A synthetic cryIA(b) gene coding for an insecticidal crystal protein of Bacillus thuringiensis (Bt) was transferred to cabbage cultivar 'Golden Acre' by co-cultivating hypocotyl explants with Agrobacterium tumefaciens. Transformed plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and mRNA expression. Immunoblot analysis revealed high-level expression of Bt toxin protein in the transgenic plants. The expression resulted in a significant insecticidal activity of transgenic cabbage plants against the larvae of diamondback moth (Plutella xylostella). The results also demonstrated that a synthetic gene based on monocot codon usage can be expressed in dicotyledonous plants for insect control

Bhat, S. R.

Bhattacharya, R. C.

Chopra, V. L.

Kirti, P. B.

Viswakarma, N.

English

Publications of the IAS Fellows

RESEARCH COMMUNICATIONS  
 
CURRENT SCIENCE, VOL. 83, NO. 2, 25 JULY 2002 146
*For correspondence. (e-mail: ramcharan99@yahoo.com) 
Development of insect-resistant 
transgenic cabbage plants expressing 
a synthetic ryIA(b) gene from Bacillus 
thuringiensis 
 
R. C. Bhattacharya†,*, N. Viswakarma†,  
S. R. Bhat†, P. B. Kirti# and V. L. Chopra† 
†National Research Centre on Plant Biotechnology, Indian Agricultural 
Research Institute, New Delhi 110 012, India 
#Department of Plant Science, University of Hyderabad,  
Hyderabad 500 46, India 
 
A synthetic cryIA(b) gene coding for an insecticidal 
crystal protein of Bacillus thuringiensis (Bt) was 
transferred to cabbage cultivar ‘Golden Acre’ by  
co-cultivating hypocotyl explants with Agrobacterium 
tumefaciens. Transformed plants resistant to kanamy-
cin were regenerated. Hybridization experiments 
demonstrated gene integration and mRNA expression. 
Immunoblot analysis revealed high-level expression of 
Bt toxin protein in the transgenic plants. The expres-
sion resulted in a significant insecticidal activity of 
transgenic cabbage plants against the larvae of dia-
mondback moth (Plutella xylostella). The results also 
demonstrated that a synthetic gene based on monocot 
codon usage can be expressed in dicotyledonous plants 
for insect control. 
 
BACILLUS thuringiensis (Bt) produces a variety of insect-
cidal crystal proteins (ICPs) upon sporulation. These pro-
teins, called d-endotoxins, are highly toxic to 
lepidopteran, dipteran and coleopteran insects1. Diff rent 
ICP genes of Bt have been successfully engineered into 
many crop- lants to obtain resistance against lepidop-
teran insects2. Poor expression of native Bt genes 
necessitates the use of codon-m dified and truncated 
version of the genes to achieve higher expression3. When 
expressed in plants, these modified genes confer 
significant protection against insects to important crops 
such as cott n, maize, rice, tomato and potato2,4. 
 Cabbage (Brassica oleracea v r. capitata) is an impor-
tant vegetable crop grown extensively throughout the 
world, including India. One of the major limitations in 
cabbage production is damage due to insect pests. The 
most important among them, the lepidopteran diamond-
back moth (Plutella xylostella) lone causes a worldwide 
damage of up to one billion dollars annually5. The larvae 
bore into the cabbage head and even a little damage into 
the head reduces marketability of the crop. Synthetic in-
secticides used to control the pest have raised concerns 
about food safety and environmental pollution. Evolution 
of insecticide resistance further adds to the problem of 
chemical control. 
 We have transformed a leading cabbage cultivar 
‘Golden Acre’ with a lepidopteran-specific, synthetic 
cryIA(b) gene extensively modified for high expression 
in plant cells. The transgenic lines were demonstrated to 
have significant resistance to the larvae of P. xylostella. 
 Plasmids pBT1291 (ref. 1), pBinAR (ref. 6) and deri- 
vatives were transformed into E. coli strain DH5a 
(Stratagene). For plant transformation, the binary vector 
was mobilized into Agrobacterium tumefaciens strain 
GV2260 (ref. 7) by freeze–thaw transformation method8. 
Single transformed colony was transferred into liquid 
bacterial medium (YEM medium with 50 mg/l rifam-
picin, 100 mg/l carbenicillin and 50 mg/l kanamycin) and 
shaken at 240 rpm at 28°C for two days. The suspensions 
were then mixed with sterile glycerol (1 : 1vol/vol) and 
stored at –80°C until used. 
 About 5 mg purified DNA of pBT1291 was restricted 
with XbaI. On restriction the 2.4 kb fragment, which inc- 
ludes full length synthetic cryIA(b) sequence along with a 
castorbean intron upstream to structural region, was puri-
fied through Wizard DNA Cleanup system (Promega) 
and ligated into the XbaI site of the binary vector pBi-
nAR, which is flanked by CaMV 35S promoter and oct - 
pine synthase poly(A) sequences. 
 The most popular Indian cultivar of cabbage (B. oler- 
acea var. capitata) ‘Golden Acre’ was used. Di infected 
seeds were germinated under aseptic condi ions on solid 
MS medium9 with 2% sucrose. Hypocotyl segments of 
approximately 5 mm length from four-day-old seedlings 
were used for plant transformations. The incubation con-
ditions for germination and in vitro culture were 25 ± 
1°C and 16 h photoperiod of approximately 28 mEm–2 s–1. 
 One ml glycerated culture was inoculated in ABMM 
broth10 containing appropriate antibiotics and grown to 
saturation. A secondary culture, initiated from the satu-
rated primary culture, was grown in the same slectiv  
medium supplemented with 50 mm acetosyringone. The 
ulture was grown to OD600
 = 0.6. Cells from 1.5 ml of 
culture were harvested by centrifugation at 5000 rpm for 
10 min at 22°C and resuspended in 15 ml CI liquid  
medium (MS basal salts with 2% sucrose + 1 mg/l 2,4-
D + 0.5 mg/l kinetin). This suspension was used for in-
fection of hypocotyl explants, preconditioned for one day 
on solid CI medium (CI liquid medium + 0.8% agar) and 
co-cultivated for three days at 24°C in dark. The explants 
were incubated on SI medium (MS basal medium with 
2% sucrose + 0.5 mg/l IAA + 2 mg/l BA + 3.5 mg/l silver 
nitrate + 250 mg/l cefotaxime + 0.25% phytagel) for a 
d lay period of one week, and transferred on S2 selection 
medium (SI medium + 25 mg/l kanamycin). The regener-
ated shoots were grown on root induction medium (MS 
asal medium + 0.1 mg/l IAA + 250 mg/l cefotaxime + 
25 mg/l kanamycin). The rooted plants were transplanted 
into small pots containing vermiculite. After initial estab-
lishment, the plants were shifted to earthen pots in the 
greenhouse.  
RESEARCH COMMUNICATIONS 
 
CURRENT SCIENCE, VOL. 83, NO. 2, 25 JULY 2002 147
 Isolation of total genomic DNA was performed by the 
procedure described by Doyle and Doyle11. DNAs were 
restricted with EcoRV, electrophoresed on 0.8% agarose 
gel and transferred onto nylon N+ membrane (Amer-
sham). Southern hybridization was carried out with a 32P-
dCTP labelled XbaI insert (2.4 kb) seperated from 
pBT1291 according to Sambrook et al.12. Total RNA was 
isolated from young leaf tissues with guaninidine thiocy-
anate-based RNeasy Plant Total RNA kit (QIAGEN) acc- 
ording to the manufacturer’s specifications. RNAs were 
eletrophoresed on 1.2% agarose formaldehyde gel and 
transferred onto nylon membrane by wet-blotting12. The 
membrane was hybridized with 32P-labelled cryIA(b) seq- 
uence restricted and purified from pBT1291.  
 The purified CryIA(b) protein and the rabbit anti-
CryIA(b) serum were kindly provided by Swapan K. 
Dutta, IRRI, Philippines. Total cellular protein was iso-
lated following the procedure by Laemmli13. Total pro-
tein from Bt-transgenics and untransformed wild-type 
plants was fractionated by SDS–PAGE (10% poly-
acrylamide) and transferred to PVDF (Amersha ) as 
described by Asubel et al.14. The Bt-proteins were dte- 
cted using the rabbit anti-CryIA(b) serum and a goat-anti 
rabbit IgG coupled to alkaline phosphatase as secondary 
antibody. 
 The leaf discs (1.5 cm diameter) from young leaves 
were cut and placed in small petri dishes containing 
moistened filter paper. On each leaf disc, five late sec-
ond-instar (6-day-old) larvae of P. xylostella were relea- 
sed and reared at 26–28°C with 60% relative humidity. 
Each assay consisted of three replicates using larvae 
originating from the same egg cluster. For each replicate 
of either transformed or control plants, the percentage of 
larval mortality was calculated. Leaf area damaged was 
determined after 24 h of feeding by plotting the total leaf 
area against the damaged leaf area on a graph paper, and 
expressed as percentage. Corrected per cent mortality 
was calculated according to the formula: 
 
 Per cent corrected mortality =  
  [(T – C)/(100 – C) ´  100], 
 
where T is per cent mortality in transgenics and C the per 
cent mortality in wild-type plants. 
 Small-scale insect-feeding trials were carried out by 
releasing 20 to 25 late second-instar larvae of P. xylos-
tella on each plant grown in a glasshouse at 28 ± 2°C.
Plants were individually covered with three-rings bags, 
after release of the larvae. 
 The chimeric gene expression cassette employed in the 
present study consisted of CaMV 35S promoter, a syn-
thetic cryIA(b) gene and the 3¢ polyadenylation signal 
from octopine synthase gene of Agr bacterium (Figure 
1). Fifteen plantlets regenerated on S2 medium were sub-
jected to rooting under kanamycin selection. Within two 
weeks the true transformed shoots pr duced normal 
roots, whereas escapes failed. Total genomic DNA from 
each of the six normal-looking plants, rooted in presence 
of kanamycin, was isolated and used for molecular analy-
ses. Presence of the cryIA(b) sequence was initially det- 
ected through PCR analysis using cryIA(b)-specific  
internal primers (results not shown). The Southern 
hybridization analysis clearly demonstrated integration of 
the transgene sequence into the target genome (Figure 2). 
The DNA from untransformed plant did not show any 
PCR amplification or signal. Since the cloned transgene 
cryIA(b) does not have any internal site for EcoRV, DNA  
 
 
npt I
cry1A(b) npt II
intron
RB
LB
nosterminator
CaMV 35S promoter
ocs terminator
12.80 kb
nos promoter
Xba I
Sal I
Hind III
pRC-Bt
Xba I
 
Figure 1. Plant transformation vector pRC-Bt containing the ICP 
gene cryIA(b). npt, Neomycin phosphotransferase; LB, Left border; 
RB, Right border. 
 
 
 
Figure 2. Southern analysis of Bt-transgenics probed with cryIA(b) 
sequence. Five mg of total DNA was restricted with EcoRV, electro-
phoresed on 0.8% agarose gel, blotted on nylon membrane and South-
ern hybridized using 32P-labelled cryIA(b) sequence. Lane C, wild type; 
lane 1, BT1; lane 2, BT2; lane 3, BT3, lane 4, BT4. 
C 1 2 3 4 
RESEARCH COMMUNICATIONS 
 
CURRENT SCIENCE, VOL. 83, NO. 2, 25 JULY 2002 148
 
 
Figure 3. Northern analysis of Bt-transgenics. Twenty-five mg of 
total RNA from young leaves was electrophoresed on 1.2% formalde-
hyde–agarose gel and probed with cryIA(b) sequence. Lane C, wild 
type; lane 1, BT2; lane 2, BT3; lane 3, BT4. 
 
 
 
Figure 4. Expression of crystal protein in Bt-transgenics. Eighty mg 
total protein from leaves was separated on SDS–PAGE and Western-
blotted using rabbit anti-Cry1A(b) serum. Forty mg of purified 
Cry1A(b) protein was used as positive control. Lane M, Molecular 
weight marker; lane PC, Purified Cry1A(b) protein (ositive control); 
lane C, Wild-type; lane 1, BT2; lane 2, BT3; lane 3, BT4. 
 
 
Table 1. Mean percentage of larval mortality of Plutella xylostella 
  and leaf area damage in leaf disc bioassay  
 Per cent Corrected Percentage leaf 
 mortality mortality area damage 
Plant no. (mean ± SD) per cent (mean ± SD) 
 
WT   10 ± 8.16 – 59.66 ± 9.10 
BT2 76.66 ± 4.71 74.06  6.0 ± 0.81 
BT3 56.66 ± 4.71 51.84  26.3 ± 2.49 
BT4   70 ± 8.16 66.66  18.3 ± 2.05 
The experiments were conducted in three independent sets and each 
corresponds to 10 larvae independently reared on leaf discs from wild 
type or transgenic plants. 
 
 
 
from putative transgenic plants when restricted with 
EcoRV for Southern blot transfer, is expected to be res- 
tricted outside the integrated gene. Consequently, the 
number of bands obtained in the autoradiogram indicates 
independent events of transgene-integration. Three inde-
pendent lines, BT2, BT3 and BT4, showed desirable sin-
gle-copy insertion of the transgene. In Northern analysis 
of total RNA from Southern-co firmed plants, presence 
of a single 2.2 kb band in the autoradiogram confirmed 
the trancription of cryIA(b) sequence in BT2, BT3 and 
BT4 lines (Figure 3). No degradation of the mRNA was 
detected. BT2 and BT4 showed higher level of transgene 
expression at the transcriptional level compared to BT3. 
Appropriate quantities of total cellu ar proteins from 
transgenics and untransformed control plants were frac-
tionated by SDS–PAGE, immunoblotted on PVDF mem-
brane and analysed for the presence of Bt-protein. A 
81.3 kDa Cry protein was detected when rabbit anti-
CryIA(b) serum was used as primary antibody (F gure 4). 
 Bt-transformants, which showed detectable levels of 
mRNA expression for the transgene and stable presence 
of Cry protein in immunoblotting, were assessed for their 
tolerance to late, second-instar larvae of P. xylostella. 
Larval mortality was recorded in order to test the influ-
ence of Cry protein ingestion on larvae survival. Extent 
of leaf damage effected before cessation of larval feeding 
was measured to assess the Bt-m diated resistance. Bio-
assay on detached leaf discs showed significant larval 
mortality ranging from 51.84 to 74.06% (Table 1). The 
highest larval mortality of 74.06% with a minimum leaf 
damage of 6.0% was obtained on transgenic BT2. The 
damage on young leaves due to insect feeding was also 
significantly less on the transformed leaves compared to 
untransformed wild type (Figure 5). The difference in 
mortality and leaf area damage observed among the dif-
ferent transgenic lines could be correlated to differences 
in Bt gene expression. 
 The larvae fed on transgenic leaf discs were severely 
stunted in growth when compared to larvae fed on wild 
type leaf (Figure 6). At whole plant level also, transgenic 
plants showed less insect damage compared to wild type 
plants (Figure 7). The result demonstrated that Bt-
mediated resistance was functionally active n planta. 
 It has been convincingly demonstrated by several 
groups that the genes encoding d-endotoxins from Bt
impart resistance to lepidopteran insects pests, when  
introduced into different crop plants15,16. Improvement in 
the expression of endotoxin gene throughcodon modifi-
cation is crucial for effective Bt-mediated resistance 
against major insect pests. High-level expression was 
important for our project of imparting pest resistance to 
cabbage cultivar ‘Golden Acre’. A synthetic version of 
cryIA(b) modified extensively on the basis of codon  
usage in plants1 was chosen. In this synthetic cryIA(b), 
the overall G + C content has been changed to 59.2% 
from the 37.6% of the native gene. This modification was 
more extensive than that previously describe  for cryIA(b) 
(ref. 3) and cryIA(c) (ref. 17). In constructing plant-
expression vector pRC-Bt, the coding sequence of syn-
thetic cryIA(b) was fused to CaMV 35S promoter, the 
castorbean catalase-1 intron and the polyadenylation sig-
nal of the octopine synthase gene. The chimeric pRC-Bt 
C 
RESEARCH COMMUNICATIONS 
 
CURRENT SCIENCE, VOL. 83, NO. 2, 25 JULY 2002 149
is similar to pBin-Bt described by Kumar et al.18. Cab-
bage is a leafy vegetable and effectiv  control strategy 
will require that the resistance operates throughout plant 
growth and development. The constitutive promoter 35S 
was therefore chosen. The combination of 35S promoter 
and the castorbean intron has previously been demon-
strated to confer high-level expression of foreign genes in 
transgenic plants19,20. The Bt-transformants isolated 
through the described selection regime of plant transfor- 
 
 
 
 
Figure 5. Insect-feeding bioassay of Bt-transgenics. Late second-
instar larvae of P. xylostella were released on leaf-discs of transgenic 
and wild type (WT) plants. Insect mortality and leaf area damage were 
recorded at 24h after release. 
 
 
 
 
Figure 6. Inhibition of larval growth due to feeding on Bt-tra genics. 
Growth of the larvae after 24 h feeding on transgenics and wild type 
leaf-discs was compared. T, Larvae fed on transgenic leaf-discs; WT, 
Larvae fed on wild type leaf-discs. 
 
 
Figure 7. In planta bioassay of Bt-transgenics. Twenty to twenty-five 
late second-instar larvae of P. xylostella were released on transgenics 
and wild type plants, grown in a contained environment. C, Wild type; 
T, transgenic (BT2). 
 
 
mation showed stable integration of cryIA(b) sequence in 
the genome. Fragment sizes in the Southern blot and  
detection of expected 2.2 kb mRNA in Northern hybridi-
zation indicated integrity of cryIA(b)-expression cassette. 
Northern analysis demonstrated substantially high level 
of cryIA(b) expression. Detection of cry transcripts in 
transgenic plants is known to be difficult and toxin 
mRNAs are often present in amounts below the level of 
detection in Northern blots21,22. The higher expression 
may be due to extensive codon modification carried out 
in the synthetic version of the gene. Although the codon 
modification was originally carried out based on monocot 
codon usage1, our study indicated that the same gene 
could be expressed in dicotyledonous plants also. How-
ever, no comparative study was made in this regard. The 
integrity of cryIA(b) transcripts was confirmed by the 
presence of a corresponding single 81.3 kDa Cry protein 
in immunoblot analysis. The size of the Cry protein cor-
responded to the expected size of the truncated CryIA(b). 
In insect-feeding bioassay conducted on leaf discs, the 
Bt-protein produced in transge ic plants caused rapid 
cessation of larval-feeding activity and subsequent inhi- 
bition of their development. Under controlled laboratory 
conditions, this provided an estimate of insecticidal 
effects before larval release could be assessed for damage 
on control vs transgenic plants. At the whole plant stage, 
Bt-expressing plants were sufficiently protected against 
damage from diamondback moth attack. The degree of 
tolerance among different independent transformants 
varied significantly and the maximum level was obtained 
in BT2 line. 
 While the tolerance of transgenics has still to be dem-
onstrated under field conditions, transgenesis in combina-
tion with other IPM strategies has the potential of 
preventing a significant part of the yield losses and qual-
ity deterioration caused by dimondback moth. 
 
1. Fujimoto, H., Itoh, K., Yamamoto, M., Kyozuka, J. and Shima-
moto, K., Bio/Technology, 1993, 11, 1151–1155. 
2. Kumar, P. A. and Sharma, R. P., J lant Biochem. Biotechnol., 
1994, 3, 3–9. 



Development of insect-resistant transgenic cabbage plants expressing a synthetic cryIA(b) gene from Bacillus thuringiensis

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Development of insect-resistant transgenic cabbage plants expressing a synthetic cryIA(b) gene from Bacillus thuringiensis

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