Rumesh cdt seminar2

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TRANSGENIC COTTON FOR INSECT CONTROL:

TRANSGENIC COTTON FOR INSECT CONTROL Rumesh Ranjan Adm No. 2010A244M Dptt.Genetics & Plant Breeding CCSHAU, Hisar .

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INTRODUCTION

Cotton: Trends in India :

Cotton: Trends in India India has a largest area of cotton than any country in the world. In 2010, the total hectarage of cotton in India was at 11 million hectares approximately and farmed by 6.3 million farmers(Indian stat.com). A crop of significant economic importance, valued at over Rs. 15000 crores.

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continued Damage by Insect pests reduce yields by 50%. Farmers spend most money on controlling bollworms; up to 15 sprays and over Rs. 12 billion. ( Gupta et.al., 2001.) Insects resistance to chemicals is increasing.( Ramasubramanyam,2004). Yields are reduced in spite of sprays.

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55% of total insecticides used is on Cotton mainly on bollworm control. Helicoverpa a common major pest, causing epidemics resulting into calamity. Other pests: Jassids, Aphids, White flies and Thrips. Rs. 33.8 billion value pesticide are used in agriculture(Agrilook,2001),with Rs 16 billion worth on cotton alone. Pesticide Usage

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Change in pest Scenario a. Excessive and indiscriminate use of pesticides. b. Improper choice, quantity and application of pesticides. c.  Use of pesticide mixtures . (Has lead to) a. Resurgence of minor pests. b. Resistance to pesticides. c. Increase in cost of protection. Constraints of Cotton Production

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COMMON INSECT PESTS IN COTTON AMERICAN BOLLWORM SPOTTED BOLLWORM PINK BOLLWORM COTTON STEM WEEVIL

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COMMON INSECT PESTS IN COTTON WHITE FLY COTTON GRASSHOPPER RED COTTON BUG COTTON LEAF ROLLER DUSKY COTTON BUG COTTON LEAF HOPPER

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CONVENTIONAL BREEDING Vs NON-CONVENTIONAL BREEDING

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Insect Resistance Host Plant Character Non preference Preference Bollworms Smooth Leaves Hairy Leaves Nectariless Nactaried Thick Boll Rind Thin Boll Rind Hard Boll Rind Soft Boll Rind Okra Leaf Normal Leaf Long Pedicel Short Pedicel Boll Weevil Frego Bract Normal Bract Red Plant Body Green Plant Body Hairy Leaves Smooth Leaves Leathery Leaves Succulent Leaves Jassids Hairy Leaves Smooth Leaves Leathery Leaves Succulent Leaves White Fly Smooth Leaves Hairy Leaves Thin Leaves Normal Leaves Table: Non preference mechanism of insect resistance in cotton

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INSECT PESTS SOURCE OF RESISTANCE/ TOLERANCE BOLL WORMS G. somalense , G. sturtianum , G. australe , G. bickii , G. harknesii , G. thurberi , G. tomentosum , G. davidsonii JASSIDS G.anomalum , G. davidsonii , G. tomentosum , G. lobatum WHITEFLY G. longicalyx , G.armourianum THRIPS G. davidsonii , G. tomentosum SPOTTED BOLLWORMS G. anomalum Table: Wild species as a source of insect resistance www.cicr.org.in

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Conventional plant breeding Desired gene Commercial variety Source variety / species Result By comparison, biotechnology offers more precise plant breeding... X Modern biotechnology

TRANSGENIC COTTON:

TRANSGENIC COTTON

Transgenic Plant:

Transgenic Plant The plant obtained through genetic engineering contain a gene or genes usually from an unrelated organism; such gene are called transgenes, and the plant containing transgene are known as transgenic plant. are also known as genetically modified or GM crops.

Application of Transgenic:

Application of Transgenic Improving yield Nutritional improvement Increasing shelf life of fruits and vegetables by delayed ripening Conferring resistance to insects, pests and viruses Tolerance to abiotic stresses (drought, salt, water-logging) Herbicide tolerance Edible vaccines

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Plant Transformation Methods Physical Chemical Biological Microinjection Biolistics - gene gun/ particle bombardment Electroporation Macroinjection Silica/carbon fibers Lipofection PEG DEAE-dextran Calcium phosphate A. Tumefaciens Rhizogenes

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BIOLISTIC GENE GUN METHOD

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What is Bacillus thuriengiensis or Bt ?

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In 1901, Japanese biologist, Ishiwata Shigetane discovered this bacillus from diseased Bombyx mori larvae. ♣ Background & History of Bt In 1950 , Studies showed that proteins produced by Bt bacteria kill insects . In 1938 , experimental Bt as a spray for insect control began in France . It was later called Bacillus thuringiensis or Bt by Ernst Berliner in 1911 ,who isolated it as the cause of a disease called schlaffsucht in flour moth caterpillars. .

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In 1958 , the first commercial Bt formulations were made available for field testing in the USA . The use of Bt toxin genes in genetically modified plants for pest control became an established field of research in the mid-1980s . From the mid-1990s , plants genetically modified to express the Bt toxin have become increasingly common, and are now grown widely in countries like USA, China, Australia, India, South Africa, Indonesia, Pakistan etc . In 1990 , Monsanto company in USA developed first transgenic plant of cotton . ☻

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It is an ubiquitous gram positive, rod shaped, aerobic, spore-forming bacterium. What is Bt ? Bacillus thuringiensis (Bt) is a spore forming bacterium that produces crystal protein (cry protein), which is toxic to many species of insects ( Lepidoptera, Diptera , Coleoptera ). Bacillus thuringiensis viewed by phase contrast microscopy. The vegetative cells contain endospores (phase bright) and crystals of an insecticidal protein toxin (delta endotoxin ). Most cells have lysed and released the spores and toxin crystals (the structures with a bipyramidal shape). Fig. :

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Bt crystals, also referred as insecticidal crystal proteins (ICP) , are protein crystals formed during sporulation in some Bt strains. Bt produces proteins that aggregate to form a crystal. What are Bt crystals ? These crystal proteins are toxic to very specific species of insects yet harmless to humans and the natural enemies of many crop pests (benenificial insects).

Cry Protein Structure:

Cry Protein Structure Domain I = responsible for inserting into the gut membrane and creating a pore where ions can pass freely Domain II = responsible for recognition of the receptors on the epithelial lining of the midgut Domain III = responsible to binding and protect the endotoxin from cleavage by gut proteases and insect specificity

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Gene Crystal shape Protein size (kDa) Insect activity cry I [several subgroups: A(a), A(b), A(c) , B, C, D, E, F, G,H,I,J,K ] bipyramidal 130-138 lepidoptera larvae cry II [subgroups A(b) , B, C ] Cuboidal 69-71 lepidoptera and diptera cry III [subgroups A, B, C,D,E ] flat/irregular 73-74 coleoptera cry IV [subgroups A, B, C, D ] bipyramidal 73-134 diptera cry V-IX Various 35-129 Nematodes Bt toxins and their classification (1) Cry (crystal) toxins, encoded by different cry genes, and according to Cry gene Bt are classified. (2) Cyt ( cytolytic ) toxins, which can augment the Cry toxins, enhancing the effectiveness of insect control. Over 50 of the genes that encode the Cry toxins have now been sequenced and Crickmore (1998) has proposed nomenclature of endo -toxin based on amino acid homology . Source: Singh, B.D., Expanding Horizon.

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T ransgenic B t C otton

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Transgenic Bt Cotton Bt cotton is genetically altered crop created by inserting a synthetic version of a gene from the naturally occurring soil bacterium, Bacillus thuringiensis (Bt). ► Technology Involved Bt gene – Cry1Ac 35S promoter – Cauliflower mosaic virus Antibiotics resistance against Kanamycin or Streptomycin, Spectinomycin respectively. (Anonymous, 2006 ) 1.The Bt gene that produces the desired lethal portion is joined to a marker gene for Antibiotics resistance. 2. Bt gene and marker is inserted into Plant Cell. 3. Plant cell are grown in the presence of antibiotics. 4. Cells that carry the Bt gene + antibiotic resistance gene survive and are grown into plant. Marker – npt ii gene (neomycin phosphotransferase II ) or aad (3”(9)-O- amino glycoside adenyltransferase

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How does Bt work? Bt has to be eaten to cause mortality. The Bt toxin dissolve in the high pH insect gut and become active. The toxins then attack the gut cells of the insect, punching holes in the lining. The Bt spores spills out of the gut and germinate in the insect causing death within a couple days. Even though the toxin does not kill the insect immediately, treated plant parts will not be damaged because the insect stops feeding within hours. Bt spores do not spread to other insects or cause disease outbreaks on their own. http:// www.bt.ucsd.edu/index.html Source:

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How does Bt work? Insect eats Bt crystals and spores The toxin binds to specific receptors in the gut and the insects stops eating. The crystals cause the gut wall to break down, allowing spores and normal gut bacteria to enter the body. The insect dies as spores and gut bacteria proliferate in the body. http:// www.bt.ucsd.edu/index.html Source:

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How does Bt work? Bt action is very specific Different strains of Bt are specific to different receptors in insect gut wall. Bt toxicity depends on recognizing receptors, damage to the gut by the toxin occurs upon binding to a receptor. Each insect species possesses different types of receptors that will match only certain toxin proteins, like a lock to a key. http:// www.bt.ucsd.edu/index.html Source:

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How does Bt work? Mismatching Receptor Toxins are released Matching Receptors http://www.bt.ucsd.edu/index.html Source:

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The major advantage of Bt cotton are summarized below: Inbuilt genetic resistance to bollworms and is very effective in controlling the yield losses caused by bollworms to a considerable extent . Use of Bt cotton reduces use of pesticides against bollworm resulting in reducing the cost of cultivation. It results in improvement of yield levels and also improves margin of profit to the farmers. It provides opportunities to grow cotton in areas of severe bollworm incidence. It promotes eco friendly cultivation of cotton and allows multiplication of beneficial insects i.e. parasites and predators of bollworms . It also reduces environmental pollution and risk of health hazards associated with use of insecticides because in Bt cotton the insecticides are used under control.

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Chronology of Events Following several years of field trials with Bt cotton, based on the recommendations of RCGM, GEAC in its 32nd meeting on 26 March, 2002 approved the commercial cultivation of three Bt cotton hybrids: MECH-12 Bt, MECH-162 and MECH-184 Bt (Barwale et al., 2004). The sequence of events that led to the development and approval of these is listed below: 1996: Mahyco imported 100 g of Cocker-312 seed containing the cry1Ac gene from Monsanto, USA . Crossing with Indian cotton breeding lines to introgress cry1Ac gene and 40 elite Indian parental lines were converted for Bt trait. 1996–1998 : Greenhouse, risk-assessment studies, pollen escape studies, aggressiveness and persistence studies, biochemical analysis, toxicological studies and allergenicity studies. 1998–1999: Multi-location field trials were conducted at 40 locations in nine states to assess agronomic benefits and safety.

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1999–2000: Field trials repeated at 10 locations in six states. July 2000: GEAC gave approval for conducting large-scale field trials on 85 ha and also to undertake seed production on 150 ha. 2001: Large-scale field trials were conducted covering 100 ha. Field trials were also conducted by All India Coordinated Cotton Improvement Project of the ICAR. 2002: GEAC approved three Bt-cotton hybrids for commercial cultivation after taking into account the data on their performances. Source-Bt cotton in India:A staus report CONTINUED:

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PROBLEM INVOLVED

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2. Development of Resistance In Insect 4. Change In Pest Complex 5. Genetic Pollution 6. I llegal Cultivation of Bt Cotton 3. Low Bt Toxin Level 1. Refuge Crop

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(1) Pest larvae emerge and attempt to feed on Bt Crop (2 ) Pests ingest protein, and vast majority die. (3) A few rare resistant (RR) pests live and become adult (4 ) The RR pests encounter susceptible (SS) adult pests from a nearly refuge. When they mate, their RS offspring are susceptible. (5) The offspring mate and lay eggs, and the cycle begins again. Fig : Basic theory of Refuge and resistance for a genetically modified Bt crop As per recommendation of GEAC, each field of Bt cotton is to be surrounded by a belt of non-Bt cotton of the same variety to serve as ‘Refuge’ for bollworm. The size of the refuge belt should be 20% of total sown area with non-Bt Cotton. 1. REFUGE CROP: www.bt.ucsd.edu

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In India due to small land holdings with farmers, these norms are not followed in practice which could lead to rapid build-up of Bt toxin resistance in bollworm. The farmers are also not knowing about the actual mechanism behind this, so they do not follow this method, and it increases resisatance in insects indirectly. Fig. : Different types of Refuge in field www.bt.ucsd.edu

2. Development of Resistance In Insect. :

2 . Development of Resistance In Insect. Development of resistance is the major problem with Bt cotton. The most frequently observed mechanism of resistance among insect pests is the modification of the protease enzyme or by receptor site.

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Inactive Bt protoxin Enzymes from Insect gut Active delta - endotoxin Change the shape by Mutation Plant DNA with Bt gene

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3. Low Bt Toxin Level Sustainable expression of Cry 1Ac in Bt-cotton is crucial for its effectiveness in the control of lepidopteron pests, especially bollworm. The critical expression level of Cry1Ac is 1.9 μ g / g in tissues, below which H. armigera would be able to survive ( Manjunath et al . 2005 ).

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4. Change in Pest Complex Bt cotton provides protection only for lepidoptera pests but It not give protection against the other pests like aphids, jassids , thrips , mites etc and apperance of leaf reddening and parawilt ( Nagrare et al.,2009). In 2006, Minor pest like Myriads one type of sucking pest become major problem in china, this results in 20% extra pesticides spray to control it. The same pest is also found in Karnataka first time on Bt cotton (Anonymous,2006). Anonymous, 2006.

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5. Genetic Pollution The term “Genetic pollution” has been coined for the spread of transgenes from their home crops to the surrounding plants, whether crops or wild relatives. It creates two types of problem in nature: In future, it may be change the wild relatives & local cultivars due to interspecific hybridization via gene flow between related species. Such spread worries producers of non- GM cotton like organic farmers.

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6. I llegal Cultivation of Bt cotton The high demand of Bt cotton has spawned a parallel industry of unapproved Bt cotton seed which is dubious origin and quality. In fact , illegal Bt cotton seed was in the market even before the first approved of Bt cotton for commercial cultivation was granted by GEAC (Jayaraman, 2004). Against 90,000 packet of legal Bt cotton sold in yavatmal district of Maharastra, the number of illegal packet sold was 250,000 (Sainath,2005). I llegal Bt cotton sold under 32 different names was sown in 2004 in India (Sainath,2005).

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controversy & Reality

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Studies on environmental safety pollen escaping and out crossing : Multi-location experiments conducted by GEAC revealed that out-crossing occurred only up to 2 meters, and only 2 % of the pollen reached a distance of 15 m. As the pollen is heavy and sticky in Bt, the range of pollen transfer is limited. The studies conducted revealed that there is essentially no chance that the Bt gene will transfer from cultivated tetraploid species such as the present Bt hybrids to traditionally cultivated diploid species. Aggressiveness and weediness : The result demonstrated that there are no substantial difference between Bt and non Bt cotton for germination and vigour. Hence, there is no difference between Bt and non-Bt cotton with regard to their weediness potential. Bt protein in soil : Bt protein was not detected in soil samples indicating that the Cry1Ac protein was rapidly degraded in the soil in both the purified form of the protein and as part of the cotton plant tissues. The half-life for the purified protein was less then 20 days. The half-life of the Cry1Ac protein in plant tissues was calculated to be 41 days. There is no adverse effect on microbes and soil invertebrates like earthworms.

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Continued… Effect on non target organism :- Studies reveal that Bt cotton hybrid don’t have any toxic effect on non target species , namely sucking pest like aphid ,jassid, white fly and mites. The beneficial insect lady bird beetle and spider remain active in Bt varieties. Bt toxin level : Expression of Bt Cry gene decreased consistently as the plant aged & it’s going below 1.9 μ g / g of tissue after 95 days, but does not affect cotton production because 70 to 80 % of harvesting is completed at this time in Bt. Bollworm starts its life cycle in the leaves and the newly hatched larvae feed on chlorophyll in the tender leaves, the presence of high level of Cry toxin in the leaves ensures the death of a large no. of larvae (Manjunath,2005).

Studies on Food safety:

Studies on Food safety Compositional analysis : Studies reveal that there was no change in the composition of Bt and non Bt seed, with respect to protein , carbohydrate, oil ,calorie and ash content. Toxicology : A goat feeding study was conducted and found no significant difference between animal feed with Bt and non Bt cotton seed. Studies have indicated that Cry 1 Ac protein was not found in refined oil obtained from Bt cotton seed.

Disease infection:

Disease infection As per the report of CICR (Central Institute of Cotton Research, Nagpur), wilt was found in Bt cotton field, was not a pathogenic wilt and is not caused by fungi, bacteria or viruses. The wilting is a characteristic of Para wilt and is a physiological disorder which normally occurs when the cotton hybrids in the field are exposed to prolong dry spell followed by heavy showers.

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Yield In 2009-10, more than 57 lakhs small and medium farmers in India grown Bt cotton. The significant increase in cotton yield was observed during the last five years reportedly higher than the cumulative increase in the last five decades. Yield, that directly measured the income of poor farmer, has increased from 309 kg/ha in 2001-02 to 560 kg/ha in 2009-10.

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Achievements

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A list of the Bt cotton event approved for cultivation in India. Event name Event number Source Company Gene Year of approval Bollgard I MON 531 Monsanto Cry 1Ac 2002 Bollgard II MON 15985 Monsanto Cry 1Ac and Cry 2Ab 2006 Event 1 Event 1 IIT Kharagpur Truncated Cry 1 Ab 2006 GFM Cry 1A GFM Cry 1A Chinese Academy of science Cry 1 Ab + Cry 1 Ac 2006 Dharwad Event Dharwad Event UAS Dharwad Truncated Cry 1 Ac 2008 9124 9124 Metahelix Cry 1c 2009 Bt Cotton in India –A Status Report

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Number of Hybrid / Varieties per event approved for cultivation in India (Till August 2009). Event number Source company Number of hybrids/ Varities MON 531 Monsanto 205 MON 15985 Monsanto 309 Event 1 IIT , Kharagpur 33 GFM Cry 1A Chinese Academy of Science 69 Dharwad Event UAS, Dharwad 1 Event 9124 Metahelix 2 Bt Cotton in India –A Status Report

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Bt cotton event currently undergoing field test in India. Bt cotton in India: A status report

Number of small farmer adopting Bt-cotton in India from 2002-2009.:

Number of small farmer adopting Bt-cotton in India from 2002-2009. year

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Bt cotton is effective in bollworm control . It help in reduction the cost of cultivation by reducing the use of pesticides for bollworm control. Insect resistance problem can be minimized to some extent by many integrated approaches like using Refuge crop, insertion of more than one resistant genes . Statements like cry protein residual, adverse effect on non-target insects etc . are not true but there are chances that microbes will acquire these transferred resistance and may create problem in future. Controversies have less scientific basis / evidences. CONCLUSION

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FUTURE THRUST

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Gene stacking for durable and broad spectrum. Awareness among the people, farmers and society. To facilitate the use of Refuge crop by the farmer. Bt transgenic seeds should be provided at cheaper rate, which can be afforded by small and marginal farmers . In case of hybrids, the farmer has to purchase fresh seed every year at a very high cost. Efforts should be made to develop Bt cotton varieties, the seed of which can be used by the farmers for 3-4 years. There is need to improve the quality of proteins and oil through genetic engineering besides fibre quality improvement.

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The spider and scorpion venom genes can also be used for developing insect resistant genotypes of cotton. Besides, Bt gene, several other genes can be used in future for developing resistant genotypes of cotton to various insects. For eg:-cholesterol oxidaze gene from Streptomycetes fungus can be used for developing boll weevil resistant genotypes. Diploid cottons cover about 10% of cotton area in India. Hence there is need to develop transgenic Bt varieties and hybrids of diploid cotton to remove monopoly of hirsutaian cotton.

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THANKS 61

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Insects 330 M yrs. Dinosaurs 160 M yrs Man 1.5 M yrs We inherited this planet from insects

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Bt action is very specific. Different strains of Bt are specific to different receptors in insect gut wall. Bt toxicity depends on recognizing receptors, damage to the gut by the toxin occurs upon binding to a receptor. Each insect species possesses different types of receptors that will match only certain toxin proteins, like a lock to a key. Fig. Mechanism of toxicity of Bt

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MAY MAA DURGA FULFILL ALL YOUR WISHES AND BRING PEACE, PROSPERITY IN YOUR LIFE.

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