Karnataka J. Agric. Sci., 22(3-Spl.

Issue ) :(467-470) 2009

Genetic enhancement of chickpea for pod borer resistance through expression of CryIAc protein
SUMA S. BIRADAR, O. SRIDEVI AND P.M. SALIMATH

Department of Genetics and Plant Breeding,

University of Agricultural Sciences, Dharwad-580 005, Karnataka, India
E-mail: biradar.suma@gmail.com

Abstract: The use of genetically modified (Bt) crops expressing lepidopteron-specific Cry proteins derived from the soil
bacterium Bacillus thuringiensis is an effective method to control the polyphagous pest Helicoverpa armigera (Hubner). As H.
armigera potentially develops resistance to Cry proteins, Bt crops should be regarded as a tool in integrated pest management.
Therefore, they should be compatible with biological control. Such technologies developed need to be tested for their efficacy.
Insect feeding bioassay on transformed chickpea plants with CryIAc (T-1 and T-2 generation of ICCV-2 and A-1 plants) with
larvae of pod borer, H.armigera showed high level of toxicity to insects and protection of transgenic plants. Transformed
chickpea plants expressing CrylAc protein had high mortality (> 60%) while, surviving insects exhibited significant loss in per
cent gain in body weight (upto 5.3%). Expression of Bt crylAc gene in chickpea showed effective resistance in transgenic plants
to the major pod borer insect to illustrate the effectiveness of Bt gene against Helicoverpa.

Key words: Chickpea, CryIAc, pod borer, insect bioassay

Introduction transformation with Bt gene CryIAc by in planta method through

Chickpea, one of the most important pulse crops, is
attacked by about 57 insect species in India, half a dozen of
which are considered to be of economic significance. ICRISAT
has made extensive survey on the insect pest problems in
chickpea. The survey showed that Helicoverpa spp. are the
dominant pests in most of the areas causing damage to about
34-40 per cent. The use of chemical insecticides has traditionally
been the primary management option for Helicoverpa control
on chickpea (Reed et al., 1987). Unfortunately, extensive and
very often indiscriminate use of chemical pesticides has resulted
in environmental degradation, adverse effects on human health
and other organisms and eradication of beneficial insects. In
recent years, however, the development of insecticide resistance
in H. armigera (Daly and Murray, 1988; Forrster et al., 1993)
renewed emphasis on sustainable environment-friendly crop
protection practices and highlighted the need to develop
alternative pest management strategies. Both economically and
ecologically, breeding chickpea cultivars having resistance to
the pest is the most important component of integrated pest
management. Inspite of concentrated breeding efforts, notable
achievements have not been made. This herculean task can be
very well addressed by genetic transformation which provides
a complementary means for the betterment of field crops. The
success story of which is very well demonstrated in cotton and
presently, about 80 per cent of area is under Bt-cotton. The
present study aimed to demonstrate potentiality of such
technologies in chickpea.
Material and methods
The studies were carried out at the Department of
Genetics and Plant Breeding, UAS, Dharwad during 2005-06.
The popular cultivars of chickpea viz., A-1 and ICCV-2 highly
susceptible to pod borer were employed for genetic
Agrobacterium mediated transformation. T1 generation plants
which were confirmed to be PCR positive for both marker gene
(kanamycin) and gene of our interest (CryIAc) were advanced
to T2 by selfing. Further, in T2 generation plants segregating
for gene of interest were subjected to PCR analysis and
transgene expression analysis through Bt-expression strips and
bioassay studies.
Bt-express normal sticks supplied by Desigen, Mahyco
Seeds Limited, Maharashtra were used in this study. Plants
carrying cry gene, express cry protein residues in their tissues.
Leaf disc of 5 mm diameter was crushed in 500 µL of extraction
buffer supplied by the manufacturer and used as tissue extract
for observing the Bt-expression. The strips work on the principle
of antigen-antibody specificity. Protein present in the tissue
will specifically bind to the antibody on the strip and band will
be visible within 10 min.
For insect bioassay studies, confirmed plants for the
presence of CryIAc and positive expression analysis were
employed for detached leaf technique. For this, eggs of H.
armigera were obtained from National Bureau of Agriculturally
Important Insects, Bangalore. After hatching, larvae were reared
on control (negative plants) chickpea plants. For the treatment,
individual five leaves from PCR positive plants were placed in
cultured bottles containing 2 per cent agar and covered with
blotting paper. With the help of a moistened camel hair brush,
the second instar larvae were carefully placed on the leaves @
5 per bottle (10/treatment). The mouth of the culture bottle was
then sealed with muslin cloth to avoid contamination, and also
to facilitate aeration to both larvae and leaves. The observation
on the mortality rate and gain in weight of larvae was taken after
72 h and per cent gain in weight was calculated by the following
formula.

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Karnataka J. Agric. Sci., 22(3-Spl. Issue ) : 2009
Per cent gain = Final weight – Initial weight x 100
in weight Initial weight
Results and discussion
H. armigera is one of the most important insect pests in
the old world due to its mobility, high polyphagy, short
generation duration, and high reproductive rate (Sharma, 2005).
Currently, the application of chemical spray insecticides is the
most common method of controlling this pest on crops, including
chickpea (Sharma et al., 2007) and cotton (Durairay et al., 2005).
However, H. armigera is known to develop resistance to almost
all the insecticides used for its control (Kranthi et al., 2002). The
chemical sprays are also of environmental concern and are
responsible for human health problems (Qaim et al., 2008). Thus,
alternative control methods are increasingly being employed.
The use of genetically modified (GM) crops that express
insecticidal genes, such as those derived from the soil bacterium
Bacillus thuringiensis, provide a powerful option to control
Lepidopteran pests (Shelton et al., 2002). Therefore, the
expression of B. thuringiensis cry genes is an option to protect
chickpeas from damage by H. armigera.
There have been great efforts for transformation of
chickpea; however, not much progress is reported due to lack of
Table 1. PCR and expression analysis of progenies of T1 plants of A-1 cultivar of chickpea
Number of T1 plants PCR analysis
No. of progenies tested
18 46
Control band
Positive band for cryIAc
Fig. 1: Dip stick assay showing band for CryIAc in transgenic chickpea
The Bt expression strips positive plants were employed
for insect bioassay using neonate larvae of chickpea pod borers.
Thirty-seven and twenty-six transgenics plants of A-1 and ICCV-
2 cultivars, respectively, were assessed for their tolerance to
second instar larvae of H. armigera. Observations on mortality
after 72 h of treatment and gain in body weight of larvae were
recorded in all the treated plants along with negative control.
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in vitro transformation protocol. By considering the fact that
recalcitrance nature of chickpea to in vitro regeneration,
transformation protocols are required which by pass the tissue
cultural procedures. In the present study, transgenic plants were
generated by in planta methods through Agrobacterium
mediated transformation successfully (Biradar et al., 2008).
Large number of transgenics produced were initially screened
for kanamycin resistance through leaf paint assay then through
PCR analysis. The kanamycin resistant plants showing
amplification for both the nptII and CryIAc were advanced to
T2 generation (Table 1). Such T2 putative transgenic plants
were analysed for physical integration of the gene (by PCR
analysis) and expression studies (using Bt expression strips
and insect bioassay). Out of 46 progenies obtained from 18 T1
plants, thirty six were found to have gene of interest and
production of crystal protein detected by antigen and antibody
interaction (Fig. 1). The results indicated that of thirty six
transgenic plants carrying the cry gene, only 28 plants produced
cry protein. Further, gene expression to the level of lethal dose
is important and identification of such plants demands further
studies. In the present study, instead of quantification of cry
protein, directly leaf samples were subjected to insect infestation
to identify plants causing 100 per cent mortality.
Total number of positive
+ve for both npt II plants for Bt stick
and CryIAc gene
36 28
1
2
3
Fig. 2: Comparison of larval growth upon feeding on transgenic
(1&2) and control plants (3)
Seventy two hours after infestation, the transgenic plants
showed notable resistance to larvae compared to non-
transformed plants. Insect mortality ranged from 0 to 60 per cent
in case of A-1 (Table 2) while, it ranged from 0 to 40 per cent in
ICCV-2. The larvae surviving exhibited drastic reduction in body
weight as compared to those fed on control plants and
phenotypically severely stunted in growth, inactive and were
Genetic enhancement of chickpea....................
expected to die after one or two days. Deterrence to feeding was
noticed with the advance in duration of feeding and the damage
was static. The feeding inhibition was accompanied by
decreased larval weight, which was quantified in terms of per
cent gain in body weight. Wide range was observed in case of
A-1 (5.30 to 614.3%) when compared to kabuli cultivar ICCV-2
(100-990%). In one of his studies Sanyal et al. (2005) reported
plants expressing cryIAc protein showed high mortality (>80%)
of insects and significant loss in weight (45-55%) (Fig. 2).
Statistically, highest gain in body weight differed significantly
from that of control in both the cultivars. From the results, it is
clear that transgenic plants in both cultivars exhibited varied
level of response to pod borer, which can be attributed to quantity
of cry protein present in the leaf tissue. Such kind of differential
feeding patterns and growth of larvae in control and transgenic
leaf discs of cabbage was reported by Chakrabarty et al. (2002).
Further these transgenics need to be evaluated under field
conditions and characterized with respect to level of expression
of cry protein at different developmental stages and the level
resistance to pod borer.

Table 2. Inhibition of growth and development of larvae as a consequence of feeding on T2 generation plants of A-1 cultivar
Generation T2 generation of A-1 plants T1 generation of ICCV-2 plants
Transgenics Initial weight Percentage of Per cent gain in Initial weight Percentage of Per cent gain
of larva (g) mortality weight of larva (g) mortality in weight
after 72 h after 72 hrs
P1 0.0013 50 307.7 0.0010 10 500.0
P2 0.0011 0 336.4 0.0009 0 455.5
P3 0.0014 40 350.0 0.0011 20 990.9
P4 0.0015 0 413.0 0.0011 0 745.4
P5 0.0014 0 264.3 0.0010 10 480.0
P6 0.0020 0 355.0 0.0012 0 533.3
P7 0.0012 20 108.3 0.0014 0 400.0
P8 0.0019 0 231.2 0.0010 20 536.4
P9 0.0015 10 233.3 0.0011 10 592.8
P 10 0.0016 30 225.0 0.0013 0 207.7
P 11 0.0016 30 168.7 0.0015 10 100.0
P 12 0.0017 40 135.3 0.0013 10 546.2
P 13 0.0018 20 177.7 0.0013 10 515.4
P 14 0.0022 0 127.3 0.0019 20 315.7
P 15 0.0017 30 270.6 0.0012 10 466.6
P 16 0.0013 40 130.7 0.0013 20 346.2
P 17 0.0015 0 166.6 0.0013 20 592.3
P 18 0.0014 0 257.1 0.0014 20 328.6
P 19 0.0012 20 300.0 0.0010 40 570.0
P 20 0.0018 30 155.5 0.0011 40 172.7
P 21 0.0014 30 257.1 0.0016 20 731.3
P 22 0.0017 30 135.3 0.0019 20 584.2
P 23 0.0012 20 150.0 0.0020 10 485.0
P 24 0.0019 40 5.30 0.0014 20 507.1
P 25 0.0016 50 87.5 0.0017 0 482.4
P 26 0.0014 30 371.4 0.0015 30 326.6
P 27 0.0019 50 110.5
P 28 0.0021 30 114.2
P 29 0.0023 0 230.4
P 30 0.0021 20 614.3
P 31 0.0017 10 194.1
P 32 0.0014 0 392.8
P 33 0.0014 10 478.6
P 34 0.0014 30 414.3
P 35 0.0013 60 361.5
P 36 0.0021 30 171.4
P 37 0.0012 50 191.6
Control 0.0018 0 688.9 0.0010 0 1300.0
S.Em.± 23.65 S.Em.± 47.32
C.D. at 1% 64.33 CD at 1% 132.02

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Karnataka J. Agric. Sci., 22(3-Spl. Issue) : 2009
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