M.Sc. (Ag.) Course SeminarM.Sc. (Ag.) Course Seminar

THE SCIENCE BEHIND THE THE SCIENCE BEHIND THE DEVELOPMENT OF Bt COTTONDEVELOPMENT OF Bt COTTON

Speaker: Koteswara Rao. Potla M.Sc. (Ag.) Final

Supervisor : Prof. L. C. PrasadCo-supervisor : Prof. J. P. Lal

Dept. of Genetics & Pl. BreedingInst. 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

Cultivation Year Total cotton cultivation area million acres

Transgenic cotton area million acres

Percentage of 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 5.922005-06 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

INCREASE IN ADOPTION OF Bt COTTON AREA IN INDIA

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

LIMITATIONS• High cost • Development of resistance in insect population

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

• 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.

Bacillus thuringiensis

Source: Herman HOFTE et.al1986

Cry genes Classification Based On Insect Specificity & Sequence Homology

Genes Proteins size (KDa)

Target

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

• Sensitivity to UV radiation and heat desiccation• Performance is not always consistent (affected by

environmental fluctuations)• Incomplete coverage

Limitations of Bt Sprays

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

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

CRY Protein Structure

Source: Herman HOFTE et.al1986

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

MODE OF ACTION OF Bt TOXIN

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

Mechanism of Toxicity of Bt

Cont……

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

The Science Behind The Development of Bt Cotton

1. cry1AC gene

2. CaMV 35S promoter

3. npt II antibiotic resistance gene (genetic marker)

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

Three primary components of the genetic package inserted into Bt cotton plants include.

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

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

Cont……

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

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

Gene Cloning

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

Tumor-producing genes

Virulence region

Opine catabolism

ORI

T-DNAregion DNA between

L and R borders is transferred to plant as SsDNA;

T-DNA encoded genes can be substituted by target genes

1. 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.

2. 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. 3. T-DNA encodes genes for opine synthesis and for tumor production.

4. 4. oc (opine catabolism) genes carried on the pTi and allows the bacterium to utilize opines as nutrient.

Ti plasmids and the bacterial chromosome act in concert to transform the plant

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

Important genes encoded by Ti plasmid

Vir Genes and their Functions

Vir Gene

Function

Vir A,

Vir G

Sense phenolic compounds from wounded plant cells and induce expression of 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 theT-complex passes

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

Vir B & Vir D4

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

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

Particle Gun Method

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

I. The plant DNA was extracted from transformed plants and control plants by a modified method of peterson et al., 1993

II. 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’

PCR amplification consisted of 3steps

1. De-naturation at 94 0c 2min.

2. Annealing at 55 0c, 1min.

3. 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

Cont……

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

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 armigera1. 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

loxsite

inducibleCRE

recombinase

unwanted

gene

gene to beretainedLB RB

LB RB

inducer

INHEREITENT OF TRANSGENE

Stable integrated cry1AC gene inherits in Mendelian fashion

and usually show dominance.

Transfer of cry1AC gene from transformed cotton to

Elite cultivarBack 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 monitoring1. Director of Extension, SAU, Nodal person Team Leader

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

3. 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

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

Insect Resistance To CRY Proteins-Management Plans

Seed Companies

MAHYCO. Ankur Seeds Ltd J.K. Agri Genetics Seeds

Ltd. Nath Seeds Ltd. Nuziveedu Seeds Ltd Rasi Seeds Ltd Ajeet Seeds Ltd. Ganga Kaveri Seeds Pvt.

Ltd

Krishidhan Seeds Pvt. Ltd. Pravardhan Seeds Ltd. Vikki Agro tech Pvt. Ltd Emergent Genetics Krishidhan Seeds Pvt. Ltd. Prabhat Seeds Ltd. Tulasi Seeds Pvt. Ltd. Vikram Seeds Pvt. Ltd

Pre Release Monitoring: MLT & LST

Director of Research, SAU, Nodal person - Team LeaderPlant Breeder (concerned crop), SAU - MemberEntomologist- Head of the Department - Member or Nominee SAUAgronomist- Head of the Department - Member or Nominee SAUPathologist- Head of the Department - Member or Nominee SAUSubject matter specialist - Member Relevant to the transgene (Biotechnologist).Joint Director/ Deputy Director, State - Member Agriculture DepartmentAgriculture Officer of the concerned district/ - Member State Agriculture DepartmentNominee of RCGM - MemberNominee 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…Thank You…