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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 hisconstant support in the Design, Implementation and Evaluationof this presentation. I am thankful to him for his constantconstructive criticism and invaluable suggestions, whichbenefited me a lot while preparing presentation. He has been aconstant source of inspiration and motivation for hard work. Hehas been very co-operative throughout this project work.Through this column, it would be my utmost pleasure to expressour warm thanks to him for his encouragement, co-operation.

I would also like to thanks my friends Rumeet Kaur & SahilSharma who helped me throughout this term paper. Withoutwhose 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 differentpests

•

% 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, aseven helix bundle, (Domain I), a triple anti-parallel beta sheetdomain (Domain II) and a beta-sheet sandwich (Domain III). (1)

• The core of the molecule is built from five sequence blocks, whichare a highly conserved feature of all the Bt toxins indicating that allthe proteins in this Cry family will adopt the same general fold.

• The long, hydrophobic and amphipathic alpha helices of Domain I isequipped for transmembrane pore formation. The seven alpha helixdomain I structure resembles the pore forming domain of Colicin Aand 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 oligomerichelical bundle pore with a radius of 5-10 Angstroms.

• Before one or more of these Cry helices can insert into themembrane 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 helicalbundle 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 poreformation.

• The three beta sheet structure (beta prism) of domain II is involved inreceptor binding and specificity determination. This is further supported by reports that domain II shared the same structural foldwith three carbohydrate binding proteins: the vitelline membraneouter layer protein I from hen's eggs, the plant lectin jacalin andthe 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 tripleanti-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 toxinsand the three dimensional structures of two known lectins suggests thatinsecticidal 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 couldbe 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 ishighly specific to only certain insects. The family of genes coding for thistoxin 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 geneticallymodified crops.

CRY PROTIENS AND THEIR ACTION ON PEST

• These toxins can be categorized under the d-endotoxins, which ishighly 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 torelease the spore and the inclusion. In these inclusions, the toxinsexist as inactive protoxins.

• When the inclusions are ingested by insect larvae, the alkaline pHsolubilizes the crystal.

• The protoxin is then converted in to an active toxin after processingby the host proteases present in the midgut.

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• It has been indicated that the activated toxin binds to insect-specificreceptors exposed on the surface of the plasma membrane of midgutepithelial cells and then inserts into the membrane to createtransmembrane pores that cause cell swelling and lysis andeventually death of the insect.

•Due to their high specificity for these unique receptors on themembrane of the gut epithelial cells, these toxins (delta-endotoxins)are harmless to non-target insects and the end-user and arecompatible with integrated pest management programs. The fact thatthey 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 todevelop 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, likeAgrobacterium tumefasciens, &transferred to cotton cells grown intissue cultureThe cells are grown into a plant & then,after testing, plants are back-crossed

into commercial lines to make newvarieties

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>>>Spectrum of Activity of Cry1Ac for differentpests

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

different pests

>>>% Efficiency of bt cotton plants

ExcellentControl

NoControl

Heliothis virescens

Helicoverpa zea(pre-bloom)

Bucculatrix thurberiella

Spodoptera exigua

Estigmene acrea

Trichoplusia ni

Spodoptera frugiperda

Spodoptera ornithogalli

Pseudoplusia includens

Pink Bollworm(PBW), our principal pest

ExcellentControl

NoControl

Heliothis virescens

Pectinophora gossypiella

Helicoverpa zea

Bucculatrix thurberiella

Spodoptera exigua

EstigmeneacreaTrichoplusiani Pseudoplusiaincludens

Agrotis &Feltia spp.

BeneficialInsects

Marmara spp.

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Cry1Ac Cry2Ab Variety

+ - DP50B

- + 985X

+ + 985BX

+ - DP33B

+ - DP448B

+ - DP458BR

+ + DP33BX

+ - SG215BR

- + SG125X

+ + SG125BX

Adjusted

100

99.591

100

100

100

100

100

100

99.758

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 soilmicrobial 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.

Years Studies undertakenGovernment of Indiaoversight committeesa

1995-1996 Application and permit for importation of Bt cotton seedcontaining the Cry1Ac gene

DBT

1996-2000 Greenhouse breeding for integration of the Cry1Ac gene intoIndian germplasm, seed purification, and stock increase

DBT

1996-2000 Limited field studies for potential of pollen escape,aggressiveness, and persistence

RCGM (DBT)

1998-2001 Biochemical and toxicology studies RCGM (DBT), GEAC

1998-2000 Multilocation field trials: agronomic and entomology performanceof first-generation Bt cotton hybrids, conducted by Mahyco and

State agriculture universities

RCGM (DBT), MEC

2000-2001 Soil rhizosphere evaluations and protein expression analysesfrom multilocation field trials

RCGM (DBT), GEAC

2001 Advanced stage multilocation field performance trials of first-generation Bt cotton hybrids, conducted by ICAR

GEAC, ICAR, DBT,MEC

2002 Submission of final biosafety, environmental safety, gene efficacyand performance documentation to GEAC; commercial release of first-generation Bt cotton hybrids by GEAC

GEAC

2002-ongoing

Continued field performance trials of second-generation Btcotton hybrids for regulatory approval

RCGM (DBT), GEAC,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 andRCGM).

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 Rs200-800.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, Monsantoscientists found that the pink bollworm had become resistant to Btcotton in parts of Gujarat, India. In four regions, Amreli, Bhavnagar,Junagarh and Rajkot the crop is no longer effective at killing thepests.

2. Secondary pests - Chinese farmers have found that after seven yearsof growing BT cotton the populations of other insects other thanbollworms, such as mirids, have become significantproblems.Similar problems but with mealy bugs have been reportedin 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 13regions,AUUUA sequence instabilize the coding mRNA.

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Possible problems

1. Lepidopteran toxicityThe most publicised problem associated with Bt crops is the claim thatpollen from Bt maize could kill the monarch butterfly This report waspuzzling 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-collectionprocedure; researchers fed non-toxic pollen mixed with anther wallscontaining Bt toxin. The weight of the evidence is that Bt crops do not posea risk to the monarch butterfly.

2.Wild maize genetic contamination

A study in Nature reported that Bt-containing maize genes wascontaminating maize in its center of origin.Nature later "concluded that theevidence available is not sufficient to justify the publication of the originalpaper." However, there still remains a controversy over the highlyunorthodox retraction on the part of Nature. In 1998, Chapela, one of theoriginal 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 initialfindings 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) isaffecting bee hives all over North America. Initial speculation on possiblecauses ranged from cell phone and pesticide use to the use of Bt resistanttransgenic crops. The Mid-Atlantic Apiculture Research and Extension

Consortium published a report in March 2007 that found no evidence thatpollen from Bt crops is adversely affecting bees. The actual cause of CCDremains unknown, and scientists believe that it may have multiple causes.

Advantages of bt-cotton

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http://en.wikipedia.org/wiki/Monarch_butterfly

http://en.wikipedia.org/wiki/Anther

http://en.wikipedia.org/wiki/Novartis

http://en.wikipedia.org/wiki/Transgenic_maize

http://en.wikipedia.org/wiki/Colony_Collapse_Disorder

http://en.wikipedia.org/wiki/Honey_bee

http://en.wikipedia.org/wiki/Mid-Atlantic_Apiculture_Research_and_Extension_Consortium


http://en.wikipedia.org/wiki/Monarch_butterfly

http://en.wikipedia.org/wiki/Anther

http://en.wikipedia.org/wiki/Novartis

http://en.wikipedia.org/wiki/Transgenic_maize

http://en.wikipedia.org/wiki/Colony_Collapse_Disorder

http://en.wikipedia.org/wiki/Honey_bee





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 sufficientdosage to the pest.2. The toxin expression is contained within the plant system and henceonly those insects that feed on the crop perish.3. The toxin expression can be modulated by using tissue-specificpromoters, and replaces the use of synthetic pesticides in the environment.

Conclusions

• The use of Bt cottons has provided the first larvicidal and selectiveapproach to controlling pest of cotton.

• The control provided by Bt cottons approaches immunity. Nosurvivors have been found in field studies.

• Bt cotton has revolutionized our ability to reduced insecticide inputsby over 60%.

• Future transgenic products for insect control in cotton should beindependently & scientifically tested.

• Problems like insect resistance against cry proteins can beovercome by using combination of cry genes (Cry1Ac + Cry2Ab).

At the end, it is concluded that use of Bt cotton is best environmentfriendly method to control pest and can be further modified to overcomethe problems related to it.

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References

http://www.agbioforum.org/v7n12/v7n12a04-zehr.htmhttp://fbae.org/2009/FBAE/website/our-position-bt-cotton.htmlhttp://fen.wikipedia.org/wiki/Bacillus_thuringiensishttp://www.dawn.com/2008/12/15/ebr3.htmhttp://ww.nature.com/nbt/journal/v27/n1/full/nbt0109-9.html

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http://www.agbioforum.org/v7n12/v7n12a04-zehr.htm

http://fbae.org/2009/FBAE/website/our-position-bt-cotton.html

http://www.dawn.com/2008/12/15/ebr3.htm

http://www.agbioforum.org/v7n12/v7n12a04-zehr.htm

http://fbae.org/2009/FBAE/website/our-position-bt-cotton.html

http://www.dawn.com/2008/12/15/ebr3.htm



and many more…..BOOK-Biotechnology by-B.D.SINGH

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