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, …

5

The case Papaya

Da Gonsalves et al 2000 APSnet)

1940 discovery of the Papaya RingSpot Virus (PRSV)1950 abandon of the production on Oahu1960 production on Hawaii region of Puna1980 95% of the production in Puna1992 PRSV in Puna1994 heavy damages with about 50% of the plants infected

Gonsalves et al 2000 APSnetPersonal communication 2012)

1991 Insertion of the Capsid protein gene of the PRS-virus1994 Field trials1998 Release for commercialization, delivery of seeds2000 first production2002 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 )

The case Papaya

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?

• 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

• 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

10

Paese

data Varietà Gene Origine Fenotipo

Italy 05/1999 09/2006

Thompson seedless; Silcora

Auxin synthesis P. savastanoi (batterio) Parthenocarpic

Australia 06/2003 06/2008

Shiraz; Sultana; Cabernet; Sauvignon; Chardonnay.

ppo Ufgt dfr inv

V. vinifera V. vinifera. V. vinifera V. vinifera

Altered browning Altered berry skin Altered anthocyanin/tannin synthesis Altered sucrose content of fruit Altered flowers and fruit

Canada

Cabernet Franc Superoxid dismutase Arabidopsis thaliana Cold tolerance

France 06/2005 10/2009

Roostok 41 B

CP genes from GFLV

GFLV Virus resistant

Romania 01/2003 30/2006

Russalka; Roostok 41B.

CP genes from: GFLV, GVA and GVB

GFLV; GVA; GVB.

Virus resistant

Germany 07/1999 10/2009 (Interrupted)

Dornfelder; Riesling; Seyval Blanc.

chi26; bgl32; rip-30

Hordeum vulgare Fungal resistant

11

United State

03/2006 12/2011

Bacteria resistant

04/2000 04/2010

Chardonnay; Merlot.

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.

Fungal resistant; Bacteria resistant

04/2006 04/2007

Chardonnay; Merlot.

PGL; MagII A. caudatus; X. leavis.

Bacteria resistant

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

11/2007 09/2008

V. vinifera Fungal resistant Bacteria resistant

Resistance against virus diseases

• Complex of Grapevine Fanleaftransmitted by nematodes – Gene of the coat protein Virus (GFLV), inserted in Nebbiolo (Italy) and in rootstocks (France, USA).

12

Transgenic grapes (2000)

• Gene coding for chitinase (ThEn42) derived from the fungus Trichoderma,

• Two bacterial genes coding for antimicrobial peptides (MagII and MSI-99)

• Marker gene nptII origin bacteria

13

Increment of powdery mildew resistance; little effect, sever somaclonal variation, in the field increase in resistance against Botrytis.Research abandoned.

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 Xylellafastidiosa

15

• 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

16Research group of Prof Gray, Uni Florida

Grape genes associated to the defense against pathogens

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

19

Resistance against powdery mildew

Dhekney, Li & Gray. 2011. Plant 47: 458-466

Thomson -seedless Not transformed

Thomson trasnsformed VVTL-1

Development of powdery mildew

20

Tampa, resistant hybrid

Transgenic lines

Thomson control

21

Syrah prima della veraison

Resistance against black rot

22

Transgenic lines Thomson control

Progression of black rot

23

control TS-ck (▲ )

Resistance against sour rot during storage

24

One week

Three weeks

Thomson controlTransgenic lines

Resistance against sour rot during storage

25

26

27

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

30

• 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

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 WageningenOctober 2011Lines : 7, 11 ,12 (8 plants each line)

CG 3429.10.2013

Fire Blight

Fire blight of apple and pear

35

Origine of resistanceM. evereste, M. robusta 5

CG 3629.10.2013

Representative plants of each plant line 39 days after leaf inoculation with Erwinia amylovora. Gala, Malus robusta 5 , T lines carrying FB_MR5.

37

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 ! ?

38

Conclusions

• 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»

39

• Public acceptance?

Summary

• Most transformation with an exotic target gene

• Grape genes in overexpression using promoter (VVTL-1) 35S

40

Transformant have unnatural characteristicsGrape Taumatin is only expressed after infection and not present at active concentration constitutively Effect on the productivityEffect on the vinification?Effect of a high concentration on the environment? Consumer?

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.

R-LBD Recombinase CodA-NptII fusionRB LBRs Rs

HcrVf2prom

Insert T-DNA

term

Chemical activation of the recombinase

R-LBD Recombinase CodA-NptII fusionRB LBRs Rs

HcrVf2prom term� �

T-DNA insert after the recombination

RB LBRs

HcrVf2prom term

Inserzione attraverso Agrobacterium