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Ashwin jayale hrtp.ppt

  1. 1. Genetic transformations- herbicide resistant plants Ashwin Jayale Id no-PALB 12221
  2. 2. Contents Introduction Herbicide resistant plants. Herbicide and mode of action on plants. Genetic engineering approach. GM plants and statistical data Case studies Controversies Conclusion2
  3. 3. Introduction In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s). Genetically modified foods or biotech foods are foods derived from genetically modified organisms (GMOs). Genetically Modified foods (GM foods) were first put on the market in 1996. Typically, genetically modified foods are transgenic plant products: soybean, corn, canola, rice, and cotton seed oil. The first commercially grown genetically modified whole food crop was a tomato called FlavrSavr Afterwards in 1995, a biotech company Monsanto introduced herbicide immune soybean also known as the Roundup ready . 3 In 1996, the first genetically modified Canola was available on the market.
  4. 4. Herbicide resistant plants Herbicide resistance: "Herbicide resistance is the inherited ability of a plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type. BY WASSA. In a plant, resistance may be naturally occurring or induced by such techniques as genetic engineering or selection of variants produced by tissue culture. Source: Weed Technology Volume 12, Issue 4 (October-December) 1998. p. 789. weed science society of America. Taking in the entire crop-growing period from 1996 to 2004, the US used some 62.7 million kg more herbicides on herbicide-resistant crops compared with conventional management (Benbrook 2004). 4
  5. 5. Importance Excessive weed growth forces crops to compete for sunlight and nutrients, often leading to significant losses. Because herbicides cannot differentiate between plants that are crops and plants that are weeds, conventional agricultural systems can only use selective herbicides. Such herbicides do not harm the crop, but are not effective at removing all types of weeds If farmers use herbicide resistant crops, non-selective herbicides can be used to remove all weeds in a single, quick application. Herbicide resistant crops also facilitate low or no tillage cultural practices, which many consider to be more sustainable. Broad-spectrum, or non-selective herbicides are effective at killing a wide range of weeds. The problem is, they can also kill valuable crops. 5
  6. 6. Properties of the genetically Food Modification modified variety Herbicide resistant gene taken Resistant to Soybeans from bacteria inserted into glyphosate herbicides soybeanCorn, field (Maize) Resistant to glyphosate New genes, some from the   herbicides. bacterium Bacillus thuringiensis, New genes added/transferred into plant genome. Resistant Alfalfa to glyphosate herbicides Variety is resistant to the papaya New gene added/transferred into Hawaiian papaya ringspot virus. plant genome Variety in which the production A reverse copy of the (an antisense gene) of the gene Tomatoes enzyme polygalacturonase (PG) is responsible for the production of suppressed, retarding fruit PG enzyme added into plant softening after harvesting. genome Resistance to herbicides New genes added/transferred into Canola (glyphosate or glufosinate), high plant genome laurate canola 6
  7. 7. Herbicide and mode of action Glyphosate (N-phosphonomethyl-glycine) is the most widely used herbicide in the world: glyphosate-based formulations exhibit broad-spectrum herbicidal activity . The extraordinary success of this simple, small molecule is mainly attributable to the high specificity of glyphosate for the plant enzyme enolpyruvyl shikimate-3-phosphate synthase in the shikimate pathway, leading to the biosynthesis of aromatic amino acids. herbicide glyphosate (N-phosphonomethyl-glycine) has had the greatest positive impact. Developed by the Monsanto Co. and introduced to world agriculture in 1974. 7
  8. 8. Shikimmic acid pathway Glyphosate works by blocking the plants ability to produce certain proteins and it disrupts amino acid synthesis. It blocks the shikimic acid pathway found only in certain plants .8
  9. 9. Roundup is the brand name of a systemic, broad-spectrum herbicide produced by the U.S. company Monsanto, and contains the active ingredient glyphosate.Monsanto developed and patented the glyphosate molecule in the 1970s, and marketed Roundup from 1973.Herbicide propertiesMain active ingredient=isopropylamine salt of GlyphosateMode of action=5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) inhibitorMonsanto also produces seeds which grow into plants genetically engineered to be tolerant to glyphosate, which are known as Roundup Ready crops. The genes contained in these seeds are patented. Soy was the first Roundup Ready crop, and was produced at Monsantos Agracetus Campus located in Middleton, Wisconsin. 9
  10. 10. Genetic engineering approach Genetic engineering, also called genetic modification, is the direct human manipulation of an organisms genome using modern DNA technology. An organism that is generated through the introduction of recombinant DNA is considered to be a genetically modified organism. It involves the introduction of foreign DNA or synthetic genes into the organism of interest. GMOs Express traits not normally found in nature  Result of introducing foreign DNA  Highly controversial  Safety concerns 10  Environmental implications.
  11. 11. Mechanism GM herbicide resistant crop Some microorganisms contains genes for producing 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS);5-enolpyruvylshikimate-3-phosphate synthatase ; phosphoenolpyruvate:3- phosphoshikimate 5-O-(1-carboxyvinyl)-transferase these resistant to glyphosate inhibition. These used in genetically modified crops, and isolated from Agrobacterium strain CP4 (CP4 EPSPS) that was resistant to glyphosate. The CP4 EPSPS gene was cloned and inserted into soybeans. The CP4 EPSPS gene was engineered for plant expression by fusing the 5 end of the gene to a chloroplast transit peptide derived from the petunia EPSPS. The plasmid used to move the gene into soybeans was PV-GMGTO4. It contained three bacterial genes, two CP4 EPSPS genes, and a gene encoding beta- glucuronidase (GUS) from Escherichia coli as a marker. The DNA was injected into the soybeans using the particle acceleration method or gene gun. 11
  12. 12. GENE CONSTRUCT12
  13. 13. introduction, herbicide-resistant soybeans have been quickly adopted. In 2010, 93% of all soybeans grown in the USA were herbicide-resistant, as were 78% of all cotton and 70% of all maize varieties ( ).13
  14. 14. Soybean Industry Portfolio Quality/Food Agronomic Pipeline of biotech events and novel trait releases Commercialized Omega-3 High-Oleic, High Oleic / Stearidonic Acid Stearate Omega-3 Low-Sat (Monsanto) (Pioneer/DuPont) EPA/DHA (Monsanto) (Pioneer/DuPont) High Beta- Modified 7S High-Oleic Conglycinin High Protein FF (Pioneer/DuPont) (Pioneer/DuPont) Stearate (Pioneer/DuPont) (Monsanto; Pioneer/DuPont) Low-Linolenic Feed: High Low Raff- (Syngenta) Low-Phytate (Pioneer/DuPont) Protein Soybean Stach (Pioneer/DuPont) (Virginia Tech) 2010 2012 2020 Glufosinate & HPPD Isoxaflutole Tolerant Tolerant (Bayer/MS Technologies) (Syngenta) 2,4-D Tolerant Disease (Dow) Higher Yield II Resistance Glufosinate & (Monsanto; (Syngenta; Imidazolinone Tolerant Isoxaflutole Tolerant Brazil only. Higher Yield I Pioneer/DuPont) Pioneer/DuPont) RR2Y (BASF/Embrapa Brazil) & LibertyLink (Monsanto; (Bayer/MS Technologies) Pioneer/DuPont) (Monsanto) Rust (Monsanto; Syngenta; Nematode LibertyLink GAT/Glyphosate-ALS Pioneer/DuPont) Resistance (Bayer) Soybean (Monsanto; Dicamba (Pioneer/DuPont) Bt/RR2Y Syngenta; Brazil only Tolerant Aphid Resistance Pioneer/DuPont) (Monsanto) (Monsanto) (Monsanto)Source: Pipeline from Industry Sources; prepared by ASA, USSEC, USB. Updated January, 2010 RVSD Biotech Pipeline 02-04-10 V3
  15. 15. 15
  16. 16. Statistical data16
  17. 17. Transgenic crops Grown in:  Traits: Argentina, Australia, Canada, Herbicide-tolerant Chile, China, France, Insect resistant Germany, India, Mexico, Viral resistant South Africa, Spain, Uruguay, Male sterile/restorers USA Delayed ripening Oil content Vitamin A, vaccines Not yet grown?: Brazil, Egypt, other EU, Japan, Kenya, Korea, Switzerland  GM-crops: 53 million ha (2001); 62.3% RR-soybean17
  18. 18. Regulation of Plant Biotechnology inthe United States The U.S. Coordinated Framework (1986) • The potential risks posed by genetically engineered organisms are not fundamentally different from those posed by conventional products • Regulation should be science-based and oversight conducted on a case-by-case basis • Existing laws provide adequate authority 18
  19. 19. Coordination of U.S. Agencies USDA Products are regulated according to their intended use, with some products Safe for agriculture being regulated under more than one and the environment agency. FDA EPA Safe for use Safe for use food and feed in pesticides19
  20. 20. Regulation under the CoordinatedFramework New Trait/Crop Agency ReviewInsect resistance in food crop USDA Agricultural and environmental(Bt corn) EPA safety FDA Environmental, food safety of pesticide Food/feed safetyHerbicide tolerance in food USDA Agricultural and environmentalcrop EPA safety(Roundup Ready soybeans) FDA New herbicide use Food/feed safetyHerbicide tolerance in USDA Agricultural and environmentalornamental crop EPA safety New herbicide useModified oil in food crop USDA Agricultural and environmental(High oleic acid soybeans) FDA safety Food/feed safetyModified flower color USDA Agricultural and environmental(Blue roses)20 safety
  21. 21. Regulation under the Coordinated Framework • Corn - HT, IR, AP  Tomato - PQ • Soybean - HT, PQ  Chicory – AP • Cotton - HT, IR  Potato - IR, VR • Canola - HT, AP, PQ  Rice – HT • Papaya – VR  Flax – AP • Squash – VR  Plum - VR • Tobacco – PQ • Sugar beet - HT HT – herbicide tolerance IR – insect resistance AP – agronomic properties VR – virus resistance PQ – product quality21
  22. 22. Transgenic technology: from test tubes tofarmers fields  Transgenic crops: cotton, cucumber, melon, maize, tomato, papaya, potato, soybean, canola, sugar beet, tobacco, carnation  In pipeline: sweetpotato, cassava, banana/plantain, groundnut, chickpea, pigeonpea, pea, cowpea, sorghum, wheat22
  23. 23. Case study#1Nature Biotechnology 22, 204 - 209 (2004)Published online: 18 January 2004 | doi:10.1038/nbt934Assessing the survival of transgenic plant DNA in the human gastrointestinaltractTrudy Netherwood1,2, Susana M Martín-Orúe1, Anthony G ODonnell2, SallyGockling1,2, Julia Graham1,2, John C Mathers3,4 & Harry J Gilbert1 23
  24. 24. IntroductionThe inclusion of genetically modified (GM) plants in the human diet has raised concernsabout the possible transfer of transgenes from GM plants to intestinal microflora andenterocytes. The persistence in the human gut of DNA from dietary GM plants isunknown. Here they study the survival of the transgene epsps from GM soya in thesmall intestine of human ileostomists (i.e., individuals in which the terminal ileum isresected and digesta are diverted from the body via a stoma to a colostomy bag).The amount of transgene that survived passage through the small bowel varied amongindividuals, with a maximum of 3.7% recovered at the stoma of one individual. Thetransgene did not survive passage through the intact gastrointestinal tract of humanGM soya. Three of seven ileostomists showesubjects fed d evidence of low-frequencygene transfer from GM soya to the microflora of the small bowel before their involvementin these experiments. 24
  25. 25. Methodsrate of DNA degradationDNA survival in humans with an intactgastrointestinal tract We fed the testmeal containing GM soya to 12 humanvolunteers (with intact gastrointestinaltracts) and quantified the presence ofthetransgene in feces by PCR. For allvolunteers, 90–98% of the indi-gestiblemarker was recovered in the feces butthe transgene was not detected. 25
  26. 26. Result 26
  27. 27. DNA survival in humans with an intact gastrointestinal tractThey fed the test meal containing GM soya to 12 human volunteers(with intact gastrointestinal tracts) and quantified the presence of thetransgene in feces by PCR. For all volunteers, 90–98% of the indi- gestiblemarker was recovered in the feces but the transgene was not detected. This wasnot the result of PCR inhibition because a 180 bp product was amplified byPCR when the fecal material was spiked with 400 copies of the transgene inthe aliquot used in the PCRs (data not shown). Thus, although the epspstransgene can survive passage through the small bowel of ileostomists, it iscompletely degraded in the large intestine27
  28. 28. Case study # 228
  29. 29. IntroductionTransgenic rice (Oryza sativa L.) containing two agronomically useful genes wasgenerated using Agrobacterium LBA4404 with an additional virulence plasmid,virG(pTiBo542)/virE1virE2(pTiA6).The plants were transformed withphosphinothricin acetyl transferase (pat) gene for herbicide resist-ance andBacillus thuringiensis (Bt) crystal insecticidal protein gene for insect resistance. The herbicide application test of the progenyfrom the three sets of primary plants showed that the transferred pat gene was stablyexpressed in the T1generation. The insect feeding bioassay with T1generation plantsconferring resistance to her-bicide and established that the transgenic plants having acomplete Bt gene were toxic to tobacco bud-worm (Heliothis virescens) larvae 29
  30. 30. Materials and MethodsMature seed-derived callus from tropical rice (Oryza sativa L. var. ja-ponica) cv. Maybelle was used for Agrobacterium-mediated transfor-mation.Bacterial strain and plasmids Agrobacterium strain, LBA4404 (octopine, Hoekema etal. 1983), wasused. The plasmid construct used was pAGM281 (provided by Myco-gen Plant Sciences) containing the ubi/pat/orf25bt(4Ocs)∆mas ex-pression vecto30
  31. 31. Results31
  32. 32. ResultsIn summary, mature seed from a commercial rice cultivar,Maybelle, was used to generate callus. Callus was trans-formed using A. tumefaciens LBA4404 carrying two agronom-ically usefulgenes, herbicide and insect resistance and the pCH32 helper plasmid withvirulence genesThis study demonstrates that a commercial rice cultivarhas been engineered with economically significant genesconferring resistance to a herbicide and insect feeding usingAgrobacterium-mediated transformation. 32
  33. 33. ControversiesViolations of human rights as a result of GM soyWednesday, 23 November 2011 12:34GMOs in agriculture again a theme on UN-committee of human rights:Violations of Human Rights as a result of the Genetic-Modified Soya-Monocultures --the Right to Food and Health - for the Argentinean Population, Farmers and Bee-KeepersGRR wrote report for UN-committee – Hearing in Geneva 14 November.on the consequences of a highly perturbing global development, the use of geneticallymodified GM-soya in Argentina. This seriously violates the economic, social andcultural rights of the Argentinean Population, Farmers and Bee-KeepersArgentina in particular is one of the most affected countries worldwide by thisdevelopment.The GM agro-biotechnology accelerates the extinction of small farmers around theworld. Deforestation, an increasing use of pesticides, destruction of livelihoods ofindigenous peoples, small farmers and peasants, land concentration, slave labour, therural exodus and increasing poverty in the countryside are the effects alreadyabundantly demonstrated by the consequences of an agro-industrial model oftransgenic monoculture. 33
  34. 34. Nature Biotechnology | NewsGlyphosate resistance threatens Roundup hegemony Nature Biotechnology28,537–538,(2010) doi:10.1038/nbt0610-537 Corrected online13 October 2010 These marestail plants infesting a crop of wheat in Tennessee are resistant to glyphosate herbicide Roundup. Weeds are becoming increasingly resistant to glyphosate, a report from the US National Academy of Sciences (NAS) released in April has found. The driving force, according to the report, is farmers dependence on the weed killer accompanied by the widespread adoption of genetically modified (GM) herbicide-tolerant crops. Seed makers are hoping to forestall the problem by developing GM crops with stacked traits that tolerate multiple herbicides. But weed scientists warn that if farmers manage these new crops in the same way as they managed their glyphosate-tolerant predecessors, weeds will simply become resistant to the new technologies. 34
  35. 35. SOYBEAN TRANSGENIC EVENT MON87705 AND METHODS FORDETECTION THEREOF1977/CHENP/2011 A (1977/CHENP/2011)Filed on 2011-03-21Publication date 2011-12-02Thepresent invention provides a transgenic soybean event MON87705, and cells, seeds, andplants comprising DNA diagnostic for the soybean event. The invention also providescompositions comprising nucleotide sequences that are diagnostic for said soybean event ina sample, methods for detecting the presence of said soybean event nucleotide sequencesin a sample, probes and primers for use in detecting nucleotide sequences that arediagnostic for the presence of said soybean event in a sample, growing the seeds ofsuchsoybean event into soybean plants, and breeding to produce soybean plants comprisingDNA diagnostic for the soybean event.ApplicantMONSANTO TECHNOLOGY LLCMAIL ZONE E1NA, 800 N. LINDBERGH BLVD., ST. LOUIS, MO 63167 U.S.A.InventorWAGNER, NICHOLAS, BURNS, WEN C., GODSY, ERIC J., ROBERTS, PETER D. International Information Classification A01H1/02 Publication number WO 2010/037016 A1 Application date 2009-09-28 Application number 35 PCT/US2009/058591
  36. 36. Conclusion GM crops gives protection from biotic factors like insect, pathogen as well as abiotic factors like drought, salinity, flood etc. In advance agriculture herbicide resistant GM crops plays vital role. Now a days two agronomically important genes transfer into single plant leads to increase potential of the crops. It really helps in growth and development of agriculture sector. 36
  37. 37. References Albert l. Lehninger., biochemistry, second edition, pg no. 708- 709. R.C.Dubey., A text book of biotechnology, fourth edition, pg no.323-327. Nature Biotechnology 22, 204 - 209 (2004) Published online: 18 January 2004 | doi:10.1038/nbt934 P lant P hysiol. 158. 1221–1226(2001) Urban&F ischerVerlag International Journal of Molecular Sciences ISSN 1422-0067 37
  38. 38. Thank you38