Euphytica 123: 343–351, 2002.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

343

Characterization of resistance to Helicoverpa armigera in three lines of

transgenic Bt Upland cotton

Jing Sun1,∗ , Canming Tang1,∗ , Xiefei Zhu1 , Wangzhen Guo1 , Tianzhen Zhang1,∗∗ , Weijun
Zhou2 , Fengxia Meng2 & Jingliang Sheng2
1 Key Laboratory for Crop Genetics & Germplasm Enhancement, Ministry of Education, and Department of Genet-

ics and Crop Breeding, Nanjing Agricultural University, Nanjing 210095, China; 2 Department of Plant Protection,
Nanjing Agricultural University, Nanjing 210095, China; ∗ Sun Jing and Tang CM contributed equally to this work;
∗∗ Author for correspondence: E-mail: cotton@njau.edu.cn

Received 21 February 2000; accepted 1 April 2001

Key words: expression, genetic stability, Helicorverpa armigera, managing resistance, resistance, tolerance to
transgenic Bt cotton, transgenic Bt cotton

Summary
In the present study, different Bt cotton lines, Shanxi94-24, Zhongxin94 and R19, showed good efficacy against
populations of Helicoverpa armigera, in both laboratory and field assays. Laboratory bioassays, however, indicated
that there was a declining level of efficacy with plant age, as the mortality (%) of Helicoverpa decreased gradually
when fed leaves sequentially from the first to the last developing node from the main stem. The mortality of neonate
insects fed leaves from the three transgenic lines was as high as 100% before flowering, but only 50%–70% or so
by peak flowering and boll set. The temporal difference in resistance of these transgenic Bt cotton lines was also
demonstrated in a second set of bioassays of transgenie leaves simultaneously collected from the transgenic plants
planted at different times from March 1 to September 11, 1999 at intervals of twenty days. No difference in efficacy
was observed over 3 or 4 successive generations of three transgenic lines, i.e., resistance was stable in different
generations. This confirms the stable transmission and expression of the Bt gene after being integrated into the
genome of the cotton plant. The relationship between the temporal difference in efficacy of transgenic cotton lines
and the possible risk of resistance development in bollworm larvae to Bt cotton are also discussed.

Introduction (Peferoen, 1997). The transgenic Bt cotton cultivars

It was a tremendous breakthrough in biotechnology
when exogenous genes were transferred into the gen-
ome of plants and shown to be stably inherited and
expressed. Cotton is an important and worldwide fiber
crop that is mainly damaged by larvae of the insect
Helicoverpa armigera Hübner (cotton bollworm). The
development of cultivars resistant to the insect is of
paramount importance to cotton production through-
out the world. Since 1987, the full or truncated con-
struct of the CryIA(b) δ-endotoxin gene from Bacillus
thuringienesis (Bt) has been transferred to many crops
(Perlak et al., 1990; Benedict et al., 1992; Fearing et
al., 1997; Williams et al., 1997). Transgenic Bt cot-
ton, corn and potato are extensively grown in the USA
developed by the Monsanto Company, and Deltapine,
USA, have also been grown in China since 1996.
In China, although transformed Bt cotton plants
were developed quite early (Xie et al., 1991),
the expression levels were extremely low and the
plants showed no significant insecticidal activity. Guo
(1993) at the Biotechnology Research Institute, China
Academy of Agricultural Science (CAAS) isolated,
synthesized and fused a crystal protein gene, GFM
CryIA (CryIA(b)+CryIA(c)). A construct with the
cauliflower mosaic virus (CaMV) 35S promoter was
made and transferred into several commercial cultivars
such as Simian 3 and Jingmian 7 through pollen tube
and Agrobacterium-mediated transformation methods
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in collaboration with the Industrial Crop Research
Institute, Jiangsu Academy of Agricultural Sciences
(JAAS), and the Cotton Research Institute, Shanxi
Academy of Agricultural Sciences (SAAS). Through
traditional pedigree selection, the commercial trans-
genic Bt cultivars such as Guokang 1, 12 and Jinmian
26 resistant to bollworm have been bred and are be-
ing grown in areas of the Yangtze and Yellow River
Valleys. Additionally, a transgenic Bt commercial hy-
brid cotton, Zhongmiansuo 29, and cultivars such as
Zhongmiansuo 30, 31, and 32 were developed at the
Cotton Research Institute, CAAS from this same ma-
terial. It can be expected that transgenic Bt varieties
resistant to bollworm will be grown quite extensively
in China in the future.
Currently, Bt cotton is licensed directly to growers
in the USA, Australia and China and, has a potential
application on over two million hectares in the USA
alone for the control of Heleothis virescens (Peferoen,
1997). However, the first large-scale production in the
USA has tempered the euphoria around Bt cotton. It
appeared that an exceptionally high level of bollworms
(Helicoverpa zea) caused severe feeding damage and
some insecticide spraying was required to supplement
the control provided by the Bt gene in 1996 (Fox,
1996; Kaiser, 1996). Sutton (1998) even reported an
average of $25 per acre net income loss attributable to
the Bollgard varieties in Arkansas, USA. The field tri-
als of transformed cottons generated by the Monsanto
Company showed good efficacy of the plants against
field populations of Helicoverpa armigera and H.
punctigera in Australia, but there were indications of
a declining level of Bt expression once plants began to
set fruit (Fitt et al., 1994). These results prompt us to
investigate the production of transgenic Bt cotton and
the ‘high-dose/ refuge’ strategy being adopted in USA
and Australia to reduce the potential for bollworm res-
istance. In this report, three transgenic cotton lines,
Zhongxin 94, R19 and Shanxi 94-24, developed in
China, were grown in the field and monitored for their
resistance to H. armigera using a laboratory bioassay.
Larval mortality and leaf feeding damage by bollworm
were evaluated throughout the growing season. The
genetic stability of the CryIA expression over three or
four successive selfing generations was also examined.
The temporal differences in the resistance of Bt cotton
to bollworm at various growing stages presented here
will provide useful information in developing future
insect management programs in China for transgenic
cottons containing the Bt δ-endotoxin.
Materials and methods
Materials
Three transgenic Bt cotton lines were used in the
present study. Shanxi 94-24 was developed directly
by transferring a synthetic cryIA gene (Guo, 1993)
into Jinmian 7 through Agrobacterium-mediated trans-
formation (Jiao et al., 1997). Seeds of this line were
supplied to us by Prof Chen Z.X. of the Cotton Re-
search Institute, SAAS. Zhongxin 94 was provided by
the Biotechnology Research Center, CAAS in 1995.
It was developed using the same gene construct as
Shanxi 94-24 and was produced through the pollen
tube transformation method in the Simian 3 cultivar
(Ni et al., 1996). The cryIA gene transformed into
both Shanxi 94-24 and Zhongxin 94 included a fusion
between the cryIA(b) and cryIA(c) gene were resyn-
thesized chemically with optimized codon usage for
plants. This hybrid gene was placed under the con-
trol of the CaMV 35S promoter and NOS terminater
(Guo, 1993; Ni et al.,1996). The line R19 was obtained
from the Cotton Research Institute, CAAS. It was de-
veloped by transferring a synthetic truncated cryIA(b)
gene into Coker 312. This Bt gene was also driven
by the CaMV 35S promoter and NOS terminater (Xia
et al., 1995; and private communication, Cotton Re-
search Institute, CAAS, Anyang, Henan Province,
China 455112). These three transgenic Bt cotton lines
were bred true for agronomic traits and resistance to
bollworm by pedigree selection and individual plant
bollworm feeding bioassays. The transformed Shanxi
94-24 and Zhongxin 94 lines were also confirmed as
homozygous by PCR amplification of a specific 800
bp DNA fragment of the synthetic Bt gene (results
not shown). Genetic analysis indicated that they all
contained single insertions (Tang et al., 2000).
Methods
Bioassays were conducted using a laboratory culture
(SuS2) of H. armigera sensitive to CryIA Bt tox-
ins. The young seedling plants of transgenic Bt lines
and non-transgenic cultivars, such as Sumian 12 were
picked at the same seedling ages. After the hypocotyl
of cotton seedlings was clipped off, leaves, stems, or
leafstalks were put into a jelly jar or plastic cup sealed
with porous plastic wrap. Individual leaves were also
used for laboratory assay. The containers were held at
a constant temperature 27 ± 1 ◦ C with relative humid-
ity 60–85%, and light for 14 h. Five neonate larvae per
leaf were generally added to each container. After 5

days in culture, the number of surviving larvae, larval
age, and the leaf damage index were recorded. The
leaf damage index was divided into four grades: 1: leaf
damaged area below 10%, with only small needle-like
damaged parts being observed; 2: damaged area about
11–50%, damaged parts distributed in small patches;
3: damaged area 51–90%, but the mesophyll tissues
still present in connected patches; and 4: damaged area
over 90%, mesophyll tissues not in connected patches
(Tang et al., 1997).
In our first experiment, resistance to bollworm was
temporally measured for each expanded leaf taken
from a defined site on the main stem of three trans-
genic Bt cotton lines during the entire cotton-growing
season in 1997. Plants of the three transgenic Bt
lines were grown at Jiangpu Cotton Breeding Station,
Nanjing Agricultural University (NAU) and the leaves
from the 2nd to 6th were sampled for bioassay. Forty-
five seedlings of each line with 3 or 4 expanded leaves
were divided into 3 groups, each was transplanted
into one block. Each plant was individually tagged
and numbered for identification throughout the season.
Each leaf from the main stem was taken from June 20
to July 19 (leaves of three blocks were picked by turns)
for bioassay. The last 2nd and 3rd node young leaves
on the fruiting branches were also taken to test their
resistance level on July 22 and August 5 before the top
bud had been cut from the cotton plant.
In order to reduce the environmental effects due
to different time of harvesting of leaves, the second
experiment was done in 1999. We planted seeds of
transgenic Bt cotton line in our genetic nursery field
in Nanjing from March 1 to Sept 11, at intervals of 20
days. All leaf samples were collected simultaneously
for laboratory assay on Sept 20 and for ELISA meas-
urement of Bt toxin level on Sept 23. Assessment of Bt
toxin level in transgenic plants was done using ELISA
according to the procedures described (Wang et al.,
1998).
To assess the stability of CryIA expression over
successive generations, some seeds of the three trans-
genic Bt lines were stored under refrigeration after
they were harvested in both Nanjing and the Hainan
province in winter. They were grown at Jiangpu
Breeding Station, three generations of R19 in 1997,
and 3 or 4 generations of all three transgenic lines and
non-transgenic Sumian 12 in 1998. More than twenty
leaves were harvested from the transgenic plants of
each generation for laboratory assay. The first fully
expanded leaf on each plant was collected for bioas-
says for our two experiments. The field was fertilized
345
and irrigated as for commercial cotton. However, no
pesticides were applied for bollworm or other pest
throughout the season. Cotton seed is usually grown at
the beginning of April, transplanted to the field at the
middle of May, and the plant has its peak flowering at
the end of July in Yangtze river valley.
In our laboratory assay, the sensitive commercial
cultivar, Sumian 12, was always added as a control.
We did preliminary screening using non-transgenic
cultivars, Simian 3, Jinmian 7, Coker 312 and Sumian
12 from 1995 to 1996 using our laboratory bioassay.
We could not find any difference in the efficacy among
these cultivars (there are no observable morphological
differences). In assays where the larval mortality on
the control cultivars was greater than 30% (mainly due
to an infection by an insect virus), the results of that
batch of assays were discarded.
Results
Temporal fluctuation of resistance of transgenic Bt
resistant lines
Resistance of transgenic Bt cotton lines and non-
transgenic commercial cultivars, Simian 3, Sumian
12, and Jinmian 7, planted at the Jiangpu Cotton
Breeding Station, NAU, at seedling stage was assayed
using whole young plants and individual young leaves
picked from the main stem at the seedling stage. The
larval mortality fed with the commercial cultivars was
normally below 20% from more than 100 tests con-
ducted over four years (data not shown). Bioassays
indicated that the bollworm larval mortality after 5
days in culture was 100% when fed with young cotton
plants and with the individual 5th and 6th leaves from
the main stem from the three Bt cotton lines at seedling
stage. This level of efficacy remained high under dif-
ferent growing conditions in both field and greenhouse
experiments (data not shown). It was concluded that
all three transgenic Bt cotton lines were highly resist-
ant to bollworm at the seedling stage. However, during
our genetic studies and pedigree-selections of hem-
izygous transgenic lines, we found that there was a
significant temporal difference in resistance depending
on when the plants were bioassayed. The morality de-
creased from 100% at seedling stage to approximately
50–60% after flowering in the trials conducted in 1995
and 1996. Therefore, we were interested to know if a
similar temporal difference existed in resistance of the
homozygous transgenic Bt cotton lines used as parents
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in these crosses. The first experiment was conducted
in 1997, in which the efficacy against bollworm was
measured over plant growth for each newly developed
leaf taken from defined sites on the main stem of the
three transgenic Bt lines and non-transgenic commer-
cial cultivars during the entire cotton-growing season
in 1997.
The larval mortality when fed with leaves from the
main stem of the control cultivar (Sumian 12) during
the whole growing season is given in Table 1. The
survival of bollworms in the bioassays was very high.
It was clear that there was no resistance to bollworm
for this commercial cultivar. Among the surviving lar-
vae, the third instar larvae was the most abundant,
while second and fourth instar larvae were much less
abundant.
Compared with the commercial cultivar, the trans-
genic Bt cotton had significant resistance against boll-
worm and confirmed previous reports (Tang et al.,
1997). Additionally, we observed from mortality and
instar survival data that there exists a temporal dif-
ference in the efficacy of these transgenic Bt cotton
lines. The mortality of bollworms fed with R19 newly
expanded leaves from the main stem over the entire
growing season is presented in Table 2. Effcacy of
R19 could be divided into two stages. During the ve-
getative stage, the field tolerance (observed against
bollworm on the 2nd–10th leaves) was at its best, as
the mortality of the larvae after five days was as high
as 99.2% (the average mortality caused by 2nd–8th
leaves was 100%). The leaf damage index was on
average 1.1 over this time. Among the few surviving
larvae, 68.2% were 2nd instar. At the same stage, the
mortality of larvae fed with leaves from the control
cotton variety Sumian 12 was only 8.9%, and most
of the surviving larvae were 3rd instar (71.1%). The
average leaf damage index reached 3–4 on the control
variety during this same period (Table 1). During the
second stage, the efficacy of the 11th–15th leaves of
R19 transgenic Bt line decreased significantly. During
this time, mortality of larvae fed with the leaves de-
creased to on average 52.4 ± 11.6%. Surviving larvae
were predominantly 2nd instar (78.4 ± 11.4%), and
the leaf damage index increased to on average 1.7 ±
0.3 for the transgenic plants. At this time, the larval
mortality on the control variety Sumian 12 was only
10.3 ± 7.3%, and the surviving larvae were predom-
inantly 3rd instar (94.88 ± 5.59%). The average leaf
damage index of Sumian 12 was very high, 3.6 ± 0.4
(Table 1). From these results, we can see clearly that,
the developmental age of the transgenic cotton plants
had a significant impact on the efficacy of the intro-
duced insecticidal transgene. By the time the plants
had flowered larval survival had increased signific-
antly. The proportion of 1st instar larvae among the
surviving larvae had decreased gradually, while that
of the 2nd and 3rd instar larvae had increased, and
the leaf damage index had increased. This indicates
that the overall efficacy of the transgenic Bt leaves
to bollworm showed a tendency for gradual decrease
following the growth and development of the plants
into the reproductive phase.
The temporal difference in resistance to bollworm
neonate larvae fed with cotton leaves from the main
stem of two other independently generated lines,
Shanxi 94-24 and Zhongxin 94, is summarized in
Tables 3 and 4. Their pattern of resistance to boll-
worm is almost the same as that of R19. The mortality
of the larvae fed with 3rd-13th leaves from the main
stem of Shanxi 94-24, was 99.3–100%; that fed with
14th–15th leaves was 77.5%, while that fed with 16th–
17th leaves was only 35.1% (Table 3). Two individual
sub-lines (8081 and 8082) pedigree-selected from the
cotton line Zhongxin 94 were monitored in 1997. The
expression pattern of resistance is the same between
the two lines and R19 and Shanxi 94-24. Additionally,
among the surviving larvae, the proportion of 1st instar
larvae had decreased gradually, while that of the 2nd
and 3rd instar larvae had increased gradually (Table
4).
The resistance of the leaves on the intervarietal
F1 hybrids produced between any of our three trans-
genic Bt lines and commercial non-transgenic cul-
tivars showed the same temporal diference of efficacy
in hybrid plants (hemizygous) as that of the parents
(homozygous). The mortality of neonate larvae fed
with the 5th leaf on the main stem was 98.2–100%,
while that with the 10th leaf was 64–80% (data not
shown).
The mortality, leaf damage index and Bt toxin con-
tent in our second experiment carried out in 1999 are
presented in Figures 1 and 2. These confirmed our res-
ults from the first experiment, i.e., that there did exist a
temporal difference in efficacy of transgenic Bt cotton
lines and that this correlated with a decline in the ex-
pression of Bt protein. As presented before, the three
transgenic Bt lines had high resistance to bollworm in
the seedling stage (seeds planted later). However ef-
ficacy declined sharply in leaves of plants planted on
August 1 or earlier. Not surprisingly, among the three
lines, Zhongxin 94 declined in efficacy earlier and to a
greater extent with the mortality of bollworm decreas-
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Table 1. Survival of bollworm fed with leaves of a commercial non-transgenic cultivar, Sumian 12

Position No. assayed Mortality ± SE % Surviving instars % Leaf damage

of leaves plants 1st 2nd 3rd 4th index ± SE

2 5 12.0 ± 17.9 0 63.2 36.8 0 3.6 ± 0.5

3 5 8.0 ± 11.0 13.0 39.0 47.9 0 2.8 ± 0.4
4 5 8.0 ± 11.0 0 17.4 82.6 0 3.3 ± 0.5
5 5 16.0 ± 16.7 4.8 0 95.2 0 3.2 ± 0.8
6 5 24.0 ± 26.1 4.5 54.5 50.0 0 3.0 ± 0
7 5 0 ±0 0 6.7 93.3 0 3.6 ± 0.5
8 5 4.0 ± 8.9 0 0 95.8 4.2 4.0 ± 0
9 5 4.0 ± 8.9 0 14.8 70.4 14.8 4.0 ± 0
10 5 4.0 ± 8.9 0 16.7 83.3 0 3.2 ± 0.4
11 5 12.0 ± 17.9 0 13.0 87.7 0 3.0 ± 1.0
12 5 0 ±0 0 3.8 96.2 0 4.0 ± 0
13 5 12.0 ± 11.0 0 0 100.0 0 3.8 ± 0.4
14 5 7.3 ± 10.1 0 0 100.0 0 3.8 ± 0.4
15 5 20.0 ± 20.0 0 9.5 90.5 0 3.2 ± 0.4

Table 2. Temporal difference in the resistance of R19 leaves to Bollworm

Position No. assayed Mortality ± SE % Surviving instars % Leaf damage

of leaves plants 1st 2nd 3rd index ± SE

2 17 100 0 0 0 1.0 ± 0.0

3 35 100 0 0 0 1.1 ± 0.3
4 6 100 0 0 0 1.0 ± 0.0
5 17 100 0 0 0 1.0 ± 0.0
6 34 100 0 0 0 1.0 ± 0.3
7 7 100 0 0 0 1.1 ± 0.4
8 17 100 0 0 0 1.1 ± 0.2
9 35 99.4 ± 3.4 100 0 0 1.1 ± 0.2
10 59 93.6 ± 15.9 31.6 68.4 0 1.1 ± 0.3
11 16 67.5 ± 31.7 26.9 65.4 7.7 1.4 ± 0.6
12 36 45.4 ± 19.6 12.1 80.8 7.1 1.9 ± 0.5
13 8 37.5 ± 22.5 0.0 96.0 4.0 2.0 ± 0.5
14 16 52.5 ± 34.9 23.1 76.9 0.0 1.7 ± 0.9
15 60 59.0 ± 27.2 27.4 72.6 0.0 1.5 ± 0.5

Table 3. Temporal difference of resistance of the transgenic Shanxi 94-24 line to bollworm

Position No. Mortality Surviving larvae % Leaf Assay date

of leaves assayed % 1st instar 2nd instar 3rd instar damage
leaves index

3rd–13th 155 99.3 99.3 6.7 0 1.0 June 20–July 4

14th–15th 17 77.5 55.3 44.7 0 1.1 July 4–8
16th–17th 16 35.1 6.9 93.1 0 2.1 July 8–16
FB∗ leaf 66 44.3 1.1 98.7 0.2 2.0 July 22
Top leaf 22 38.8 16.9 64.6 18.5 2.1 July 29

∗ FB = fruiting branch.
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Table 4. Temporal difference of resistance of Zhongxin 94 line to bollworm

Lines Position No. Mortality Surviving larvae % Leaf Assay date

of leaves assayed % 1st instar 2nd instar 3rd instar damage
leaves index

8081 3rd–8th 73 96.2 16.7 83.3 0 1.1 June 20–July 4

9th–12th 68 80.3 49.7 43.9 6.4 1.2 June 28–July 4
13th–15th 20 44.8 10.2 83.2 6.6 2.3 July 4–8
16th–17th 14 11.4 6.7 93.3 0 2.6 July 19
8082 3rd–8th 76 98.0 70.0 30.0 0 1.0 June 20–July 4
9th–12th 69 76.8 39.1 57.6 3.3 1.2 June 28–July 4
13th–15th 24 54.2 1.6 94.9 3.5 1.8 July 4–8
16th–17th 15 18.8 6.4 92.4 1.2 2.2 July 19

Figure 1. Mortality of bollworm and leaf damage index of three transgenic Bt lines (a. Zhongxin 94; b. Shanxi 94-24; c. R19) and
non-transgenic cotton (d. Sumian 12) sown at different dates. Standard errors was indicated as the bar.

ing to 50–60%. Zhongxin 94 had considerably less Bt
protein than the other two transgenic cottons, even at
the seedling stage (Figure 2). Shanxi 94-24, on the
other hand, had a good level efficacy throughout de-
velopment although there was still a small decrease in
efficacy during later stages. The leaf damaged index,
also followed the temporal differences in larval mor-
tality. There was a good correlation between mortality
of bollworm and the leaf damage index. The higher
the mortality of bollworm, the lower the leaf damage
index. It was clear that as development of the cotton
plant proceeded, the leaf damage index increased (Fig-
ure 1). The measurement of Bt protein levels revealed
that the decrease of resistance to bollworm might res-
ult from the decrease in Bt toxin expression since it
had decreased after the reproductive stage when lar-
val survival had begun to increase (Figure 2). The
Bt protein levels were low and very variable in the

Figure 2. Mortality and Bt toxin content of three transgenic Bt lines sown at different dates. a. Zhongxin 94; b. Shanxi 94-24; c. R19. Standard
errors was indicated as the bar.
young seedling stage (seeds planted later on Sept 11,
and August 21), however, the mortality of bollworm
was highest. We think that this may be due to high
secondary metabolites in cotyledon (planted on Sept
11) and the first and second expanded leaves, and
this affects the accuracy of the measurement of the Bt
content. Although Zhongxin 94 and Shanxi 94-24 are
transformed with the same vector, there was a 30–40
fold difference in the content of Bt protein between
these two transgenic lines. Position effects may be
the reason since they were inserted at different sites
in the cotton genome (Tang et al., 2000). Due to the
difference in the growing stage, it was very difficult
to harvest leaves of consistent size for the bioassay,
although best efforts were made. That may be why
our standard error of the mortality of bollworm in our
bioassay was relatively high.
Compared with the first experiment, the mortal-
ity in the second experiment decreased relatively little
for three transgenic Bt lines (Table 2 and Figure 1).
A temperature effect on efficacy of the transgenic Bt
lines was likely since the first experiment was carried
out during summer when it is usually over 30 ◦ C, and
sometimes 37–39 ◦ C, in July and August in Nanjing.
349
In contrast the second experiment was conducted in
late September.
Genetic stability of different generations resistant to
bollworm
The seeds from three consecutive generations of cot-
ton line R19 after the selection of homozygotes were
planted simultaneously in 1997. At the end of July at
peak flowering stage the tender leaves on the top of
the main stems of the plants were plucked and used
in a bioassay for testing their resistance to bollworm
(Figure 3). After the neonate larvae were fed with
the leaves on the plants for five days respectively, the
mortality of larvae was 51.5%, 48.0%, and 57.3%.
The surviving larvae were mainly 1st and 2nd instar.
On the other hand, the mortality of larvae fed with
tender leaves of the control cultivar Sumian 12 was
only 11.7%, and all the surviving larvae reached 3rd
instar. Therefore the three consecutive generations of
cotton line R19 all showed significant resistance to
bollworm, and the resistance had been maintained at
a uniform level. In 1998, the seeds of Shanxi 94-24,
Zhongxin 94, and R19 for 3 or 4 consecutive gen-
350
Figure 3. Resistance of shoot tips of three transgenic Bt lines bioassayed over 3–4 generations of selfing.
erations were planted in May 5 1998 at the Jiangpu
Cotton Breeding Station. At the beginning of Septem-
ber the resistance to bollworm of the tender leaves on
the main stems of the plants was tested in our labor-
atory bioassay method as described above. The results
indicated that larval mortality, age of surviving larvae,
and leaf damage index of all three transgenic lines re-
mained relatively constant from one generation to the
next (Figure 3). The average mortality of larvae caused
by the leaves on the plants of varieties Shanxi 94-24,
Zhongxin 94, and R19 were 72.2 ± 4.3%, 61.3 ± 5%,
and 68.2 ± 6.7% respectively. The first instar larvae
among the survival larvae amounted to 9.5 ± 8.9%,
7.7 ± 6.0%, 13.2 ± 16.7%; and 2nd instar larvae
90.5 ± 8.9%, 92.2 ± 6.0%, and 86.8 ± 16.75% re-
spectively. These results were in agreement with the
results acquired in 1997. It indicated that Bt gene in-
tegrated into the genome of Upland cotton is stable
and can be reliably transferred to the progeny through
a conventional breeding program. The expression of
the resistance to bollworm by the cotton plants was
not influenced by successive passage through meiosis.
Discussion
In this paper, we show that, as compared with a com-
mercial non-transgenic cultivar (Sumian 12), three
different transgenic Bt cotton lines, Shanxi 94-24,
Zhongxin 94 and R19, which were developed in
China, showed high resistance to cotton bollworm.
However, the efficacy decreased as plants developed.
The temporal differences in expression were observed
for all three transgenic Bt lines, although the extent of
decline differed from event to event. This result is in
agreement with that reported by Fitt et al. (1994) for
the Bt cotton being used in Australia. We expect that
this differential expression was caused primarily by
both the cotton’s developmental programme and the
activity of the transgene’s promoter, although envir-
onmental factors such as temperature may also have
adverse effects. This could explain why both Bt toxin
and the leaf resistance to bollworm decreased as the
plant grows. Mortality of bollworm fed with trans-
genic cotton flower buds did not decrease as much
as that fed leaves (data not shown). Hence it is still
necessary, especially under higher insect pressure, to
spray chemical insecticides at the later stage of grow-
ing transgenic Bt cotton cultivars. This may be also the
reason that transgenic Bt cotton did not perform up to
expectations in USA and Australia in 1996 (Kaiser,
1996; Jeffery, 1996; Forrester & Pyke,1997), although
the increase in the requirement for spraying may be the
result of unusually hot weather and the fact that more
Southern farmers planted corn, a breeding ground for
(H. zea) bollworms (Kaiser, 1996). In the cotton grow-
ing area of the Yangtze River Valley, China, there
are generally 4, sometimes 5 generations of bollworm
reproduction in one year. So from our research, we
still estimate that at the first and second generations
of bollworm development, while cotton is just at the
seedling to initial flowering stage, the resistance of the
leaves from transgenic Bt lines will attain 100% larval
mortality. It should not be necessary to spray chemical
insecticides for controlling larvae on the cotton at this
stage. As soon as the 3rd generation of bollworm de-
velops after the peak stage of blooming, the resistance
of the young cotton leaves to the bollworm decreases
significantly and the surviving larvae will cause the
cotton buds and bolls to be damaged. It would be ne-
cessary to control the bollworms with supplementary
applications of chemical insecticides.
Several major pest species, including Heliothis
virescens, Leptinotarsa decemlineata, Plodia in-
terpunctella, and Plutella xylostella, have already

demonstrated the ability to adapt to Bt in the labor-
atory. Plutella xylostella has evolved widespread res-
istance in the field. A Bt-resistant laboratory line
(YHD2) of Heliothis virescens, was selected on ar-
tifical diet containing the Bt toxin, CryIA(c) (Gould
et al., 1995). Thus, one might expect insect resist-
ance to be a significant problem as Bt usage increases
(McGaughey & Whalon, 1992). Our results reported
here indicated that there was a declining level of effic-
acy with plant age as the mortality (%) of Helicoverpa
decreased gradually, when fed with leaves from the
main stem, sequentially from the first to the last de-
veloping node. The mortality of neonate insects fed
leaves from the three transgenic lines was as high
as 100% before flowering, but only 50–70% or so
by peak flowering and boll set. The temporal differ-
ence in resistance of these transgenic Bt cotton lines
was also demonstrated in a second set of bioassays
of transgenic leaves simultaneously collected from
the transgenic plants planted at different times from
March 1 to September, 11, 1999, at intervals of twenty
days. So, it may be that insect resistance may develop
more easily on these plants since some bollworms can
survive on transgenic Bt cotton during the later stages
of growth of the cotton. We should pay a great deal
of attention to the management of resistance when
transgenic Bt cotton is extensively grown in the field.
The efficiency of the ‘high-dose/refuge’ strategy in
delaying or preventing the production of bollworm res-
istance to Bt cotton which shows a temporal variation
in efficacy remains to be confirmed and highlights the
need for the development of plants with more than one
insecticidal activity.
Acknowledgements
We are grateful to Prof J.J. Pan in Nanjing Agricultural
University, and Dr R.J. Kohel in USDA ARS, Crop
Germplasm Research Unit, College Station, USA,
for valuable discussions on the topics discussed here
and editing. This research is supported in part by the
National 863 High-tech Program and Trans-century
Training Program Foundation for the Talents by the
State Education Commission, China, to Dr Zhang TZ.
351
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