Cong Nghe Sinh Hoc Thuc Vat

7/29/2019 Cong Nghe Sinh Hoc Thuc Vat

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Plant Biotechnology

Two broad categories

New techniques for plant

propagation

Already a profitable enterprise

Works with existing germplasm

Mutation breeding

The creation of transgenic

plants


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Transgenic Plant Biotechnology

Made possible about 30 years ago by thedevelopment of ways to introduce and expressforeign genes in plants.

Commercial ventures have been funded byChemical companies; Dupont (Pioneer), Monsanto,

Dow, Syngenta (Zeneca, Novartis), BASF, Bayer

(Aventis)

Seed Companies; Pioneer, Delta-Pine Land, Seminis

Venture Capital has funded many smaller companies.


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Endless Possibilities

The ability to express any gene in any part of aplant means that the possibilities for crop

improvement are theoretically limitless.

In reality many potential improvements are

beyond our present capabilities.

Pleitropic effects are often a fatal flaw


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Disease Resistance: Practicalities

Its easy to create a disease resistant transgenic

plant.

Its very hard to make one that is commercially

viable


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Why cant famers buy this product?

Dont know for sure

Didnt work in larger scale field trials?

Didnt work well enough to make it economically

viable?

Other issues (toxicity etc.)?


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Issues Need a suitable gene and promotor

Legal issues

Ability to transform plant Technical

Legal

Pleiotropic Effects

Yield depression Economic Impact

Does it save the farmer money or time? Does it increase yield or obviate the need for fungicide applications?

And/or would consumers be willing to pay more for it?

Regulatory Allergenic, toxic

weedy

Public Perception


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Requirements -1

Techniques for the introduction of DNA into

plants. Available for most plants.

Agrobacterium

Particle Bombardment


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Agrobacterium

Agrobacterium tumefaciens cause tumorous

growth on plants.

Does this by transferring large extra-

chromosomal piece of DNA (T-DNA) into host

genome. T-DNA encodes several oncogenes.


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Agricultural

biotechnology:Gene

exchangebydesign

StantonB.

Gelvi

n

Nature433,

583-584(10Feb

ruary2005)
http://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.html


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Agricultural

biotechnology:Gene

exchangebydesign

StantonB.

Gelvi

n

Nature433,

583-584(10Feb

ruary2005)
http://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.htmlhttp://www.nature.com/nature/journal/v433/n7026/full/433583a.html


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Biolistics

DNA is bound to metal (often gold) particles and

literaly shot into the cell (gunpowder or

compressed gas).

The rare events in which foreign DNA has been

incorporated into the host genome are identifiedusing screening for selectable marker phenotypes

(e.g. herbicide resistance)


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http://www.plant.uoguelph.ca/research/homepages/raizada/Equipment/RaizadaWeb%20E ui ment%20Ima es/8.%20PDS100HE%20 un.


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Requirements-2

Components for expressing a gene in a plant

Promoter: Controls when where and how

much gene is turned on. For disease

resistance we might want a disease or

pathogen-inducible promoter

Gene: Codes for protein.

Can also be designed to turn

off an endogenous gene

Terminator:

Needed at the

end of a gene


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Requirements-3

Access to the appropriate intellectual property

Transformation techniques

IP specific to plant or class of plant

Genes, promoters, selectable markers etc.

Approaches -e.g. RNAi


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Problems-1

Genetic engineering can be unpredictable Somewhat analogous to traditional plant breeding- dealing with a

very complex situation

Every gene/plant combination is unique. Every transformed

plant is unique- need to screen through lots of transformants tofind a good one

Results seen in the laboratory often cannot be replicated under

field conditions.


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Problems-2

Intellectual property is an extremely complex area

Often several different pieces of IP involved in a

single transgenic plant

Includes IP on the transformation method, each

element of the expression construct

Especially difficult for smaller companies


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Problems-3

Very strict regulations

Transgenic plants need to go through an extremely

stringent process of analysis.

Regulated by USDA, EPA and FDA

The exact position of all the inserted DNA has to bedocumented.

Difficult to prevent pollen spread in certain cases

E.g. Prodigene caseA large problem for BioPharming


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Ultimately this regulation is probably a good

thing for the industry but it creates a LARGE

FINANCIAL HURDLE

~~$60m to create a commercial transgenic plant


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NY TIMES

Spread of Gene-Altered Pharmaceutical Corn Spurs $3 Million FineBy ANDREW POLLACK

Published: December 7, 2002A biotechnology company will pay the government about $3 million to settle chargesthat it did not take proper steps to prevent corn that was genetically engineered to produce

pharmaceuticals from entering the food supply.

Aventis Says More Genetically Altered Corn Has Been

FoundBy DAVID BARBOZA

Published: November 22, 2000The Aventis Corporation said yesterday that a genetically altered

protein unapproved for human consumption had been discovered in

a variety of corn that could be headed toward the nation's food supply.


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Problems-5

Development of product is very expensive.

Transgenic plant needs to be equal of elite

varieties for all other traits.

New germplasm may supercede variety into which

construct has been introduced


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Problems-6

Public acceptanceMost criticisms are unsubstantiated but public is

skeptical.

European restrictions mean its hard for US farmers toexport

Labeling requirement, although ostensibly fair, is

tantamount to a complete ban

A h t G ti E i i


24/61

Approaches to Genetic Engineering

for Disease Resistance

Commercially available virus and insect resistant

plants are available

No commercially available Fungal or bacterial

resistant plants (see above!)


25/61

Possible Approaches

Using R-genes

Many R-genes cloned

Could simply introduce cloned R-gene to a new

species or line by transformation.

This works in some cases but:

R-genes tend to be quite specific and easily overcome

Often dont work in other species anywayThis might be a good way to rapidly pyramid R-genes

or to introduce different alleles of the same gene into a

single background


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Using R-genes continued

R-gene expression can cause growth reductions

E.g Rpm1

This is likely to be a case-by-case issue though

and may be remedied by using weaker promoters


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Using R-genes- continued

R-genes provide very effective resistance whentriggered. The trick is to trigger them at the right

time and place.

How about if you could activate R-genes at an

appropriate time when the plant is attacked by a

broad variety of pathogens?

Overcomes the disease/race-specificity problem


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De Wit 1992, Ann. Rev. Phytopathology 30.391-41


29/61

This approach is dependent of finding appropriate

promoters.

Genomics approaches are ideal for finding such

promoters


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Using R-genes- Custom Design

Can you design an R-gene to interact with and be

activated by your molecule of choice?

Ideally it would interact with a molecule essential

for fungal pathogenicity

This would mean the fungus would incur a large

penalty if it evolved to overcome the R-gene.

Di t d l l l ti G Sh ffli


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Directed molecular evolution- Gene Shuffling

Lassner and BedbrookCurrent Opinion in Plant Biology

Volume 4, Issue 2, 1 April 2001, Pages 152-156
http://www.sciencedirect.com/science/journal/13695266http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%236252%232001%23999959997%23229523%23FLA%23&_cdi=6252&_pubType=J&_auth=y&_acct=C000015398&_version=1&_urlVersion=0&_userid=290868&md5=11b690b72f3b80f14f495f296f31612chttp://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%236252%232001%23999959997%23229523%23FLA%23&_cdi=6252&_pubType=J&_auth=y&_acct=C000015398&_version=1&_urlVersion=0&_userid=290868&md5=11b690b72f3b80f14f495f296f31612chttp://www.sciencedirect.com/science/journal/13695266


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Gene shuffling has been used to identify

recognition determinants in R-genes Wulff BBH, Thomas CM, Smoker M, Grant M, Jones JDG: Domain

swapping and gene shuffling identify sequences required for induction of anAvr-dependent hypersensitive response by the tomato Cf-4 and Cf-9

proteins. Plant Cell 2001, 13:255-272.

Attempts have been made to shuffle for specific novelrecognition

No success (that I know of).

This would really be the dream scenario if possible.

Using R genes finding novel R


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Using R-genes- finding novel R-genes from other species

See Wroblewski et al Plant Phys 2009 150:1733-1749.

Identified and cloned 171 putative bacterial effector

genes, from a variety of bacterial pathogens, using a set

of predictive rules.

Expressed them in 59 different plant genotypes including

accessions of lettuce, tomato, pepper, Arabidopsis,tobacco

Used an agrobacterium transient assay

10,089 different combinations.

More than a third elicited a reaction


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More than a third elicited a reaction

in at least one genotype

Can express effectors in plant


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Can express effectors in plantpathogens infecting the target host

Cant do transient agro infection in all plants

Sohn et al 2007 Plant cell 19:4077-4090

Expressed effectors fromH. parasitica in P.

syringae and got appropriate response in the host.


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So what?

The thought here is:

1.In the target plant/pathogen system; Identify

effectors that are widespread within the pathogen

species. These are likely important for

pathogenesis

2.By expressing these effector genes in

heterologous hosts can find R-genes that

recognize them.

3.Clone the R-genes and move them into target host


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There R-genes stand a chance of being durable

since they target conserved effectors that are

likely to be hard for the pathogen to lose.


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This is essentially what was done in:

Vleeshouwers, et al. 2008. Effector genomics acceleratesdiscovery and functional profiling of potato disease resistance and

Phytophthora infestans avirulence genes. PLoS ONE, 3:e2875

Expressed 54 predicted P. infestans effector

genes in a set of wild potatoes using a PVX

system

Found a couple of R-genes


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Gene Silencing -RNAi

A technology that uses a basic plant process to

silence selected genes

From Wikipedia


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RNAi

This has been used extensively to silence plant

genes.

This the basis of most transgenic viral resistance

in plants.

Viral protein gene is silenced in plant cells

Virus cant reproduce, spread

A transgenic success story (more-or-less)

G i i i S S i


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Genetic Engineering Success StoriesTransgenic Papaya, resistant to Papaya Ring Spot


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RNAi

What about other uses of RNAi

RNAi is a process common to most higher

organisms including plants, fungi, insects,vertebrates.

Can you get the small RNAs into Fungi, nematodes,bacteria to silence important genes?

In some cases yes

D RNAi h b d li ti f


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Does RNAi have broader applications for

Transgenic Disease Resistance?

Fungi/Oomycetes Resistance toPhytophthora nicotianae

in Tobaccowww.venganzainc.com


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Insects

Silencing a cotton bollworm P450

monooxygenase gene by plant-mediated RNAi

impairs larval tolerance of gossypolNature Biotechnology 25, 1307 - 1313 (2007)

Control of coleopteran insect pests through RNA

interferenceNature Biotechnology 25, 1322 - 1326 (2007)


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Nematodes

Engineering broad root-knot resistance in

transgenic plants by RNAi silencing of a

conserved and essential root-knot nematodeparasitism gene

PNAS | September 26, 2006 | vol. 103 | no. 39 |

14302-14306


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Trends in Biotechnology

Volume 25, Issue 3, March 2007, Pages 89-92
http://www.sciencedirect.com/science/journal/01677799http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%235181%232007%23999749996%23644784%23FLA%23&_cdi=5181&_pubType=J&_auth=y&_acct=C000015398&_version=1&_urlVersion=0&_userid=290868&md5=ca59930fff846db43d9cadd86eef16achttp://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%235181%232007%23999749996%23644784%23FLA%23&_cdi=5181&_pubType=J&_auth=y&_acct=C000015398&_version=1&_urlVersion=0&_userid=290868&md5=ca59930fff846db43d9cadd86eef16achttp://www.sciencedirect.com/science/journal/01677799


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This is a really great approach if it works

Very specific

Can target essential processes so very difficult toovercome

No growth penalty for plant

Other Approaches to Engineering


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Other Approaches to EngineeringDisease Resistance in Plants

Manipulate components of the existing defence

reponse

Introduced preformed anti-fungal metabolites

from other species

Also could manipulate cell-death pathways

i i f


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Existing Defence Response

Can try to manipulate single defensive proteins

E.g Anti Fungal Peptides (Defensins), PR proteins

Or can try to work with Master Switches

Genes that effect the expression or function of whole

suites of other genesKinases, Transcription Factors and other signalling

molecules

Th l f b h h


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There are numerous examples of both these

approaches working in the literature

E.g NPR1 overexpression

Generation of broad-spectrum disease resistance by

overexpression of an essential regulatory gene insystemic acquired resistance

Proc Natl Acad Sci U S A. 1998 May 26; 95(11):

65316536.

But growth peanlties are frequently observed


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ProcNatl

AcadSciUSA

.1998May26;

95(11):65316536.


52/61

Why are there no commercial transgenic


53/61

Why are there no commercial transgenic

products for fungal or bacterial resistance?

IP Results cant be replicated

Growth Penalty

Greenhouse = Field

Arabidopsis = Corn

No feasible economic model

Regulatory Issues


54/61

Three factors need to be present the technical

solution to a problem which has no other obvious

alternative, the economic incentive forimplementing the solution, and therefore market

and public acceptance. Collinge et al Eur J Plant Pathol (2008) 121:217231- required

reading

I would add access to IP

P l Vi


55/61

Personal View Possible that transgenic plants for fungal/bacterial disease

resistance will still be largely unavailable in 25 years. On the one hand:

In most crops a large amount of natural variability is available

The main exceptions are clonally propagated crops like banana.

I dont think public perception is going to be an issue in the long run.

On the other From an evolutionary standpoint, we are trying to do the same thing as the

plant- improve disease resistance without effecting yield.

Can we improve on millions of years of evolution?

In most cases, for a viable financial outcome we need to engineer broad-spectrum resistance

As weve seen often conferring resistance to biotrophs confers susceptibility tonecrotrophs and vice-versa

S h t th P i t?


56/61

So whats the Point?

While plant biotech has been successful/lucrative

for some applications, transgenic diseaseresistance is largely a failure commercially (sofar).

Is anything of practical use going to come fromresearch into the molecular genetics of diseaseresistance?Quite possibly. Technologies like RNAi are poised to

exploit our molecular knowledge.Our knowledge is still rapidly growing

Understanding is a goal in itself.

Molecular markers for MAS


57/61

A major crop for the developing and developed

world

85 million tonnes produced annually

More the 2/3 of this production is for local

consumption

Export market worth ~$5 billion annually

Great candidate for genetic engineering

Banana


58/61

Bananas come in all shapes and sizes- this

diversity is very hard to exploit.


59/61

Black Sigatoka


60/61

Fields sprayed

with fungicide up

to once a week

Genetic Transformation- the best method


61/61

Genetic Transformation the best method

for Banana Improvement

No realistic alternative for plant improvement

Conventional Breeding Difficult

Commercial varieties need to have very precisecharacteristics.

No chance of gene escape into the environment.

Traits developed in commercial varieties can beeasily transferred to non-commercial varieties.

Commercially produced bananas have a single

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Cong Nghe Sinh Hoc Thuc Vat