lecture 7 herbicide resistance
TRANSCRIPT
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Growth of Transgenic Crops
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Growth of Transgenic Crops
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Growth of Transgenic Crops
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2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
Year
P e r c e n t G E
c r o
p s
All GE Corn Varieties
All GE Cotton Varieties
All GE Soybean Varieties
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Impact of 6 GMO Trait and Crop
Combinations
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Soybeans are different
• Non-GMO soybeans are getting to be hardto find
• Europe buys its Soybean from Brazil
which SAYS it is a non-GMO producer• Much of Brazil’s Soybean production is
GMO in reality
• China, a big customer for US soybeans,still working out terms of trade re GMO
---Randall Fortenbery, UW-Madison Ag Econ
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In crops, two main kinds of geneshave been commercialized
• Insect resistance genes, called “Bt”
– Bt specific for corn borers
– Bt specific for rootworms
• Herbicide resistant genes
– Resistance to the herbicide “Roundup”
– Resistance to the herbicide “Liberty”
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Growth of Transgenic Crops by Trait
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The genetic manipulation
of herbicide resistance
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Weed
Weed control is essential to modern crop
production practices. Without control,weeds can
– prevent establishment of a good crop stand
– compete with the crop for:
• water and nutrients in the soil
• sunlight limiting the photosynthetic activity of thecrop.
– harbor pathogens in some circumstances
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How can weeds be controlled in
crops?
• by cultivation and tillage
– this has a number of disadvantages including beinglabor intensive, use of fossil fuel, compaction of soil
and soil erosion• using physical barriers
– use of black plastic mulch in vegetable production
– shredded bark or wood chips in lanscape applications
• currently there is interest in developing covercrops.
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Herbicides are now the most
widely used
• Herbicides are undoubtedly very effective
• They do not require large amounts of laborfor application
• They are quite cost effective
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Herbicides function
• interfere with various pathways of amino
acid biosynthesis
• disrupt photosynthesis
• interfere with lipid biosynthesis
• block synthesis of carotenoid pigments
• affect cell division• interact with other metabolic pathways
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What makes a "good" herbicide?
• must have herbicidal activity, kill plants
• compound must be readily taken up by theplant
• chemistry to synthesize the compoundmust be adaptable to large scaleproduction at an economic cost
• the compound must have low toxicity tonon-targets, including farmers, applicatorsand other organisms
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Herbicides can be classified in a
number of ways• When they are effective
– Before the weeds emerge from the soil, pre-emergent herbicides block processes essential toseed germination or early seedling development.
– After weeds have emerged, post-emergentherbicides can affect many metabolic processes thatare essential to plant growth.
• What plants are killed by the herbicide. – Broad spectrum herbicides are effective on
essentially anything that is green. – In a cropping system, herbicides must be selective,
having a minimal effect on the crop plant whilecontrolling the majority of the weeds.
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Herbicide-resistant crops
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What the herbicide must do in
order to be effective?
1. The herbicide must be taken up by the plant,get across the cuticle and absorbed into cells
2. The herbicide must be transported throughout
the plant (not necessary for 'contact'herbicides)
3. The herbicide must remain in its active form inthe plant
4. The herbicide must finally inhibit the metabolictarget within the plant cells.
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Herbicide-resistant plants
• Herbicides are generally non-selective (killingboth weeds and crop plants) and must be appliedbefore the crop plants germinate
• Four potential ways to engineer herbicideresistant plants
1) Inhibit uptake of the herbicide
2) Overproduce the herbicide-sensitive target
protein3) Reduce the ability of the herbicide-sensitive target
to bind to the herbicide
4) Give plants the ability to inactivate the herbicide17
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Herbicide Tolerant Crops
• Herbicide tolerant crops can be treated with anonselective herbicide (e.g. Roundup, Liberty)that controls most weeds without damage to the
crop.• Conventional weed control requires:
– Two or more herbicides
– Narrow application window, as little as one day
– Higher cost
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Growth of Herbicide Tolerant Traits
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The Roundup Ready Story
• Glyphosate is a broad-spectrum herbicide• Active ingredient in Roundup herbicide• Kills all plants it come in contact with
• Inhibits a key enzyme (EPSP synthase ) in an aminoacid pathway
• Plants die because they lack the key aminoacids
• A resistant EPSP synthase gene allows cropsto survive spraying
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+ Glyphosate
X
Roundup Sensitive Plants
X
X
Shikimic acid + Phosphoenol pyruvate
3-Enolpyruvyl shikimic acid-5-phosphate
(EPSP)
Plant
EPSP synthase
Aromatic
amino acids
Without amino acids,
plant dies
X
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Why glyphosate is such an effective
and relatively safe herbicide?• all plants (and microbes) possess this enzyme
and pathway to synthesize aromatic aminoacids, therefore all plants are susceptible toglyphosate.
• glyphosate is not metabolised very quickly byplants.
• glyphosate is quickly immobilized in soil, where itis metabolized by soil microorganisms
• the enzyme that is inhibited by glyphosate is notpresent in animals - animals do not have ashikimate pathway but obtain aromatic aminoacids in their diet.
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How could we make glyphosate
resistant plants?
• as glyphosate is a broad-spectrum herbicide, all(or nearly all) plants are killed by this herbicide.
• one approach is to identify or select organisms
with increased resistance to glyphosate.• "How come these organisms are now able to
survive this herbicide?".
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What is the reason for their
resistance?
• They produce more of the target enzyme, EPSPsynthase. The enzyme is still inhibited byglyphosate, but producing more of the enzyme
allows the plant cells to still make amino acids inthe presence of glyphosate.
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How can we make plants thatproduce more EPSP synthase?
• isolate an EPSP synthase gene froma plant (petunia)
• modify the promoter to give higher level of
expression (use the 35S promoter from cauliflowermosaic virus, CaMV 35S promoter).A highly active promoter will result in more mRNAfor EPSP synthase, and then more EPSPsynthase.
• produce transgenic plants containing this modifiedEPSP synthase gene
• analyze glyphosate resistance of transgenic plants
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How can we make plants thatproduce more EPSP synthase?
• isolate the mutant EPSP synthase gene fromSalmonella
• replace the bacterial promoter with regulatory
elements that would give high level expression intransgenic plants, such as using the CaMV 35Spromoter
• produce transgenic plants that express this gene• study their tolerance to glyphosate
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How can we make plants that
produce more EPSP synthase?
• Both of the approaches outlined above weresuccessful in that the transgenic plants were
more tolerant of glyphosate than theuntransformed plants
• However, the level of tolerance was notsufficient for this glyphosate resistance gene to
be used in crops.
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Features
• high level of expression of a plant EPSPsynthase
• an enzyme that was insensitive to glyphosate,
like the bacterial EPSP synthase• correct targeting of this protein to the chloroplast
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The last approach
In screening through bacterial collections, astrain of Agrobacterium (CP4) was identified thatcontained a suitable EPSP synthase. The genewas cloned and sequenced. From this a
chimeric gene was assembled from the followingcomponents: – the open reading frame for EPSP synthase from
Agrobacterium tumefaciens strain CP4 – the coding sequence for a chloroplast transit peptide,
to target the protein to the chloroplast – a highly expressed promoter that is expressed in
essentially all cells, in this case a modified form of theCaMV 35S promoter
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Bacterial
EPSP synthase
Shikimic acid + Phosphoenol pyruvate
3-enolpyruvyl shikimic acid-5-phosphate
(EPSP)
Aromatic
amino acids
Roundup Resistant Plants
+ Glyphosate
With amino acids,
plant lives
RoundUp has no effect;
enzyme is resistant to herbicide
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Market penetration of Roundup
Ready in US Soybeans
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Resistance to Glufosinate
• This herbicide blocks this synthesis of
glutamine. Because this enzymaticreaction utilizes ammonia, when the
pathway is blocked by the herbicide itresults in the accumulation of ammonia totoxic levels that kill the plant.
• This is another broad spectrum herbicide.
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Strategy
• the gene encoding this detoxifying enzymewas cloned
• the gene was modified so that it would be
expressed in plants, primarily by alteringthe promoter
• plants were then transformed with this
gene by a variety of methods• transformed plants were evaluated fortolerance to glufosinate
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Benefits and Advantages of
Herbicide Tolerant Crops
• Increased yields
• Reduced input costs
• Improved net returns
• Reduced herbicide use and environmentalimpact
• Performance – excellent weed control
• Simplicity – single product• Rotation crop flexibility
• Fewer weather and timing problems
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• Approximately 100 seed companies are in the process ofdeveloping, and in some cases already have marketed, Liberty-Link corn, which is resistant to glufosinate. This is in collaborationwith AgrEvo, the manufcturer of Liberty.
• Liberty-Link soybeans will be available in 1998, providing some
competition for Roundup Ready beans.• Garst is planning to introduce corn with multiple herbicideresistances, to imidazolinones and glufosinate, in the near future.
• Roundup Ready corn will be available in 1998. This will beavailable only through Dekalb in 1998, but the technology will belicensed to other seed companies for release the following year.
• Roundup Ready cotton was introduced in 1997.• Canola varieties with resistance to Roundup, Liberty or
imidazolinones were introduced in Canada in 1997.• Asgrow will introduce soybeans with resistance to Roundup and
sulfonylureas in 1998.
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Biotechnology Research Platforms
CRW = corn rootworm; LL = Liberty Link; RR- Roundup Ready; IR = Insect resistant 37
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Controversies surrounding the
development of herbicide tolerant crops
HTCs will result in an increase in the acreagethat is treated with herbicides by farmers andgrowers.
– 97% of agronomic production acreage is alreadytreated with herbicides, so there is essentially noroom to increase this figure, unless herbicides can beapplied in situations, such as forestry or golf courses,
where herbicides are not currently used.
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Controversies surrounding the
development of herbicide tolerant crops
HTCs will lead to increased use of herbicides. – if the HTCs are extremely tolerant, the farmer might be
encouraged to use herbicides more frequently, safe in theknowledge that his/her transgenic crops are extremely resistantand will not suffer any stress from repeated application of the
herbicide. However, each herbicide application is an additionalexpense, reducing potential profit. It unlikely that farmers willincur extra costs for the sake of a really clean field.
– on the other hand, this development might actually decreaseoverall herbicide use. The farmer could plant without using anypre-emergence herbicide, and take a wait-and-see position to
see where and when weeds will grow. Herbicides might onlyneed to be applied to selected areas rather than throughout thefield in the preemptive, precautionary strategy that is currently inuse
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Controversies surrounding the
development of herbicide tolerant crops
HTCs will become weeds - a weed is any plant that isgrowing in the wrong place at the wrong time, from thegrower's point of view. – It is interesting that this concern has not been raised previously
with non-engineered crops. However, there are some special
circumstances created here that are worthy of comment. Thereis always some carryover of seed from year to year in a field.You have all seen "volunteer corn" in a field of soybeans. If thatcorn was Roundup Ready and resistant to glyphosate, whathappens in the next year when you plant Roundup Readysoybeans? The volunteer corn comes up but cannot be
controlled with Roundup, so you have to find an alternativeherbicide just to control the volunteer corn. This indicates thatthere may be a need for greater planning and selection ofappropriate crop rotations.
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Controversies surrounding the
development of herbicide tolerant crops
HTCs will lead to a reliance on herbicides forweed control and abandonment, or failure todevelop alternative techniques.
– this is certainly possible, but it does depend on howHTCs are accepted, the share of the market theytake, and the pressures (or lack of them) to moveaway from pesticide use. There may be problems witha reliance on herbicides for weed control, it is not the
development of HTCs that have led to this situation. – HTCs may allow, for the first time, a chemical method
to control pests like parasitic weeds, where at themoment killing the weed would also kill the host.
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Controversies surrounding the
development of herbicide tolerant crops
There will be increased use of particularherbicides on HTCs.
– this is obviously the hope of Monsanto for glyphosate,
and similarly for other herbicide companies. – however, HTCs will also allow new combinations of
crops and herbicides to be tested. Correctmanagement of these combinations could also lead to
development of methods to reduce the chances thatweeds will develop resistance, by rotating herbicideuse.
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Controversies surrounding the
development of herbicide tolerant crops
Some HTCs may be developed for herbicidesthat are regarded as more damaging to theenvironment. – while this is certainly possible, and is being pursued
for some crops, herbicides will still be regulated byEnvironmental Protection Agency (EPA), Food andDrug Administration (FDA) and Department ofAgriculture (USDA) regulations. It is unlikely thatherbicides that are more toxic than those currently in
use would be developed as a result of HTCdevelopments.
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Controversies surrounding the
development of herbicide tolerant crops
HTCs will reduce the level of biological diversity. – after all, that is the whole point of using herbicides, to
produce a field of corn, soybean, whatever, withoutany other plants growing in the field.
– a possible problem is that weeds that, for whateverreason, survive herbicide treatment will have greatlyreduced competition and be able to rapidly propagate.
– whenever cultural practices are changed, there is thepotential for new weed species to develop and
endure. HTCs might provide new niches for newweeds to fill.
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Controversies surrounding the
development of herbicide tolerant crops
Herbicide resistance genes will be transferredfrom HTCs to wild relatives of the crops – potential to develop "superweeds". One of the most
widely quoted examples is shattercane. This is thesame species as sorghum, and the two are quitecross-fertile. If sorghum was developed withresistance to glyphosate, it is almost certain that theresistance gene would be transferred to the wild
shattercane weed. This would lead to shattercanethat could no longer be controlled with glyphosate.
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Controversies surrounding the
development of herbicide tolerant crops
HTCs will result in the development of newvarieties of herbicide resistant weeds – the most serious threat of increasing use of one, or a
small number of, herbicide is that this will promotedevelopment of herbicide resistance in weedpopulations. Almost without exception, introduction ofany new pesticide, for weeds, insects, etc., haseventually led to the development of resistance to that
pesticide. Continual use of the same herbicide in thesame location, perhaps with more than oneapplication per year, will accelerate the developmentof resistant weeds.
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