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Agricultural Sciences in China

November 2011

2011, 10(11): 1693-1700

Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt


cry1Ba3 Gene for Control of Diamondback Moth
YI Deng-xia, CUI Lei, LIU Yu-mei, ZHUANG Mu, ZHANG Yang-yong, FANG Zhi-yuan and YANG Li-mei
Key Laboratory of Horticultural Crops Genetic Improvement, Ministry of Agriculture/Institute of Vegetables and Flowers, Chinese Academy
of Agricultural Sciences, Beijing 100081, P.R.China

Abstract
To obtain transgenic cabbage line with broad insect resistance, a new synthetic Bacillus thuringiensis cry1Ba3 gene was
introduced into white cabbage via Agrobacterium tumefaciens-mediated transformation and 37 transformants were obtained.
Polymerase chain reaction (PCR) and Southern blot analyses confirmed that cry1Ba3 was successfully inserted into the
genome of cabbage. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that cry1Ba3 was expressed.
Western blot results confirmed the production of insecticidal protein encoded by cry1Ba3. Insect bioassays showed that
transgenic cabbages effectively controlled both susceptible and Cry1Ac-resistant diamondback moth (DBM) larvae.
Key words: Bacillus thuringiensis cry1Ba3, cabbage, diamondback moth, resistance

INTRODUCTION
Cabbage (Brassica oleracea L. var. capitata) is one of
the most important vegetables in the world. However,
the cultivation of this vegetable is severely challenged
by infestation of pests, including the major insect,
Plutella xylostella (diamondback moth, DBM), which
results in great loss of the yield and damage to the quality of cabbage production (Shelton et al. 1982; Gould
et al. 1984; Theunissen et al. 1995). The conventional
insect control method heavily relies on the intensive
and extensive use of chemical pesticides. Chemical
insecticides cause severe environmental pollution and
bring about adverse effects on people and beneficial
insects. Moreover, DBM have evolved resistance to
chemical insecticides (Hama 1992; Shelton et al. 1993b).
More recently, genetic engineering has provided a promising method for breeders to obtain insect resistant plants
(Qaim et al. 2003). In 2009, 14 million farmers planted
Received 30 January, 2011

134 million ha of transgenic crops in 25 countries (James


2009). The adoption of transgenic crops has saved
pesticide costs and reduced environmental impact from
pesticides (Qaim et al 2003; Kleter et al. 2007).
Bt transgenic crops express insecticidal crystal (Cry)
proteins from Bacillus thuringiensis (Bt). Various Cry
proteins are highly toxic to lepidopterans, coleopterans
or dipterans, but do not harm people or the environment (Crickmore et al. 1998; Bravo et al. 2007). Although there are many Cry toxins, only a small number
of Cry proteins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C,
etc.) have been used commercially in Bt crops (Bravo
et al. 2008). Agrobacterium tumefaciens-mediated
transformation of cabbage has been reported with some
Cry toxins such as Cry1Ab, Cry1Ac, and Cry1C. Bioassays of Bt cabbage plants showed that DBM was
effectively controlled by these Bt proteins (Metz et al.
1995; Jin et al. 2000; Bhattacharya et al. 2002). Insect
resistance genes are very limited in availability for plant
breeders. Hence, novel Bt genes will be new resource

Accepted 3 June, 2011

Correspondence YANG Li-mei, Tel: +86-10-82108756, Fax: +86-10-62174123, E-mail: yanglm@mail.caas.net.cn

2011, CAAS. All rights reserved. Published by Elsevier Ltd.


doi:10.1016/S1671-2927(11)60167-3

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for the transformation of plants.


A cry1Ba3 gene obtained from the Bt strain UV17
was recently cloned by Wang et al. (2008). Its predicted molecular weight is 140 kD. Bioassays showed
that the Cry1Ba3 protein possessed distinct insecticidal
activities against DBM, and there was no cross resistance with Cry1Ac (Liu et al. 2010). Cry1Ba3 will be
available for use in gene pyramiding to inhibit development of resistant insects. The objective of this study
was to obtain transgenic cabbage expressing cry1Ba3
via A. tumefaciens-mediated transformation. Deployment of the resulting plants may expand the insect resistance gene spectrum for cabbage pests.

MATERIALS AND METHODS


Plant materials
Cabbage inbred line (B. oleracea L. var. capitata) CA213 was used as transformation material.

Diamondback moths
Susceptible and Cry1Ac-resistant DBM were reared on
cabbage plants in an environmental chamber at (271)C,
(352)% RH, and photoperiod of 16 h/8 h (light/dark).
The DBM colonies were started with material collected

YI Deng-xia et al.

from America in 2005 and since then reared continuously in the Laboratory of Entomology, Institute of
Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Newly molted second instar larvae were
used for insect bioassays. At the time of the bioassay,
the Cry1Ac-resistant population was over 1 000-fold
more resistant to Cry1Ac protoxin than the susceptible
one.

Binary vector
The A. tumefaciens strain EHA105 harboring a binary
vector pCSBaN with the synthetic cry1Ba3 and the
neomycin phosphotransferase-II (npt II) genes was used
for cabbage transformation. The cry1Ba3 and npt II
genes were both regulated by the enhanced cauliflower
mosaic virus 35S promoter and the nopaline synthase
terminator. The npt II gene was a selectable marker
gene allowing the transformed shoots to grow in media
containing kanamycin. The cry1Ba3 gene fragment
was introduced into the expression vector
pCAMBIA2300, which carried the npt II gene, and then
transformed into Escherichia coli strain JM110. The
resulting plasmid was introduced into Agrobacterium
tumefaciens strain EHA105 by electroporation
(Mersereau et al. 1990). The T-DNA region of the
binary vector pCSBaN is shown in Fig. 1.

Fig. 1 The binary vector pCSBaN used in cabbage transformation. LB is T-DNA left border, RB is T-DNA right border, eCaMV35 is
enhanced CaMV35S promoter, Tnos is nopaline synthase terminator. The Hind III sites were used for DNA digestion in Southern blot. A
1.222 kb PCR fragment containing part of the cry1Ba3 gene was used as a probe for DNA hybridization.

Plant regeneration
Various factors that might influence shoot regeneration were tested with non-inoculated explants. Tested
parameters included types of explant (hypocotyl and
cotyledon), age of explant (3, 5, 7, and 9 d), and

concentration of 6-BA (1, 2, and 3 mg L-1) in combination with NAA ( 0.01, 0.05, and 0.1 mg L-1). Explants were cut and placed on MS medium
(Murashige et al. 1962). All treatments were carried
out in triplicate. Regeneration frequency of buds was
scored after 30 d.

2011, CAAS. All rights reserved. Published by Elsevier Ltd.

Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth

Transformation procedure
The transformation was following the procedures of
Cao et al. (2008) and Metz et al. (1995). Full-strength
MS salts and 28 g L-1 sucrose were present in all the
following media except in Luria-Bertani (LB) medium.
Seed germination and pre-culture: Cabbage seeds were
surface-sterilized in 70% ethanol for 2 min and then in
10% Clorox for 10 min. The seeds were rinsed four
times with sterile water and placed on hormone-free
MS medium for germination at 25C under 16 h/8 h
light/dark regime. 5-d-old hypocotyls were cut into 10
mm segments and placed on MS medium. They were
cultured at 25C under 16 h/8 h light/dark regime for 2 d.
Agrobacterium cells derived from a single colony
harboring pCSBaN were suspended in LB medium containing 100 mg L-1 kanamycin and cultured at 28C
with shaking at 250 r/min until the OD600 reached to
0.6-0.9. Pre-cultured explants were immersed in the
Agrobacterium suspension for 10 min and then rinsed
once with MS liquid medium. Inoculated explants were
placed on MS medium containing 2 mg L -1 6benzylaminopurine (6-BA) and 0.1 mg L -1 naphthaleneacetic acid (NAA). They were incubated
at 28C in the dark for 3 d. Explants were then transferred to regeneration medium supplemented with 150
mg L-1 timentin, 2 mg L-1 6-BA, and 0.1 mg L-1 NAA
and cultured at 25C under 16 h/8 h light/dark regime
to eliminate Agrobacterium. 7 d later, explants were
transferred to selective medium that contained 150 mg L-1
timentin, 20 mg L-1 kanamycin, 2 mg L-1 6-BA, and 0.1
mg L-1 NAA. Resistant plantlets emerged 5 wk later.
The green plantlets were excised from the calli and put
into MS medium that had a higher concentration of
kanamycin (25 mg L-1) to reduce the number of escapes.
Plantlets that remained green were placed into rooting
medium containing 0.1 mg L-1 NAA and 0.1 mg L-1 3indolebutyric acid. Rooted plants were transplanted
into soil.

Molecular analysis
PCR and Southern blot analyses Genomic DNA was
extracted from fresh leaves of putative transformants
and non-transformed plants by the CTAB method (Doyle
et al. 1987). Specific forward and reverse primers

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were designed for PCR analysis. The sequences of the


primers for cry1Ba3 were 5-CCAGAGACTA
CTCCGACTATTGCG-3 (forward) and 5-TACA
CTTCCCCATTGCCACTAA-3 (reverse). PCR was
carried out for cry1Ba3 in 20 L in a thermal cycler
with the following amplification program: 94C for 4
min, 1 cycle; 94C for 60 s, 60C for 60 s, 72C for 90
s, for 30 cycles; 72C for 10 min, 1 cycle. The amplified products of cry1Ba3 were electrophoresed in 1%
agarose gels in 0.5TBE buffer.
For Southern blot analysis, 15 g genomic DNA per
sample was digested with Hind III (there are many Hind
III restriction sites in the cabbage genome). The digested DNA was electrophoresed in 1% agarose gel
and transferred onto Hybond N+ nylon membrane. The
DNA probe was prepared from a PCR-amplified 1.222
kb fragment covering the part of the cry1Ba3 gene.
Blots were obtained following the manufacturers instruction with the DIG High Prime DNA Labeling and
Detection Kit II from Roche Molecular Biochemicals
(Basel, Switzerland).
After hybridization, the membrane was washed sequentially with stringency solutions to blocking solution.
Finally, the membrane was washed with colour substrate solution (NBT/BCIP mixture) for colour detection.
RT-PCR analysis Total RNA was isolated from fresh
leaves of transformed plants and non-transformed plants
using a Total RNA Extraction Kit from Biomed-tech
company (Beijing, China) and the manufacturers
protocol. Synthesis of first-strand cDNA was performed with Revert AIDTM First Strand cDNA Synthesis Kit from Fermentas company (Burlington, Ontario,
Canada), and then PCR analysis was carried out using
the cry1Ba3 specific forward and reverse primers for
the detection of transcripts using procedures outlined
previously. -actin was used as an internal reference.
The PCR products of cry1Ba3 were electrophoresed in
1% agarose gels in 0.5TBE buffer.
Western blot analysis Total protein was extracted
from fresh leaves of transformed and non-transformed
plants with a protein extraction buffer as described by
Sambrook et al. (1989). The protein extraction buffer
contained 200 mmol L-1 TrisHCl (PH 8.0), 100 mmol L-1
NaCl, 400 mmol L -1 sucrose, 14 mmol L -1 mercaptoethanol, 1 mmol L-1 phenylmethyls ulfonyl fluoride and 0.05% Tween-20. After grinding, the mixture

2011, CAAS. All rights reserved. Published by Elsevier Ltd.

1696

was centrifuged at 12 500 r/min for 20 min at 4C.


Protein concentrations in the supernatant were determined using a Lowry protein assay (Larson et al. 1986).
The supernatant was subjected to electrophoresis in
10% SDS-polyacrylamide gels as described by
Sambrook et al. (1989). Then protein was electrotransferred onto polyvinylidene-fluoride membrane.
Western blot analysis was carried out according to the
enhanced chemiluminescence Western blotting protocol described by Bradd and Dunn (1993). The primary
antibody was raised in rabbit against proteins isolated
from Bt. This antibody had the reactivity to Cry1Ba3
protein. It was diluted at 1:500 for probing. Secondary antibody against rabbit IgG (whole molecule) was
produced in goat and purchased from Sigma company
(Saint Louis, Missouri, USA).

Insect bioassay
For detached leaf bioassays a leaf of each cry1Ba3 plant
and non-transformed cabbage was cut into two sections,
each 50 mm in diameter. One section was used for
bioassay of susceptible DBM larvae while the other was
used for bioassay of Cry1Ac-resistant DBM larvae.
Each leaf section was placed in a 90-mm Petri dish (a
moist filter paper was put on the bottom of the Petri
dish beforehand to maintain moisture). Ten 2nd instar
DBM larvae were placed on the surface of each leaf
section, and then the Petri dishes were sealed with
Parafilm. All insect bioassays were carried out in
triplicate. Leaf damage (visual estimate) and mortality
of larvae were scored after 6 d.

RESULTS
Plant regeneration and transformation
Plant regeneration Effects of seedling age, types of
explant, and hormones on shoot regeneration were tested
to facilitate transformation of cabbage. Based on these
results, the hypocotyls from 5-d-old seedlings and the
combination of 2 mg L-1 6-BA with 0.1 mg L-1 NAA
were used for subsequent transformation experiments.
Selection of transformants Kanamycin-resistant
plantlets emerged 5 wk later after transformation. The

YI Deng-xia et al.

majority of the initially green shoots gradually turned


white or brown and some became necrotic after subsequent subcultures. After rooting healthy plants were
transplanted into soil. A total of 37 kanamycin-resistant plantlets were recovered of which 29 survived.
The transgenic plants were morphologically normal.

Molecular analysis of transgenic plants


PCR and Southern blot analyses Kanamycin-resistant plants were subjected to PCR analyses to confirm
the presence of the cry1Ba3 gene. The expected 1.222
kb band representing the cry1Ba3 fragment was amplified from the genomic DNA of transgenic plants and
the pCSBaN plasmid. No band was amplified from the
genomic DNA of non-transformed plants. Representative results of the PCR analysis are shown in Fig. 2.
Lane 5 shows a kanamycin-resistant plant in which the
cry1Ba3 gene was not integrated.

Fig. 2 PCR analysis of genomic DNA from kanamycin-resistant


plants. Lane 1, 2 kb molecular weight marker; lane 2, pCSBaN
carrying cry1Ba3 gene; lane 3, blank lane; lane 4, non-transformed
cabbage; lanes 5-9, kanamycin-resistant plants.

Southern blot analysis provided additional evidence


of integration of cry1Ba3 into the cabbage genome. DNA
isolated from the leaves of PCR-positive plants and nontransformed plants was digested with Hind III and hybridized with the probe from the PCR products of cry1Ba3
gene. Most of the transformed plants showed hybridization signals to the probe. No signal was detected for
the non-transgenic plants. Representative results of the
Southern blot are shown in Fig. 3. There was a single
insertion site in some transgenic plants while others possessed two or more insertion sites.
RT-PCR analysis Transgenic cabbages confirmed by
PCR and Southern blot were analyzed by RT-PCR to
evaluate the presence of cry1Ba3 transcripts. For all
the transgenic cabbages, RT-PCR amplified the expected
1.222 kb cry1Ba3 band and the 0.196 kb band for the
-actin internal reference. Only the 0.196 kb internal

2011, CAAS. All rights reserved. Published by Elsevier Ltd.

Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth

reference band was amplified from non-transformed


cabbage (Fig. 4).
Expression of cry1Ba3 Western blots were performed
on the RT-PCR-positive plants to identify the production of insecticidal crystal protein encoded by cry1Ba3.
100 g of total soluble protein extracted from transformed and non-transformed cabbage was subjected
to Western blot analysis. The results demonstrated the
presence of the expected 140 kD Cry1Ba3 protein in
some transformed cabbages while non-transformed
plants did not express the protein. Some transformed
plants did not express the expected protein though they
were confirmed by RT-PCR (data not shown). Representative results are shown in Fig. 5.

Insect bioassay
Transgenic plants expressing Cry1Ba3 protein were

Fig. 3 Southern blot analysis of PCR-positive plants. Lane 1,


plasmid pCSBaN; lane 2 , non-transformed cabbage; lanes 3-11,
transformed cabbages.

Fig. 4 RT-PCR analysis of transformed cabbages. Lane 1, 2 kb


molecular weight marker; lane 2, is pCSBaN carrying the cry1Ba3
gene; lane 3, non-transformed cabbage; lanes 4-7, transformed
cabbages.

Fig. 5 Western blot analysis of transformed cabbages. Lane 1,


protein marker; lane 2 , Cry1Ba3 protein; lane 3, non-transformed
cabbage; lanes 4-6 are transformed cabbages.

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tested for insect resistance by exposing detached leaves


individually to susceptible and Cry1Ac-resistant DBM.
The results showed that transgenic plants were resistant to susceptible DBM larvae and could also control
DBM larvae resistant to Cry1Ac toxin (Table). There
was no mortality on untransformed plants. Some
transgenic plants showed no leaf damage and caused
complete mortality of larvae within 2 to 3 d. After
infestation, leaf disks from control cabbages were completely eaten, and live larvae were present (Fig. 6).

DISCUSSION
The kind and age of explant, concentration of 6-BA in
combination with NAA, length of inoculation with
Agrobacterium, length of pre-cultivation and cocultivation, usage of AgNO 3 , concentration of
kanamycin, and some other parameters have been reported to influence transformation of cabbage (Metz
et al. 1995; Pius et al. 2000; Rafat et al. 2010). In our
study, various factors that might influence shoot regeneration were tested to obtain transgenic cabbage.
We mainly focused on the effects of 6-BA and NAA on
regeneration and transformation because the combination of 6-BA with NAA has been reported to increase
the frequency of Brassica shoot regeneration (Metz et al.
1995; Paul et al. 1999; Cao et al. 2008).
Western blots indicated that some plants did not express Cry1Ba3 protein although the presence of the gene
in these plants was confirmed by PCR and Southern
blot. The cause of this lack of expression is unknown.
It has been reported that gene silencing occurs at transcriptional and post-transcriptional levels due to introduction of two or more similar genes, promoters or
enhancers into a plant genome (Meyer 1998; Matzke et al.
2000). The synthetic cry1Ba3 gene was modified to
change the codon usage to fit plant codon preference,
and the gene was controlled by the enhanced cauliflower
mosaic virus 35S promoter for efficient transcription.
The expression of cry1Ba3 gene in cabbage is worthy
of further study.
CA21-3 is a cabbage inbred line that possesses the
features of superior quality and high yield. Molecular
analyses proved that cry1Ba3 gene has been inserted
into the genome of CA21-3. Insect assays demonstrated
that some Cry1Ba3 plants could cause complete mor-

2011, CAAS. All rights reserved. Published by Elsevier Ltd.

1698

YI Deng-xia et al.

Table The results of detached leaf bioassay of transgenic cabbages after 6 d


Plant
Control
YA-1
YA-2
YA-3
YA-4

Mortality (%)
0b
100 a
96.77 a
93.33 a
100 a

Susceptible DBM
Estimated defoliation (%)
90-95
0-1
0-1
1-2
0-1

Mortality (%)

Cry1Ac-resistant DBM
Estimated defoliation (%)

0b
100 a
100 a
96.77 a
96.77 a

90-95
0-1
0-1
0-1
0-1

Different letters following mortality values indicate statistically significant difference based on the Duncans new multiple range test at P<0.05.

Fig. 6 In vitro bioassay of non-trangenic and transgenic cabbage with DBM. A, assays with DBM susceptible to Cry1Ac. B, assays with
DBM resistant to Cry1Ac. 1, non-transformed control; 2-6, transgenic plants.

tality of susceptible and Cry1Ac-resistant DBM. Homozygous insect-resistant plant lines can be obtained
by self-pollination, test-crossing, and molecular
analyses. Such lines may be useful for insect resistance breeding in the future.
DBM has evolved resistance to Cry1Ab, Cry1Ac and
some other Bt toxins in the field (Shelton et al. 1993a;
Perez et al. 1996; Tang 1997; Bravo et al. 2008). Thus,
Bt crops are threatened by the evolution of insect
resistance. Various strategies have therefore been suggested to delay insect resistance evolution: (I) high dose
plus refuge strategy; (II) use of two or more insectresistance genes; (III) deployment of transgenic plants
expressing toxin genes at a moderate level so that not
all susceptible insects are killed; (IV) transgenic plants
expressing toxin in certain organs, during a certain period or in response to an environment signal. A high
dose of a single Bt gene in conjunction with refuges of
non-Bt plants has been applied in several countries
(Gould 1998; Tabashnik et al. 2003, 2009). Pyramiding
two or more Bt toxins in plants is a promising way to
postpone insect resistance evolution (Zhao et al. 2003;

Bates et al. 2005; Halpin 2005; Tabashnik et al. 2008,


2009). Cui et al. (2009) obtained Bt cry1Ia8 cabbage,
and the results of bioassays showed that cry1Ia8 plants
were resistant to the larvae of both susceptible and
Cry1Ac-resistant DBM. Cry1Ba3 was successfully
inserted into the genome of cabbage in this research.
Therefore, cry1Ia8 and cry1Ba3 genes can be pyramided
by sexual crosses or by sequential transformation.
Deployment of resulting plants may inhibit the evolution of insect resistance.

Acknowledgements
We thank Prof. Huang Dafang, Biotechnology Research
Institute, Chinese Academy of Agricultural Sciences,
China, Dr. Zhang Jie,Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China,
and Dr. Lang Zhihong, Biotechnology Research
Institute, Chinese Academy of Agricultural Sciences,
Beijing, China, for their help in providing gene and
vector. We are grateful to Dr. Zhang Youjun, Institute
of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China. for test larvae supply

2011, CAAS. All rights reserved. Published by Elsevier Ltd.

Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth

and help in bioassays. Thanks are due to Prof. Elizabeth Earle, Department of Plant Breeding and genetics,
Cornell University, USA, and Dr. Cao Jun, Athenix
Corporation, USA, for providing the Brassica genetic
transformation protocol and constructive advice on
writing the manuscript. This work was supported by
the grants from the National High Technology Research
and Development Program of China (863 Program,
2008AA10Z155), the National Natural Science Foundation of China (31071697), and the earmarked fund
for the Modern Agro-Industry Technology Research
System, China (nycytx-35-gw01).

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