Download Biotechnology for a pesticide free Vineyard? - IOBC-WPRS

Biotechnology for a pesticide
free Vineyard?
GM-plants in the World agriculture
• >99.5 % (excluding unknown-unregistered
virus resistant crops in China)
• Products for which seeds can be sold and /or
also a specific herbicide
– (advantage to product developer and to “user”)
• User/Benefit: convenience, reduction of
complexity, reduction of labor, …
The case Papaya
1940 discovery of the Papaya RingSpot Virus (PRSV)
1950 abandon of the production on Oahu
1960 production on Hawaii region of Puna
1980 95% of the production in Puna
1992 PRSV in Puna
1994 heavy damages with about 50% of the plants infected
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Da Gonsalves et al 2000 APSnet)
The case Papaya
1991 Insertion of the Capsid protein gene of the PRS-virus
1994 Field trials
1998 Release for commercialization, delivery of seeds
2000 first production
2002 16’000 Tons (37% of the total production)
2008 about 24‘000 T (50%)
2011 Trans 80% (Export to Japan reduced from 16’000 T to 1000T )
Gonsalves et al 2000 APSnet
Personal communication 2012)
Constraints in classical breeding
• Always a new cultivar
• Several generation needed to eliminate wild non
target genome
• Long generation time (from seed to seed 4- more
years)
• Pyramid several resistance loci (genes) against the
same and different pathogens difficult/improbable
• Marker assisted selection
How to add few traits without changing
everything?
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First: what traits? –
– disease resistance
Based on what? –
– as nature does it - on recognition genes, so that the defense
reaction is only activated when necessary
What is the source of such genes?
– The wild gene pool of the “species” (Vavilov).
With which method? –
– currently by Agrobacterium, in near future with technologies
allowing site targeted introduction
What is the results?
– Cisgenic lines of the desired cultivar with specific additional traits:
example apple - scab resistance, fire blight resistance
GM-Grape
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Fungal, virus and bacterial resistance (claim)
Field trials
EU 7
USA 59,
Approval
None
Perspectives No immediate commercial use
of GM-grape vines is expected.
Main field experiments with GM-grapes
Paese
Italy
Australia
data
Varietà
05/1999
09/2006
06/2003
06/2008
Thompson
seedless; Silcora
Shiraz; Sultana;
Cabernet;
Sauvignon;
Chardonnay.
Origine
Fenotipo
Auxin synthesis
P. savastanoi (batterio)
Parthenocarpic
ppo
Ufgt
dfr
inv
V. vinifera
V. vinifera.
V. vinifera
V. vinifera
Cabernet Franc
Superoxid dismutase
Arabidopsis thaliana
Altered browning
Altered berry skin
Altered anthocyanin/tannin synthesis
Altered sucrose content of fruit
Altered flowers and fruit
Cold tolerance
06/2005
10/2009
01/2003
30/2006
Roostok 41 B
GFLV
Virus resistant
GFLV; GVA;
GVB.
Virus resistant
07/1999
10/2009
(Interrupted)
Dornfelder;
Riesling;
Seyval Blanc.
CP genes from
GFLV
CP genes from:
GFLV, GVA
and GVB
chi26; bgl32;
rip-30
Hordeum vulgare
Fungal resistant
Canada
France
Romania
Germany
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Russalka;
Roostok 41B.
Gene
United
State
03/2006
12/2011
chi
Trichoderma harzianum
(fungo)
Fungal resistant
05/1998
11/2008
chi
T. harzianum
Fungal resistant
04/2007
04/2008
En42; MSI-99;
MagII; PGL;
Synthetic antimicrobial peptide.
T. harzianum;
Xenopus laevis (rana
africana);
Amaranthus caudatus.
A. caudatus;
X. leavis.
Fungal resistant;
Bacteria resistant
04/2000
04/2010
Chardonnay;
Merlot.
04/2006
04/2007
Chardonnay;
Merlot.
PGL; MagII
07/2007
07/2008
NR Rootstock
CP gene
GLRaV-2;
GLRaV-3; GFLV
Virus resistance
05/2005
05/2009
Niagara;
Concord
Drr206
Pisum sativum
Fungal resistant
06/2004
06/2014
Chardonnay
Thompson
Seedless;
Rootstock Saint
George.
Polygalacturonase inhibitor
protein
P. communis
Fungal resistant
V. vinifera
Fungal resistant
Bacteria resistant
11/2007
09/2008
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Bacteria resistant
Bacteria resistant
Resistance against virus
diseases
• Complex of Grapevine Fanleaf
transmitted by nematodes
– Gene of the coat protein Virus (GFLV),
inserted in Nebbiolo (Italy) and in
rootstocks (France, USA).
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Transgenic grapes (2000)
• Gene coding for chitinase (ThEn42)
derived from the fungus Trichoderma,
• Two bacterial genes coding for
antimicrobial peptides (MagII and MSI99)
• Marker gene nptII origin bacteria
Increment of powdery mildew resistance; little effect, sever
somaclonal variation, in the field increase in resistance
against Botrytis.
Research abandoned.
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Grape:
University of Florida grape researcher Dennis Gray has
been awarded a five-year, $2.2 million specialty crop
grant from the U.S. Department of Agriculture's
National Institute of Food and Agriculture to continue
his work toward creating disease-resistant, seedless
muscadine grape varieties.
• Florida is the second state in
consummation of viticulture products in
the USA (after California)
• Florida does almost not produce grapes
(< 1%)
• All varieties of V. vinifera will be
destroyed through the «Pierce disease»
caused by the bacterium Xylella
fastidiosa
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Grape genes associated to the
defense against pathogens
• Grape responds to an attack of a
pathogen with the formation of several
specific proteins
• Taumatin similar protein (gene VVTL-1)
inhibits in vitro several pathogens.
• Gene VVTL-1 isolated from Chardonnay
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Research group of Prof Gray, Uni Florida
Transformants
• Variety: Thomson seedless
• Construct: VVTL-1, promoter 35S,
marker genes gfp/nptII
• Constitutive expression high (105-107 >
than in Chardonnay)
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Field Testing Transgenic Grapevine
for Disease Resistance
• V. vinifera thaumatin-like protein (VVTL−1)
resitance to the bacterial Pierce’s disease (PD)
and a range of fungal diseases endemic to
Florida.
• In 2007, transgenic V. vinifera ‘Merlot’ and
‘Thompson Seedless’ and hybrid scion ‘Seyval
Blanc’ and rootstock ‘Freedom’ were planted.
D. J. GRAY, Z. T. Li, S. A. Dhekney, D. L. Hopkins,
and T. W. Zimmerman
Resistance against powdery
mildew
Thomson seedless
Not
transformed
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Thomson
trasnsformed VVTL-1
Dhekney, Li & Gray. 2011. Plant 47: 458-466
Development of powdery mildew
Thomson control
Transgenic lines
Tampa,
resistant hybrid
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Syrah prima della veraison
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Resistance against black rot
Transgenic lines
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Thomson control
Progression of black rot
control TS-ck (▲ )
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Resistance against sour rot
during storage
One week
Three weeks
Thomson control
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Transgenic lines
Resistance against sour rot
during storage
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Summary of D. Gray’s work
• ‘Thompson Seedless’ grapevines genetically
engineered with synthetic lytic peptide
transgenes (lima-a or lima-b) or cisgene Vitis
vinifera thaumatin-like protein gene (vvtl-1),
• GM-lima plants showed little or no Pierce’s
disease
• GM-plants containing vvtl-1 show significant
resistance to anthracnose, black rot, and sour
bunch rot diseases,
Resistance to P. viticola (Rpv)”
• Rpv1 and Rpv2 are located on chromosome 12
and chr 18,
• Rpv3 locus on chr 18 (Hypersensitivity),
– Corresponding to QTL peaks for downy mildew
resistance in the grapevine ‘Bianca’,
• Rpv12 from the Asian Species Vitis amurensis
Example apple and apple diseases
• Problem similar to grape
– Breeding for resistance results in a new
variety
– Disease problem is resolved through fungicide
Apple: Classical breeding produced scab
resistant cultivars by introgressing the gene Vf
Vf (scab) and FB_MR5 (fire blight) have been
cloned and demonstrated to function as
recognition gene of the pathogen
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Patocchi, Broggini Vanblaere; Fahrentrapp, Kost
Schorf verursacht
durch den Pilz
Venturia inaequalis
In Europe between 10 and 15
specific fungicide treatments/year
Line 11.1.53
Gala
Field trials Wageningen
October 2011
Lines : 7, 11 ,12 (8 plants each line)
Fire Blight
CG 34
29.10.2013
Fire blight of apple
and pear
Origine of resistance
M. evereste, M. robusta 5
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Representative plants of each plant line 39 days after leaf inoculation with
Erwinia amylovora. Gala, Malus robusta 5 , T lines carrying FB_MR5.
CG 36
29.10.2013
Cisgenesis
• Risk and dangers:
– Specific risk due to the gene product are
already examined in classical crosses.
– Insertion site effect? Epigenic effect?
– Use of “ pathogen recognition genes” no side
effects ! ?
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Conclusions
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Classical breeding: new variety
Cisgenic: adds missing trait
Risk evaluation
Ethic questions
Environmental and consumer benefit?
Main element in our discussion: we have to
consider/evaluate the product and not the use of the
technology «genetic engineering»
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• Public
acceptance?
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Summary
• Most transformation with an exotic target
gene
• Grape genes in overexpression using
promoter (VVTL-1) 35S
Transformant have unnatural characteristics
Grape Taumatin is only expressed after infection and not
present at active concentration constitutively
Effect on the productivity
Effect on the vinification?
Effect of a high concentration on the environment?
Consumer?
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Wrong information
• ( found in :http://www.ask.com/)
• Are Seedless Grapes Genetically Modified?
• Answer: Seedless grapes are the result of
genetically modified technology. When
growing, these seeds normally undergo
selective breeding in order to develop
varieties with little or no seeds at all.
Insert T-DNA
Rs
RB
prom
HcrVf2
R-LBD Recombinase
CodA-NptII fusion
Rs LB
term
Inserzione attraverso Agrobacterium
Rs
RB
prom
HcrVf2
R-LBD Recombinase
CodA-NptII fusion
Rs LB
term
Chemical activation of the recombinase
Rs LB
RB
prom
HcrVf2
term
T-DNA insert after the recombination