The safety of a project is always a very important part to take into account, especially if the project involves living organisms. For that reason in our project we have taken all the safety precautions while working in the lab. Things like working on sterile conditions, using gloves or disposing waste in the corresponding container were the bases of our diary work lab. Moreover transient transgene expression in Nicotiana benthamiana was carried out in laboratory and greenhouse facilities equipped with containment measures compliant with National and EU regulations.

The control and regulation of the GMO are stablished by a collection of laws. The general aim of all this laws is to protect people and the environment avoiding any incident cause by a GMO and is based in the prevention principle.

There are two different sections when treating a GMO. The first one regulates the intentional liberation of the GMO into the environment to mere test it and see if it has any environmental repercussion. The second one is done if the GMO is going to be commercialized.

There are slightly differences between European and Spanish GMO legislation. Spanish law is controlled by the Ministerio de Agricultura, AlimentaciÛn y Medio Ambiente. It is in charge of controlling the voluntary liberation of the GMO in the environment in case that this organism produce medicines for human and veterinary use, as it is in our case. (Chapter 2, article 3 from law 9/2003, de 25 April)

In order to carry out an intentional liberation the next documents will be necessary:

• The corresponding authorization request.
• A technical study, which includes information and data prescribed by regulation.
• An assessment of the risks to human health and the environment, which should include the methodology used and the conclusions on the potential impact in the environment.

The applications will be evaluated "step by step" and "case by case" as is indicated in the law

The second part of general legislation comprises the commercialization of the GMO. They are administrated by the country of departure but it will be sent to the State Members of the EU, also it will be a public information that anyone can comment and submit their opinion to the Regulatory Comitee. Point 46,DIRECTIVE 2001/18/EC

The request form asks for the taxonomy of the GMO, phylogenetic relation with some local crop, genetic information that is introduce into the GMO, method of reproduction of the specie. You can find all the specification in here.

Finally Cartagena Protocol needs to be mentioned. It is the international Biosafety Protocol that try to unify all legislation to regulate efficiently the biotechnology work and the GMO acceptation, minimizing the possible risks to humans and the environment. All the concepts aforemention are in the link above.

AladDNA is not designed for environmental release. The production phase takes place under contained conditions (the lamp device) and disposal of biological material under safe conditions does not require special equipment, as a simple tissue disruption mechanism will avoid seed viability after the production phase.

An important advantage of the plant chassis is the multiplication capacity of the seed biofactory. Seed banks should be produced under contained conditions in centralized locations at large scale, whereas small and medium scale contained facilities can also be easily established in remote locations for local multiplication.

However it is worth to evaluate the risks associated with the eventual scape of transgenic seeds from contained facilities into the environment and propose additional genetic containment measures for reducing risk associated with this contingency.

Implementation of strict indoors cultivation conditions for the multiplication phase, and automatic disposal after the production phase should be sufficient measures to ensure that no release into the environment takes place. However, unintended release of medicine-producing seeds could ultimately led to contamination of the food chain, and therefore the risk evaluation of this unlikely event needs to be considered. Unintended exposure with vaccine antigens, antibodies or other drugs at low (occasional, dietary) doses is not expected to led to severe health effects on most healthy individuals. Most seeds are consumed after heat treatment (cooking), at conditions that severely reduce or even abolish the biological activity of the drugs. However in certain circumstances and for certain drugs it cannot be discarded that drug exposure could severely affect immunocompromised or allergic individuals.

As far as the environment is concerned, failures in containment conditions could lead to cross-pollination with wild species during seed batch production bringing the traits outside the boundaries of the target species. Therefore, although the enforcement of contained production measures should be sufficient to maintain AladDNA contamination risks at acceptable low levels, adoption of additional safety measures is advisable.

Taking into consideration the risks discussed above, here we propose a number of additional measures that could be implemented. The introduction of one or more of the following measures, combined with physical containment measures described above, would bring the risk associated with AladDNA implementation virtually to zero.

Selection of plant species attending to safety considerations:

Use of non-food, Generally Accepted As Safe (GRAS) seeds as e.g. Medicago truncatula will bring the risk of food contamination to zero.

Use of seeds of self-pollinating crops to avoid transgene scape by cross-pollination (e.g. pea, rice)

Implementation of additional genetic containment measures

Introduction of an additional genetic module in the genetic design for male sterility.

Introduction an additional genetic module in the genetic design that activates lethality in the hybrid progeny, avoiding propagation.

Introduction of identity-preservation traits (e.g. distinctive colour/shape) using metabolic engineering.

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