Journal of Integrative Agriculture 2017, 16(3): 551–558

Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Development of glyphosate-tolerant transgenic cotton plants

harboring the G2-aroA gene

ZHANG Xiao-bing1, 2*, TANG Qiao-ling1*, WANG Xu-jing1, WANG Zhi-xing1

1
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
2
Biology Institute, Hebei Academy of Sciences, Shijiazhuang 050051, P.R.China

Abstract
Given that glyphosate weed control is an effective strategy to reduce costs and improve economic outcomes of agricultural
production in China, the development of glyphosate-resistant cotton holds great promise. Using an Agrobacterium-medi-
ated transformation method, a new G2-aroA gene that encodes 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)
was transformed into cotton cultivar K312. The transgenic cotton plants were regenerated from a callus tissue culture via
kanamycin selection. Ten regenerated cotton plants were obtained and allowed to flower normally to produce fruit. The
results from polymerase chain reaction (PCR) and Southern and Western blot analyses indicated that the target gene was
integrated into the cotton chromosome and was expressed effectively at the protein level. The glyphosate tolerance analysis
showed that the transgenic cotton had a high resistance to glyphosate. Further, even cotton treated with 45.0 mmol L–1 of
glyphosate was able to slowly grow, bloom and seed. The transgenic cotton may be used for cotton breeding research of
glyphosate-tolerant cotton.

Keywords: cotton (Gossypium hirsutum L.), Agrobacterium-mediated method, glyphosate, G2-aroA, genetic transformation

weeds adversely affect the yield and quality of cotton (Awan

1. Introduction
Cotton is an important fiber crop and is a major resource of
plant protein and edible oil in the world (Zhang 2013; Chakra-
varthy et al. 2014; Guo et al. 2015). Because excessive
Received 15 March, 2016 Accepted 18 July, 2016
ZHANG Xiao-bing, E-mail: zhangxiaobing9@126.com;
Correspondence WANG Xu-jing, Tel: +86-10-82106124,
E-mail: xujingwang0514@126.com; WANG Zhi-xing,
Tel: +86-10-82106102, E-mail: wangcotton@126.com
*
These authors contributed equally to this study.
© 2017, CAAS. Published by Elsevier Ltd. This is an open
access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/)
doi: 10.1016/S2095-3119(16)61458-2
et al. 2015), chemical weed control is an effective approach
to reduce costs and improve economic benefits. One com-
monly used herbicide is glyphosate, which is non-selective
and has the advantages of broad-spectrum effects, high
efficiency, low toxicity, and an absence of residue (Ma et al.
2016). Glyphosate inhibits 5-enolpyruvylshikimate-3-phos-
phate (EPSP) synthase (EC 2.5.1.19), which is the enzyme
that catalyzes the critical step of the shikimate pathway in the
biosynthesis of aromatic amino acids, and its functionality is
absolutely required for the survival of microorganisms and
plants (Steinrucken and Amrhein 1980; Funke et al. 2006;
Gong et al. 2016). However, when glyphosate kills weeds
in a field, it also damages the crops. Therefore, efforts to
cultivate and promote glyphosate-resistant cotton varieties
via genetic engineering technology are the most effective
approaches for controlling glyphosate weeds in cotton fields.
552 ZHANG Xiao-bing et al. Journal of Integrative Agriculture 2017, 16(3): 551–558
There are primarily three types of strategies to achieve
resistance to glyphosate, which include overexpression of
sensitive EPSP synthase (Gaines et al. 2010), detoxification
of glyphosate (Malherbe et al. 2003; Castle et al. 2004)
and expression of an insensitive form of EPSP synthase
(Padgette et al. 1995; Tian et al. 2010; Yan et al. 2011). Cur-
rently, multiple genetically modified cotton varieties, which
have obtained glyphosate resistance via the introduction
of their chromosomes to insensitive EPSPS, have been
developed and are widely used in several developed coun-
tries, providing enormous economic and societal benefits
(Zhang 2013; Vats 2015). Several national research groups
(Xie et al. 2004; Zhao et al. 2006; Zhang et al. 2014; Yan
et al. 2015) obtained glyphosate-resistant cotton cultivars.
However, domestic herbicide-resistant cotton, which could
be used for commercial production, has not been studied.
Therefore, studies on glyphosate-resistant genes and her-
bicide-resistant crops are still critically needed and are of
great significance in China.
In this study, a glyphosate-resistant G2-aroA gene was
isolated and cloned from the Pseudomonas fluorescens
strain G2 in the Prof. Lin Min’s Laboratory at the Biotechnolo-
gy Research Institute of the Chinese Academy of Agricultural
Sciences (Zhu et al. 2003; Wang et al. 2014) via indepen-
dent intellectual property rights. The gene was introduced
into an upland cotton cultivar using K312 as a receptor via
the Agrobacterium-mediated transformation. Thus, several
glyphosate-resistant transgenic cotton lines were obtained.
Meanwhile, resistance identification and molecular biological
analysis of the cotton provides a scientific basis for breeding
and commercial production in the future.
2. Materials and methods
2.1. Plant materials
Cotton seeds (Gossypium hirsutum L. cv. K312), which were
stored in our laboratory, were used for this study. The seeds
were manually dehusked, sterilized with 75% (v/v) ethanol
for 5 min, washed three times with sterile distilled water,
soaked in a 1.8% (w/v) sodium hypochlorite solution, and
P-nos P-rubisco
Left boder nptII T-nos HindIII (2 898)
7 100 bp
Fig. 1 Structure of the plant expression vector of p-G2-aroA. P-nos, nos promoter; nptII, neomycin phosphotransferase gene;
T-nos, nos terminator; P-rubisco, Daisy Rubisco small subunit promoter; CTS, Daisy Rubisco small subunit chloroplast signal
peptide; G2-aroA, 5-enolpyruvyl-shikimate-3-phosphate synthase gene; T-rubisco, Daisy Rubisco small subunit terminator.
washed 6 times with sterile distilled water. The sterile seeds
were germinated on a solidified MS medium (Murashige
and Skoog 1962) that was supplemented with 1.5% (w/v)
glucose and 0.8% (w/v) agar at pH 5.8. Hypocotyl segments
(5–6 mm), which were excised from 5- to 6-d-old seedlings,
were used for the transformation.
2.2. Cotton transformation and regeneration of
transformants
The hypocotyl segments were infected with Agrobacterium
tumefaciens LBA4404 that contained the nptII and G2-aroA
genes, which were derived using a nos promoter and a
daisy rubisco small subunit promoter (Fig. 1), respectively.
Infected hypocotyls were transferred onto a piece of filter
paper using co-cultivation medium. After co-cultivation, the
hypocotyls were rinsed thoroughly with sterile distilled water
prior to transfer to a callus induction medium. Embryogenic
calli were induced for 4 wk and transferred onto fresh me-
dium in which the concentration of kanamycin was gradu-
ally increased to 100 mg L–1 for further selection until they
were vigorously growing and until friable, loose and white
healthy calli were obtained. After approximately 3–4 wk,
the embryogenic calli were excised and transferred onto
embryo differentiation medium and maintained on this
medium until somatic embryos developed and geminated.
One subculture was performed every 4 wk, and shoot re-
generation occurred within 7–9 wk. When selected shoots
grew to 3–4 cm in height, they were transferred to an induced
root culture medium. Selected shoots with 3–4 pieces of
leaves and roots were acclimatized and then transplanted.
2.3. DNA extraction and PCR detection of the trans-
formed cotton plant
Cotton genomic DNA was isolated from young leaves of
non-transformed control plants and transgenic cotton plants
using an improved cetyltrimethylammonium ammonium
bromide (CTAB) extraction protocol (Paterson et al. 1993;
Chaudhry and Yasmin 1999). The quality and quantity of
DNA was analyzed using a spectrophotometer (NanoDrop
Probe
Right boder
CTS G2-aroA T-rubisco
4 204 bp
 ZHANG Xiao-bing et al. Journal of Integrative Agriculture 2017, 16(3): 551–558
2000, Thermo Fisher Scientific, Wilmington, DE, USA) with
260/280 nm and 260/230 nm ultraviolet (UV) absorption
ratios and then examined using 1% agarose gel electro-
phoresis.
The conventional polymerase chain reaction (PCR) was
performed in a Bio-RAD T100TM Thermal Cycler (Bio-Rad
Hercules, CA, USA). The amplification of the G2-aroA gene
was performed using the following pair of specific primers:
5ˊ-GGCTCCAAATCCATTACCAACC-3ˊ and 5ˊ-CGCAG
GTTCGCCAGTTCA-3ˊ, which resulted in a 896-bp product.
Each reaction mixture contained 1× PCR buffer, 0.5 mmol
L–1 of dNTP, 0.3 μmol L–1 of each primer, 3 ng of each DNA
sample, and 2.5 U of Taq DNA polymerase; the final reac-
tion volume was 30 μL. The amplification protocol was run
as follows: one cycle of denaturing at 95°C for 5 min, 34
cycles of 45 s at 95°C, 45 s at 60°C, 60 s at 72°C, and a
final extension at 72°C for 10 min. Amplified products were
analyzed via 1% (w/v) agarose gel electrophoresis and de-
tected by staining with GelStain (Beijing TransGen Biotech
Co., Ltd., China). The results were evaluated and recorded
using a Molecular Imager Gel Doc Ex System (NEWBIOTM
INDUSTRY, China).
The quantitative real-time PCR (qRT-PCR) assays with
SYBR Green chemistry were conducted using an ABI 7500
Real Time PCR System (Applied Biosystems, Singapore) in
final volumes of 30 μL. The assays were performed using a
TransStart® Top Green qPCR SuperMix Kit (Beijing Trans-
Gen Biotech Co., Ltd., China) and the Sadl gene (GenBank
no. AJ132636) as the endogenous reference gene.
The qRT-PCR amplification of the Sad l gene was
performed using the following pair of specific primers:
5ˊ-CCAAAGGAGGTGCCTGTTCA-3ˊ and 5ˊ-TTGAGGT
GAGTCAGAATGTTGTTC-3ˊ, which resulted in a 108-bp
product (Yang et al. 2005). The qRT-PCR amplification of
the G2-aroA gene was performed using the following pair
of specific primers: 5ˊ-GCGTGTTTGCCTGATGATT-3ˊ
and 5ˊ-AAGTTGGGCGGTGTAACG-3ˊ, which resulted in
a 116-bp product. The fluorescence was monitored at the
annealing step of every PCR cycle. Each reaction mixture
contained 1× TransStart® Top Green qPCR SuperMix,
1× Passive Reference Dye, 0.2 μmol L–1 of each primer,
90 ng of DNA template, and the final reaction volume was
30 μL. The qRT-PCR reactions were run according to the
following procedure: one cycle of 2 min at 50°C, 30 s at
94°C, 40 cycles of 5 s at 95°C, and 34 s at 60°C.
Standard curves were obtained using a plasmid con-
taining a single copy of the Sadl and G2-aroA genes as the
standard material.
2.4. Southern blot analysis
The stable integration of G2-aroA was confirmed via South-
553
ern hybridization. In total, 60 mg of each genomic DNA from
non-transgenic and transgenic cotton was completely digest-
ed with EcoRI and HindIII, respectively. The digested DNAs
were resolved using 0.8% (w/v) agarose gel electrophoresis
and then transferred onto a nylon membrane. A 515-bp DNA
fragment amplified with the primers of 5ˊ-CAGAAAACCGT
GACCGTTAC-3ˊ and 5ˊ-GATACG TACTGGCTGGACAA-3ˊ
of the G2-aroA gene, which was labeled with DIG-dUTP us-
ing a PCR DIG Probe Synthesis Kit (Roche, Germany), was
used as the hybridized probe. Hybridization was performed
at 42°C for 24 h, and the filter was washed at 42°C with 2×
saline sodium citrate (SSC)/0.1% SDS for 5 min twice and
at 65°C with 0.5×SSC/0.1% SDS for 15 min twice. DNA
markers (DNA molecular weight marker II, Digoxigenin-
labeled, Roche) were run on the same gel.
2.5. Western blot analysis
The expression levels of the G2-aroA gene within the
transgenic cotton plants were monitored using Western blot
analysis. Young leaves from control and transgenic cotton
plants were collected and ground in liquid nitrogen. Aliquots
of approximately 400 mg of crushed tissue samples were
separately mixed with 400 μL of protein extraction reagent
(Plant Protein Extraction Kit, Beijing Cowin Bioscience Co.,
Ltd., Beijing, China). The suspensions were centrifuged,
and the clarified supernatants were transferred to new
tubes. Protein concentrations were ascertained using a
BCA Protein Assay Kit (Beijing Cowin Bioscience Co., Ltd.)
and calculated as follows: y=0.2146x+0.011, where, x is the
EPSPS protein concentration. The protein (20 mg) was
mixed with an equal volume of 2× sample loading buffer
that contained 100 mmol L–1 of Tris·HCl at pH 6.8, 4% (w/v)
SDS, 20% (v/v) glycerol, 0.02% (w/v) Bromophenol blue,
and 200 mmol L–1 DTT, and it was boiled for 10 min. The
prepared protein samples were then separated on a 12%
(w/v) polyacrylamide SDS gel and transferred to PVDF
membranes using a transfer unit (Bio-Rad Hercules, CA,
USA). The PVDF membrane was blocked with 1% (w/v)
BSA in Tris-buffered Tween 20 (TBT) overnight at 4°C,
followed by incubation with a polyclonal antibody against
EPSPS, which was kindly provided by Prof. Lin Min (Bio-
technology Research Institute, Chinese Academy of Agri-
cultural Sciences). Protein detection was performed using
5-bromo-4-chlore-3-indolyl phosphate (BCIP) and nitro-blue
tetrazolium (NBT), and photos were taken.
2.6. Transgenic plant tests for resistance to glypho-
sate
Transgenic and untransformed control seeds were sowed
in a flowerpot. After approximately 4 wk, plants at the 3- to
554 ZHANG Xiao-bing et al. Journal of Integrative Agriculture 2017, 16(3): 551–558
4-leaf stage were sprayed with effective concentrations
of 0, 4.5, 9.0, 18.0, 36.0, and 45.0 mmol L–1 glyphosate
(Monsanto Co., USA). The plants were grown in growth
chambers at a day/night temperature of (30/22±3)°C with
60–70% relative humidity and a photosynthetic photon flux
density of 350–450 μmol m–2 s–1 for a 15-h photoperiod.
The growth status and viability of the transgenic plants
were evaluated and recorded once every 3 wk following
the treatments. Data analysis was performed using SPSS
(SPSS for Windows, ver. 16.0, USA).
3. Results and discussion
3.1. Transformation and regeneration of cotton plants
with kanamycin as a mode of selection
The hypocotyl explants were infected with the A. tumefaciens
strain that included the G2-aroA gene construct. During the
initial stage of selection, infected explants (Fig. 2-A) were
plated on a calli induction medium that was supplemented
with 50 mg L–1 kanamycin. Following subculture in the
same induction medium, kanamycin concentrations were
increased to 75 and 100 mg L–1 for cellular proliferation. After
several rounds of selection, tender white granular embryos
(Fig. 2-B) were obtained and carefully transferred to the
embryonic callus induction medium. A beige embryogenic
callus (Fig. 2-C) was gradually produced, and regenerated
shoots (Fig. 2-D) were developed. The regenerated shoots
were transferred to rooting medium (Fig. 2-E). Using the
kanamycin selection system, 10 independent plantlets
(Fig. 2-F) were obtained that exhibited a normal morphology
and flowered. T1 (the first generation of transgenic plant)
seeds were obtained via self-fertilization of T0 (the regener-
ated plant of tissue culture) mature ones. The 10 putative
transgenic plants, each representing an independent trans-
formation event, were designated as BG2-1, BG2-2, BG2-3,
BG2-7, BG2-11, BG2-12, BG2-14, BG2-17, BG2-18, BG2-
20, etc. In the follow-up study, these 10 transgenic lines’
agronomic traits were not identical from each other in the
field. Some emergence was very slow, some growth was
retardation, and some plant type was dwarf, and so on. By
M 1 2 3 4 5
bp
1 000
Fig. 3 Results of the PCR for transgenic cottons with G2-aroA. M, marker 5K; 1–6, transgenic cottons (BG2-1, BG2-2, BG2-3,
BG2-7, BG2-12, BG2-14); CK1, ddH2O; CK2, K312; P, plasmid DNA.
screening, the line of BG2-7 is the best one. So, naturally,
it has become the focus of this study.
3.2. Molecular detection of transgenic cotton
PCR detection of the putative transgenic plants was per-
formed. The 896-bp expected target stripe size was ampli-
fied in all of the regeneration plants, and the electrophoretic
migration distance of the stripe was consistent with that of
the plasmid. The PCR results of some of the transgenic
plants are shown in Fig. 3. The target stripe was picked out
and sequenced. The sequencing results were identical to
the target sequence. These preliminary findings showed that
the G2-aroA genes were integrated into the cotton genome.
In transgenic plants, the number of transgene copies can
significantly influence the level of expression and genetic
stability of the target gene (Weng et al. 2004). The qRT-
PCR exhibits a high flux and fast and good repeatability,
and it is particularly useful as a preliminary screening tool
for estimating copy numbers of a large number of transfor-
mants (Bubner and Baldwin 2004). To accurately detect
the number of copies of the target gene in the transgenic
samples, standard curves were developed. The numbers
A B C
D E F
Fig. 2 Obtaining transgenic cotton. A, callus. B, resistant
callus. C, embryogenic callus. D, regeneration of cotton. E,
rooting culture. F, transplant.
6 CK1 CK2 P
bp
896
 ZHANG Xiao-bing et al. Journal of Integrative Agriculture 2017, 16(3): 551–558
of copies of the sadl and G2-aroA genes were calculated
using the following equations: G=43.79–3.80log(cn) and
G=41.45–3.54log(cn), respectively. The correlation coeffi-
cients of the curves were good (0.990–0.992), and the PCR
efficiencies were all above 0.90. According to the equations,
the number of copies of the G2-aroA gene was obtained
(Table 1), and the results showed that the target gene was
a single copy inserted into the transgenic cottons.
To further confirm the integration of the G2-aroA gene,
the independent cotton was analyzed using Southern blot
hybridization. The number of bands and their hybridization
pattern in the plant represented an estimate of the transgene
copy numbers in the cotton genome (Zhao et al. 2006). The
cotton genomic DNA was completely digested with EcoRI
or HindIII. EcoRI and HindIII cut the genomic DNA at a
unique site where the genomic DNA is restricted and where
the number of independent loci of transgenic integration is
determined (Leelavathi et al. 2004). HindIII was the single
restriction enzyme site in the T-DNA region between the
G2-aroA and nptII cassettes (Fig. 1). There were 4 204-bp
bases between the HindIII enzyme loci and the right border
end; therefore, the displacement distance of the band from
the HindIII enzyme treatment should be at least larger than
4 kb. According to the T-DNA region with 7 100-bp bases,
the displacement distance of the band from EcoRI enzyme
treatment should be at least larger than 7 kb. A 515-bp
G2-aroA DNA fragment was used as the hybridized probe.
The DNA fragments were labeled with DIG-dUTP. EcoRI
and HindIII restriction digests of genomic DNA, which were
extracted from the transgenic plants, yielded one single
band, and their displacement distances were in agreement
with the ones suggested (Fig. 4). These results indicated
the presence of a single copy of the G2-aroA gene inte-
grated into the transgenic cotton genome. According to
the flanking sequence information (Zhang 2015; Zhang
et al. 2016), there was a HindIII enzyme locus in the right
flanking sequence, and the length of bases between the
two HindIII enzyme loci was 4 289 bp, which also confirms
the previous results.
Table 1 The copy number of G2-aroA in transgenic cotton gene
CT value1)
Sample code
sadl
BG2-1 21.81±0.19
BG2-2 21.67±0.28
BG2-3 21.72±0.10
BG2-7 21.63±0.11
BG2-12 21.65±0.07
BG2-14 21.93±0.09
BG2-17 21.92±0.22
BG2-20 22.71±0.42
1)
All CT (number of amplification cycles) values represent means±SD of triplicate samples.
555
To examine the G2-aroA gene expression, the transgen-
ic cotton plants were further analyzed for EPSPS protein
production via immunoblotting with a polyclonal antibody
that was specific to EPSPS. Transgenic cotton expressed
the EPSPS protein with an expected molecular mass of
59 kDa. However, the non-transgenic control did not express
the EPSPS protein (Fig. 5). Thus, the transgenic cotton
plants that correctly expressed the EPSPS protein were
successfully obtained.
3.3. Glyphosate tolerance analysis of transgenic
cotton
In the field, the agronomic traits of BG2-7 were the best
among the obtained transgenic cotton lines in which the gly-
phosate tolerance test was conducted. T2 transgenic plants
at the 3- to 4-leaf-stage were sprayed with solutions that
had different concentrations of glyphosate. Three days after
treatment, the leaves and stem apex of the non-transgenic
cottons began to wilt with a treatment of 36.0 and 45.0 mmol
L–1 glyphosate, and no difference was apparent among the
transgenic cotton plants that had different treatments. Ten
days after treatment with glyphosate concentrations greater
than 9.0 mmol L–1, the stem apex of the non-transgenic cot-
tons wilted completely and stopped growing. With further
increase in the glyphosate concentration, the vulnerability
of the transgenic and non-transgenic cotton plants both
increased gradually. During the third week of treatment
(Fig. 6), the transgenic cotton showed no significant effects
due to treatment with 9.0 mmol L–1 glyphosate, which is the
recommended working concentration in the field (Zhao et al.
2006; Wang 2015). The transgenic cotton plants treated
with 45.0 mmol L–1 glyphosate still grew slowly, and with
increased time, the growth vigor of the transgenic cotton
recovered gradually. Thus, the plants were able to blossom
and seed. The glyphosate resistance test showed that the
transgenic cotton in this study displayed a high resistance
to glyphosate.
Glyphosate is a non-selective herbicide, which has a
G2-aroA copy number
G2-aroA
22.64±0.28 1
22.18±0.24 1
23.00±0.08 1
22.88±0.17 1
22.96±0.18 1
23.20±0.04 1
23.22±0.12 1
23.48±0.11 1
556 ZHANG Xiao-bing et al. Journal of Integrative Agriculture 2017, 16(3): 551–558

kb M 1 2 3 4 P
23.13

9.42
6.66
kDa M CK 1 2 3
4.36
70
kDa
2.32 59
2.02 55

Fig. 4 Southern blot analysis of the transgenic and control Fig. 5 Western blot analysis of the transgenic and control
plants. M, marker; 1, K312 DNA/EcoRI; 2, K312 DNA/HindIII; plants. M, protein marker; CK, K312; 1–3, transgenic cottons
3, BG2-7/EcoRI; 4, BG2-7/HindIII; P, plasmid DNA. (BG2-1, BG2-7, BG2-20).

Fig. 6 Reaction of the glyphosate-resistant cotton lines to different concentrations of glyphosate. CK1, K312 was sprayed with
0 µmol L–1 glyphosate; CK2, BG2-7 was sprayed with 0 mmol L–1 glyphosate; I-1, K312 was sprayed with 4.5 mmol L–1 glyphosate;
II-1, BG2-7 was sprayed with 4.5 mmol L–1 glyphosate; I-2, K312 was sprayed with 9.0 mmol L–1 glyphosate; II-2, BG2-7 was sprayed
with 9.0 mmol L–1 glyphosate; I-3, K312 was sprayed with 18.0 mmol L–1 glyphosate; II-3, BG2-7 was sprayed with 18.0 mmol L–1
glyphosate; I-4, K312 was sprayed with 36.0 mmol L–1 glyphosate; II-4, BG2-7 was sprayed with 36.0 mmol L–1 glyphosate; I-5,
K312 was sprayed with 45.0 mmol L–1 glyphosate; II-5, BG2-7 was sprayed with 45.0 mmol L–1 glyphosate.

widespread application in China. The herbicide remains
and accumulates in plant meristems, where it can hinder
reproductive development and lower crop yield (Pline et al.
2002). The effect of the herbicide is closely related to its
applied concentration. Additionally, the expression of foreign
genes and the accumulation of target products are depen-
dent on gradually increasing concentrations; thus, the timing
of herbicide treatment is an extremely important factor.
Up to date, researchers cloned lots of genes related to
glyphosate tolerance, such as cp4-epsps, G6-epsps, G10-
aroA and G2-aroA (Padgette et al. 1995; Zhu 2002; Zhao
2008; Li 2013). Among these genes, cp4-epsps was widely
commercial used in glyphosate tolerance rice, maize, soy-
bean and cotton. Compared with cp4-epsps gene, G2-aroA
gene has 24.53% nucleotide sequence homology and does
not contain the sequences and mutant sites that protected
by patent. Also, G2-aroA gene has higher glyphosate
tolerance than cp4-epsps at enzyme level (Zhu 2002; Dun
et al. 2014). In this study, we found that transgenic cotton
line G2-7 can tolerate 45.0 mmol L–1 glyphoste and meet
production needs. This research results will provide a new
resource for cotton glyphosate-tolerant genetic engineering.
4. Conclusion
In this study, glyphosate-resistance transgenic cotton was
developed which displayed a high resistance to glyphosate.
Even the cotton plants treated with 45.0 mmol L–1 glyphosate
were still able to slowly grow, bloom and seed. Therefore,
these plants could be used for research regarding the
breeding of glyphosate-tolerant cotton.
Acknowledgements
This work was supported by the Genetically Modified Major
Projects, China (2012ZX08011-003 and 2014ZX08011-
 ZHANG Xiao-bing et al. Journal of Integrative Agriculture 2017, 16(3): 551–558
004B). We thank Prof. Lin Min (Biotechnology Research
Institute, Chinese Academy of Agricultural Sciences) for
providing the G2-aroA gene.
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