M.Sc. (Ag.

) Course Seminar

THE SCIENCE BEHIND THE

DEVELOPMENT OF Bt COTTON

Supervisor : Prof. L. C. Prasad Speaker: Koteswara Rao. Potla

Co-supervisor : Prof. J. P. Lal M.Sc. (Ag.) Final
Dept. of Genetics & Pl. Breeding
Inst. Of Agril. Sciences,
Banaras Hindu University
Cotton is a leading commercial fiber crop
• India-
• Has largest area under cotton cultivation ( 22.4M acres,25%
of world’s cotton area)
• Ranks 2nd in world’s cotton production (31.5 million bales,
2007-08)
• Average cotton yield is 485 kg/ha compared to world’s
average of 680 kg/ha
• 80 % of total cotton acreage is under Bt cotton
INCREASE IN ADOPTION OF Bt COTTON AREA IN INDIA
Cultivation Year Total cotton cultivation Transgenic cotton area Percentage of
area million acres million acres Transgenic cotton area
2002-03 18.94 0.07 0.37
2003-04 18.8 0.21 1.21
2004-05 22.13 1.30 5.92
2005-06 22.23 3.29 14.83
2006-07 22.57 8.54 37.9
2008-09 22.40 17.2 80.0 Source: ISAAA, 2007
 Cotton is highly susceptible to insect pests especially to
the larvae of Lepidoptera
 Bollworms cause an estimated loss of 50 to 60 %
potential yield in cotton.
 55% of total insecticides consumed in India are used for
cotton improvement.
 Pesticides account 1/3rd of cultivation cost in cotton.
 Chemical control to suppress these insect pest developed
highly level of resistance for most of the chemicals used.
 High level of resistance required,
 Repeated application of insecticides leading to
• Heavy expenditure.
• Crop failure.
• Viscous cycle of debt for farmers.
• Adverse effect on the beneficial organisms.
• Environmental pollution .
• Pesticide residues.
Source: ISAAA, 2007
 Major challenge is to increase and sustain
crop productivity with less use of chemicals.
 IPM has historically placed great hopes on
Host Plant Resistance (HPR).
 Conventional host plant resistance to insects
involve quantitative traits at several loci.
 Until recently pest resistance varieties are
developed through conventional plant
breeding

Source: H.C. Sharma et.al.2000

 Genetically Modified Crops
 GM crops represent a promising opportunity to make an
important contribution to
 IPM & offers the possibility of developing entirely new
biological insecticides.
 Unique opportunities of genetic engineering.
• widened pool of useful genes
• Allows the use of several desirable genes in a single
event
• Reduces the time to introgress novel genes into elite back
ground
• Ability to change the level of gene expression
• Develop transgenics with different insecticidal genes
LIMITATIONS
• High cost
• Development of resistance in insect population
Source: H.C. Sharma et.al.2000
 Bacillus thuringiensis

• Gram positive soil dwelling bacteria.

• Serves as an important reservoir of cry genes for
production of biological insecticide an insect resistant
genetically modified crops.
• Forms proteinaceous crystals upon sporulation .
• Plasmids encode at least 90 genes for protoxins.
Source: Herman HOFTE et.al1986
 Cry genes Classification Based On Insect
Specificity & Sequence Homology

Genes Proteins Target

size (KDa)
cry - I 130 Lepidopteron larvae
cry - II 70 Lepidopteron &Dipteran
larvae
cry - III 70 Coleopteran larvae
cry - IV 130 Dipteran larvae
cry - V ------- Lepidopteron &coleopteran
grubs

Source: Mohan Babu .R. et.al 2003

 Limitations of Bt Sprays

• Sensitivity to UV radiation and heat desiccation

• Performance is not always consistent (affected by
environmental fluctuations)
• Incomplete coverage
Source: K.R. Ostlie.et.al 2007
 What is Bt cotton ?
• Genetically engineered form of cotton.
• Produced by inserting a synthetic version of
a Bt gene.
• Produces its own Bt toxin to destroy the
bollworm.
• Causes the production of Bt toxin in all parts
of cotton plant through out its life span.

Source: F.J. Perlak . et.al.2001

 CRY Protein Structure

 Domain I is responsible for

inserting into the gut
membrane and formation of
pore where ions can pass
freely
 Domain II helps in
recognition of receptors on
the epithelial lining of the
mid gut
 Domain III binds the
receptor

Source: Herman HOFTE et.al1986

 MODE OF ACTION OF Bt TOXIN

Ingestion of Bt plant material

Solubilization of crystalline proteins in mid gut (pH 12 )

Mid gut proteases activate endo toxins by proteolytic processing

Active toxins bind to amino peptidase receptors

Formation of a pore resulting in swelling and eventually cell lysis

Death of larvae

Source: H.C. Sharma .et.al 2000

Mechanism of Toxicity of Bt
Cont…
…
The Science Behind The Development of Bt Cotton
Basic requirements of Bt cotton development involves,
 A target genome.
 A candidate gene.
 A vector to carry the gene.
 Modification of foreign DNA to increase the level
of gene expression.
 Method to deliver the plasmid into the cell.
 Protocols to Identify the transformed cell.
 Tissue culture and procedures to transformed cells.
 Characterization of the putative transgenic plants at
molecular and genetic level.
Source: H.C. Sharma .et.al 2000
 Three primary components of the genetic
package inserted into Bt cotton plants include.

2. cry1AC gene

4. CaMV 35S promoter

6. npt II antibiotic resistance gene (genetic marker)

Source: I.S.Katageri .et.al 2007

 Cont…
Cry1 AC gene
• Derived from Bacillus thuringiensis …
sub spcies kurstaki
srtain HD73
• Modified for improved expression for cotton

CaMV 35S promoter

• Effective in driving cry1Ac gene expression in cotton
• Most widely used promoter for cotton transformation
• Constitutive promoter (odell et.al 1985)
• Provides site for binding of RNA polymerase and hence
involved in the transcription initiation

npt II ANTIBIOTIC RESISTANCE GENE(genetic marker)

• Encodes neomycin phospho transferase (NPT II)
• Used to indentify transformed cells containing cry1AC
gene
Source: I.S.Katageri .et.al 2007
 Structure of cry1 Ac gene cassette

LOW LEVELS OF EXPRESSION OF cry1AC GENE

• Due to the presence of sequences not commonly found in plants
coding regions.
• In plants transcription termination is signaled by polyadenylation
sequence AAUAAA or AAUAAU.
• cry1AC gene has potential polyadenylation sites responsible for
low accumulation of cry1AC gene transcripts in Bt cotton plants.
• The mRNA of cry1AC gene has sites recognized in plants as
introns and are used for splicing of this mRNA. The spliced mRNA
is rapidly degraded .
• Codon usage defined as non random selective use of 1 or more
codons during translation in preference to other codons specifying
the same amino acids
Source: F.J. Perlak . et.al.2001
 Gene Cloning
 cry1AC gene cassette is initially cloned in modified
E.coli plasmid
 pTi plasmid cannot be used for cloning due to
• Large size of pTi plasmid
• Non availability of unique restriction sites within
T-DNA
The subsequent gene transfer in to cotton tissue
utilizes
 Co-integrate vectors
 Binary vectors

Source :G.Sunil Kumar et.al 2001

 Co-integrate Vector
 Produced by Homologous recombination between pTi plasmid&
modified E.coli plasmid
 pBR 322 is suitably modified to produce an intermediate
vector(IV)
Vector IV must contain
 Origin of replication of E.coli
 pBR sequences present in the T-region of the disarmed pTi T-
DNA FROM pTi plasmid
 Neo gene for selection of recombinant T-DNA
 kanr for selection of co-integrate vector in Agro bacterium
 Cry1AC gene cassette is inserted with in the T-DNA to yield a
recombinant IV
 tra genes of helper plasmid pRK 2013 transfer recombinant IV
plasmid in to Agro bacterium with disarmed pTi plasmid
 Homologous recombination between IV Plasmid & pTi plasmid
produce co-integrate pTi vector
Source :G.Sunil Kumar et.al 2001
 Co-integrated vectors (hybrid Ti-plasmids)

DISADVANTAGES:
 1) Long homologies required between the Ti plasmid and the E.
coli plasmids (pBR322 based Intermediate vectors) making them
difficult to engineer and use
 2) Relatively inefficient gene transfer compared to the binary
vector
 Construction of Agro Bacterium Mediated Binary Vector
Binary vector consists of a pair of plasmids
Disarmed T-DNA Plasmid (mini Ti or micro Ti)
 Has deleted oncogenes
 Lack vir genes
 Has only left and right borders of T-DNA sequences
 Has origin of replication of both E coli and Agro bacterium
 cry1AC gene cassette is integrated within T region b/w left and right borders
with BamHI Restriction Enzyme.
 Recombinant mini Ti is cloned in E coli.
 Transfer of mini Ti into Agro bacterium by heat shock or elctroporation
method.
Helper Plasmid
 Ti plasmid have functional vir region.
 Lacks T-DNA region including the border sequences.
 Vir region induce transfer of T-DNA of the mini Ti into plant cells.
Source :G.Sunil Kumar et.al 2001
 Ti Plasmid Vector Systems Often Working As
Binary Vectors

DISADVANTAGE:
Depending on the orientation, plasmids with two different origins of replication
may be unstable in E. coli.
ADVANTAGE:
Small vectors are used, which increases transfer efficiency from E. coli to Agro
bacterium. No intermolecular recombination is needed.
Source :G.Sunil Kumar et.al 2001
 Plant Material
 Selection of variety.
 De linting seeds with concentrated H2SO4
 Soaked in Hgcl2 (50 mg/lit) for 3 minutes
 Seeds washed 3 times germinated at 280c
 Hypocotyl segments 5 to 6 mm excised from 10 to
20 days old seedling
 Plant material is placed on P1 AS medium containing
2,4-D

Source :G.Sunil Kumar et.al 2001

 Methods of Delivering Plasmid DNA In To
Cotton Tissues

 Two methods are used

 Agrobacterium mediated gene transfer
method.
 Direct Gene Transfer method.
Mechanism of infection of Agrobacterium in plants
 Ti Plasmid

T-DNA
region DNA between
L and R borders is
transferred to plant as
SsDNA;
Tumor-
producing
genes
Opine catabolism T-DNA encoded genes
Virulence region can be substituted by
ORI target genes
Ti plasmids and the bacterial chromosome act in
concert to transform the plant
• Agro bacterium tumefaciens chromosomal genes:
chvA, chvB, pscA required for initial binding of the
bacterium to the plant cell and code for polysaccharide
on bacterial cell surface.
• Virulence region (vir) carried on pTi, but not in the
transferred region (T-DNA). Genes code for proteins
that prepare the T-DNA and the bacterium for transfer.
• 3. T-DNA encodes genes for opine synthesis and for
tumor production.
• 4. oc (opine catabolism) genes carried on the pTi and
allows the bacterium to utilize opines as nutrient.
 Important genes encoded by Ti plasmid
1. Cytokinins
(plant hormone for cell plant division and tumorous growth)
2. Enzymes for indoleacetic acid (auxin) synthesis
Another plant hormone (inducing stem and leaf elongation,
inducing parthenocarpy and preventing aging)
3. Enzymes for synthesis and release of novel plant
metabolites:
The opines (uniques amino acid derivatives) the
agrocinopines (phosphorylated sugar derivatives) .
 Opines and agrocinopines are NUTRIENTS for
A.tumefacies.
 They can not be used by other bacterial species It provides
unique niche for A.tumefaciens
 Vir Genes and their Functions
Vir Function
Gene
Vir A, Sense phenolic compounds from wounded plant cells and induce expression of
Vir G other virulence genes
VirD2 Endo nuclease; cuts T-DNA at right border to initiate T-strand synthesis

Vir D1 Topiosomerase; Helps Vir D2 to recognise and cleave within the 25bp
border sequence
Vir D2 Covalently attaches to the 5I end of the T-strand, thus forming the T-DNA
Complex. Also guides the T-DNA complex through the nuclear pores

Vir C Binds to the 'overdrive' region to promote high efficiency T-strand Synthesis

Vir E2 Binds to T-strand protecting it from nuclease attack, and intercalates with lipids
to form channels in the plant membranes through which the
T-complex passes

Vir E1 Acts as a chaperone which stabilises Vir E2 in the Agro bacterium

Vir B & Assemble into a secretion system which spans the inner and outer bacterial
Vir D4 membranes. Required for Export of the T-complex and Vir E2 into the plant
cell
 Particle Gun Method

 Mix 3 micro meter of gold

particles with 2.5 micro gram of
plasmid DNA in a 2.5M cacl2 &
0.1 M Spermidine solution.

 Mixture is vertexed for uniform

coating of DNA on gold particles.

 Gold particles with precipitated

DNA molecules is transferred on
to macro membrane

Source: I.S. Kategeri et.al 2007

TRANSFORMATION AND REGENERATION SCHEME FOR COTTON

Hypocotyl Ex plant Time Transformation /regeneration

stage

P1-AS 3days Co-cultivation

P1-c4k50 3-4 weeks Selection and growth of individual

transgenic events on the ex plant
P1c4K50 2 weeks Selection / proliferation of individual lines

P7-c4k50 4 weeks Selection / proliferation of individual lines

P7-c4k50 4 weeks Embryonic callus induction/proliferation

P7-C4k50 4 weeks Early embryogenesis

MSBOK 4 weeks Embryogenesis , embryo formation

& maturation &maturation

EG3 4 weeks Embryo germination

MS3 3-6 weeks Plantlet development

SOIL 2-6 weeks Plant

Source : E.Firoozabady et.al. 1987

 Characterization of Putative Transgenic Cotton
Plants at Molecular and Genetic level

Confirmation of cry1AC Gene Transformation In

Cotton Plants by
• DNA Isolation & PCR Analysis.
• Southern Blot Analysis.
• Western Blot Analysis.
• Insect Bio-Assays
 GENOMIC DNA ISOLATION AND PCR
ANALYSIS
• The plant DNA was extracted from transformed
plants and control plants by a modified method of
peterson et al., 1993
• The genomic DNA of T0 (primary transformants) and
T1 (first generation) plants was analyzed for the
presence of cry1 AC gene by performing PCR
( saiki et al., 1988). cry1 AC gene was PCR amplified
using following primers.
FORWARD PRIMER
5’ ACA GAA GAC CCT TCA ATA TC 3’
REVERSE PRIMER
5’ GTT ACC GAG TGA AGA TGT AA 3’
 Cont…
…
PCR amplification consisted of 3steps

• De-naturation at 94 0c 2min.
• Annealing at 55 0c, 1min.
• Extension at 720c, 1min.

This is repeated for 30 cycles

The amplified product was analyses on 1% agarose gel
Non transformed plants were used as negative control
SOUTHERN BLOT ANALYSIS
 Genomic DNA was isolated from leaf tissues according to
peterson et al 1993 from transgenic plants DNA was digested
with Hind III fractionated separately on 1% agarose gel.
 Transferred on to nylon membrane .
 DNA was fixed to the membrane by baking at 800c for 30
minutes and hybridized to DNA fragments’ labeled with
(P32) dCTP suing a random primer DNA labeling system.
 After hybridization reaction the membrane is washed to
remove the unbound probes.
 The membrane is now placed in close contact with an X-ray
film and incubated for desired period to allow images due to
radioactive probes formed on the film.
 The film is then developed to reveal distinct bands indicating
positions in the gel of the DNA fragments that are
complementary to the radioactive probe used in the study
SOUTHERN BLOT ANALYSIS
PROTIEN EXPRESSION ANALYSIS
The transformed plants were studied for the expression cry1
AC gene through
1. Bio assay against Helicoverpa armigera.
2. Western blot analysis.
Bio assay against Helicoverpa armigera
1. Insect bio assay of cry1 AC.
2. Fully expanded young leaves from transgenic cotton
plants was detached and placed in Petri dish 9 cm in
diameter.
3. Non transgenic parent plants were used as control
each leaf was inoculated with 8 first in star of larvae
of Helicoverpa armigera 5 days later the amount of
leaf consumed and larval mortality was find out.
WESTERN

BLOT

ANALYSIS
 Removal of Superfluous Genes Using
Inducible CRE/lox
inducer

gene to be unwanted
retained gene
LB RB

inducible lox
CRE site
recombinase

LB RB
INHEREITENT OF TRANSGENE
Stable integrated cry1AC gene inherits in Mendelian fashion
and usually show dominance.
Transfer of cry1AC gene from transformed cotton to
Elite cultivar
Back cross method is followed.
 cry1AC gene is dominant
Elite cultivar(rr) susceptible to boll worms with good
agronomic performance used as recurrent parent.
Transformed cotton9(RR) with poor agronomic
performance is used as non-recurrent parent.
Approval Process Of Bt-Cotton/ Any GMO
Applicant

IBSC - To note, approve, recommend to RCGM

RCGM –MLT, LST & Biosafety data

GEAC – Approve for large scale use & release

ICAR - Agronomic data & Commercial release.

Final Release for Commercial Agriculture

 Post release monitoring
1. Director of Extension, SAU, Nodal person Team Leader

3. Plant Breeder (concerned crop), SAU - Member

5. Entomologist- Head of the Department - Member

or Nominee State Agriculture University

4. Agronomist- Head of the Department - Member

or Nominee State Agriculture University

5. Pathologist- Head of the Department - Member

or Nominee State Agriculture University

6. Subject matter specialist relevant to - Member

transgene (Biotechnologist)

7. Biostatistician - Member
 Benefits of Bt Cotton Varieties

•Safety benefits:
Reduction in the use of chemical insecticides.
Reduced trips across the field.
Reduced worker exposure to insecticides.
Reduced chemical load on the environment.

•Economic benefits:
Increased yields.
Decreased insect control costs.

•Limitations of Bt cotton varieties:

Development of resistance to Bt toxin in insect population.
Varied expression of Bt gene.
Silencing of Bt gene.

Source: F.J. Perlak . et.al.2001

Insect Resistance To CRY Proteins-
Management Plans
Crop refuge method:
 Includes cultivation of Bt varieties with Non Bt
varieties
 Two choices of refuge planting include.
 5% of non Bt cotton crop area.
 No pesticide is sprayed.
 20% or more non Bt cotton crop area.
 Chemicals are sprayed except Bt formulations.
 CRY protein susceptible bollworm genotypes (SS, Ss)
 CRY protein resistance boll worm genotypes (ss)
Source: F.J. Perlak . et.al.2001
 Seed Companies
 MAHYCO.  Krishidhan Seeds Pvt. Ltd.
 Ankur Seeds Ltd  Pravardhan Seeds Ltd.
 J.K. Agri Genetics Seeds  Vikki Agro tech Pvt. Ltd
Ltd.  Emergent Genetics
 Nath Seeds Ltd.  Krishidhan Seeds Pvt. Ltd.
 Nuziveedu Seeds Ltd  Prabhat Seeds Ltd.
 Rasi Seeds Ltd  Tulasi Seeds Pvt. Ltd.
 Vikram Seeds Pvt. Ltd
 Ajeet Seeds Ltd.
 Ganga Kaveri Seeds Pvt.
Ltd
 Pre Release Monitoring: MLT & LST
Director of Research, SAU, Nodal person - Team Leader
Plant Breeder (concerned crop), SAU - Member
Entomologist- Head of the Department - Member
or Nominee SAU
Agronomist- Head of the Department - Member
or Nominee SAU
Pathologist- Head of the Department - Member
or Nominee SAU
Subject matter specialist - Member
Relevant to the transgene (Biotechnologist).
Joint Director/ Deputy Director, State - Member
Agriculture Department
Agriculture Officer of the concerned district/ - Member
State Agriculture Department
Nominee of RCGM - Member
Nominee of GEAC - Member
 Conclusion
• Bt cotton provides control of bollworms that is superior to
chemical insecticides.
• It allows the grower the option of reducing his insect
control costs & increasing his cotton yield.
• The reduction of chemical insecticides use results in less
insecticidal exposure for the farmer and his surrounding
community.
• Reduction in production costs through use of Bt cotton
allows growers to remain competitive in the global market,
provides stability and sustainability in cotton production.
• Thus Bt cotton is a glimpse of providing safe, improved
alternatives for agriculture production through the use of
new technology.
Thank You…