lecture 7 herbicide resistance

8/6/2019 Lecture 7 Herbicide Resistance

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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



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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http://en.wikipedia.org/wiki/File:Pink_petunias.jpg



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



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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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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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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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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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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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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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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C i di h





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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C i di h





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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lecture 7 herbicide resistance

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