Professional Documents
Culture Documents
November 2011
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
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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.
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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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.
Transformation of Cabbage (Brassica oleracea L. var. capitata) with Bt cry1Ba3 Gene for Control of Diamondback Moth
Insect bioassay
Transgenic plants expressing Cry1Ba3 protein were
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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-
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YI Deng-xia et al.
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;
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
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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capitata) for insect resistance by Agrobacterium tumefaciensmediated transformation. In Vitro Cellular & Developmental
thuringiensis Cry and Cyt toxins and their potential for insect
control. Toxicon, 49, 423-435.
References
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of transgenic Brassica developed through Agrobacteriummediated gene transfer. Current Science (India), 76, 15691573.
Perez C J, Shelton A M. 1996. Field applications, leaf-dip assay,
and diet incorporated diagnostic assays used against Bacillus
thuringiensis-susceptible and resistant diamondback moth.
Journal of Economic Entomology, 89, 1364-1371.
Pius P K, Achar P N. 2000. Agrobacterium tumefaciens-mediated
transformation and plant regeneration of Brassica oleracea
var. capitata. Plant Cell Reports, 19, 888-892.
Qaim M, Zilberman D. 2003. Yield effects of genetically modified
crops in developing countries. Science, 299, 900-902.
Rafat A, Aziz M A, Rashid A A, Abdullah S N A, Kamaladini H,
Sirchi M H T, Javadi M B. 2010. Optimization of
Agrobacterium tumefaciens-mediated transformation and
shoot regeneration after co-cultivation of cabbage (Brassica
oleracea subsp. capitata) cv. KY Cross with AtHSP101 gene.
Scientia Horticulturae, 124, 1-8.
Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning:
A Laboratory Manual. Cold Spring Harbor Laboratory, New
York.
Shelton A M, Andalora J T, Barnards J. 1982. Effects of cabbage
looper, imported cabbageworm, and diamondback moth on
fresh market and processing cabbage. Journal of Economic
Entomology, 75, 742-745.
Shelton A M, Roberson J L, Tang J D, Perez C, Eigenbrode S D,
Preisler H K, Wilsey W T, Cooley R J. 1993a. Resistance of
diamondback moth (Lepidoptera: Plutellidae) to Bacillus
thuringiensis subspecies in the field. Journal of Economic
Entomology, 86, 697-705.
Shelton A M, Wyman J A, Cushing N L, Apfelbeck K, Dennehy
YI Deng-xia et al.