Benefici e rischi di OGM dalla ricerca pubblica Italiana Prof. Bruno Mezzetti Departimento di Scienze Agrarie, Alimentari ed Ambientali Università Politecnica delle Marche b.mezzetti@univpm.it Mola di Bari, 08/07/2015
BIOTECHNOLOGY IN THE ANCIENT GREEK A grafted plant it is already a combination of 2 genomes Teofrasto Philosopher and botanist of the ancient greek, disciple of Aristotle. 371 b.c., : 287 b.c. 10/01/2016 Bruno Mezzetti 2
Agrobacterium tumefaciens: the natural biotechnologist
GM Research Trend in Italy 1992-2003 Source CIV Type of Genes Insect resistance 50 Fungi resistance 6 Virus resistance 38 Plant yield 3 Plant Habitus 15 Fruit development-quality Nutritional Value 2 25 Herbicide resistance 70 Male sterility Male Ster.+ Herbicide Res. 2 9 Insect + Herbicide Res. 27 Other 6 0 10 20 30 40 50 60 70 10/01/2016 Bruno Mezzetti 5
GM Research Trend in Italy 1992-2003 Source CIV REGIONS where GM trials were carried out Basilicata Calabria Campania milia Romagna riuli Ven.Giulia Lazio Liguria Lombardia Marche Piemonte Puglia Sardegna Sicilia Toscana Umbria Veneto 1 1 2 4 8 8 10 15 17 17 14 14 31 64 69 72 0 10 20 30 40 50 60 70 80 10/01/2016 Bruno Mezzetti 6
0 10 20 30 40 50 60 70 80 90 100 Sugarbeet Calendula Canola Cicory S.cherry W.Melon Strawberry Geranio Ginestrino 1 Weat Kiwi Raspberry 1 Lettuce Mais Eggplant Melon Oliv Potato Tomato Rice Soia Statice Tobacco Grape Zucchina 1 1 1 1 1 1 1 2 2 10/01/2016 Bruno Mezzetti 7 2 3 3 4 4 5 7 7 7 8 12 38 45 24 different GM crops 95 The most studied were maize (95 notifications) and tomato (45 notifications), but as a peculiarity of our country, there were also research activities carried out on many horticultural crops, such as fruit, vegetables and flowers.
Fruit without Fertilization 1.Novelty - especially for fruits such as cherry and raisin tomatoes, squash, eggplant, pepper, strawberry, melon and watermelon. 2. Improved taste - since seedless fruits often have higher Total Soluble Sugar (TSS) levels than seeded fruit. 3.Greater reliability of crop yield under varying climatic conditions - due to elimination of pollination as a factor in fruit set. 4.Cost reductions of greenhouse grown fruits, since pollination-related expensed are avoided. 5.Increased profitability for the fruit processing industry: a) because the longer harvesting period of parthenogenic crops enables processing equipment to be more efficiently used, b) the elimination of the seed removal stage reduces expenses (for instance in tomato paste production or in melon canning), or increases the value of the product (as in oleoresin extraction from paprika). Eggplant has been produced using Auxin gene (iaam) under the control of Antirrhinum majus ovule-specific promoter
EcoRI HindIII BamHI EcoRI KpnI EcoRI DefH9-IaaM Gene (Spena UniVR) LB Terminator IaaM coding DefH9 Promoter RB P. savastanoi Antirrhinum majus It confers expression within the placenta and the ovules and it has been used to confer parthenocarpic fruit development to several plant species and varieties (Rotino et al., 1997) From regeneration and selection to the open field (2001)
The experimental trial: Thompson Seedless Control and GM line: 32 plants each Silcora Control and GM lines (line A and line B): 16 plants each Vines were spaced at 2.5 x 1.5 m, trained with the double guyot system
1. Thomphson seedless yield N clusters/plant 8 7 6 5 4 3 2 N clusters/plant Cluster weight (g) b b a a 225 200 175 150 125 100 75 50 Cluster weight (g) DefH9-iaaM gene effects on plant production, fruit and berry size cv. Thompson seedless 1 25 0 Th CT Production cycles 2004-05-06 Mean+SE Duncan s statystical test, P < 0.05 Th DefH9-iaaM Berry weight (g) 0,98 0,96 0,94 0,92 0,90 0,88 0,86 0,84 0,82 0 b a Berry weight N berries/cluster b a 300 250 200 150 100 50 N berries/cluster 0,80 0 Th CT Th DefH9-iaaM
DefH9-iaaM - Table grape Effect on shoot fruitfulness (Duncan s Multiple Range Test; p < 0.01) 2003 Bud Fertility and Position - Laboratory Field fruitfulness CLONES Total bud with shoots % Buds with fertile shoots % Buds without fertile shoot Thompson seedless Control Buds fertility position TH - CT 141.00 14.90 85.10 12.62 0.08 TH - GM 108.00 25.00 75.00 10.81* 0.26* SI - CT 176.00 50,00 50.00 11.89 0.33 SI - GM 166.00 59.64 40.36 10.51* 0.46* Total number of bunches per shoot Thompson seedless DefH9-iaaM 10/01/2016 Bruno Mezzetti 12 Laboratory Inflorescence on a shoot arose from a basal bud of DefH9-iaaM Thompson seedless, unusual for this variety. Field fruitfulness
2. DefH9-iaaM gene expression and effects on IAA content and on VvHT1 gene DefH9-iaaM gene expression during berry development DefH9-iaaM thomp DefH9-iaaM silcora relative expression 70 60 50 40 30 20 10 cv. Thompson seedless relative expression 70 60 50 40 30 20 10 cv. Silcora 0 0 PB* A FS SH LH V H PB* A FS SH LH V H Fruit developmental phases: PB= pre-blooming; A= antesys; FS= fruit-set; SH= small hard fruit; LH= large hard fruit. Values are referred to PB, arbitrary set to 1. Elisa Costantini - Ph.D. discussion - Marche Polytecnic University 26/01/07
2. DefH9-iaaM gene expression and effects on IAA content and on VvHT1 gene IAA content during berry development GC-MS analysis on tissues samples Cv Thompson seedless and Silcora (control and GM line) IAA pmol/g 45 40 35 30 25 20 15 10 5 Thompson CT Thompson DefH9-iaaM line IAA pmol/g 140 120 100 80 60 40 20 Silcora CT Silcora DefH9-iaaM line A 0 0 IS PB A FS SH IS PB A FS SH Fruit developmental phases: IS=inflorescences separated; PB=pre-blooming; A= antesys; FS=fruit-set; SH=small hard fruit Elisa Costantini - Ph.D. discussion - Marche Polytecnic University 26/01/07
DefH9-iaaM - Table grape Effect on yield capacity (0ne plot production) TH Control TH GM SI Control SI GM
GMO at Marche Polytechnic University Strawberry and raspberry fruits from control and DefH9-iaaM plants (a) F. vesca - Control, Line 1, Line 2 (b) F. x ananassa - Control and Line 1 (c) R.idaeus Control and Line 1
GMO at Marche Polytechnic University Raspberry fruits from control and DefH9-iaaM plants Ruby Control Ruby Line 1 Raspberry - Plant Fecundity DefH9-iaaM raspberry differs for Increased No. of Inflorescence and of No. Fruits per laterals.
NEW PLANT BREEDING TECHNIQUES CISGENESIS/INTRAGENESIS T-DNA Agrobacterium tumefaciens Cell and Tissue culture Cisgenic plants (Solanum tuberosum) Biolistic techniques DNA from - the species itself - a cross-compatible species Wild Solanum
(PNAS, 20 gennaio 2004, vol. 101, n.3, fig.3) difference among gm clones. Same symptoms
Papayas are grown in many tropical countries. But papaya cultivation is being threatened by Papaya Ringspot-Virus, a disease that is sharply lowering yields. Transgenic VS Non transgenic Papaya Since 1998, genetically modified papayas have been cultivated in Hawaii, USA. They are resistant to a domestic viral disease. In the EU, genetically modified papayas are not approved.
NEW PLANT BREEDING TECHNIQUES RdDM: RNA-DEPENDENT DNA METHYLATION SEQUENCE HOMOLOGOUS TO THE PROMOTER Plant cell Plant genome SMALL DOUBLE STRANDED RNA SILENCED GENE x RdDM plants
VIRUS Resistance an emergency in Prunus cultivation
NEW PLANT BREEDING TECHNIQUES GRAFTING ON GM ROOTSTOCK Non- Transgenic scion Non- Transgenic fruits Agrobacterium tumefaciens Transgenic plant Transgenic rootstock
Why we need the open field trials with GM plants: Assessment of risks and benefits Assessment of benefits and risk Which risks: environment?, Human Health?, the agricultural economy???? Which benefits: increased production efficiency and quality 10/01/2016 Bruno Mezzetti 29
The European Commission adopted Directive 2001/18/EC (repealing Directive 90/220/EEC) to govern the deliberate release of GMOs into the environment. APPLICARE LE DIRETTIVE COMUNITARIE E LE L.G. EFSA Rischio Salute Uomo Rischio Ambiente Rischio sistemi agricoli CASO PER CASO (Gene-Specie-Pianta) SU BASE SCIENTIFICA DA METODO A PRODOTTO 10/01/2016 Bruno Mezzetti 30
DA METODO A PRODOTTO
Protection nets: One with holes of 5 x 5 cm max, 180 cm high; and one with holes 1,5 x 1,5 cm and 90 cm high, but half under soil (40 cm). PLANNING A GMO FIELD EXPERIMENTAL TRIAL 10 m non cultivated Border 10 m border with trap plant TRANSGENIC TRIAL A Screen House net type of protection can be requested depending to the type of crop. 10/01/2016 Bruno Mezzetti 32
TO CREATE A NET OF RESEARCH ON OPEN FIELD GMO RISK AND BENEFITS EVALUATION Strawberry Grape Notification Costs for Public research: Italy 1500 Euro The Netherlands, Poland, Spain = 0