Genetic transformations- herbicide resistant plants

Ashwin Jayale Id no-PALB 1222

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Contents Introduction Herbicide resistant plants. Herbicide and mode of action on plants. Genetic engineering approach. GM plants and statistical data Case studies Controversies Conclusion

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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 .

In 1996, the first genetically modified Canola was available on the market.3

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).

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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.

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Food Properties of the genetically modified variety Modification

Soybeans Resistant to  glyphosate herbicides

Herbicide resistant gene taken from bacteria inserted into

soybean Corn, field (Maize) Resistant to glyphosate

  herbicides. New genes, some from the bacterium Bacillus thuringiensis,

Alfalfa Resistant to glyphosate  herbicides

New genes added/transferred into plant genome.

Hawaiian papaya Variety is resistant to the papaya ringspot virus.

New gene added/transferred into plant genome

Tomatoes

Variety in which the production of the

enzyme polygalacturonase (PG) is suppressed, retarding fruit softening after harvesting.

A reverse copy (an antisense gene) of the gene

responsible for the production of PG enzyme added into plant

genome

CanolaResistance to herbicides

(glyphosate or glufosinate), high laurate canola

New genes added/transferred into plant genome

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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

Shikimmic acid pathwayGlyphosate 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 .

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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 properties

Main active ingredient=isopropylamine salt of Glyphosate

Mode of action=5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) inhibitor

Monsanto 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 Monsanto's Agracetus Campus located in Middleton, Wisconsin.

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Genetic engineering approach Genetic engineering, also called genetic modification,

is the direct human manipulation of an

organism's 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

Environmental implications.10

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.

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GENE CONSTRUCT

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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 (http://www.ers.usda.gov/Data/BiotechCrops/).

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Soybean Industry Portfolio Quality/Food

Agronomic

2020

Modified 7S Protein FF

(Pioneer/DuPont)

High-Oleic, Stearate

(Pioneer/DuPont)

Source: Pipeline from Industry Sources; prepared by ASA, USSEC, USB. Updated January, 2010

Nematode Resistance

(Monsanto; Syngenta;

Pioneer/DuPont)

LibertyLink (Bayer)

Imidazolinone TolerantBrazil only.

(BASF/Embrapa Brazil)RR2Y(Monsanto)

Dicamba Tolerant

(Monsanto)

Omega-3 Stearidonic Acid

(Monsanto)

High Beta- Conglycinin

(Pioneer/DuPont)

Low-Phytate(Pioneer/DuPont)

Feed: High Protein Soybean

(Pioneer/DuPont)

Omega-3EPA/DHA

(Pioneer/DuPont)

2010 2012

GAT/Glyphosate-ALS Soybean

(Pioneer/DuPont) Bt/RR2YBrazil only

(Monsanto)

HPPD Tolerant (Syngenta)

High Oleic / Low-Sat

(Monsanto)

HighStearate(Monsanto;

Pioneer/DuPont)

Pipeline of biotech events and novel trait releases

2,4-D Tolerant(Dow)

Disease Resistance

(Syngenta; Pioneer/DuPont)

Glufosinate & Isoxaflutole Tolerant (Bayer/MS Technologies)

Low Raff-Stach

(Virginia Tech)

RVSD Biotech Pipeline 02-04-10 V3

Commercialized

Glufosinate & Isoxaflutole Tolerant

& LibertyLink(Bayer/MS Technologies)

High-Oleic(Pioneer/DuPont)

Low-Linolenic(Syngenta)

Higher Yield I(Monsanto;

Pioneer/DuPont)

Higher Yield II(Monsanto;

Pioneer/DuPont)

Rust(Monsanto; Syngenta;

Pioneer/DuPont)

Aphid Resistance(Monsanto)

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Statistical data

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Grown in:Argentina, Australia, Canada, Chile, China, France, Germany, India, Mexico, South Africa, Spain, Uruguay, USA

Not yet grown?:Brazil, Egypt, other EU, Japan, Kenya, Korea, Switzerland

Traits:Herbicide-tolerant Insect resistantViral resistantMale sterile/restorersDelayed ripeningOil contentVitamin A, vaccines

GM-crops: 53 million ha (2001); 62.3% RR-soybean

Transgenic crops

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Regulation of Plant Biotechnology in the 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

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Coordination of U.S. Agencies

FDA Safe for usefood and feed

USDASafe for agriculture and the environment

EPA Safe for usein pesticides

Products are regulated according to their intended use, with some products being regulated under more than one agency.

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Regulation under the Coordinated Framework

New Trait/Crop Agency ReviewInsect resistance in food crop(Bt corn)

USDAEPAFDA

Agricultural and environmental safety Environmental, food safety of pesticideFood/feed safety

Herbicide tolerance in food crop(Roundup Ready soybeans)

USDAEPAFDA

Agricultural and environmental safetyNew herbicide useFood/feed safety

Herbicide tolerance in ornamental crop

USDAEPA

Agricultural and environmental safetyNew herbicide use

Modified oil in food crop(High oleic acid soybeans)

USDAFDA

Agricultural and environmental safetyFood/feed safety

Modified flower color (Blue roses)

USDA Agricultural and environmental safety20

Regulation under the Coordinated Framework

• Corn - HT, IR, AP• Soybean - HT, PQ• Cotton - HT, IR• Canola - HT, AP, PQ• Papaya – VR• Squash – VR• Tobacco – PQ• Sugar beet - HT

Tomato - PQ Chicory – AP Potato - IR, VR Rice – HT Flax – AP Plum - VR

HT – herbicide toleranceIR – insect resistanceAP – agronomic propertiesVR – virus resistancePQ – product quality

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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, wheat

Transgenic technology: from test tubes tofarmers fields

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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 gastrointestinal tractTrudy Netherwood1,2, Susana M Martín-Orúe1, Anthony G O'Donnell2, Sally Gockling1,2, Julia Graham1,2, John C Mathers3,4 & Harry J Gilbert1

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Introduction The inclusion of genetically modified (GM) plants in the human diet has raised concerns

about the possible transfer of transgenes from GM plants to intestinal microflora and

enterocytes. The persistence in the human gut of DNA from dietary GM plants is

unknown. Here they study the survival of the transgene epsps from GM soya in the

small intestine of human ileostomists (i.e., individuals in which the terminal ileum is

resected 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 among

individuals, with a maximum of 3.7% recovered at the stoma of one individual. The

transgene did not survive passage through the intact gastrointestinal tract of human

GM soya. Three of seven ileostomists showesubjects fed d evidence of low-frequency

gene transfer from GM soya to the microflora of the small bowel before their

involvement in these experiments. 24

Methods rate of DNA degradation

DNA survival in humans with an intact gastrointestinal tract We fed the test meal containing GM soya to 12 human volunteers (with intact gastrointestinal tracts) and quantified the presence of the transgene in feces by PCR. For all volunteers, 90–98% of the indi-gestible marker was recovered in the feces but the transgene was not detected.

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Result

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DNA survival in humans with an intact gastrointestinal tract

They fed the test meal containing GM soya to 12 human volunteers

(with intact gastrointestinal tracts) and quantified the presence of the

transgene in feces by PCR. For all volunteers, 90–98% of the indi- gestible

marker was recovered in the feces but the transgene was not detected. This was

not the result of PCR inhibition because a 180 bp product was amplified by

PCR when the fecal material was spiked with 400 copies of the transgene in

the aliquot used in the PCRs (data not shown). Thus, although the epsps

transgene can survive passage through the small bowel of ileostomists, it is

completely degraded in the large intestine

Case study # 2

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Introduction Transgenic rice (Oryza sativa L.) containing two agronomically useful genes was

generated using Agrobacterium LBA4404 with an additional virulence plasmid,

virG(pTiBo542)/virE1virE2(pTiA6).The plants were transformed with

phosphinothricin acetyl transferase (pat) gene for herbicide resist-ance and

Bacillus thuringiensis (Bt) crystal insecticidal protein gene for insect resistance.

The herbicide application test of the progeny

from the three sets of primary plants showed that the transferred pat gene was stably

expressed in the T1generation. The insect feeding bioassay with T1generation plants

conferring resistance to her-bicide and established that the transgenic plants having a

complete Bt gene were toxic to tobacco bud-worm (Heliothis virescens) larvae

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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 et al. 1983), wasused. The plasmid construct used was pAGM281 (provided by Myco-gen Plant Sciences) containing the ubi/pat/orf25\bt\(4Ocs)∆mas ex-pression vecto

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Results

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Results In 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 useful

genes, herbicide and insect resistance and the pCH32 helper plasmid with

virulence genesThis study demonstrates that a commercial rice cultivar

has been engineered with economically significant genes

conferring resistance to a herbicide and insect feeding using

Agrobacterium-mediated transformation.

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Controversies Violations 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 genetically modified GM-soya in Argentina. This seriously violates the economic, social and cultural rights of the Argentinean Population, Farmers and Bee-KeepersArgentina in particular is one of the most affected countries worldwide by this development.The GM agro-biotechnology accelerates the extinction of small farmers around the world. Deforestation, an increasing use of pesticides, destruction of livelihoods of indigenous peoples, small farmers and peasants, land concentration, slave labour, the rural exodus and increasing poverty in the countryside are the effects already abundantly demonstrated by the consequences of an agro-industrial model of transgenic monoculture.

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Nature Biotechnology | News Glyphosate 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.

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SOYBEAN TRANSGENIC EVENT MON87705 AND METHODS FOR DETECTION THEREOF1977/CHENP/2011 A (1977/CHENP/2011)Filed on 2011-03-21Publication date 2011-12-02

The present invention provides a transgenic soybean event MON87705, and cells, seeds, and plants comprising DNA diagnostic for the soybean event. The invention also provides compositions comprising nucleotide sequences that are diagnostic for said soybean event in a sample, methods for detecting the presence of said soybean event nucleotide sequences in a sample, probes and primers for use in detecting nucleotide sequences that are diagnostic for the presence of said soybean event in a sample, growing the seeds ofsuch soybean event into soybean plants, and breeding to produce soybean plants comprising DNA 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 InformationClassificationA01H1/02Publication numberWO 2010/037016 A1Application date2009-09-28Application numberPCT/US2009/058591

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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.

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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 http://www.urbanfischer.de/journals/jpp International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijm

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Thank you

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