Biotecnologie ambientali aa 2012-2013 Rischi ambientali e Protocollo di Cartagena

PROGRAMMA Le piante coltivate e la sindrome da domesticazione: shattering e dormienza Rischi e benefici ambientali delle piante transgeniche in paragone a quelle convenzionali. Convenzione di Rio, Protocollo di Cartagena e normativa sulle piante create tramite ingegneria genetica Piante per una maggiore sostenibilità ambientale (es. plastiche biodegradabili), per il risanamento (fitodepurazione) e come biosensori di contaminazione. Interazione pianta-microrganismo: le risposte di difesa delle piante e generazione di specie resistenti. Interazione simbiotiche pianta-microrganismo: fissazione dell’azoto (batteri azoto fissatori)

La convenzione di Rio o sulla Biodiversità (CBD) http://www.cbd.int/ The Convention on Biological Diversity is an international treaty to sustain the rich diversity of life on Earth. http://www.cbd.int/doc/legal/cbd-en.pdf in ita: http://www.admin.ch/ch/i/rs/0_451_43/ 42 articoli e 2 annex

Its objectives are: the conservation of biological diversity, the sustainable use of its components, and the fair and equitable sharing of benefits arising out of the utilization of genetic resources. Article 2. Use of Terms "Biotechnology" means any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use. Article 8. In-situ Conservation (g) Establish or maintain means to regulate, manage or control the risks associated with the use and release of living modified organisms resulting from biotechnology which are likely to have adverse environmental impacts that could affect the conservation and sustainable use of biological diversity, taking also into account the risks to human health: (h) Prevent the introduction of, control or eradicate those alien species which threaten ecosystems, habitats or species:

 Nasce il Protocollo di Cartagena Article 19. Handling of Biotechnology and Distribution of its Benefits 1. Each Contracting Party shall take legislative, administrative or policy measures, as appropriate, to provide for the effective participation in biotechnological research activities by those Contracting Parties, especially developing countries, which provide the genetic resources for such research, and where feasible in such Contracting Parties. 2. Each Contracting Party shall take all practicable measures to promote and advance priority access on a fair and equitable basis by Contracting Parties, especially developing countries, to the results and benefits arising from biotechnologies based upon genetic resources provided by those Contracting Parties. Such access shall be on mutually agreed terms. 3. The Parties shall consider the need for and modalities of a protocol setting out appropriate procedures, including, in particular, advance informed agreement, in the field of the safe transfer, handling and use of any living modified organism resulting from biotechnology that may have adverse effect on the conservation and sustainable use of biological diversity. 4. Each Contracting Party shall, directly or by requiring any natural or legal person under its jurisdiction providing the organisms referred to in paragraph 3 above, provide any available information about the use and safety regulations required by that Contracting Party in handling such organisms, as well as any available information on the potential adverse impact of the specific organisms concerned to the Contracting Party into which those organisms are to be introduced.  Nasce il Protocollo di Cartagena

Conference of the Parties (COP) : governing body of the Convention The governing body: Conference of the Parties to the Convention serving as the meeting of the Parties to the Protocol (COP-MOP) Storia della CBD e del Protocollo di Cartagena

1700 M$ in 12 years from GEF + 3400 in cofinancing of biodiversity projects. GEF=Global Environment Facility

The Cartagena Protocol on Biosafety to the Convention on Biological Diversity It was adopted on 29 January 2000 and entered into force on 11 September 2003. An additional protocol to the Convention of Biological Diversity è frutto delle decisioni prese all’interno della CBD

Eventuale testo in italiano (non necessariamente corretto) http://www.rfb.it/csa/links/archivio/protocollo-cartagena-traduzione.htm http://bch.cbd.int/database/attachment/?id=10694

The Cartagena Protocol on Biosafety to the Convention on Biological Diversity An international agreement which aims to ensure the safe handling, transport and use of living modified organisms (LMOs) resulting from modern biotechnology that may have adverse effects on biological diversity, taking also into account risks to human health. It was adopted on 29 January 2000 and entered into force on 11 September 2003. The Protocol promotes biosafety by establishing rules and procedures for the safe transfer, handling, and use of LMOs, with specific focus on transboundary movements of LMOs. It features a set of procedures including one for LMOs that are to be intentionally introduced into the environment called the advance informed agreement procedure, and one for LMOs that are intended to be used directly as food or feed or for processing.

Il Protocollo di Cartagena “A Protocol is a binding international instrument, separate from, but related to, another treaty.” Preamble The Parties to this Protocol, Being Parties to the Convention on Biological Diversity, … Recalling Article 19, paragraphs 3 and 4, and Articles 8 (g) and 17 Recalling also decision II/5 of 17 November 1995 of the Conference of the Parties to the Convention to develop a Protocol on biosafety, specifically focusing on transboundary movement of any living modified organism resulting from modern biotechnology that may have adverse effect on the conservation and sustainable use of biological diversity, setting out for consideration, in particular, appropriate procedures for advance informed agreement, Reaffirming the precautionary approach contained in Principle 15 of the Rio Declaration on Environment and Development, Aware of the rapid expansion of modern biotechnology and the growing public concern over its potential adverse effects on biological diversity, taking also into account risks to human health,

Recognizing that modern biotechnology has great potential for human well-being if developed and used with adequate safety measures for the environment and human health, Recognizing also the crucial importance to humankind of centres of origin and centres of genetic diversity, Taking into account the limited capabilities of many countries, particularly developing countries, to cope with the nature and scale of known and potential risks associated with living modified organisms, Recognizing that trade and environment agreements should be mutually supportive with a view to achieving sustainable development, Emphasizing that this Protocol shall not be interpreted as implying a change in the rights and obligations of a Party under any existing international agreements, Understanding that the above recital is not intended to subordinate this Protocol to other international agreements, Have agreed as follows:

Paragonate con CBD Article 8. In-situ Conservation (g) Establish or maintain means to regulate, manage or control the risks associated with the use and release of living modified organisms resulting from biotechnology which are likely to have adverse environmental impacts that could affect the conservation and sustainable use of biological diversity, taking also into account the risks to human health: (h) Prevent the introduction of, control or eradicate those alien species which threaten ecosystems, habitats or species: but Article 19  “…may have adverse effects”

I virus normalmente NON sono considerati “living organisms”

AIA The Primary focus of the Cartagena Protocol is the regulation of transboundary movement of living modified organisms. The main instrument is the Advanced Informed Agreement (AIA) This Protocol shall not apply to the transboundary movement of living modified organisms which are: - pharmaceuticals - in transit - destined for contained use (research) Separate AIAs apply to: - LMOs intended for direct use as food or feed, or for processing - LMOs for deliberate release (= cultivation)

It includes four components: The "Advance Informed Agreement" (AIA) procedure applies to the first intentional transboundary movement of LMOs for intentional introduction into the environment of the Party of import. It includes four components: -notification by the Party of export or the exporter, -acknowledgment of receipt of notification by the Party of import, -the decision procedure, and -opportunity for review of decisions. The purpose of this procedure is to ensure that importing countries have both the opportunity and the capacity to assess risks that may be associated with the LMO before agreeing to its import. The Party of import must indicate the reasons on which its decisions are based (unless consent is unconditional). A Party of import may, at any time, in light of new scientific information, review and change a decision. A Party of export or a notifier may also request the Party of import to review its decisions.  Art.8  Art.9  Art.10 & 11 (food, feed, processing)  Art.12 There is a simplified procedure (Art.13) - Immissione contemporanea alla notifica - Esenzioni  MAI USATA

ANNEX I  Informazioni richieste dall’AIA per il rilascio ambientale

ANNEX II  Informazioni richieste dall’AIA uso per Food, feed, processing

Valutazione del rischio

Probabilità di conseguenze negative Rischio = Pericolo x Probabilità di conseguenze negative Risk is defined as the probability that exposure to a hazard will lead to a negative consequence, or more simply, Risk = Hazard x Dose (Exposure) Tante parole, ma come si fa in pratica una valutazione del rischio?

Decision tree to determine nontarget effects of Bt plants on natural enemies (parasitoids and predators) feeding on either host/prey species or directly on plant material (e.g. pollen) in studies under confined conditions. Transgenic crops expressing Bacillus thuringiensis toxins and biological control Romeis (2006) Nature Biotech. 24:63-71 doi:10.1038/nbt1180 Tiered approach Tiered – sequenziale? Gerarchica?

In the case of nontarget risk assessment of insecticidal transgenic crops, early tier (laboratory) tests are conducted to determine whether an organism is susceptible to the toxin under worst case conditions, that is, organisms are directly exposed to high doses of the toxin. These tests are relatively simple in design, easy to standardize, repeatable and the results are easy to interpret. The risk assessment can stop here if risks under these worst case conditions are considered negligible or acceptable. However, if risks have been identified or cannot be ruled out with some certainty, higher tier tests will follow that expose nontarget organisms to the toxin under more realistic conditions.

Conclusions and recommendations Although the effects of Bt plants have been investigated for a limited number of predator and parasitoid species under confined conditions, two general trends are evident: first, there is no indication of direct effects of Bt plants on natural enemies, either in direct plant feeding assays or when natural enemies have been provided with unsusceptible hosts/prey containing the Cry toxin; second, adverse effects on natural enemies have been observed only in studies with susceptible herbivores as hosts/prey. These effects are most likely due to reduced host/prey quality. One exception to this is the reported impact of Bt potatoes on an aphid parasitoid, which was probably due to affected aphid hosts. In contrast, applications of conventional insecticides have usually resulted in negative impacts on biological control organisms. Because Bt-transgenic varieties can lead to substantial reductions in insecticide use in some crops, they can contribute to integrated pest management systems with a strong biological control component. David A. Andow et al. (2006) Ecological risk assessment for Bt crops. Nature Biotechnology 24:749-751. doi:10.1038/nbt0706-749 Jörg Romeis et al.(2006) Reply to Ecological risk assessment for Bt crops. Nature Biotechnology 24:751-753. doi:10.1038/nbt0706-751

http://bch.cbd.int/

Solo approvazioni a livello europeo

Risk assessment

Laws and regulations

Le rimanenti sono queste 17 su 23 sono di tipo EU Le rimanenti sono queste In realtà mancano una serie di norme

Sito Italiano (in inglese)

Normativa nazionale

 Tutti queste buone intenzioni e articoli si scontrano con una concezione viziata della transgenesi

FAQs, http://www.biodiv.org/biosafety/faqs.asp Mackenzie (2003) Mackenzie (2003) An Explanatory Guide to the Cartagena Protocol on Biosafety. IUCN Environmental Law Centre (www.iucn.org/bookstore)

 Relazione di W. Parrott alla PAS Mito: “I transgenici sono innaturali mentre le piante convenzionali sono naturali e non sono modificate dal punto di vista genetico” Nuovi geni vengono continuamente creati. In riso hanno identificato 898 retrogeni, 55% sembrano funzionanti e 35% sono chimere (costruiti a partire da geni diversi). La mutazione Wp in soia ha semi più grandi (22%), più proteine (4%) e i fiori rosa pallido. La mutazione è dovuta a un retrotransposone, inserito in una calcone sintasi, che ha catturato 5 esoni da 4 geni differenti fusi in un nuovo gene ‘‘Insertion of DNA can cause deletions and rearrangements of the original DNA at the insertion site. This information helps us understand that GE is significantly different from conventional breeding techniques.’’ Sono meglio i geni creati consciamente o quelli creati inconsciamente (dalla natura)?  Relazione di W. Parrott alla PAS

gene bz Organizzazione della regione che contiene il gene bronze (bz) in 7 diverse linee di mais 5 linee inbred i diversi trasposoni sono rappresentati da triangoli colorati 2 razze locali Wang e Dooner (2006) PNAS 103:17644-9.

Esempi di variegazione indotta da trasposoni nei semi di mais (b) Esempi di variegazione indotta da trasposoni nei semi di mais a) Immagine cortesia di W.P. Amstrong (Palomar college, California, USA); b) Immagine cortesia di K. Petroni, Univ. di Milano. c) Immagine cortesia di A. Marocco, Univ. Cattolica di Piacenza. (c)

Il Grande Mito del mais: Il mais Bt contaminerà la biodiversità nel centro di origine mettendo a rischio la sovranità alimentare di centinaia di milioni di persone ‘‘The introduction of genetically engineered varieties also threatens the indigenous cultures and the very knowledge that indigenous peoples have developed for millennia. Genetic contamination of maize threatens the food sovereignty for hundreds of millions of people that rely on maize as their primary source of food.’’ Pannocchie di mais bianco, nero e giallo stese ad asciugare sui tetti delle case di San Juan Ostuncalco, Guatemala. I contadini sanno come mantenere l’identità delle diverse varietà Foto: cortesia di Eduardo Roesch

Non per questo le varietà hanno perso la loro identità Foto: cortesia di W. Parrott, Università della Georgia, AL (USA) Pannocchie raccolte nei campi in Guatemala; le varietà locali si incrociano Non per questo le varietà hanno perso la loro identità i contadini sanno bene come mantenere l’identità delle diverse varietà Il genoma del “Palomero Toluqueno” (varietà indigena) è 78% del B73

Arca di Noé - S. Maurizio al Corso (Mi)

I transgenici sono utili per i PVS? Mais Mais infestato da Striga hermonthica http://4.bp.blogspot.com/_9MXPl2EjFG4/TRycNxyqCsI/AAAAAAAAAbs/qfpB67p6xMU/s1600/OGM+-+Striga+4.jpg  Relazione di J. Gressel e M. Qaim

Il desiderio: Risultato???? THE BIOSAFETY PROTOCOL WILL ENABLE PEOPLE EVERYWHERE TO ENJOY THE BENEFITS OF BIOTECHNOLOGY WHILE AVOIDING UNNECESSARY RISKS. Biosafety and the environment (An Introduction to the Cartagena Protocol on Biosafety) Risultato????

Varietà convenzionale La realtà: Varietà transgenica Per la coltivazione occorre un permesso sulla base di documentazione scientifica Varietà convenzionale

Regulatory frameworks Cartagena countries-- which includes the EU-- do not regulated GMOs as such. They regulate the products of modern biotechnology, defined as a crop obtained by injecting nucleic acids into a cell. Nothing says the nucleic acid has to stay there. Thus, all products from genome editing are GMOs unless they change the definition or make an exception. USDA does not regulate transgenes as such, either. They regulate plant and animal pests. Thus in the US it depends on the presence or absence of pathogen-derived sequences and whether transformation is done with the gun or with agro. Canadian regulatory trigger is based on novelty of the trait. It is the only current regulatory trigger compatible with OECD, NAS, ACRE science-based regulatory trigger recommendations.

Herbicide tolerant sunflower Herbicide tolerant sunflower Two methods, one result Herbicide tolerant sunflower mutagenesis transgenesis Herbicide tolerant sunflower Sunflower spont. mutation Same result using different methods:  is one evil and the others benign? Focus on product risks, not on method used * Allergen inactivation (soybean) * Toxin inactivation (cotton ) *... Any loss of function… Morandini (2010) New Biotechnol.

Nel frattempo... ZFNs, TALENs, CRISPS Figura da: http://ko.cwru.edu/services/directtargeting.shtml Tabella da Wikipedia: http://en.wikipedia.org/wiki/Genome_editing

http://ko.cwru.edu/services/directtargeting.shtml

Gaj T, Gersbach CA, Barbas CF 3rd Gaj T, Gersbach CA, Barbas CF 3rd. (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31:397-405.

Gaj T, Gersbach CA, Barbas CF 3rd Gaj T, Gersbach CA, Barbas CF 3rd. (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31:397-405.

Tutti questi nuovi metodi (e la mutagenesi classica Tutti questi nuovi metodi (e la mutagenesi classica...) pongono seri problemi ai regolatori! http://ko.cwru.edu/services/directtargeting.shtml

Segnalato predone Tusken Scene di panico a Milano  Qual è la credibilità di un protocollo che dovrebbe normare una tecnologia di cui non comprende i fondamenti? Avvistati extraterrestri nel Nord-Italia Segnalato predone Tusken Scene di panico a Milano

“In Reports 1 and 2 we conclude that the framework for controlling GMOs in the EU is out-dated and not fit for purpose. Regulatory frameworks need to evolve along with the evidence, especially when dealing with novel technology. This includes the EU’s legislation for controlling GMOs. ACRE’s recommendation is that the current framework should be replaced by a system that regulates organisms based on the novelty of their characteristics rather than on how they are produced. It should also take benefits, as well as risks, into account to allow for more informed decision-making.” http://www.defra.gov.uk/acre/files/Report-1.pdf

Refreshing the vocabulary 2 petal 4 carpel 1 sepal 3 stamen

Identity crisis Mutant: Apetala Petal  flower Sepal  leaf

More problems Pistillata Apetala3 Agamous petal  sepal petal  sepal stamen  petal stamen  carpel stami  carpelli carpel  flower Images courtesy of L. Colombo (Unimi)

and Cauliflower… …as well as Arabidopsis

Even cucumbers have problems One of the most aggressive insects damaging cucumbers in greenhouses in Canada & USA Western flower thrips Frankliniella occidentalis Female cucumber flower Images courtesy of M. Kater (Unimi) http://bugguide.net/node/view/252440/bgimage

egg Adult larva Pupa meeting place mating place Life cycle of Frankliniella occidentalis Female flower egg Adult larva Pupa Images courtesy of M. Kater (Unimi)

Mutante Green petals: B (CUM26) S S --- C 1 2 3 4 cum26 Petal  sepal Images courtesy of M. Kater (Unimi)

total damage mm2 thrips larvae wt gp mutant 8000 4000 200 100 C D Basal leaves Apical Take home message: genetic engineering is a more predictable, more precise, faster and less expensive method to introduce a character Green petals mutant Wild-type Images courtesy of M. Kater (Unimi)

Bibliografia Nicholas Kalaitzandonakes (2004) The Potential Impacts of the Biosafety Protocol on Agricultural Commodity Trade PRRI (2005) Guide for notifications and risk assessments for releases into the environment of genetically modified organisms Mackenzie (2003) An Explanatory Guide to the Cartagena Protocol on Biosafety. IUCN Environmental Law Centre (www.iucn.org/bookstore) PRRI(2003) Introductory seminar for the participation of public research scientists in the second meeting of the parties to the Cartagena protocol on biosafety Biosafety and the environment (An Introduction to the Cartagena Protocol on Biosafety)

Altri problemi all’orizzonte TALENs are pathogen derived. ZFNs and CRISPRs could be embodied without pathogen sequences, but the research community first needs to break its dependance on 35S promoters, SV40 nuclear target sequences, and NOS terminators. The USDA has indicated that once pathogen derived sequences are segregated out, the item is not regulated, which I interpret to say ZFN and CRISPR-modified plants should be exempt. However, there have been a couple of cases now where the plants with no pathogen-derived sequences were regulated anyway b/c Agro was used. So, my take is ZFN and CRISPR derived plants should be exempt as along as Agro is not used, and as long as they are not used to add pathogen-derived sequences. I am hopeful USDA will give a blanket exception even if Agro is used.