Lab safety rules

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. Working on sterile conditions or following laboratory security rules- using gloves or disposing waste in the corresponding container were the bases of our diary work lab- it is necessary. 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.

Legal considerations on the release of GMO

In not a too distant future, society must respond at the foreseeable increasing demand of food production. Plant breeding has the aim of reaching nutritional quality improvement, increase of crop production and even avoid the use of plaguicides. Until recently, these improvements were carried out using mutation breeding techniques, with chemical mutagens or ionizing radiation followed by screening of mutated populations, searching for the desirable traits. Few years ago in the story of plant breeding, transgenic techniques to produce new plant varieties emerged. This strategy is based in the insertion of new resistance genes against plant pests, diseases or herbicides, as well as any kind of improvement. Despite the development of this approach has created great opportunities at this field, public opinion is against them. In fact, that position led in part to the formulation of the Cartagena Protocol on Biosafety in 2000.(1)
The control and regulation of the GMO are established by a collection of laws. Their general aim is to “protect human health and environment” and a “high level of protection of human life and health, animal health and welfare, environment and consumer interests”, avoiding any incident caused by a GMO. These regulations are based in the prevention principle.

In the last few years, several revolutionary new plant breeding techniques (NPBTs) have been developed offering a great technical potential to modify plant’s genomes much faster and with higher precision than with traditional techniques used to obtain new varieties. They are often indistinguishable from classically bred plants and are not expected to imply higher risks for health and the environment. (2). One of these techniques is the revolutionary CRISPR/Cas9 genome editing system. While in the USA some CRISPR/Cas9 edited plants have been approved by the FDA, there is currently an unspecific information about current regulation in the EU, as there is not a specific legislation for these kind of crops. Given that our project is based in CRISPR/Cas9, we find that it is mandatory to consider how and when they are going to be regulated by the legislation concerning genetically modified organisms (GMOs).

Many European countries have established their own interpretation of the general directive and have decided not to apply the same legislation that for transgenesis because these plants do not contain any foreign part of DNA and cannot be distinguished from mutations that can be produced naturally. Meanwhile, some organizations, mainly non-governmental, are supporters of applying the directive because they are concerned about the process by which the mutation has been produced (3) (4).

Risk for the population and the environment

In our project transient transgene expression in Nicotiana benthamiana was carried out in a fully equipped laboratory for plant biotechnology. It has to be clear that transient expression is not the same as stable. We have never created or used a transgenic plant. The plants that we have used do not have flowers, so there is no risk of spreading modified phenotypes to the environment through pollen, having no risk of contamination. In the laboratory we always work in laminar flow cabinet in order to maintain sterile conditions. After finishing the analysis and wet-lab procedures of the plant, we discard it as a biological waste following autoclave protocol in order to maintain security conditions.

It must be outlined that plant virus do not affect in any way humans or animals.

When we are working with plant viral vectors in the laboratory, the optimal conditions to carry out different experiments are treating them as plant viral vectors. They are not viral particles (observe difference between particle and vector) so they cannot be accidentally released. When we introduce viral vectors in Agrobacterium tumefaciens, it still doesn’t replicate inside the bacteria. When we finally infect the plant with Agrobacterium (with the viral vector inside) in the greenhouse, the viral vector will replicate itself and the viral particles will be synthetized. At that point, it is possible the transmission from plant to plant. To avoid that, the infected plants are stored in a separate room in the greenhouse. Again, these plants don’t have flowers and we do not obtain seeds from them. Finally, we also autoclave these plants, because viral particles are destroyed with high temperatures.
As far as the environment is concerned, failures in containment conditions could lead to infection of wild species during expression of proteins by viral vectors, bringing the traits outside the boundaries of the target species. Therefore, although the enforcement of contained production measures should be sufficient to maintain HYPE-IT contamination risks at acceptable low levels, adoption of additional safety measures is advisable.
Finally, it is important to highlight that the virus used do not express the coat protein, so they are not able to spread from plant to plant. This is relevant for avoiding contamination of the environment with the edited plant.

References

1. Schaart, J., van de Wiel, C., Lotz, L. and Smulders, M. (2016). Opportunities for Products of New Plant Breeding Techniques. Trends in Plant Science, 21(5), pp.438-449.
2. Hartung, F. and Schiemann, J. (2014). Precise plant breeding using new genome editing techniques: opportunities, safety and regulation in the EU. The Plant Journal, 78(5), pp.742-752.
3. Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC - Commission Declaration.
4. Hartung, F. and Schiemann, J. (2014). Precise plant breeding using new genome editing techniques: opportunities, safety and regulation in the EU. The Plant Journal, 78(5), pp.742-752.
5. Abbott, A. (2015). Europe’s genetically edited plants stuck in legal limbo. Nature, 528(7582), pp.319-320.
6. European Parliamentary Research Service, (2016). New plant-breeding techniques. Applicability of GM rules.