Published January 25, 2017

CROP ECOLOGY & PHYSIOLOGY

Changed Growth Characteristics with Bacillus thuringiensis Gene

Introduction and Nitrogen Regulation in Bt Cotton
Eltayib Abidallha, Leila I. M. Tambel, Li Heng, Xiang Zhang,* De-Hua Chen*

S
ABSTRACT ince commercial cotton cultivars producing
insecticidal Bt proteins were planted in 1997, Bt cotton has
Changes in vegetative and reproductive growth have been
been planted widely in China and other areas of the world
widely observed in Bacillus thuringiensis (Bt) transgenic cotton
(Clive, 2012; Huang et al., 2010).The Bt technology improved
(Gossypium hirsutum L.). The objectives of this study were to
growers’ profitability, reduced environmental pollution from
determine the characteristics of vegetative and reproductive
synthetic insecticides, and increased workers’ safety (Huang et
growth after the Bt gene was introduced into cotton and to
al., 2001; Kranthi et al., 2005; Pray et al., 2001). However, unsta-
study the effect of N on vegetative and reproductive growth to
ble lint yields have been observed frequently among farms in
determine if N nourishment could provide a potential mechanism
cotton production regions; some farms have a higher lint harvest,
for changed growth characteristics in Bt cotton. Three Bt
while other farms growing the same Bt cultivars have a lower
cultivars (Sikang1, Ccsi41, and Ccsi45), with their current parents
lint harvest under similar ecological conditions (Dong et al.,
(Simian3, Ccsi23, and Jimian7, respectively), were chosen to
2011; Li et al., 2005). Lint reduction has been attributed to the
compare the characteristics of vegetative and reproductive growth
application of traditional cultural technology resulting in heavy
and N distribution, in 2011 to 2012; the same six cultivars with
abscission of reproductive organs in Bt cultivars. Because vegeta-
two N fertilizer application rate treatments (0 and 450 kg/ha)
tive and reproductive growth characteristics were changed by the
were further designed to investigate the N regulation impact on
introduction of the Bt gene, management practices also require
growth in 2013. The results indicated that the Bt cotton cultivars
change. Tian et al. (2000) reported that Bt cultivar CSC30 had
had higher growth rates of main stem leaf area, sympodium leaf
smaller bolls and a lower lint percentage than its parent. Higher
number, and plant height and a lower growth rate of sympodium
vegetative growth, including increased plant height and higher
leaf area than their parents. Higher numbers and development
relative growth rate and biomass, were also observed in Bt cot-
rates of fruiting nodes, fruiting branches, bolls, and retained boll
ton (Chen et al., 2002; Tian et al., 2000). In contrast to their
rates were also observed but lower fruiting branch positions than
parents, Bt cultivars showed earlier boll opening, more fruiting
their parents. The N-deficit and N-rich treatments affected both
nodes, and a boll-setting rate that was maintained during repro-
vegetative and reproductive growth of Bt cultivars. The Bt cultivars
ductive growth. However, the causes of the changed characteris-
had reduced numbers and rates of vegetative and reproductive
tics during growth and development were unclear. Chen and Gu
organs under N deficit compared with their parents. These
(2004) and Zhang et al. (2007) measured a changed N metabo-
results show that the introduction of the Bt gene changed growth
lism in Bt cotton. We, therefore, hypothesized that the changed
characteristics, including reduced vegetative and reproductive
vegetative and reproductive growth in Bt cotton was related to a
organ development under N deficit.
changed N distribution.
The objectives of this study were: (i) to determine the
characteristics of vegetative and reproductive growth after the
Core Ideas
introduction of the Bt gene into cotton; and (ii) to study the effects
• Bt cotton cultivars had higher sympodium leaf number and
plant height. of N on vegetative and reproductive growth to determine if N
• Bt cotton cultivars had higher growth rates of main stem leaf and nourishment could account for the changed growth characteristics
lower growth rate of sympodium leaf area. in Bt cotton.
• Bt cotton cultivars had higher numbers of fruiting branches,
fruiting nodes, bolls, and retained boll rates. MATERIALS AND METHODS
• N-deficit and N-rich treatments affected number and rates of Plant Material and Experimental Design
vegetative and reproductive organs.
The study was conducted on the farm of Yangzhou
University (32°30¢ N, 119°25¢ E) in 2011 to 2013. Three Bt
transgenic cotton cultivars, Sikang1, Ccsi41, and Ccsi45,
Published in Agron. J. 109:168–174 (2017) widely grown in China, and their parents were used in this
doi:10.2134/agronj2016.04.0209
Received 12 Apr. 2016 E. Abidallha, L.I.M. Tambel, L. Heng, X. Zhang, and D.H. Chen, Jiangsu
Accepted 22 Sept. 2016 Provincial Key Lab. of Crops Genetics and Physiology, Yangzhou Univ.,
Available freely online through the author-supported open access option Yangzhou 225009, P.R. China; E. Abidallha, Dep. of Forest Management,
Faculty of Forestry, Univ. of Khartoum, 13314, Khartoum, Sudan.
Copyright © 2017 American Society of Agronomy
*Corresponding author (zhangxiang@yzu.edu.cn, cdh@yzu.edu.cn).
5585 Guilford Road, Madison, WI 53711 USA
This is an open access article distributed under the CC BY-NC-ND Abbreviations: Bt, Bacillus thuringiensis FP, first fruiting branch
license (http://creativecommons.org/licenses/by-nc-nd/4.0/) position.

168 A g ro n o my J o u r n a l • Vo l u m e 10 9, I s s u e 1 • 2 017
study. Sikang1, Ccsi41, and Ccsi23 were the conventional Bt
cultivars developed by introducing Bt genes into Simian3,
Ccsi23, and Jimian7 (non-transgenic cultivars), respectively.
The use of the three Bt cultivars with their current parents was
designed to compare the characteristics of vegetative growth,
reproductive growth, and N distribution between the Bt cultivar
and its parent during cotton growing seasons in 2011 and 2012.
The plot dimensions were 5.4 by 8 m, containing six rows spaced
at 0.90 by 0.30 m. Cultivar was used as the experimental factor.
The experiments were arranged according to a randomized
complete block design with three replications. Seeds were sown
on 4 April in a warm room and covered with plastic film in
both years. Seedlings were transplanted to the field on 17 May.
The field had a sandy loam soil (Typic Fluvaquents according to
the US taxonomy), which contained 24.5 g/kg organic matter,
and available N, P, and K at 108, 40.5, and 82.0.0 mg/kg,
respectively. Nitrogen (60 kg/ha as urea), P (300 kg/ha as single
superphosphate), and K (120 kg/ha as KCl) were applied and
incorporated before transplanting. Nitrogen (54 kg/ha as urea),
P (300 kg/ha as single superphosphate), and K (120 kg/ha as
KCl) were applied again at the early flowering stage. Nitrogen
was also applied at early boll development (126 kg/ha as urea)
and peak boll (30 kg/ha as urea) stages. Mepaquat chloride
(1,1-dimethyl piperidinium chloride, C7H16ClN) was applied at
peak square (15 g/ha), early flowering (30g/ha), peak flowering
(45g/ha), and peak boll (60g/ha) stages.
Based on the results of 2011 and 2012, the same six cultivars
with two N fertilizer application rate treatments were further
tested on Yangzhou University Farm in 2013. The 2 N fertilizer
treatments consisted of 0 (N deficit) and 450 kg/ha (N rich)
The experiment was arranged in a randomized complete block
design, with three replications for each treatment. The sowing
date was 5 April, and the transplanting date was 18 May. The
seedlings were planted at a spacing of 0.90 by 0.30 m. The plot
dimensions were also 5.4 by 8 m. With the exception of the N
treatments, the same management practices were used in both
years according to local recommendations.
Measurements
In 2011 and 2012, the growth characteristics of vegetative
organs and reproductive organs were investigated every 20 d
from square appearance to boll opening. The indices for the
vegetative organs included number and area of the main stem
leaf and sympodium leaf, main stem nodes, plant height, and
growth rate. Ten leaf samples for each experimental cultivar
from each plot were harvested. Leaf samples were the four
top leaves of the main stem plus 10 sympodial leaf samples
collected from the first nodes of the fruit branches at mid-
plant. All the leaf samples were dried at 80°C to constant
weight. The H2SO4 –H2O2 Kjeldahl digestion method
was used for the measurement of total leaf N concentration
(Richards, 1993). Nitrogen content was expressed as the
product of concentration and dry weight.
Growth indices of the reproductive organs were investigated
every 15 d from peak boll setting to boll opening and included
the position of the first branch, the numbers of fruit branches
and fruit nodes, boll number, and their growth rates. At 10 d
after flowering, 10 bolls were harvested from the first nodes of
the fruit branches at mid-plant. All the boll samples were dried
and used for measurement of N content.
In 2013, the growth characteristics of vegetative organs
and reproductive organs were investigated under different N
treatments for Bt cultivars and their parents.
Statistical Analysis
A Student’s t-test was conducted for each characteristic of
growth and N distribution between Bt cultivars and their
parents, using proc ANOVA in SAS 6. The data are presented
separately by year.
Results
Effect of the Introduction of the Bt Gene
into Cotton on Vegetative Organ Growth
The number of main stem leaves per plant was similar for all
the studied cultivars during the cotton growing seasons of 2011
and 2012 (Table 1)—approximately 25 leaves. No significant
differences in main stem leaf number were detected between
Bt cultivars and their corresponding parents. However, there
were differences in main stem leaf area (Table 1). Cultivars
containing the Bt gene (Sikang1, Ccsi41, and Ccsi45) had
higher leaf area than their corresponding parents (Simian3,
Ccsi23, and Jimian7), and the differences were significant
between Bt cultivars and their parents in both years (2.4 and
3.3% increase between Sikang1 and Simian3, 3.0 and 2.4%
between Ccsi41 and Ccsi23, and 4.1 and 2.6% between Ccsi45
and Jimian7 for 2011 and 2012, respectively).

Table 1. The number and leaf area of main stem leaves and sympodium leaves and plant height for Bt cotton cultivars and their parents in
the 2011 and 2012 cotton growing seasons.
Main stem leaf Sympodium leaf Plant height
Cultivar 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012
––– leaves/plant––– ––– cm2/leaf ––– ––– leaves/plant ––– ––– cm2/leaf ––– ––––– cm –––––
Sikang1 25.9 26.2 4520 4573 103.2 105.5 4216 4167 108.4 112.3
Simian3 25.6 26.0 4415 4426 92.5 95.3 4394 4275 102.3 105.2
  Significance ns† ns 0.014 0.036 0.043 <0.01 <0.01 <0.01 <0.01 <0.01
Ccsi41 25.1 25.4 4640 4710 99.6 102.6 4298 4212 112.3 116.7
Ccsi23 24.7 25.1 4505 4602 90.4 94.7 4452 4398 104.6 107.1
  Significance ns ns <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
Ccsi45 25.4 25.6 4705 4736 95.2 97.4 4315 4265 115.1 120.4
Jimian7 25.2 25.5 4520 4615 87.3 91.9 4521 4472 105.6 109.1
  Significance ns ns <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
† ns, not significant at the 0.05 level.

Agronomy Journal   •  Volume 109, Issue 1  •   2017 169

 The number of sympodium leaves per plant was different for
all the studied cultivars during the cotton growing seasons of
2011 and 2012 (Table 1). The Bt cultivars had more sympodium
leaves than did their corresponding parents. Significant
differences were observed between each Bt cultivar and its
parent: Sikang1 had 10.7 and 10.2 more leaves than Simain3,
Ccsi41 had 9.2 and 7.9 more leaves than Ccsi23, and Ccsi45
had 7.9 and 5.5 more leaves than Jimian7 in 2011 and 2012,
respectively. In contrast to the number of sympodium leaves,
the Bt cultivars had lower sympodium leaf areas than their
corresponding parents (Table 1). Differences in sympodium
leaf areas were also significant between each Bt cultivar and its
parent: the leaf area of Sikang1 had 178 and 108 cm2 less leaf
area than Simain3, Ccsi41 had 154 and 186 cm2 less leaf area
than Ccsi23, and Ccsi45 had 206 and 207 cm2 less leaf area than
Jimian7 in 2011 and 2012, respectively.
Plant height was also different for all the studied cultivars
during the cotton growing seasons of 2011 and 2012 (Table 1).
The differences in plant height were significant between Bt
cultivars and their parents in both years, with 6.0 and 6.7%
increases in Sikang1 compared with Simian3, 7.4 and 9.0%
increases in Ccsi41 compared with Ccsi23, and 9.5 and 10.4%
increases in Ccsi45 compared with Jimian7 in 2011 and 2012,
respectively.
Main stem leaf growth rates showed no significant differences
in leaf number per day between a Bt cultivar and its parent
during the two cotton growing seasons (Table 2). However,
higher increments of the leaf area were measured during the
expansion period of main stem leaves for Bt cultivars compared
with their parents in both years. The growth rates were enhanced
by 8.8 and 9.0 cm2/d for Sikang1 compared with Simian 3, by
18.5 and 22.1 cm2/d for Ccsi41 compared with Ccsi23, and
by 14.5 and 14.8 cm2/d for Ccsi45 compared with Jimian7 in
2011 and 2012, respectively. These differences detected in the
leaf area increment per day between the Bt cultivars and their
corresponding parents were significant (Table 2).
There were also significant differences in the growth rate
and number of sympodium leaves between the Bt cultivars and
their corresponding parents (Table 2). Bt cultivars had higher
sympodium leaf growth rates. Sikang1 produced 0.23 and 0.17
more leaves per day than Simian3, Ccsi41 produced 0.17 and
0.21 more leaves per day than Ccsi23, and Ccsi45 produced 0.1
and 0.11 more leaves per day than Jimian7 in 2011 and 2012,
respectively. In contrast to the results of the growth rate of
sympodium leaf number, the increment of leaf area of the Bt
cultivar was lower than that of the corresponding parent (Table
2). Leaf area increments were reduced by 3.9 and 4.6 cm2/d
for Sikang1 compared with Simian3, by 5.4 and 5.9 cm2/d for
Ccsi41 compared with Ccsi23, and by 6.3 and 7.6 cm2/d for
Ccsi45 compared with Jimian7 in 2011 and 2012, respectively.
There were no significant differences in growth rates of main
stem nodes between a Bt cultivar and its parent in 2011 and 2012
(Table 2). They grew 0.26 to 0.27 nodes per day for both the Bt
cultivars and their parents in 2011 and 0.28 to 0.30 nodes per
day for both the Bt cultivars and their parents in 2012.
Faster growth rates for plant height were observed for Bt
cultivars during the 2-yr field experiment (Table 2). Sikang1
grew 0.11 and 0.16 cm/d higher than Simian3, Ccsi41 grew
0.13 and 0.19 cm/d higher than Ccsi23, and Ccsi45 grew
0.14 and 0.24 cm/d higher than Jimian7 in 2011 and 2012,
respectively.
Effect of Introducing the Bt Gene into Cotton
on Reproductive Organ Development
First fruiting branch position (FP) was different for all the
studied cultivars during the cotton growing seasons of 2011
and 2012 (Table 3). The Bt cultivars had lower FP values than
their corresponding parents. Significant differences were
observed between each Bt cultivar and its parent: Sikang1 had
an FP 0.7 and 0.4 nodes lower than that of Simain3, Ccsi41
had an FP 0.9 and 1.1 nodes lower than that of Ccsi23, and
Ccsi45 had an FP 0.9 and 1.1 nodes lower than that of Jimian7
in 2011 and 2012, respectively.
More fruiting nodes were observed in Bt cultivars compared
with those in the corresponding parents in both years. The
differences in the fruiting nodes were significant between Bt
cultivars and their parents, with 13.1 and 13.6% increases
between Sikang1 and Simian3, 11.3 and 11.8% increases
between Ccsi41 and Ccsi23, and 8.2 and 7.2% increases
between Ccsi45 and Jimian7 in 2011 and 2012, respectively.
Similarly, there were higher fruiting branches, bolls, and
retained boll rates for Bt cultivars, and the differences were all
significant between the Bt cultivars and their corresponding
parents (Table 3).

Table 2. The growth rate of vegetative organs for Bt cotton cultivars and their corresponding parents in the 2011 and 2012 cotton grow-
ing seasons.
Growth rate Growth rate Nodes of main Plant
of main stem leaf of sympodium leaf stem height
Cultivar 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012
––– leaves/d ––– ––– cm2/d ––– ––– leaves/d ––– ––– cm2/d ––– ––– nodes/d ––– ––– cm/d –––
Sikang1 0.273 0.281 256.3 260.1 1.81 1.87 246.4 243.2 0.27 0.3 1.2 1.31
Simian3 0.268 0.28 247.5 251.1 1.58 1.6 250.3 247.8 0.27 0.29 1.09 1.15
  Significance ns† ns 0.024 0.035 0.047 0.023 0.028 0.051 ns ns 0.052 0.026
Ccsi41 0.266 0.275 270.1 278.4 1.71 1.76 250.2 248.2 0.26 0.29 1.25 1.38
Ccsi23 0.258 0.273 251.6 256.3 1.54 1.55 255.6 254.1 0.26 0.28 1.12 1.19
  Significance ns ns 0.011 <0.01 0.022 0.037 0.194 <0.01 ns ns 0.043 0.019
Ccsi45 0.264 0.277 278.6 281.3 1.62 1.64 250.9 249.6 0.27 0.28 1.29 1.45
Jimian7 0.261 0.275 264.1 266.5 1.52 1.53 259.2 256.7 0.27 0.28 1.15 1.21
  Significance ns ns <0.01 <0.01 0.039 0.052 <0.01 <0.01 Ns ns 0.024 <0.01
† ns, not significant at the 0.05 level.

170 Agronomy Journal  •  Volume 109, Issue 1  •  2017

Table 3. Characteristics of reproductive organs in Bt cotton cultivars and their corresponding parents in the 2011 and 2012 cotton
growing seasons.
First fruiting branch Fruiting Fruiting Retained boll
position nodes branches Bolls rate
Cultivar 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012
– no. of main stem nodes – –– nodes/plant –– –– branches/plant –– ––– bolls/plant ––– ––– % –––
Sikang1 6.8 6.5 108.2 112.1 19.6 20.8 29.3 31.6 37.1 38.3
Simian3 7.5 6.9 95.6 98.7 18.3 19.1 25.7 27.1 33.3 34.1
  Significance 0.046 0.043 <0.01 <0.01 0.047 0.032 <0.01 <0.01 <0.01 <0.01
Ccsi41 6.5 6.2 103.6 105.2 18.7 19.3 27.8 29.5 35.6 36.2
Ccsi23 7.4 7.3 93.1 94.1 17.9 18.5 24.2 25.6 32.4 33.5
  Significance 0.032 0.051 <0.01 <0.01 0.072 0.053 0.043 0.021 0.036 0.027
Ccsi45 7.0 6.4 98.7 101.1 17.8 18.9 26.5 28.4 33.1 34.0
Jimian7 7.9 7.5 91.2 94.3 16.4 17 24.1 25.5 30.3 31.2
  Significance 0.024 0.011 <0.01 0.01 0.041 0.025 0.028 0.032 0.049 0.043

There were significant differences in the increase of number of Effect of Nitrogen Regulation on Vegetative and
fruiting nodes and retained boll rates between the Bt cultivars and Reproductive Organ Growth and Development
their corresponding parents (Table 4). Sikang1 had 0.15 and 0.20 Higher total N contents were detected for the Bt cultivars
fruiting nodes per day more than Simian3, Ccsi41 had 0.13 and in the main stem leaves during square and boll development
0.08 more fruiting nodes per day than Ccsi23, and Ccsi45 had periods. The differences were significant between Bt cultivars
0.11 and 0.09 more fruiting nodes per day than Jimian7 in 2011 and their corresponding parents, with 0.25 to 0.39% difference
and 2012, respectively. Correspondingly, Sikang1 had 0.12 and between Sikang1 and Simian3, 0.27 to 0.55% difference between
0.14 more bolls per day than Simian3, Ccsi41 had 0.08 and 0.09 Ccsi41 and Ccsi23, and 0.26 to 0.55% difference between
more bolls per day than Ccsi23, and Ccsi45 had 0.09 and 0.08 Ccsi45 and Jimian7 in 2011 and 2012, respectively. However,
more bolls per day than Jimian7 in 2011 and 2012, respectively. the total N content in sympodium leaves showed a different
pattern than that in main stem leaves, with Bt cultivars having
Table 4. The development rate of reproductive organs in Bt cot- significantly lower N contents (Table 5).
ton cultivars and their corresponding parents in the 2011 and Nitrogen application data showed that vegetative organ growth
2012 cotton growing seasons. was different under N deficit (0 kg/ha) and under N-rich conditions
Retained fruiting Retained (450 kg/ha) between the Bt cultivars and their corresponding
node rate boll rate parents. Under N-rich conditions (Table 6), no significant
Cultivar 2011 2012 2011 2012 differences were detected for main stem leaves and leaf area between
—— nodes/d —— —— bolls/d —— Bt cultivars and their corresponding parents. However, under an
Sikang1 1.18 1.25 0.53 0.57 N deficit, lower numbers of main stem leaves and leaf areas were
Simian3 1.03 1.05 0.41 0.43 detected in Bt cultivars than in their corresponding parents. The leaf
  Significance <0.01 <0.01 <0.01 <0.01 number was reduced by 2.2, 2.6, and 2.3 leaves for Sikang1, Ccsi41,
Ccsi41 1.15 1.17 0.47 0.50 and Ccsi45, respectively, and the leaf area was decreased by 84, 108,
Ccsi23 1.02 1.09 0.39 0.41 and 141 cm2 for Sikang1, Ccsi41, and Ccsi45, respectively.
  Significance 0.022 0.035 0.023 0.044 There were no significant differences in number of sympodium
Ccsi45 1.09 1.11 0.46 0.48 leaves and sympodium leaf area for Bt cultivars under the
Jimian7 0.98 1.02 0.37 0.40 450 kg/ha N treatment (Table 6). However, lower number of
  Significance 0.039 0.043 0.052 0.047 sympodium leaves and leaf area were detected in Bt cultivars than
Table 5. Total N content in main stem and sympodium leaves in Bt cotton cultivars and their corresponding parents during the 2011 and
2012 cotton growing seasons.
Main stem leaf Sympodium leaf
25 June 15 July 5 Aug. 25 June 25 July 25 Aug.
Cultivar 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012 2011 2012
––––––––––––––––––––––––––––––––––––––––––––––––––––––––– m/d –––––––––––––––––––––––––––––––––––––––––––––––––––––––––
Sikang1 3.75 3.82 3.66 3.78 3.43 3.61 3.43 3.51 3.56 3.64 2.73 2.83
Simian3 3.43 3.5 3.41 3.49 3.05 3.22 3.66 3.72 3.74 3.88 2.85 2.97
  Significance 0.049 0.035 0.052 0.048 0.041 0.029 0.047 0.041 0.043 0.041 0.047 0.051
Ccsi41 3.83 3.89 3.72 3.76 3.51 3.65 3.58 3.67 3.68 3.79 2.87 2.91
Ccsi23 3.52 3.61 3.45 3.48 2.98 3.1 3.71 3.85 3.85 3.98 2.98 3.12
  Significance 0.034 0.042 0.413 0.039 <0.01 0.016 0.051 0.052 <0.01 0.023 0.058 0.053
Ccsi45 3.96 4.02 3.84 3.91 3.76 3.84 3.67 3.75 3.75 3.86 2.93 2.98
Jimian7 3.61 3.67 3.58 3.63 3.15 3.29 3.83 3.98 3.89 4.01 3.15 3.27
  Significance 0.019 0.021 0.022 0.024 0.023 0.018 0.064 0.052 0.055 0.013 0.032 0.024

Agronomy Journal   •  Volume 109, Issue 1  •   2017 171

Table 6. Nitrogen treatment effects on vegetative organ growth in Bt cultivars and their corresponding parents in the 2013 cotton grow-
ing season.
Cultivar Main stem leaf Sympodium leaves Nodes of main stem Plant height
leaves/plant cm2/leaf leaves/plant cm2/leaf nodes/plant cm
N fertilizer at 450 kg/ha
Sikang1 26.1 4906 96.7 4252 26.2 139.7
Simian3 26.0 4927 97.1 4226 26.2 137.8
  Significance ns† ns ns 0.053 ns ns
Ccsi41 25.5 5245 90.2 4305 25.5 152.1
Ccsi23 25.6 5268 90.5 4320 25.4 151.6
  Significance ns ns ns ns ns ns
Ccsi45 25.9 5367 85.2 4422 25.5 148.8
Jimian7 25.8 5379 86.1 4489 25.6 155.2
  Significance ns ns ns 0.057 ns ns
No N fertilizer (0 kg/ha)
Sikang1 19.5 3142 71.2 2545 19.5 82.1
Simian3 21.7 3226 75.3 2592 21.8 85.7
  Significance 0.036 0.043 <0.01 0.081 0.039 <0.01
Ccsi41 18.9 3015 66.4 2427 18.3 77.6
Ccsi23 21.5 3123 69.5 2510 21.2 80.3
  Significance 0.027 0.036 0.021 0.043 0.027 0.054
Ccsi45 18.5 2987 62.1 2513 18.6 72.3
Jimian7 20.8 3128 65.3 2587 21.1 75.5
  Significance <0.01 0.024 0.037 0.052 0.032 0.017
† ns, not significant at the 0.05 level.

in the corresponding parents under N deficit: the fruiting branch Nitrogen rates also had different effects on the reproductive
leaf number reduced by 4.1, 3.1, and 3.2 leaves for Sikang1, Ccsi41, organ development of the Bt cultivars and their corresponding
and Ccsi45, respectively, and the leaf area decreased by 47, 83, and parents under N-deficit (0 kg/ha) and -rich (450 kg/ha)
73 cm2 for Sikang1, Ccsi41, and Ccsi45, respectively. conditions. Under the N-rich condition, the FP of Bt cultivars
Main stem nodes of the Bt cultivars were similar to those of the and their corresponding parents were not significantly
corresponding parents under the N-rich treatment (450 kg/ha) different. However, under N deficit, the Bt cultivars had a
(Table 6), but lower main stem nodes were detected in Bt cultivars higher FP than their corresponding parents.
under N deficit. Similarly, plant heights were not significantly The fruiting nodes, fruiting branches, bolls, and retained
different between Bt cultivars and their corresponding parents boll rates of Bt cultivars were not significantly different than
under the N-rich treatment, but contrary results were observed for those of the corresponding parents under the N-rich treatment
Bt cultivars under N deficit. (Table 8). However, different results were measured for these
Larger total N contents were detected in bolls in Bt cultivars reproductive organs when Bt cultivars and their corresponding
during 2011 and 2012. The differences were significant between parents were compared under a N deficit. The fruiting nodes
Bt cultivars and their corresponding parents from peak flowering per plant were reduced by 3.4, 5.8, and 5.1 nodes , the fruiting
to boll opening, with a 0.26 to 0.43% increase between Sikang1 branches were decreased by 0.8, 0.9, and 1.7 layers per plant, the
and Simian3, a 0.36 to 0.51% increase between Ccsi41 and retained bolls were reduced by 1.5, 1.2, and 1.1 bolls per plant,
Ccsi23, and a 0.44 to 0.65% increase between Ccsi45 and and the retained boll rate was decreased by 2.4, 2.1, and 1.9%
Jimian7 in 2011 and 2012, respectively (Table 7). for Sikang1, Ccsi41, and Ccsi45, respectively.
Table 7. Total N in the boll after >10 d past flowering in Bt cotton cultivars and their corresponding parents during the 2011 and 2012 cot-
ton growing seasons.
30 July 15 Aug. 30 Aug.
Cultivar 2011 2012 2011 2012 2011 2012
––––––––––––––––––––––––––––––––––––––––––––––––––– m/d –––––––––––––––––––––––––––––––––––––––––––––––––––
Sikang1 3.56 3.62 3.41 3.5 3.29 3.35
Simian3 3.27 3.3 3.15 3.19 2.89 2.92
  Significance 0.024 0.015 0.015 <0.01 0.037 0.023
Ccsi41 3.63 3.7 3.59 3.62 3.35 3.38
Ccsi23 3.21 3.25 3.08 3.11 2.93 3.02
  Significance 0.016 0.022 <0.01 0.012 0.026 0.036
Ccsi45 3.75 3.79 3.64 3.69 3.42 3.5
Jimian7 3.19 3.25 2.98 3.12 2.98 3.05
  Significance <0.01 <0.01 <0.01 <0.01 0.021 0.027

172 Agronomy Journal  •  Volume 109, Issue 1  •  2017

Table 8. Nitrogen treatment effect on the reproductive organ development of Bt cultivars and their corresponding parents in 2013.
First fruiting branch Fruiting Fruiting Retained
Cultivar position nodes branches Bolls boll rate
no. of main stem node nodes/plant branches/plant bolls/plant %
N fertilizer at 450 kg/ha
Sikang1 7.9 93.5 17.2 23.5 25.2
Simian3 8.2 92.7 17.1 23.1 24.4
  Significance 0.055 ns† ns ns ns
Ccsi41 8.1 90.2 16.8 22.4 23.1
Ccsi23 8.5 88.3 16.5 22.9 23.5
  Significance ns 0.054 ns 0.058 ns
Ccsi45 8.5 85.4 16.6 21.3 21.8
Jimian7 8.6 84.5 15.9 20.2 21.2
  Significance ns ns ns 0.52 ns
No N fertilizer (0 kg/ha)
Sikang1 6.3 70.1 14.3 13.3 23.1
Simian3 5.8 73.5 15.1 14.8 25.5
  Significance 0.047 0.032 0.047 <0.01 0.026
Ccsi41 6.6 65.4 13.6 12.7 22.6
Ccsi23 5.9 71.2 14.5 13.9 24.7
  Significance 0.049 <0.01 0.031 0.019 0.032
Ccsi45 6.8 62.8 13.5 12.5 21.8
Jimian7 6.0 67.9 14.2 13.6 23.7
  Significance 0.031 0.013 0.039 0.035 0.047
† ns, not significant at the 0.05 level.

Discussion fertilizer application is important for bolstering the lint yield of

Effect of the Introduction of the Bt Gene
on Vegetative Growth and Reproductive
Development in Cotton
Some experiments have shown that the insertion of the Bt
gene affected vegetative growth and reproductive development
(Chen et al., 2002; Zhang et al., 2002), such as increased plant
height and biomass (Fu et al., 2001; Godoy et al., 1998; Zhang
et al., 2006), changed boll size, fiber quality, and lint yield
potential in cotton (Chen et al., 2006; Tian et al., 2003). In
the present experiment, we extended these findings, showing
that the Bt cotton cultivars had larger main stem leaf area and
higher sympodium leaves, but with smaller sympodium leaf
areas in single leaves. Higher plant heights were also observed
in the Bt cultivars in both 2011 and 2012. Our studies also
found that the Bt cotton cultivars had a higher growth rate of
main stem leaf area, sympodium leaf number, and plant height
than their parents and a lower growth rate of sympodium leaf
area. Therefore, growth of the main stem leaf area and plant
height were more robust, but a higher sympodium leaf number
was balanced with a smaller leaf area, which is a benefit for
the development of reproductive organs (Chen and Gu, 2004;
Zhang et al., 2002).
The results of reproductive organ development showed
that the Bt cotton cultivars were higher than their parents in
the number and development rate of fruiting nodes, fruiting
branches, bolls, and retained boll rate but lower in FP. These
findings suggest that the introduction of the Bt gene results
in more reproductive organs, which results in an increase in
fruiting nodes and retained boll number. More reproductive
organs means that a Bt cultivar has a higher lint yield potential.
However, realization of that higher lint yield needs more inputs
of fertilizers, especially N and K. Therefore, an increase in
Bt cotton (Biradar et al., 2010; Sankaranarayanan et al., 2011).
Effect of Nitrogen Application on Vegetative
and Reproductive Growth of Bt Cotton
Bt cotton has a higher total N content in leaves and bolls than its
parent (Chen et al., 2005; Dong et al., 2000). High N in Bt cotton
results in robust vegetative growth and low lint (Main et al., 2010,
2011); high N also causes strong N metabolism physiology in Bt
cotton (Chen et al., 2012). In this study, we found that Bt cultivars
had higher total N contents in main stem leaves but low total N
contents in sympodium leaves. The correlation analysis showed
there were significant positive correlations between total N content
on 25 June in main stem leaves and main stem leaf area, plant height
(correlation coefficients were 0.901 and 0.967, respectively [both
P < 0.01]). However, on the same date, negative correlations were
found between total N content in sympodium leaves and fruiting
nodes, fruiting branches, and bolls (correlation coefficients were
–0.794 [P < 0.05], –0.730 [P < 0.05], and –0.678, respectively).
The results suggest that the increase in total N content in main
stem leaves during the square period causes more vegetative growth;
the decrease in total N content in sympodium leaves during the
square period benefits the development of reproductive organs,
which may be the cause of higher numbers of reproductive organs
in Bt cotton (Xue et al., 2007).
Our studies showed further that the N-rich and N-deficit
treatments affected the vegetative and reproductive growth of Bt
cotton. There were no significant differences in vegetative organ
growth between Bt cultivars and their corresponding parents
under the N-rich condition; however, significantly reduced
vegetative organs were found under N deficit compared with their
corresponding parents. Similarly, the reproductive organs of the
Bt cultivars were not different than those of the corresponding

Agronomy Journal   •  Volume 109, Issue 1  •   2017 173

parents under the N-rich treatment, but fewer reproductive
organs were found under N deficit. Surplus N resulted in excess
vegetative growth, which caused severe abscission of squares and
bolls and reduced reproductive development (Zhang et al., 2012).
Nitrogen deficit caused a larger reduction in both vegetative and
reproductive organs, which may be related to Bt protein synthesis
in Bt cotton. Therefore, the N deficit affected both lint and Bt
protein production in the Bt cotton cultivars (Bruns and Abel,
2003; Wang et al., 2012); application of enough N is important
to supply the N nourishment for both reproductive organ
development and the insecticidal protein in Bt cotton.
Conclusion
In this study, we found that the introduction of the Bt
gene resulted in greater main stem leaf area, plant height, and
sympodium leaf number but low sympodium leaf area, higher
fruiting nodes, fruiting branches, bolls, and retained boll rate,
and lower FP. Nitrogen application affected both vegetative and
reproductive growth of the Bt cultivars. There were no differences
in vegetative and reproductive organs under the N-rich condition;
however, there was reduced vegetative and reproductive organ
development under N deficit. These findings highlight the
importance of optimum N application to maximize production in
Bt cultivars.
Acknowledgments
We are grateful for the projects no. 31301263, no. 31471435, and no.
31671613 supported by the National Natural Science Foundation of
China, Project 20113250110001 supported by the Doctoral Advisor
Foundation of Education Department of China, a project funded
by the Priority Academic Program Development of Jiangsu Higher
Education Institutions, China(PAPD), and Project SXGC(2014)317
supported by Three New Technology Foundation of Agriculture in
Jiangsu Province (China). We also thank Dr. Lianne Dwyer for her
writing assistance in this study.
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