Biotechnological Trait Enhancement in Banana - Pngg.org

clatteringlippsBiotechnology

Dec 5, 2012 (5 years and 1 month ago)

178 views

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


The Creation Of Transgenic Plants



The introduction and expression of novel genes
in plants



Creates new germplasm



Transgenic Plant Biotechnology


Made possible about
30
years ago by the
development of ways to introduce and express
foreign genes in plants.



Commercial ventures have been funded by


Chemical 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.


Endless Possibilities


The ability to express any gene in any part of a
plant 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



Disease Resistance: Practicalities


It’s easy to create a disease resistant transgenic
plant.



It’s very hard to make one that is commercially
viable

Why can’t famers buy this product?


Don’t know for sure


Didn’t work in larger scale field trials?


Didn’t work well enough to make it economically
viable?


Other issues (toxicity etc.)?


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

Requirements
-
1


Techniques for the introduction of DNA into
plants. Available for most plants.



Agrobacterium



Particle Bombardment

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”.


Agricultural biotechnology:


Gene exchange by design

Stanton B. Gelvin

Nature
433
,
583
-
584
(
10
February
2005
)


Agricultural biotechnology:


Gene exchange by design

Stanton B. Gelvin

Nature
433
,
583
-
584
(
10
February
2005
)

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 identified
using screening for selectable marker phenotypes
(e.g. herbicide resistance)


http://www.plant.uoguelph.ca/research/homepages/raizada/Equipment/RaizadaWeb%20
Equipment%20Images/8.%20PDS100HE%20gun.jpg

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

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

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 to
find a “good one”


Results seen in the laboratory often cannot be replicated under
field conditions.

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

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 be
documented.


Difficult to prevent pollen spread in certain cases


E.g. Prodigene case


A large problem for BioPharming



Ultimately this regulation is probably a good
thing for the industry but it creates a LARGE
FINANCIAL HURDLE



~~$
60
m to create a commercial transgenic plant


NY TIMES

Spread of Gene
-
Altered Pharmaceutical Corn Spurs $3 Million Fine

By ANDREW POLLACK

Published: December 7, 2002

A biotechnology company will pay the government about $3 million to settle charges

that 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
Found


By DAVID BARBOZA


Published: November
22
,
2000

The 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.

1
http%3a%
%20Sprea
%20A%20
2002120
By%20AN
The%20N
%20Natio
www%2en
TopAd%2
XU3hc0JC
1
http%3a%
%20Aven
%20The%
2000112
By%20DA
The%20N
%20Busin
www%2en
TopAd%2
UlmCf3Cs
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

Problems
-
6


Public acceptance


Most criticisms are unsubstantiated but public is
skeptical.


European restrictions mean it’s hard for US farmers to
export


Labeling requirement, although ostensibly fair, is
tantamount to a complete ban


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!)


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 don’t work in other species anyway


This might be a good way to rapidly pyramid R
-
genes
or to introduce different alleles of the same gene into a
single background

Using R
-
genes continued


R
-
gene expression can cause growth reductions


E.g Rpm
1



This is likely to be a case
-
by
-
case issue though
and may be remedied by using weaker promoters

Using R
-
genes
-

continued


R
-
genes provide very effective resistance
when
triggered
. 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


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


This approach is dependent of finding appropriate
promoters.



Genomics approaches are ideal for finding such
promoters

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.


Directed molecular evolution
-

Gene Shuffling



Lassner and Bedbrook
Current Opinion in Plant Biology


Volume
4
, Issue
2
,
1
April
2001
, Pages
152
-
156


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 an
Avr
-
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 novel
recognition



No success (that I know of).



This would really be the dream scenario if possible.


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
in at least one genotype

Can express effectors in plant
pathogens infecting the target host


Can’t do transient agro infection in all plants


Sohn et al
2007
Plant cell
19
:
4077
-
4090


Expressed effectors from
H. parasitica
in
P.
syringae
and got appropriate response in the host.


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


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.


This is essentially what was done in:


Vleeshouwers, et al.
2008
. Effector genomics accelerates
discovery and functional profiling of potato disease resistance and
Phytophthora infestans avirulence genes. PLoS ONE,
3
:e
2875



Expressed
54
predicted
P. infestans

effector
genes in a set of wild potatoes using a PVX
system



Found a couple of R
-
genes

Gene Silencing
-
RNAi


A technology that uses a basic plant process to
silence selected genes

From Wikipedia

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 can’t reproduce, spread


A transgenic success story (more
-
or
-
less)

Genetic Engineering Success Stories

Transgenic Papaya, resistant to Papaya Ring Spot

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

Does RNAi have broader applications for
Transgenic Disease Resistance?


Fungi/Oomycetes


Resistance to
Phytophthora nicotianae
in Tobacco
www.venganzainc.com

Insects


Silencing a cotton bollworm P
450
monooxygenase gene by plant
-
mediated RNAi
impairs larval tolerance of gossypol


Nature Biotechnology
25
,
1307
-

1313
(
2007
)


Control of coleopteran insect pests through RNA
interference


Nature Biotechnology
25
,
1322
-

1326
(
2007
)


Nematodes


Engineering broad root
-
knot resistance in
transgenic plants by RNAi silencing of a
conserved and essential root
-
knot nematode
parasitism gene



PNAS |
September
26
,
2006

| vol.
103
| no.
39
|
14302
-
14306




Trends in Biotechnology


Volume
25
, Issue
3
, March
2007
, Pages
89
-
92


This is a really great approach if it works


Very specific


Can target essential processes so very difficult to
overcome


No growth penalty for plant

Other Approaches to Engineering
Disease Resistance in Plants


Manipulate components of the
existing defence
reponse



Introduced preformed
anti
-
fungal metabolites

from other species



Also could manipulate cell
-
death pathways


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 genes


Kinases, Transcription Factors and other signalling
molecules


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 in
systemic acquired resistance


Proc Natl Acad Sci U S A. 1998 May 26; 95(11):
6531

6536.



But growth peanlties are frequently observed



Proc Natl Acad Sci U S A.
1998
May
26
;
95
(
11
):
6531

6536
.

Why are there no commercial transgenic
products for fungal or bacterial resistance?



IP


Results can’t be replicated


Growth Penalty


Greenhouse = Field


Arabidopsis = Corn


No feasible economic model


Regulatory Issues



“Three factors need to be present the technical
solution to a problem which has no other obvious
alternative, the economic incentive for
implementing the solution, and therefore market
and public acceptance.”


Collinge et al Eur J Plant Pathol (
2008
)
121
:
217

231
-

required
reading


I would add “access to IP”


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 don’t 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 we’ve seen often conferring resistance to biotrophs confers susceptibility to
necrotrophs and vice
-
versa


So what’s the Point?


While plant biotech has been successful/lucrative
for some applications, transgenic disease
resistance is largely a failure commercially (so
far).


Is anything of practical use going to come from
research into the molecular genetics of disease
resistance?


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



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

Bananas come in all shapes and sizes
-

this
diversity is very hard to exploit.

Black Sigatoka

Fields sprayed
with fungicide up
to once a week

Genetic Transformation
-

the best method
for Banana Improvement


No realistic alternative for plant improvement


Conventional Breeding Difficult


Commercial varieties need to have very precise
characteristics.


No chance of gene escape into the environment.


Traits developed in commercial varieties can be
easily transferred to non
-
commercial varieties.


Commercially produced bananas have a single
major pathogen.