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Transcript
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 5 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? • • • • • 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 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 10 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 11 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). 12 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. 13 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 15 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 16 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) 17 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 19 Thomson trasnsformed VVTL-1 Dhekney, Li & Gray. 2011. Plant 47: 458-466 Development of powdery mildew Thomson control Transgenic lines Tampa, resistant hybrid 20 Syrah prima della veraison 21 Resistance against black rot Transgenic lines 22 Thomson control Progression of black rot control TS-ck (▲ ) 23 Resistance against sour rot during storage One week Three weeks Thomson control 24 Transgenic 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 • 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 30 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 35 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 ! ? 37 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» 38 • Public acceptance? 39 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? 40 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