1

Write-up
Of
Microbiology
TOPIC–“BT-COTTON”

SUBMITTED TO-DR. JOGINDER SINGH PANWAR

SUBMITTED BY-MS.DAWINDER KAUR

MSC (HONS) BIOTECHNOLOGY
ROLL NO. -RP7010A11
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Acknowledgement

I am extremely grateful and remain indebted to our guide Dr.

JOGINDER SINGH PANWAR for being of inspiration and for his
constant support in the Design, Implementation and Evaluation
of this presentation. I am thankful to him for his constant
constructive criticism and invaluable suggestions, which
benefited me a lot while preparing presentation. He has been a
constant source of inspiration and motivation for hard work. He
has been very co-operative throughout this project work.
Through this column, it would be my utmost pleasure to express
our warm thanks to him for his encouragement, co-operation.

I would also like to thanks my friends Rumeet Kaur & Sahil

Sharma who helped me throughout this term paper. Without
whose co-operation this term paper cannot get completed.

Dawinder kaur

(RP7010A11)
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CONTENTS
• Introduction to BT-Cotton
• Bacillus thuringiensis
• Structure of the Cry Proteins
• The cry Gene Family
• CRY PROTIENS AND THEIR ACTION ON PEST
• Status of primary Pests- Lepidopteran
• Transformation
• Spectrum of Activity of Cry1Ac for different pests
• Spectrum of Activity of (Cry1Ac + Cry2Ab) for different
pests
• % Efficiency of bt cotton plants
• Bt Cotton in India
• Bt Cotton Study conducted by Greenpeace in Karnataka
• Some other limitations
• Possible problems
• Advantages of bt-cotton
• Conclusions
• References
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INTRODUCTION-BT COTTON
• Bt cotton is based on the crystalline protein produced by Bacillus
thuringiensis (Bt).
• Bacillus thuringiensis (Bt) are the gram positive bacteria.
• First pesticide producing crop:-Potato plants producing Bt toxin
(1995) were approved safe by the Environmental Protection
Agency.
• Other Bt crops-Bt Maize, Bt Potato and Bt cotton (1996) were
being grown by farmers in the USA.

Bt cotton=cotton plant + bt gene

Cultivated in the U.S., Australia, Mexico, South Africa, India, China,
Argentina, Indonesia

>>>Bacillus thuringiensis (Bt)

• Common soil bacterium
• Present in nature in a variety of forms (species & strains)
• Bacillus thuringiensis is a gram-positive soil bacterium, with a
genome size of 2.4 to 5.7 million basepairs.
• The prevalence of this strain is not restricted and has been
isolated worldwide from many habitats, including soil, stored-
product dusts, insects, deciduous and coniferous leaves.
• Produces proteins that are toxic to insects called as cry proteins.
• Commonly used in garden sprays & for commercial agriculture,
including organic farming.
• Extremely well-known toxin in terms of human health &
environmental safety
• B. thuringiensis is widely used as a larvicide against mosquito
larvae, where it is also considered an environment friendly
method of mosquito control.
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Structure of the Cry Proteins

• The Cry toxin has three domains which are, from N to C terminus, a
seven helix bundle, (Domain I), a triple anti-parallel beta sheet
domain (Domain II) and a beta-sheet sandwich (Domain III). (1)
• The core of the molecule is built from five sequence blocks, which
are a highly conserved feature of all the Bt toxins indicating that all
the proteins in this Cry family will adopt the same general fold.
• The long, hydrophobic and amphipathic alpha helices of Domain I is
equipped for transmembrane pore formation. The seven alpha helix
domain I structure resembles the pore forming domain of Colicin A
and is important for the membrane insertion step.
• Pore formation is initiated by insertion of a helical hairpin
(alpha4/alpha5) from domain I with subsequent association of
alpha4/alpha5 hairpins from several molecules to form an oligomeric
helical bundle pore with a radius of 5-10 Angstroms.
• Before one or more of these Cry helices can insert into the
membrane to initiate oligomerization and pore formation, a major
conformational change must occur, since in the water soluble pre-
insertion form all the hydrophobic faces of the Cry Domain I helical
bundle face inwards.
• Membrane penetration occurs in two steps: binding to a specific
receptor exposed on the membrane surface, followed by insertion of
the delta-endotoxin protein into the membrane leading to pore
formation.
• The three beta sheet structure (beta prism) of domain II is involved in
receptor binding and specificity determination. This is further
supported by reports that domain II shared the same structural fold
with three carbohydrate binding proteins: the vitelline membrane
outer layer protein I from hen's eggs, the plant lectin jacalin and
the Maclura pomifera agglutinin.
• Domain III of the Bt toxin (see below) may also be a determinant of
insect specificity/receptor binding.
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• Schematic ribbon diagram structure of the CryA toxin-The Cry toxin has three
domains which are, from N to C terminus, a seven helix bundle, (Domain I), a triple
anti-parallel beta sheet domain (Domain II) and a beta-sheet sandwich (Domain III).

The striking similarity between the structure of domain II of the Bt toxins

and the three dimensional structures of two known lectins suggests that
insecticidal specificity might be determined by the carbohydrate affinity of
the domain II lectin fold. A recent discovery that domain III is also a lectin-
like domain suggests that the insecticidal specificity of these toxins could
be determined by two lectin-like domains acting in concert or
independently.
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The Cry Gene Family:

These toxins can be categorized under the delta -endotoxins, which is
highly specific to only certain insects. The family of genes coding for this
toxin is the Cry gene family. A common characteristic of the cry genes is
their expression during the stationary phase.

Cry proteins are divided into 5 main groups on basis of

insecticidal activities :-

Gene Target
Cry I Lepidoptera(moths and butterflies

Cry II Diptera(flies and mosquitoes),

CryIII Coleoptera (beetles),

CryIV Diptera & nematodes(rats)

CryV Nematodes(rats)

Thus, B. thuringiensis serves as an important reservoir of Cry toxins for

production of biological insecticides and insect-resistant genetically
modified crops.

CRY PROTIENS AND THEIR ACTION ON PEST

• These toxins can be categorized under the d-endotoxins, which is

highly specific to only certain insects. The family of genes coding for
this toxin is the Cry gene family.
Action mechanism of cry proteins-
• During sporulation, it synthesizes a cytoplasmic inclusion containing
one or more proteins that are toxic to insect larvae.
• Upon completion of sporulation the parent bacterium lyses to
release the spore and the inclusion. In these inclusions, the toxins
exist as inactive protoxins.
• When the inclusions are ingested by insect larvae, the alkaline pH
solubilizes the crystal.
• The protoxin is then converted in to an active toxin after processing
by the host proteases present in the midgut.
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• It has been indicated that the activated toxin binds to insect-specific

receptors exposed on the surface of the plasma membrane of midgut
epithelial cells and then inserts into the membrane to create
transmembrane pores that cause cell swelling and lysis and
eventually death of the insect.
• Due to their high specificity for these unique receptors on the
membrane of the gut epithelial cells, these toxins (delta-endotoxins)
are harmless to non-target insects and the end-user and are
compatible with integrated pest management programs. The fact that
they are proteins ensures that they are readily biodegraded

>>>Status of primary Pests- Lepidopteran

• Primary pests-Pink Bollworm

• Status of Pests-Secondary Lepidopteran

Trichoplusia ni

Spodoptera exigua

Transformation-insertion of bt-gene to
develop a genetically modified cotton plant
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Cry-gene

Coker 312

Process of transformation of cry- gene

The gene of interest is spliced out of

the bacterium using a vector, like
Agrobacterium tumefasciens, &
transferred to cotton cells grown in
tissue culture
The cells are grown into a plant & then,
after testing, plants are back-crossed
into commercial lines to make new
varieties
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>>>Spectrum of Activity of Cry1Ac for different

pests
Excellent No
Control Control
Trichoplusia ni
Spodoptera exigua
Heliothis virescens Estigmene acrea Spodoptera frugiperda
Spodoptera ornithogalli
Pink Bollworm
Bucculatrix thurberiella
(PBW), our
Helicoverpa zea principal pest
(pre-bloom)

Pseudoplusia includens

>>>Spectrum of Activity of (Cry1Ac + Cry2Ab) for

different pests
Excellent No
Control Control
Pectinophora gossypiella
Heliothis virescens Beneficial
Insects
Bucculatrix thurberiella
Marmara spp. Agrotis &
Feltia spp.
Helicoverpa zea
Estigmene
acrea
Trichoplusia
ni
Pseudoplusia
includens
Spodoptera exigua

>>>% Efficiency of bt cotton plants

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Cry1Ac Cry2Ab Variety Adjusted

+ - DP50B 100
- + 985X 99.591
+ + 985BX 100
+ - DP33B 100
+ - DP448B 100
+ - DP458BR 100
+ + DP33BX 100
+ - SG215BR 100
- + SG125X 99.758
+ + SG125BX 100

It is concluded from this data –

• Cry1Ac= 100%

• Cry2Ab =99.67%

• Both Genes(Cry1Ac + Cry2Ab )=100%

Bt Cotton in India
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Bt cotton seeds were first tested in India for germination, vigor, and insect
efficacy. Other experiments were conducted to confirm the environmental
safety of Bt cotton, including tests of gene flow, persistence of the
transformed plants, weediness characteristics, crossability of the
transgenic pollen with the nontransgenic relative and near relatives, effect
of the pollen on insects and nontarget organisms, and changes in the soil
microbial flora. These studies were conducted under the unique
environmental conditions of India and with the Bt trait in Indian germplasm.
Studies of the molecular characterization and stability of the Cry1Ac gene
were also carried out, as well as feeding studies and tests of food and feed
safety, toxicity, and allergenicity.

summary for regulatory processes leading to commercial release of Bt

cotton in India.

Government of India
Years Studies undertaken oversight committeesa
1995-1996 Application and permit for importation of Bt cotton seed DBT
containing the Cry1Ac gene
1996-2000 Greenhouse breeding for integration of the Cry1Ac gene into DBT
Indian germplasm, seed purification, and stock increase
1996-2000 Limited field studies for potential of pollen escape, RCGM (DBT)
aggressiveness, and persistence
1998-2001 Biochemical and toxicology studies RCGM (DBT), GEAC

1998-2000 Multilocation field trials: agronomic and entomology performance RCGM (DBT), MEC
of first-generation Bt cotton hybrids, conducted by Mahyco and
State agriculture universities

2000-2001 Soil rhizosphere evaluations and protein expression analyses RCGM (DBT), GEAC
from multilocation field trials
2001 Advanced stage multilocation field performance trials of first- GEAC, ICAR, DBT,
generation Bt cotton hybrids, conducted by ICAR MEC
2002 Submission of final biosafety, environmental safety, gene efficacy GEAC
and performance documentation to GEAC; commercial release of
first-generation Bt cotton hybrids by GEAC

2002- Continued field performance trials of second-generation Bt RCGM (DBT), GEAC,

ongoing cotton hybrids for regulatory approval ICAR, MEC
a
DBT = Department of Biotechnology; GEAC = Genetic Engineering Approval Committee; RCGM =
Review Committee for Genetic Modification (constituted by DBT); ICAR = Indian Council of
Agriculture Research; MEC = Monitoring & Evaluation Committees (constituted by GEAC and
RCGM).

Bt Cotton Study conducted by Greenpeace in Karnataka

1. 77% of the farmers interviewed in the study reported Bollworm
infestation in the Bt cotton plants.
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2. A majority of the farmers reported an increase in pesticide costs in the

case of Bt cotton . This was despite a fall in the number of sprays
compared to the Non-Bt hybrids. This increase is cost was related to the
nature of pesticides that were used by the farmers, which were more potent
and expensive.

3. The total cost of fertilisers was higher in the case of Bt Cotton plants (Rs.
600-1200 higher) as compared to the Cotton hybrids.

4. Farmers reported an increase in the labour cost of Bt cotton as the

cotton bolls are smaller in size, tightly packed and picking takes longer.

5. The yields for Bt Cotton and non- Bt hybrids were more or less the same,
however in Raichur non Bt hybrids fared better.

6. The market value for Bt Cotton is lower than non-Bt Cotton hybrids by Rs
200-800.This is because of the shorter staple fibers and the relatively dull
colour of the cotton fiber.

7. In terms of economic viability it is clear that Bt Cotton is a much more

expensive alternative for the farmers.

• Apart from the input costs such as pesticides, fertilizers, water that
have been mentioned before, the Bt Cotton seed itself costs Rs
1600.00 /packet .The cost of the non Bt Hybrid seed is about Rs 450.

• Furthermore, the market value that the farmers are getting for Bt
Cotton is far lower than for Non Bt hybrid

Some other limitations

1. Primary insects resistance to bt cotton-In November 2009, Monsanto
scientists found that the pink bollworm had become resistant to Bt
cotton in parts of Gujarat, India. In four regions, Amreli, Bhavnagar,
Junagarh and Rajkot the crop is no longer effective at killing the
pests.
2. Secondary pests-Chinese farmers have found that after seven years
of growing BT cotton the populations of other insects other than
bollworms, such as mirids, have become significant
problems.Similar problems but with mealy bugs have been reported
in India
3. Low level expression of cry genes- For example in cry1(A)b contains
(I) localized A+T regions (ii)18 polyadenylation sites (iii) at 13
regions,AUUUA sequence instabilize the coding mRNA.
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Possible problems
1. Lepidopteran toxicity
The most publicised problem associated with Bt crops is the claim that
pollen from Bt maize could kill the monarch butterfly This report was
puzzling because the pollen from most maize hybrids contains much lower
levels of Bt than the rest of the plant and led to multiple follow-up studies.

It appears that the initial study was flawed by faulty pollen-collection

procedure; researchers fed non-toxic pollen mixed with anther walls
containing Bt toxin. The weight of the evidence is that Bt crops do not pose
a risk to the monarch butterfly.

2.Wild maize genetic contamination

A study in Nature reported that Bt-containing maize genes was
contaminating maize in its center of origin.Nature later "concluded that the
evidence available is not sufficient to justify the publication of the original
paper." However, there still remains a controversy over the highly
unorthodox retraction on the part of Nature. In 1998, Chapela, one of the
original paper's authors spoke out against Berkeley accepting a multi-
million dollar research grant from the Swiss pharmaceutical company,
Novartis.

A subsequent large-scale study, in 2005, failed to find any evidence of

contamination in Oaxaca. However, further research confirmed initial
findings concerning contamination of natural maize by transgenic maize.

3. Possible link to Colony Collapse Disorder

As of 2007, a new phenomenon called Colony Collapse Disorder (CCD) is
affecting bee hives all over North America. Initial speculation on possible
causes ranged from cell phone and pesticide use to the use of Bt resistant
transgenic crops. The Mid-Atlantic Apiculture Research and Extension
Consortium published a report in March 2007 that found no evidence that
pollen from Bt crops is adversely affecting bees. The actual cause of CCD
remains unknown, and scientists believe that it may have multiple causes.

Advantages of bt-cotton
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Bt Cotton—Agronomic Benefits

Trials conducted in several locations in 1998/99, 1999/2000, 2000/01, and

2001/02 demonstrated the following agronomic benefits of Bt cotton:

• good control of bollworm species in different growing areas;

• significantly higher yield and boll retention (compared to control or
non-Bt cotton);
• reduction in expense of insecticide application;
• additional revenue (Rs.2,500-4,000/acre) in farm income (compared to
non-Bt cotton); and
• no adverse effect on nontarget insects or adjacent non-Bt cotton
crops.

There are several advantages in expressing Bt toxins in transgenic Bt

cotton:
1. The level of toxin expression can be very high thus delivering sufficient
dosage to the pest.
2. The toxin expression is contained within the plant system and hence
only those insects that feed on the crop perish.
3. The toxin expression can be modulated by using tissue-specific
promoters, and replaces the use of synthetic pesticides in the environment.

Conclusions

• The use of Bt cottons has provided the first larvicidal and selective
approach to controlling pest of cotton.
• The control provided by Bt cottons approaches immunity. No
survivors have been found in field studies.
• Bt cotton has revolutionized our ability to reduced insecticide inputs
by over 60%.
• Future transgenic products for insect control in cotton should be
independently & scientifically tested.
• Problems like insect resistance against cry proteins can be
overcome by using combination of cry genes (Cry1Ac + Cry2Ab).

At the end, it is concluded that use of Bt cotton is best environment

friendly method to control pest and can be further modified to overcome
the problems related to it.
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References
http://www.agbioforum.org/v7n12/v7n12a04-zehr.htm
http://fbae.org/2009/FBAE/website/our-position-bt-cotton.html
http://fen.wikipedia.org/wiki/Bacillus_thuringiensis
http://www.dawn.com/2008/12/15/ebr3.htm
http://ww.nature.com/nbt/journal/v27/n1/full/nbt0109-9.html
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and many more…..

BOOK-Biotechnology by-B.D.SINGH