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

Issue ) : (609-610) 2009

Quantification of Cry1Ac protein over time in different tissues of Bt cotton hybrids

Cotton hybrids that express the Cry1Ac delta-endotoxin
of Bacillus thuringiensis (Berliner) are highly effective against
bollworms viz., Helicoverpa armigera (Hubner), Earias vitella
(Fabricius) and Pectinophora gossypiella (Saunders)
(Greenplate, 1999). The novel transgenic technology was found
to be highly beneficial in almost all parts of the world. However,
for the Bt-transgenic technology to be sustainable, it is important
that the toxin expression levels be expressed at adequate
quantities in appropriate plant parts at the requisite time of the
season to afford protection against major target insect pests,
which primarily include the bollworms. The variations in overall
expression levels of cry1Ac among bollgards have been
correlated to survival of various lepidopteran pests indicating
that cultivars do not provide the same level of control (Adamczyk
and Meredith, 2003). Similar effect has been noticed by
Greenplate (1999), Sun et al. (2002) and Olsen et al. (2005) in
different Bt cotton genotypes. The spatio-temporal variations
in eight Indian Bt cultivars have been reported by Kranthi et al.
(2005). However there is need to monitor the newly released
hybrids. Hence the present study was conducted to know the
field performance of different Bt cotton hybrids in terms of season
long expression of Cry1Ac protein.
Four Bt cotton hybrids were selected for protein profiling.
Three plant parts viz., fully opened terminal leaf, first terminal
pre-candle square and first terminal bolls were sampled at 80-90,
110-115, 135-140 and 150-165 days after sowing (DAS). Plant
tissue samples were frozen in -80°C freezer and subsequently
lypolized in a heto FD8 freeze dryer. The entire terminal leaf,
square and boll were lypholized. After complete lypolization,
the dried samples were ground into a fine powder using a paint
shaker or mortar and pestle and stored sealed containers at
freezer.
Quantitative estimation of Cry1Ac protein in interspecific
bollgard hybrids was done using commercially available Quant-
T ELISA 96- well plate kits from Desi-Gen, Jalna, Maharashtra.
About 5 mg of lyophilized tissue samples were taken in a 1.5 ml
microfuge tube, by the addition of 500 µl of ice- cold sample
extraction buffer and then macerated at 3000 rpm using motor
driven pestle for 30 seconds, later it has been chilled on ice for
10 minutes and again macerated for 30 seconds. Finally the sample
was subjected to centrifugation at 8000 rpm for 15 minutes. Then
the supernatant was extracted and stored at 4ºC. Before loading
the sample extracts to plate the positive and negative standards
were diluted with standard buffer. After loading plates were
incubated at 37ºC for 1.5 hour in a humid environment, later the
samples were discarded and washed with wash buffer and
conjugate buffer twice allowing the plate to stand five minutes
with wash buffer in the wells in between washes. The plate was
incubated at room temperature for 30 minutes for colour
development. Then plate was subjected to ELISA reader. Based
on absorbance values of the plate at 405 nm the quantification
of Cry protein was estimated with the help of sigma plot version
8.01 programme.
The results indicated that the expression of Cry1Ac was
declined over season independent of the genotype. Among the
hybrids the expression level was different. The Cry1Ac content
was high i.e., 5.51-7.85, 3.88-5.97 and 2.97-6.34 µg/g dry wt in
leaves, squares and bolls, respectively at 80 DAS (Table 1).
Later on the expression declined through out the season and

Table 1. Cry1Ac protein different plant part tissues of Bollgard cotton hybrids
Genotypes (H×B) Plant part tissue Cry1Ac protein (µg/g dry weight) Std deviation
80 DAS 110 DAS 135 DAS 150 DAS Mean ±
MRC- 7201 Leaf 7.85 8.66 6.31 6.39 7.30 1.15
Square 5.97 6.70 6.23 7.41 6.58 0.63
Boll 5.84 6.46 2.26 3.07 4.41 2.06
MRC- 6918 Leaf 7.56 5.68 3.62 4.10 5.24 1.78
Square 4.83 6.00 3.86 6.54 5.31 1.20
Boll 6.34 5.31 3.76 3.32 4.68 1.40
RASI XL -708 Leaf 5.51 7.26 5.44 4.87 5.77 1.03
Square 4.78 5.31 3.30 4.44 4.46 0.85
Boll 2.97 4.67 3.07 4.40 3.78 0.88
SP-911 Leaf 6.9 6.27 3.53 3.28 4.99 1.79
Square 3.88 4.14 2.55 2.42 3.29 0.44
Boll 4.31 4.99 4.75 4.50 4.59 0.92
Mean over Leaf 6.95 6.96 4.72 4.66
genotypes Square 4.86 5.58 3.98 5.20
Boll 4.82 5.35 3.46 3.82

DBS- Day before spray

Figures in the parentheses are arc sine transformed values
Values in the columns followed by same letters are non significant at P = 0.05 as per DMRT
* Two per cent jaggery was added for all the insecticide solution
609
Karnataka J. Agric. Sci., 22(3-Spl. Issue ) : 2009
reached minimum at 150 DAS. However, the Cry1Ac protein did
not reach to the undetectable level and maintained quiet high
i.e. above the critical limit of 1.9 µg/g in all the hybrids.
Exceptionally in squares of MRC-7201 CryIAc toxin increased
to 7.41 µg/g at 150 DAS from 5.97 µg/g at 80 DAS. It may be due
to arrest of proanthocyanin at the later stages, as the possible
cause for decline in endotoxin concentration with maturity was
attributed to interaction by proanthocyanin (Olsen et al., 2005).
The decline in expression over the season has been well
documented and present findings are in corroboration with the
early reports. Greenplate (1999) reported the decline in Cry1Ac
concentration in terminal structure from 63.4-34.5 µg/ g at 53
and 116 days after planting. Similarly seasonal variation in
Cry1Ac was observed by Adamczyk et al. (2004) where fall in
concentration from more than two ppm to one pp was quiet
evident. Further studies of Sun et al. (2002) and Wan et al.
(2005) also confirmed the fact that expression pattern vary with
in the season with higher concentration at the beginning and
least at the end
Assessment of Cry1Ac toxin through ELISA indicated
variation in expression among different tissues of Bt cotton
plants. Cry1Ac toxin was the highest in leaves, followed by
Department of Agril. Entomology, College of Agriculture,
Navile, Shimoga-577 204, Karnataka, India
References
Adamczyk, J.J., And Meredith, W.R., 2003, Variability of Bt
expression among commercial transgenic cotton varieties
in the United States. Proc. of World Cotton Res. Conf.-3:
9-13, March, 2003. Cape Town South Africa, pp. 1233-
1238.
Adamczyk, J.J., Willium, R.And Meredith, J., 2004, Genetic bases
for viability of cry1Ac expression among commercial
transgenic Bacillus thuringensis (Bt) cotton cultivars in
the Unites States. J. Insect Sci., 8: 17-23.
Greenplate, J.T., 1999, Quantification of Bacillus thuringiensis
insect control protein cry1Ac over time in Bollgard cotton
fruit and terminals. J. Econ. Ent., 92 : 1377-1383.
Kranthi, K.R., Naidu, S., Dharwad, C.S., Tatwawadi, A., Mate.
K., Patil. E., Bharose, A.A., Behera, G.T., Wasaskar, R.M.
And Kranthi, S., 2005, Temporal and intra-plant variability
of cry1Ac expressing in Bt-cotton and its influence on
the survival of cotton bollworm, Helicoverpa armigera
(Hubner) (Noctuidae: Lepidoptera). Curr. Sci., 89: 291-
298.
610
squares and bolls in all the hybrids. MRC-7201 hybrid expressed
the highest concentration of 7.85, 5.97 and 5.84 µg/g in leaves,
squares and bolls respectively at 80 DAS and similar trend was
followed during subsequent periods of crop growth. Lowest
concentration was noticed in SP-911 hybrid.
In general, similar trend of variation in expression of
Cry toxin among different tissues of Bt cotton plants was noticed
in the subsequent periods irrespective of the crop growth stages.
Thus there was considerable intra plant variability in expression
of cry toxin. The present findings are in close agreement with
the results of earlier studies. According to Greenplate (1999)
terminal leaves expressed very high level of concentration
compared to proximal fruiting structure. Udikeri (2006) reported
that the Cry protein expression was maximum in leaves followed
by squares, flowers and bolls. Further Kranthi et al. (2005)
reported variable expression in different parts of plants which
declined with the advancement of season, a critical level of 1.90
µg per g was suggested by Kranthi et al. (2005) below which
larvae would be able to survive. The present data on Cry1Ac
expression showed concentration much above this critical level
in all the tissues at all the stages of crop growth.
R. MANJUNATHA
S. PRADEEP
S. SRIDHARA
M. MANJUNATHA
MOHAN I. NAIK
B.K. SHIVANNA
VENKATESH HOSAMANI
Olsen, K.R., Daly, J.C., Holt, H.E. and Finnegan, E.J., 2005,
Seasonal-long variation in expression of Cry1Ac gene
and efficacy of Bacillus thuringensis toxin in transgenic
cotton against Helicoverpa armigera Hub. (Lepidoptera
: Nocuidae). J. Econ. Ent., 98: 1007-1017.
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R., 2002, Effect of transgenic Bt cotton on population of
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sustainability of Cry protein expression, computation of
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model for Bt cotton under rainfed condition. Ph. D.
Thesis, Univ. Agric. Sci, Dharwad (India).
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expression profiles of insecticidal protein and control
efficacy against Helicoverpa armigera for Bt cotton in
China. J. Econ. Ent., 98: 195-201.