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Write-up Of Microbiology


2 Acknowledgement I am extremely grateful and remain indebted to our guide Dr. He has been very co-operative throughout this project work. I am thankful to him for his constant constructive criticism and invaluable suggestions. which benefited me a lot while preparing presentation. JOGINDER SINGH PANWAR for being of inspiration and for his constant support in the Design. He has been a constant source of inspiration and motivation for hard work. Through this column. co-operation. I would also like to thanks my friends Rumeet Kaur & Sahil Sharma who helped me throughout this term paper. Dawinder kaur (RP7010A11) . Without whose co-operation this term paper cannot get completed. Implementation and Evaluation of this presentation. it would be my utmost pleasure to express our warm thanks to him for his encouragement.

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 .3 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.

including organic farming. storedproduct dusts. • Bt cotton=cotton plant + bt gene Cultivated in the U. thuringiensis is widely used as a larvicide against mosquito larvae. deciduous and coniferous leaves. • Extremely well-known toxin in terms of human health & environmental safety • B. • First pesticide producing crop:-Potato plants producing Bt toxin (1995) were approved safe by the Environmental Protection Agency. 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.. Bt Potato and Bt cotton (1996) were being grown by farmers in the USA. • Other Bt crops-Bt Maize. • The prevalence of this strain is not restricted and has been isolated worldwide from many habitats.4 to 5.S.7 million basepairs. insects. India. where it is also considered an environment friendly method of mosquito control. • Produces proteins that are toxic to insects called as cry proteins.4 INTRODUCTION-BT COTTON Bt cotton is based on the crystalline protein produced by Bacillus thuringiensis (Bt). including soil. South Africa. . with a genome size of 2. • Commonly used in garden sprays & for commercial agriculture. Mexico. • Bacillus thuringiensis (Bt) are the gram positive bacteria. China. Argentina. Australia.

followed by insertion of the delta-endotoxin protein into the membrane leading to pore formation. 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. (1) The core of the molecule is built from five sequence blocks. The long. The seven alpha helix domain I structure resembles the pore forming domain of Colicin A and is important for the membrane insertion step. a seven helix bundle. Domain III of the Bt toxin (see below) may also be a determinant of insect specificity/receptor binding. the plant lectin jacalin and the Maclura pomifera agglutinin. (Domain I). • • • • • . since in the water soluble preinsertion form all the hydrophobic faces of the Cry Domain I helical bundle face inwards. hydrophobic and amphipathic alpha helices of Domain I is equipped for transmembrane pore formation.5 Structure of the Cry Proteins • • • The Cry toxin has three domains which are. 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. The three beta sheet structure (beta prism) of domain II is involved in receptor binding and specificity determination. Membrane penetration occurs in two steps: binding to a specific receptor exposed on the membrane surface. from N to C terminus. 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. a triple anti-parallel beta sheet domain (Domain II) and a beta-sheet sandwich (Domain III). a major conformational change must occur.

a seven helix bundle. A recent discovery that domain III is also a lectinlike domain suggests that the insecticidal specificity of these toxins could be determined by two lectin-like domains acting in concert or independently. 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.6 • Schematic ribbon diagram structure of the CryA toxin-The Cry toxin has three domains which are. . from N to C terminus. (Domain I).

Action mechanism of cry proteins• • • • During sporulation. the alkaline pH solubilizes the crystal. Cry proteins are divided into 5 main groups on basis of insecticidal activities :Gene Cry I Cry II CryIII CryIV Target Lepidoptera(moths and butterflies Diptera(flies and mosquitoes). The family of genes coding for this toxin is the Cry gene family. Upon completion of sporulation the parent bacterium lyses to release the spore and the inclusion. Coleoptera (beetles). The family of genes coding for this toxin is the Cry gene family. it synthesizes a cytoplasmic inclusion containing one or more proteins that are toxic to insect larvae. thuringiensis serves as an important reservoir of Cry toxins for production of biological insecticides and insect-resistant genetically modified crops. B. CRY PROTIENS AND THEIR ACTION ON PEST • These toxins can be categorized under the d-endotoxins. Diptera & nematodes(rats) CryV Nematodes(rats) Thus. In these inclusions. which is highly specific to only certain insects. which is highly specific to only certain insects. the toxins exist as inactive protoxins. The protoxin is then converted in to an active toxin after processing by the host proteases present in the midgut. When the inclusions are ingested by insect larvae.7 The Cry Gene Family: These toxins can be categorized under the delta -endotoxins. A common characteristic of the cry genes is their expression during the stationary phase. .

Due to their high specificity for these unique receptors on the membrane of the gut epithelial cells.8 • 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.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 . The fact that they are proteins ensures that they are readily biodegraded • >>>Status of primary Pests. these toxins (delta-endotoxins) are harmless to non-target insects and the end-user and are compatible with integrated pest management programs.

& transferred to cotton cells grown in tissue culture The cells are grown into a plant & then.9 Cry-gene Coker 312 Process of transformation of cry. like Agrobacterium tumefasciens. after testing.gene The gene of interest is spliced out of the bacterium using a vector. plants are back-crossed into commercial lines to make new varieties .

10 >>>Spectrum of Activity of Cry1Ac for different pests Excellent Control Trichoplusia ni Heliothis virescens Spodoptera exigua Spodoptera frugiperda Spodoptera ornithogalli Bucculatrix thurberiella Helicoverpa zea (pre-bloom) No Control Estigmene acrea Pink Bollworm (PBW). Helicoverpa zea Estigmene acrea Trichoplusia ni Pseudoplusia includens Spodoptera exigua Beneficial Insects Agrotis & Feltia spp. No Control >>>% Efficiency of bt cotton plants . our principal pest Pseudoplusia includens >>>Spectrum of Activity of (Cry1Ac + Cry2Ab) for different pests Excellent Control Pectinophora gossypiella Heliothis virescens Bucculatrix thurberiella Marmara spp.

591 100 100 100 100 100 100 99.67% Both Genes(Cry1Ac + Cry2Ab )=100% Bt Cotton in India .11 Cry1Ac + + + + + + + + Cry2Ab + + + + + Variety DP50B 985X 985BX DP33B DP448B DP458BR DP33BX SG 215BR SG 125X SG 125BX A djusted 100 99.758 100 It is concluded from this data – • • • Cry1Ac= 100% Cry2Ab =99.

Bt Cotton Study conducted by Greenpeace in Karnataka 1. GEAC. ICAR. 77% of the farmers interviewed in the study reported Bollworm infestation in the Bt cotton plants. Studies of the molecular characterization and stability of the Cry1Ac gene were also carried out. GEAC Studies undertaken 1995-1996 Application and permit for importation of Bt cotton seed containing the Cry1Ac gene Years 1996-2000 Greenhouse breeding for integration of the Cry1Ac gene into Indian germplasm. MEC Submission of final biosafety. RCGM = Review Committee for Genetic Modification (constituted by DBT). vigor. effect of the pollen on insects and nontarget organisms.12 Bt cotton seeds were first tested in India for germination. MEC of first-generation Bt cotton hybrids. MEC 2002ongoing a DBT = Department of Biotechnology. . GEAC = Genetic Engineering Approval Committee. environmental safety. ICAR. and stock increase 1996-2000 Limited field studies for potential of pollen escape. and changes in the soil microbial flora. crossability of the transgenic pollen with the nontransgenic relative and near relatives. persistence of the transformed plants. DBT. seed purification. and persistence 1998-2001 Biochemical and toxicology studies 1998-2000 Multilocation field trials: agronomic and entomology performance RCGM (DBT). These studies were conducted under the unique environmental conditions of India and with the Bt trait in Indian germplasm. and insect efficacy. MEC = Monitoring & Evaluation Committees (constituted by GEAC and RCGM). conducted by ICAR RCGM (DBT). aggressiveness. toxicity. including tests of gene flow. conducted by Mahyco and State agriculture universities 2000-2001 Soil rhizosphere evaluations and protein expression analyses from multilocation field trials 2001 2002 Advanced stage multilocation field performance trials of firstgeneration Bt cotton hybrids. Other experiments were conducted to confirm the environmental safety of Bt cotton. weediness characteristics. gene efficacy GEAC and performance documentation to GEAC. GEAC GEAC. ICAR = Indian Council of Agriculture Research. commercial release of first-generation Bt cotton hybrids by GEAC Continued field performance trials of second-generation Bt cotton hybrids for regulatory approval RCGM (DBT). summary for regulatory processes leading to commercial release of Bt cotton in India. as well as feeding studies and tests of food and feed safety. Government of India oversight committeesa DBT DBT RCGM (DBT) RCGM (DBT). and allergenicity.

tightly packed and picking takes longer. In four regions. 600-1200 higher) as compared to the Cotton hybrids. Monsanto scientists found that the pink bollworm had become resistant to Bt cotton in parts of Gujarat. the market value that the farmers are getting for Bt Cotton is far lower than for Non Bt hybrid • Some other limitations 1.00 /packet . Amreli.Bt hybrids were more or less the same. This was despite a fall in the number of sprays compared to the Non-Bt hybrids. 7.This is because of the shorter staple fibers and the relatively dull colour of the cotton fiber. In terms of economic viability it is clear that Bt Cotton is a much more expensive alternative for the farmers. have become significant problems. Secondary pests-Chinese farmers have found that after seven years of growing BT cotton the populations of other insects other than bollworms. A majority of the farmers reported an increase in pesticide costs in the case of Bt cotton . which were more potent and expensive. Furthermore.The cost of the non Bt Hybrid seed is about Rs 450. The yields for Bt Cotton and non. however in Raichur non Bt hybrids fared better. such as mirids. The market value for Bt Cotton is lower than non-Bt Cotton hybrids by Rs 200-800. The total cost of fertilisers was higher in the case of Bt Cotton plants (Rs. 2. . Primary insects resistance to bt cotton-In November 2009. Low level expression of cry genes. 6. Bhavnagar.13 2. India. 3. water that have been mentioned before. fertilizers. 5.Similar problems but with mealy bugs have been reported in India 3.For example in cry1(A)b contains (I) localized A+T regions (ii)18 polyadenylation sites (iii) at 13 regions. • Apart from the input costs such as pesticides. Junagarh and Rajkot the crop is no longer effective at killing the pests. Farmers reported an increase in the labour cost of Bt cotton as the cotton bolls are smaller in size.AUUUA sequence instabilize the coding mRNA. the Bt Cotton seed itself costs Rs 1600. 4. This increase is cost was related to the nature of pesticides that were used by the farmers.

one of the original paper's authors spoke out against Berkeley accepting a multimillion dollar research grant from the Swiss pharmaceutical company. there still remains a controversy over the highly unorthodox retraction on the part of Nature. In 1998. It appears that the initial study was flawed by faulty pollen-collection procedure. However. Novartis. further research confirmed initial findings concerning contamination of natural maize by transgenic maize. 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.Wild maize genetic contamination A study in Nature reported that Bt-containing maize genes was contaminating maize in its center of origin. Chapela. Advantages of bt-cotton . A subsequent large-scale study. The weight of the evidence is that Bt crops do not pose a risk to the monarch butterfly. in 2005.Nature later "concluded that the evidence available is not sufficient to justify the publication of the original paper. 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. researchers fed non-toxic pollen mixed with anther walls containing Bt toxin. The actual cause of CCD remains unknown. 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.14 Possible problems 1." However. 3. and scientists believe that it may have multiple causes. failed to find any evidence of contamination in Oaxaca. Initial speculation on possible causes ranged from cell phone and pesticide use to the use of Bt resistant transgenic crops. 2.

The toxin expression can be modulated by using tissue-specific promoters. The toxin expression is contained within the plant system and hence only those insects that feed on the crop perish. 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. reduction in expense of insecticide application. Bt cotton has revolutionized our ability to reduced insecticide inputs by over 60%. 3. At the end.000/acre) in farm income (compared to non-Bt cotton). The level of toxin expression can be very high thus delivering sufficient dosage to the pest. . There are several advantages in expressing Bt toxins in transgenic Bt cotton: 1. Conclusions • • • • • The use of Bt cottons has provided the first larvicidal and selective approach to controlling pest of cotton.500-4. Problems like insect resistance against cry proteins can be overcome by using combination of cry genes (Cry1Ac + Cry2Ab). Future transgenic products for insect control in cotton should be independently & scientifically tested. No survivors have been found in field studies. significantly higher yield and boll retention (compared to control or non-Bt cotton). and replaces the use of synthetic pesticides in the environment.2. 2.15 Bt Cotton—Agronomic Benefits Trials conducted in several locations in 1998/99. The control provided by Bt cottons approaches immunity. 1999/2000. 2000/01. and 2001/02 demonstrated the following agronomic benefits of Bt cotton: • • • • • good control of bollworm species in different growing areas. additional revenue (Rs. and no adverse effect on nontarget insects or adjacent non-Bt cotton crops.

dawn.htm http://fbae.htm References http://fen.html .org/2009/FBAE/website/

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