seminar onbt_cotton

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Published on March 8, 2014

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PowerPoint Presentation: Wel Come PowerPoint Presentation: Speaker Rajesh C. Jeeterwal Reg.no . 10-02-02-09-10 Seminar Incharge Dr. K.C. Sharma Assoc. Professor, S K N COA Jobner S K RAU Bikaner. Major Advisor Dr. E.V.D.Sastry Assoc. Professor, S K N COA Jobner S K RAU Bikaner . Bt Cotton: Problem and Prospects Seminar on PowerPoint Presentation: CONTENT Transgenic Bt Cotton Bt Cotton in Indian Context Problems Involved Achievements Conclusion Future Prospects Breeding Methodology Introduction: History of Bt Bt Hybrids Vs Conventional Hybrids Controversy & Reality PowerPoint Presentation: Introduction PowerPoint Presentation:  1995:Mahyco sent its application to Dept. of Biotechnology (DBT) seeking permission for introducing this technology. ♣ Background & History of Bt 1996-2002: After testing the efficacy of this gene, these were used in breeding programmes and 40 elite Indian parental lines were introgressed with Cry I A(c) gene by crossing with Bt gene donar parent Demonstration the safety and benefit of Bt cotton as per regulatory requirements. 1996: Mahyco  received 100 gms of Bt-cotton seeds (variety Cocker 312) containing the Bollgard Bt gene Cry I A(c) form Monsanto, USAV ☻ ☻ ☻ PowerPoint Presentation:  2004 – 2006:20 Bt. Strains released for commercial cultivation by various seed companies 2003- 2004:Bt. Varieties failure report from Andhra Pradesh. ☻ ☻ ☻ 1996-2002:Three Bt. Varieties namely, MECH 12, MECH 162 and MECH 184 were released commercially by Mahyco seed company. 3% cotton area under Bt. Cotton. 2007– 2009:59 Bt. Strains released for commercial cultivation by various seed companies ☻ PowerPoint Presentation:  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. : 1 PowerPoint Presentation:  Bt crystals, sometimes 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). Ribbon model of Cry1Ac toxin molecule. The structure of the activated toxin is shown, demonstrating the three distinct domains. The α -helical cylinder that forms domain-I is involved in membrane insertion and pore formation. Domains II and III are involved in recognition and binding to receptors in the insect midgut wall respectively. Fig. : 2 PowerPoint Presentation: Gene Crystal shape Protein size (kDa) Insect activity cry I [several subgroups: A(a), A(b) , A(c) , B, C , D, E , F , G ] bipyramidal 130-138 lepidoptera larvae cry II [subgroups A(b) , B, C ] Cuboidal 69-71 lepidoptera and diptera cry III [subgroups A, B, C ] flat/irregular 73-74 coleoptera cry IV [subgroups A, B, C, D ] bipyramidal 73-134 diptera cry V-IX Various 35-129 various ♣ 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. PowerPoint Presentation: T ransgenic B t C otton PowerPoint Presentation: ♣ 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 B) ☻ ☻ ☻ 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 ) ☻ PowerPoint Presentation: T DNA (Bt) of Bt Fig. : 3 Insertion of Bt gene from bacteria to plant. PowerPoint Presentation:  Monsanto company (1996) approved first Bt cotton varieties for commercial cultivation in the USA. Subsequently, Bt cotton was also introduced into other countries like Australia (1996), Argentina (1997), China (1997), Mexico (1998), South Africa(1998), Indonesia(2000), Colombia (2002) and India (2002). ♠ How does Bt work ? Fig. : 4 Leaf has to be eaten to cause mortality. (A) 12 day old cotton bollworm larva raised on a diet with non- Bt cotton. (B) 12 day old cotton bollworm larva raised on a diet containing Bt proteins. (A) (B) PowerPoint Presentation:  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. : 5 Mechanism of toxicity of Bt (Prasad et al. , 2004) PowerPoint Presentation: B t Hybrids Vs Conventional Hybrids PowerPoint Presentation: 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: 1 Non preference mechanism of insect resistance in cotton Mehetre et al ., 2004 PowerPoint Presentation: Table: 2 Genetics of various characters associated with insect Resistance in cotton Character Gene Symbol Species Smoothness Sm1, Sm2, Sm3 Gossypium hirsutum, G. barbadence Okra leaf L1,L2 G. hirsutum, G. barbadence Nectariless Ne1, Ne2 G. tomentosum Gossypol glands G12, G13 G. hirsutum, G. barbadence Red plant body R1, R1 dar G. hirsutum Frego bract Fg G. hirsutum Hairiness H1, H2,H6 G. hirsutum , G. barbadence Mehetre et al ., 2004 PowerPoint Presentation: + X Desired Gene Donor Plant Commercial Plant Variety New Plant Variety ۩ CONVENTIONAL PLANT BREEDING ۩ PLANT BIOTECHNOLOGY A single gene is transferred Desired Gene Desired Gene Donor Plant Commercial Plant Variety Improved Commercial Plant Variety Undesired Gene PowerPoint Presentation: B reeding M ethodology PowerPoint Presentation:  Some crop lines are genetically more equipped to handle the stresses of tissue culture. Since these lines are typically lower yielding, recipient lines, once a plant is successfully transformed, plant breeders use backcrossing to transfer the transgene from these transformed lines into elite and high yielding lines. Also, plant breeders can screen for the presence of negative mutations that occurred during the transformation process. ♣ Developing Transgenic Line by Backcross Breeding ♣ Gene Stacking : Gene stacking is combining desired traits into one line. Plant breeders are always stacking genes by making crosses between parents that each have a desired trait and then identifying offspring that have both of these desired traits (Anonymous,2003 B). PowerPoint Presentation: Another way to stack genes is by transferring two or more genes into the cell nucleus during transformation. The use of a selectable marker in addition to the gene of interest would be considered gene stacking. The effect of the transgenes on the overall metabolism of the plant will probably be the major limitation to how many genes and what combinations of genes we can stack into a crop plant. ► ► PowerPoint Presentation: ♣ Chronology of events Based on the recommendations of RCGM, GEAC in the 32 nd meeting on 26 March, 2002 approved the commercial cultivation of three Bt cotton Hybrids : MECH-12 Bt , MECH-162 Bt and MECH-184 Bt . The sequence of events that led to the development and approved of these are listed below: Imported Cocker-312 seed (100 g) containing the cry 1Ac gene from Monsanto, USA. Crossing with 40 elite Indian Cotton breeding lines to introgress cry 1Ac. Greenhouse, risk-assessment studies, pollen escape studies, aggressiveness and persistence studies, biochemical analysis , toxicological studies and allergenicity studies. Multi-location field trials were conducted at 40 locations in nine states to assess agronomic benefits and biosafety. GEAC gave approval for conducting large-scale field trials. 1996: 1996-1998: 1998-1999: July 2000: 2001: Large scale field trials conducted, field trials were also conducted by AICRP on Cotton. 2002: GEAC approved three Bt cotton hybrids for commercial cultivation after taking into account the data on their performances. (Anonymous, 2009) Up to 2009: More than 79 approved varieties were released for commercial cultivation. PowerPoint Presentation: Table : 4 Genes for insect resistance in cotton ( Anonymous, 2009 ) PowerPoint Presentation:  Area, Production and Productivity of cotton in India (Anonymous, 2006 ) Year Area ( lakh ha) Production ( lakh bales) Productivity (kg lint/ha) 2000-01 81.5 167 319 2001-02 85.9 153 309 2002-03 73.9 158 322 2003-04 76.3 179 399 2004-05 89.2 243 463 2005-06 88.2 243 465 PowerPoint Presentation: Problems Involved PowerPoint Presentation: 2. Development of Resistance In Insect 8. High Cost of Cultivation 4. Change In Pest Complex 6. Health Hazards 5. Genetic Pollution 7 . I llegal Cultivation of Bt Cotton 3. Low Bt Toxin Level 1. Refuge Crop PowerPoint Presentation: (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 : 8 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 either five rows of non- Bt Cotton or 20% of total sown area whichever is more. 1. REFUGE CROP: (Slater et al., 2003) PowerPoint Presentation: ♠ 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. ♠ It is also argued that H. armigera , the most predominant bollworm in India, has a large number of alternate hosts and at present only 14% of the cotton area is under Bt hybrids, the rest of the Non- Bt cotton and other surrounding crops already serve as a refuge. ♠ 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. : 9 Different types of Refuge in field (Manjunath, 2004 A, 2005) PowerPoint Presentation:  2 . Development of Resistance In Insect. ♣ Development of resistance is the major problem with Bt cotton. ♣ Dr. Kranthi (CICR, Nagpur) has developed the model which shows that in 2007-08 H. armigera will become complete resistance to Bt cotton. ♣ The most frequently observed mechanism of resistance among insect pests is the modification of the protease enzyme or by receptor site. PowerPoint Presentation:  Inactive Bt protoxin Enzymes from Insect gut Active delta - endotoxin Susceptible Susceptible Susceptible Resistant Resistant ☻ ☻ ☻ ☻ ☻ ☻ Change the shape by Mutation or Selection Plant DNA with Bt gene PowerPoint Presentation: 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. In India, there is problem of intra-plant and in-seasonal variability in cry1Ac expression in Bt -cotton hybrids. The critical expression level of Cry1Ac is 1.9 μ g / g in tissues, below which H. armigera would be able to survive ( Kranthi et al . 2005 ). PowerPoint Presentation: 4. Change in Pest Complex ☻ Bt cotton provides protection only for lepidoptera pests but It not give protection against the secondary pests like aphids, jassids, thrips, mites etc. In 2003, Bt cotton had high level of infestation of thrips in central zone of India. ☻ In 2004, 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. (Prasad, 2004). ☻ PowerPoint Presentation: White Fly Incidence % no. of plots with Bt Cotton Non Bt Cotton High incidence 25 22 Medium Incidence 38 44 Low Incidence 37 35 Aphid Incidence % no. of plots with Bt Cotton Non Bt Cotton High Incidence 28.4 19.7 Medium Incidence 34.8 43.9 Low Incidence 36.8 36.5 Jassid Incidence % no. of plots with Bt Cotton Non Bt Cotton High Incidence 25.4 28.0 Medium Incidence 39.6 40.7 Low Incidence 35.0 31.3 Mite Incidence % no. of plots with Bt Cotton Non Bt Cotton High Incidence 22.5 16.6 Medium Incidence 42.9 46.3 Low Incidence 34.6 37.2 Table : 8 Sucking pest infestation in Bt and non- Bt variety at Maharashtra during 2009 Anonymous (2009) Maharashtra PowerPoint Presentation: 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. Stewart et al. ,2003 PowerPoint Presentation: 6. Health Hazards The Cotton seeds are used for increasing milk secretion; the use of Bt cotton seeds can cause serious health hazards to the animals. Similarly, The cotton roots used during the labour to enhance the uterine contracts, The ash of the cotton used to check bleeding from wound, The leaves are used as diuretic . So, the introduction of Bt cotton will have a dangerous effect on the traditional medical practices and the use of its seeds, leaves, roots, oil will be fetal for the patients (Shiva, 2004). ♣ European Union rejected Monsanto’s application for the sale of GM Cottons in Europe, because the gene aad. (Mae-Wan-Ho, 2002) ♣ ♣ PowerPoint Presentation: 7. 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). ♦ A recent news report states that against 90,000 seed packets of legal Bt cotton sold in Yavatmal district of Maharashtra, the number of illegal packets sold was 2,50,000 (Sainath,2005). ♦ I llegal Bt cotton sold under 32 different names was sown in 2004 in India (Sharma,2005). ♦ GEAC got 10 packets of such seeds from Gujarat and tested at CICR, Nagpur, for verification by PCR and ELISA tests. They found eight of these were F1 seeds while two were F2 seeds (Anonymous, 2006 A). PowerPoint Presentation: 8. High Cost of Cultivation Bt technology is not free of cost to the farmer. ITEMS BOLLGARD CONVENTIONAL Average Technology Fee $ 19.14 $ 0.00 Insect Control Cost $ 5.63 $ 18.98 % damaged bolls 4.47% 5.25% Additional Scouting Fees $ 2.50 $ 0.00 Total $ 27.27 $ 25.06 Table : 9 Costs and Damages ( $ / Acre ) of Bollgard Vs Conventional Cotton for Mississippi produces 1. Technology fees varies acco. to seed rate & row spacing. 2. Scouting requirement for bollgard typically exceed those needed for conventional cotton. Shiva et al. , 2004 PowerPoint Presentation: OVERALL ECONOMICS Bt Cotton (per acre) Non- Bt Cotton (per acre) Average Cost of Cultivation, in Rupees 6184.11 4138.16 Yield in Quintals (100 Kgs =1 quintal) 4.97 4.71 Average Rate per Quintal, in Rupees 1843.60 1898.00 Average Gross Income, in Rupees 9148.00 8929.58 Average Net Income, in Rupees (Gross Income minus Cost of Cultivation) 2963.95 4791.00 Table : 10 Comparison between Bt Cotton and non- Bt Cotton, in terms of the total cost of cultivation . Maharashtra Anonymous (2003 A) PowerPoint Presentation: ۩ State Agriculture Department Report On Bt Cotton In Different States during 2002. ● GUJARAT Hy. Cotton MECH – 184 affected by wilt. Bt. MECH – 162 variety not accepted by farmer because the bollworm infestation is more in this variety. Bt . MECH – 162 also showed susceptibility toward sucking pest . ● KARNATAKA The cost of Bt cotton seeds is very high ; it is almost 45 times higher than the normal cotton. The regular plant protection measures were necessary for other sucking pest. There is a fear that in future this condition may leads to emergence of other minor insects in to major pest. The market price of Bt cotton is less then the non- Bt cotton. PowerPoint Presentation: ● MAHARASTRA Heavy infestation of Wilt was noticed in all Bt . MECH variety, so farmer suffered heavy economic loss. Infestation of wilt is high in Amravati, Yavatmal and Nanded districted on Bt . Cotton varieties. The cost of Bt Cotton is also major point, the cost fixed by Mahyco is Rs. 1600/- packet which can not be a justifiable cost. ● ANDHRA PRADESH In A.P. 18% reduction in the yield after adoption of Bt cotton. Problem with the GM cotton variety included failure to germinate, drought damage, root-rot, leaf curl virus, brittle stems, increased pest, smaller bolls, increased labour requirement per acre and a shorter harvest season. Some farmers complained that they were not able to grow other crops after Bt because it infected their soil very badly. PowerPoint Presentation: CONTROVERSY & REALITY PowerPoint Presentation: 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 . 1. Gene Flow 2. 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. PowerPoint Presentation: 3. 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). The choice of parental background appeared to be crucial for sustainable expression of the Cry transgene (Adamezyk,2004). 4. Health Hazards GEAC studies have indicated that Cry1Ac protein was not found in refined oil obtained from Bt cotton seeds. The feeding studies on cows, buffaloes, goat, poultry and fish indicated that Bt cotton seed meal was nutritionally as wholesome and safe as the non- Bt Seed meal. PowerPoint Presentation: 5. Sucking Pest Infestation Sucking pest infestation is not due to Bt gene present in the cotton plant, but it is due to genetic background of the recipient parent and /or environmental condition. 6. 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 Parawilt 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. 7. Cost of Seeds Now, recently Govt. decided the price limit of Bt Hy. cotton Seeds. No one can sell Bt Hy. Variety more than Rs. 750 per packet . PowerPoint Presentation: 8. Yield In 2005-06, more than 10 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. Cotton production has touched 250 lac bales (1 bale = 170 Kg) in the 2005 season, higher than the projected target of 220 lac bales for the 10 th five year plan under the technology mission on cotton . Yield, that directly measured the income of poor farmer, has increased from 309 kg/ha in 2001-02 to 460 kg/ha in 2004-05 , and is estimated to reach 475 kg/ha in 2006-07 . PowerPoint Presentation: ☻ Bt cotton is effective in 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 PowerPoint Presentation: Future Prospects PowerPoint Presentation: To facilitate the use of Refuge crop by the farmer. Monitoring of susceptibility of bollworms to the Bt gene should be undertaken by an agency identified by the ministry of Environment and Forests at applicant’s cost. ۩ ۩ Awareness among the people, farmers and society. Gene stacking for durable and broad spectrum Further research to know the impact of Bt cotton on ecology (e.g., non-target insect, animal, human ) ۩ ۩ ۩ Bt can be a component of IPM for sucking pests and other non target pests ۩ Markers other than antibiotic resistance should be used. ۩ Apart from cry protein, another anti - feedant protein genes are introduced in the crop e.g. trypsin inhibitors, Juvenile hormone esterase ۩ Indigenous gene should be introduced so that cost of cultivation decrease. ۩ PowerPoint Presentation: Thanks

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