 min at 72 C.

MATERIALS AND METHODS

Collection of putative Bt cotton genotypes

Quantitative polymerase chain reaction

The seeds of 52 putative Bt cotton (Gossypium

hirsutum L.) genotypes were collected from farmers
and seed dealers. Out of 52 genotypes, 10 were purchased from seed retailers and 42 were collected from
various farmers of the Khanewal, Vehari and Multan

For quantification of Cry1Ac gene transcripts by RtPCR assay, only those samples were assessed which
had earlier been reported to harbour the transgene.
Syber green super mix (Fermentas, USA) was used to
carry out Rt-PCR assays in a fluorometric thermal

Transgene analysis in Bt cotton

Table 1. Primer pairs used for testing cotton genotypes by qualitative PCR
Amplicon
size (bp)

Event

Target gene

Mon531

Junction of transgene and host

genome
Sad1
Cry1Ac gene

654

Cry1Ac/NOS
terminator
CpTI gene

346

Mon15985 Cry2ab gene

260

Gk19
SGK321

uidA (GUS gene)

fsACP-2 gene

Sequence (53)

References

346

AAGAGAAACCCCAATCATAAAA
GAGAATGCGGTAAAGATACGTC

Yang et al. (2005b)

107

CCAAAGGAGGTGCCTGTTCA
TTGAGGTGAGTCAGAATGTTGTTC
ACAGAAGACCCTTCAATATC
GTTACCGAGTGAAGATGTAA
CTTCACTCGGTAACATCGT
ATGGGTTTTTATGATTAGAGTCC
CACTAAATCAATACCTCCTCAA
TTACTCATCATCTTCATCCCT

Yang et al. (2005a)

172

CAGCGGCGCCAACCTCTACG
TGAACGGCGATGCACCAATGTC
TTTCTTTAACTATGCCGGAATCCATC
CACCACGGTGATATCGTCCAC
CAAACAAGAGACCGTGGATAAGGTA
CAAGAGAATCAGCTCCAAGATCAAG

82
116

Bakhsh et al. (2009)

Yang et al. (2005a)
Chinese patent,
Pub. No. 1219586,
Seq. No. 4
Randhawa et al.
(2010)
Randhawa et al.
(2010)
Lee et al. (2007)

Table 2. Primers used for real time Rt-PCR

Sr.#

Gene

Primers

Cry1Ac

GhUBQ7

5-ACTGTGAATCAGGAAGAGTACGG-3
5-ACACGGAGGCATAGTCAGCAGGA-3
5-AAGCCCAAGAAGATCAAGCA-3
5-CGCATTAGGGCACTCTTTTC-3

cycler (icycler Bio-Rad, USA) with a final volume of

25 l. Fluorescence was monitored during every
cycle at the annealing step. The reaction was carried
out in triplicate for each sample using primers of
Cry1Ac and a ubiquitin gene (GhUBQ7) (Table 2)
used as a reference for normalization. The normalized
expression value in terms of Ct (Ct Ct of Cry1Ac-Ct
of GhUBQ7) was obtained from the inbuilt software
of the iCycler (Bio-Rad, USA). The fold expression of
Cry1Ac in the transgenic genotypes was determined
by using the lowest Ct value as a reference point,
which was obtained after normalization. Relative
fold transcript level was determined by 2Ct
method.
Immunoblot strip assay
The strip test was performed to detect the presence or
absence of the Cry1Ac and Cry2Ab genes. Leaf

Product size (bp)

94
114

samples of all genotypes were ground manually in

extraction buffer and tested by Immunoblot strips, following the manufacturers instructions (Envirologix
Inc., USA). Specific Immuno strips for Cry1Ac and
Cry2Ab (Catalog No. AS 012 LS) were used in the
study.
Quantification of Cry1Ac Bt-toxin
Collected genotypes were analysed by sandwich
ELISA to quantify the varying levels of Cry1Ac
protein. Leaves (45 days old) were sampled from
the 3rd and 4th node from the bottom at 70 days
after sowing (DAS). The samples were processed
immediately for sandwich ELISA according to the
manufacturers instructions (Envirologix, USA). The
optical density was calculated three times and the
mean was recorded by adjusting the wavelength at
450 nm, using a micro plate reader for each genotype

H. M. N. Cheema et al.

(Epoch BioTek). Simple regression analysis was

carried out in Microsoft Excel to calculate Cry1Ac
concentration (g/g) in the sampled leaves.

RESULTS

fresh weight basis. Three genotypes, Bt-2333, FH167 and FH-142, expressed the highest amounts of
Cry1Ac toxin, i.e. 199, 180 and 180 g/g, respectively at 70 DAS, while VH-282, FH-119 and FH-154
expressed the lowest amount of Cry1Ac toxin, i.e.
003, 004, and 009 g/g, respectively.

Qualitative analysis of transgene

Qualitative PCR (Table 3) showed the presence of only
the MON531 event in Pakistani genotypes. None of
the PCRs was able to amplify the MON15985 event
in the sampled genotypes, but the internal control
gene (SadI) was successfully amplified in all
samples. A simple and efficient multiplex PCR
method was optimized for the subsequent detection
of MON531 in the collected genotypes. In the established multiplex PCR, three primer pairs with different
amplified lengths were employed in one reaction for
the synthesis of expected amplicons of Cry1Ac,
Mon531 event and SadI gene with sizes of 655, 346
and 107 bp, respectively. These were amplified in
45 Bt cotton genotypes while only seven non-Bt genotypes could amplify the SadI gene alone.
For immunoblot strip test, all genotypes showed
negative results for Cry2Ab while 085 genotypes
showed positive reactions for Cry1Ac gene (Table 3)
and 015 of total genotypes did not show any signals
for either Cry1Ac or Cry2Ab.

Quantitative analysis by real time polymerase chain

reaction
The difference in transcript level in real time PCR
analysis might have been caused by some pipetting
errors or from the difference in initial RNA samples
used for complementary DNA (cDNA) synthesis.
Therefore, GhUBQ7 was used as a reference gene to
normalize the transcript analysis assay. The relative
quantification of the accumulated transgene transcripts showed a highly variable rate of transcription
among various genotypes. The maximum mRNA transcripts were observed in FH-142, FH-167, FH-182,
FH-183 and BT-2333 (Fig. 1).

Quantitative analysis by enzyme linked

immunosorbent assay
All collected cotton genotypes were subjected to
ELISA for determining the concentration of Cry1Ac
toxin. Results showed that Cry1Ac expression level
in leaf tissues ranged from 003 to 199 g/g on a

D I SC U SS I O N
It was observed during the collection of genotypes for
the present study that there is a long list of cotton seed,
mostly unapproved, being sold in Pakistan as Bt cotton
(Table 3). It might be speculated that these genotypes
have not undergone the quality checking procedures
usually adopted in the variety approval process.
Transgenic testing procedures should consist of
reliable, cost-effective and reproducible qualitative
and quantitative techniques. In the present study, multiplex PCR and strip tests were conducted for qualitative testing whereas Rt-PCR and ELISA were performed
for quantitative evaluation of transgenic Bt expression.
The detection of non-Bt genotypes among those
recognized as Bt by the seed providers indicated the
worst situation of quality measurements, adopted for
Bt cotton cultivation. The poor performance of transgenic technology in Pakistan might be attributed to
this prevailing situation, which could foster resistance
in target pests. Toxin concentration should not be less
than the standard lethal level, because it may create
resistance in the target pests faster than the predicted
period (Ferr & Van Rie 2002). Late approval, illegal
cultivation and unregulated adoption of Bt cotton
has encouraged the malpractice of selling non-Bt
cotton seed with the label of Bt (Ahsan & Altaf
2009). The qualitative detection of transgene products
by immunoblot strip tests further endorsed the PCR
results, except for one genotype (FH-187) that
showed negative results for the Cry1A gene, though
it was reported by PCR to harbour the transgene.
This may be due to low levels of Cry toxin, below
the detection potential of the strip test. The zero
ELISA reading further confirmed that the toxin was
below detectable concentrations in this particular
sample. Another study (Ali et al. 2010) evaluated 42
local Bt cotton genotypes by strip test and confirmed
only 34 as Bt gene carriers. This mixing of Bt and
non-Bt genotypes badly affects their yield potential
due to infestation of pests, which might not be able
to survive if pure Bt genotypes are cultivated in the
fields. The transcriptome analysis by real time RtPCR showed that the transgene promoter is not

Transgene analysis in Bt cotton

Table 3. Source, approval status and testing results of collected genotypes

Immunoblot
strip test

Qualitative PCR testing

Sr. #

Genotypes

Source

Legal status

Cry1Ac

Mon531 event

SadI

Cry1Ac

Cry2Ab

Conc. of
Cry1Ac (g/g)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49

BT-S-15
VH-148
CRS-456
FH-119
IR-4
BT-118
Bt-121
N-820
IR-3
IR-901
FH-114
FH-4243
BT-222
FH-142
VH-283
BT-2009
AS-01
VH-295
BT-2333
BT-23
FH-187
FH-177
FH-171
FH-167
FH-161
FH-182
FH-183
FH-158
FH-174
C-26
VH-282
AA-703
AA-802
MHN-886
MNH-888
IR-3701
CRS-2007
FH-113
SB-149
NS-131
KZ-181
KZ-189
KZ-191
VH-259
FH-169
FH-172
FH-154
FH-159
FH-166

F
F
F
F
F
F
F
F
F
SD
SD
F
F
F
F
F
F
SD
F
F
F
F
F
F
F
F
F
SD
SD
F
F
SD
F
F
F
F
F
F
F
F
F
F
F
F
F
SD
SD
F
F

Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Approved
Unapproved
Approved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Approved
Approved
Approved
Unapproved
Approved
Unapproved
Approved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved
Unapproved

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+

+
+
+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+

000
020
084
004
050
041
168
021
041
0149
049
055
048
180
090
000
118
054
199
116
000
050
088
180
033
140
141
058
028
000
003
019
052
039
012
030
000
037
064
088
023
016
028
031
018
016
009
000
000

H. M. N. Cheema et al.

Table 3. (Cont.)
Sr. #

50
51
52

Genotypes

FH-157
FH-170
FH-153

Source

F
SD
SD

Legal status

Unapproved
Unapproved
Unapproved

Qualitative PCR testing

Immunoblot
strip test

Cry1Ac

Mon531 event

SadI

Cry1Ac

Cry2Ab

+
+
+

+
+
+

+
+
+

+
+
+

Conc. of
Cry1Ac (g/g)

055
017
013

F, farmers; SD, seed dealer.

behaving as a constitutive type of promoter. The difference of many folds in transcription of a particular
transgene with the same promoter suggests the involvement of some other factors in attenuating the transcription of Cry1Ac gene. The quantification of toxin
protein by ELISA also revealed that Cry1Ac toxin differed significantly in all Bt cotton genotypes. A
similar trend of variation was expected for transcript
accumulation (Rt-PCR) and translated gene product
(ELISA) for a given genotype, but only a few genotypes
showed the same trend. The different location of
leaves used for RNA extraction and ELISA might be
the reason for the different trends between Cry1Ac
mRNA and protein (Kranthi et al. 2005). The Bt toxin
in different tissues of the cotton plant vary throughout
its life-cycle (Adamczyk & Sumerford 2001; Mahon
et al. 2002; Kranthi et al. 2005), so no uniformity in
toxin level in all genotypes was observed.
Another important observation was the sub-lethal
concentration of Bt toxin in most of the genotypes.
The fact is that no prior study has reported the baseline
susceptibility of local lepidoptera before the release of
transgenic cotton. In the absence of the standard lethal
level, the lethal level of 18 g/g reported in India by
Kranthi et al. (2005) was used for comparison.
Sufficient levels of Cry protein are crucial in appropriate plant parts for resistance against target insect pests
(Greenplate et al. 2001). The main factors contributing
towards the variable expression of Bt gene are its base
sequences, copy number, the promoter used and gene
incorporation point into the host genome of cotton
genotypes (Guo et al. 2001). However, in the
present study the same transgenic event was observed
in which the Cry1Ac gene is under control of the
CaMv35S promoter, a constitutive type of promoter.
So a high level of variability in expression of the
same transgene among collected genotypes might be
attributed to variations in the methylation status of
the promoter (35S) region, over-expression of the

insecticidal genes at earlier stages of plant growth

that ultimately results in gene silencing at later stages
(Xia et al. 2004), or differences in genetic background
(Adamczyk & Meredith 2004). The variation in toxin
level in different genotypes, ranging from low to
higher levels, can be harnessed by breeders to
exploit the potential of this technology by developing
genotypes that express the transgene at a higher level.
Variable expression of the insecticidal transgene
would not only increase the production cost but also
instigate resistance in target pests against a transgenic
crop. The main cause of this low level of Cry protein
might be the adoption of unapproved genotypes.
The current results also showed that even approved
varieties did not meet the standard toxin level. Most
genotypes collected from farmers fields were elite
lines leaked from various research institutes but still
unapproved. No specific trend of expression was
observed regarding the source of seed. None of the
cotton seed collected from seed dealers was found
to be non-Bt, but the smaller sample size prevents
any final verdict about the reliability of source.
The results suggest that Government agencies must
implement the Seed Act quickly to ensure that seed is
correctly labelled and prevent the cultivation of unapproved varieties, along with considering toxin level as
a quality criterion for approval and certification. The
approval of plant breeders rights together with
amendments to the Seed Act can overcome obstacles
in the involvement of the private sector for research
and development, which may improve technology
development and dissemination. Attention should
also be paid to developing consistent and stable promoters for the Cry transgene that can drive the
expression throughout cotton plant development,
especially in the tissues that are more vulnerable to
target insect-pest attack. In addition breeders should
utilize better combinations of parents, favouring
higher toxin concentration, considering that genetic

Transgene analysis in Bt cotton

background has the potential to manipulate transgene

expression in cotton. The present study also showed
the need for capacity development and creating
awareness about the application of transgenic technology to harvest maximum benefits.
Funding for this study was provided by a project
funded by USDA supported Endowment Fund
Secretariat, University of Agriculture, Faisalabad, to
Dr Hafiza Masooma Naseer Cheema. The student fellowship, provided by Mian Muhammad Afzal, Afzal
Traders Pvt Ltd, is also acknowledged.

Fig. 1. Relative fold transcription of Cry1Ac, determined by real time Rt-PCR, in transgenic genotypes.

R E F E R EN C ES
ABEL, C. A. & ADAMCZYK, J. J., JR (2004). Relative concentration
of Cry1A in maize leaves and cotton bolls with diverse
chlorophyll content and corresponding larval development of fall armyworm (Lepidoptera: Noctuidae) and
southwestern corn borer (Lepidoptera: Crambidae) on
maize whorl leaf profiles. Journal of Economic
Entomology 97, 17371744.
ADAMCZYK, J. J., JR & MEREDITH, W. (2004). Genetic basis for
variability of Cry1Ac expression among commercial transgenic Bacillus thuringiensis (Bt) cotton cultivars in the
United States. Journal of Cotton Science 8, 1723.
ADAMCZYK, J. J., JR & SUMERFORD, D. V. (2001). Potential factors
impacting season-long expression of Cry1Ac in 13 commercial varieties of Bollgard cotton. Journal of Insect
Science 1, 13. PMCID: 355897.
ADAMCZYK, J. J., JR, PERERA, O. & MEREDITH, W. R. (2009).
Production of mRNA from the cry1Ac transgene differs
among Bollgard lines which correlates to the level of
subsequent protein. Transgenic Research 18, 143149.
AGUILERA, M., QUERCI, M., PASTOR, S., BELLOCCHI, G.,
MILCAMPS, A. & VAN DEN EEDE, G. (2009). Assessing copy
number of MON 810 integrations in commercial seed
maize varieties by 5 event-specific real-time PCR validated method coupled to 2CT analysis. Food
Analytical Methods 2, 7379.
AHSAN, R. & ALTAF, Z. (2009). Development, adoption and
performance of Bt cotton in Pakistan: a review. Pakistan
Journal of Agricultural Research (Pakistan) 22, 7385.
ALI, S., HAMEED, S., MASOOD, S., ALI, G. M. & ZAFAR, Y. (2010).
Status of Bt cotton cultivation in major growing areas of
Pakistan. Pakistan Journal of Botany 42, 15831594.
BAKHSH, A., RAO, A. Q., SHAHID, A. A., HUSNAIN, T. &
RIAZUDDIN, S. (2009). Insect resistance and risk assessment
studies in advance lines of Bt cotton harboring Cry1Ac
and Cry2A genes. American-Eurasian Journal of
Agricultural and Environmental Sciences 6, 111.
BAKHSH, A., RAO, A. Q., SHAHID, A. A., HUSNAIN, T. &
RIAZUDDIN, S. (2010). CaMV 35S is a developmental promoter being temporal and spatial in expression pattern
of insecticidal genes (Cry1Ac & Cry2A) in cotton.
Australian Journal of Basic and Applied Science 4, 3744.

H. M. N. Cheema et al.

BELTRN, J., JAIMES, H., ECHEVERRY, M., LADINO, Y., LPEZ, D.,
DUQUE, M. C., CHAVARRIAGA, P. & TOHME, J. (2009).
Quantitative analysis of transgenes in cassava plants
using real-time PCR technology. In Vitro Cellular and
Developmental Biology Plant 45, 4856.
CLIVE, J. (2013). Global Status of Commercialized Biotech/
GM Crops: 2013. ISAAA Brief 46. Manila, The
Philippines: ISAAA.
CORORATON, C. B., SALAM, A., ALTAF, Z., ORDEN, D., DEWINA, R.,
MINOT, N. & NAZLI, H. (2008). Cotton-textile-apparel
Sectors of Pakistan: Situations and Challenges Faced.
IFPRI Discussion Paper no. 800. Washington, DC:
International Food Policy Research Institute.
DOYLE, J. J. & DOYLE, J. L. (1990). Isolation of plant DNA from
fresh tissue. Focus 12, 1315.
FERR, J. & VAN RIE, J. (2002). Biochemistry and genetics of
insect resistance to Bacillus thuringiensis. Annual Review
of Entomology 47, 501533.
GASKELL, G., BAUER, M. W., DURANT, J. & ALLUM, N. C. (1999).
Worlds apart? the reception of genetically modified foods
in Europe and the US. Science 285, 384387.
GORE, J., LEONARD, B. R. & ADAMCZYK, J. J. (2001). Bollworm
(Lepidoptera: Noctuidae) survival on Bollgard and
Bollgard II cotton flower bud and flower components.
Journal of Economic Entomology 94, 14451451.
GREENPLATE, J. T., PENN, S. R., MULLINS, J. W. & OPPENHUIZEN, M.
(2000). Seasonal Cry1Ac levels in DP50B: the Bollgard
basis for Bollgard II. In Proceedings of the Beltwide
Cotton Conference (Eds P. Dugger & R. Richter), pp.
10391040.
GREENPLATE, J. T., MULLINS, W., PENN, S. & EMBRY, K. (2001).
Cry1Ac levels in candidate commercial Bollgard varieties as influenced by environment, variety and plant
age: 1999 gene equivalency field studies. In
Proceedings of the Beltwide Cotton Conference (Eds P.
Dugger & R. Richter), pp. 790793. Memphis, TN, USA:
National Cotton Council of America.
GUO, W. Z., SUN, J., GUO, Y. F. & ZHANG, T. Z. (2001).
Investigation of different dosages of inserted Bt genes
and their insect-resistance in transgenic Bt cotton. Yi
Chuan Xue Bao (Acta Genetica Sinica) 28, 668676.
GUTIERREZ, A. P., ADAMCZYK, J. J., JR, PONSARD, S. & ELLIS, C. K.
(2006). Physiologically based demographics of Bt
cottonpest interactions: II. Temporal refuges, natural
enemy interactions. Ecological Modelling 191, 360382.
HOLT, H. E., MARES, C. & AKHURST, R. (2002). Determination of
the Cry Protein Content of Bt Transgenic Cotton : a
Technical Manual for Laboratory Use. Canberra: CSIRO
Australia, Division of Entomology.
JACKSON, R. E., BRADLEY, J. R., JR, VAN DUYN, J. W. & GOULD, F.
(2004). Comparative production of Helicoverpa zea
(Lepidoptera: Noctuidae) from transgenic cotton expressing either one or two Bacillus thuringiensis proteins
with and without insecticide oversprays. Journal of
Economic Entomology 97, 17191725.
KHAN, S. M., SAEED, I., SHAH, M., SHAH, S. F. & MIR, H. (2012).
Integration of tolerance of Bt cotton varieties with insecticides against spotted bollworm, Earias insulana (Boisd.)
and E. vittella (Fab.)(Noctuidae: Lepidoptera). Sarhad
Journal of Agriculture 28, 5762.

KRANTHI, K. R., NAIDU, S., DHAWAD, C. S., TATWAWADI, A.,

MATE, K., PATIL, E., BHAROSE, A. A., BEHERE, G. T.,
WADASKAR, R. M. & KRANTHI, S. (2005). Temporal and
intra-plant variability of Cry1Ac expression in Bt-cotton
and its influence on the survival of the cotton bollworm,
Helicoverpa armigera (Hubner) (Noctuidae: Lepidoptera).
Current Science 89, 291298.
LEE, S. H., KANG, S. H., PARK, Y. H., MIN, D. M. & KIM, Y. M.
(2006a). Quantitative analysis of two genetically modified
maize lines by real-time PCR. Journal of Microbiology and
Biotechnology 16, 205211.
LEE, S. H., MIN, D. M. & KIM, J. K. (2006b). Qualitative and
quantitative polymerase chain reaction analysis for
genetically modified maize MON863. Journal of
Agricultural and Food Chemistry 54, 11241129.
LEE, S. H., KIM, J. K. & YI, B. Y. (2007). Detection methods for
biotech cotton MON 15985 and MON 88913 by PCR.
Journal of Agricultural and Food Chemistry 55,
33513357.
LI, Z., HANSEN, J. L., LIU, Y., ZEMETRA, R. S. & BERGER, P. H.
(2004). Using real-time PCR to determine transgene
copy number in wheat. Plant Molecular Biology
Reporter 22, 179188.
MAHON, R., FINNEGAN, J., OLSEN, K. & LAWRENCE, L. (2002).
Environmental stress and the efficacy of Bt cotton.
Australian Cottongrower 23, 1822.
MASOOD, A., ARIF, M. J., HAMED, M. & TALPUR, M. A. (2011).
Field performance of Trichogramma chilonis against
cotton bollworms infestation in different cotton varieties
as a sustainable IPM approach. Pakistan Journal of
Agriculture, Agricultural Engineering and Veterinary
Science 27, 176184.
OLSEN, K. M., DALY, J. C., HOLT, H. E. & FINNEGAN, E. J. (2005).
Season-long variation in expression of Cry1Ac gene and
efficacy of Bacillus thuringiensis toxin in transgenic
cotton against Helicoverpa armigera (Lepidoptera: Noctuidae). Journal of Economic Entomology 98, 10071017.
RANDHAWA, G. J., CHHABRA, R. & SINGH, M. (2009). Multiplex
PCR-based simultaneous amplification of selectable
marker and reporter genes for the screening of genetically
modified crops. Journal of Agricultural and Food
Chemistry 57, 51675172.
RANDHAWA, G. J., CHHABRA, R. & SINGH, M. (2010). Decaplex
and real-time PCR based detection of MON531 and
MON15985 Bt cotton events. Journal of Agricultural and
Food Chemistry 58, 98759881.
SINGH, C. K., OJHA, A., BHATANAGAR, R. K. & KACHRU, D. N.
(2008). Detection and characterization of recombinant
DNA expressing vip3A-type insecticidal gene in GMOs
standard single, multiplex and construct-specific PCR
assays. Analytical and Bioanalytical Chemistry 390,
377387.
TARIQ, M. I., AFZAL, S., HUSSAIN, I. & SULTANA, N. (2007).
Pesticides exposure in Pakistan: a review. Environment
International 33, 11071122.
WAN, P., ZHANG, Y., WU, K. & HUANG, M. (2005). Seasonal
expression profiles of insecticidal protein and control efficacy against Helicoverpa armigera for Bt cotton in the
Yangtze River valley of China. Journal of Economic
Entomology 98, 195201.

Transgene analysis in Bt cotton

WASTI, S. E. (2013). Pakistan Economic Survey 201213.
Islamabad, Pakistan: Ministry of Finance, Government of
Pakistan.
WU, Y., WU, G., XIAO, L. & LU, C. (2007). Event-specific
qualitative and quantitative PCR detection methods for
transgenic rapeseed hybrids MS1 RF1 and MS1 RF2.
Journal of Agricultural and Food Chemistry 55,
83808389.
XIA, L., XU, Q. & GUO, S. (2004). Bt insecticidal gene and its
temporal expression in transgenic cotton plants. Acta
Agronomica Sinica 31, 197202.

YANG, L., PAN, A., ZHANG, K., GUO, J., YIN, C., CHEN, J.,
HUANG, C. & ZHANG, D. (2005a). Identification and quantification of three genetically modified insect resistant
cotton lines using conventional and TaqMan real-time
polymerase chain reaction methods. Journal of
Agricultural Food Chemistry 53, 62226229.
YANG, L., PAN, A., ZHANG, K., YIN, C., QIAN, B., CHEN, J.,
HUANG, C. & ZHANG, D. (2005b). Qualitative and quantitative PCR methods for event-specific detection of genetically modified cotton Mon1445 and Mon531. Transgenic
Research 14, 817831.