<p>The other side of the story on the other hand is that if the nitroplast is independent from the host, there is a chance that it can survive outside the plant for example in the soil and potentially spread in the environment. This could have many consequences one for example the alteration of soil microbial communities that also effects the wellbeing of plants. One solution to the spreading of a less host-dependant nitroplast is engineering a kill switch so when it gets out of the host cell somehow, due to the change of the environment or the lack of nutrient the nitroplast starts a cell death mechanism. From the engineering part (incorporation of the nitroplast by fusion) the size problem must be considered as well since other cyanobacteria species are in general bigger than UCYN-A.</p>
<divclass="h3">Engineer marker</div>
<p>We thought of a marker that we would introduce into our GM seed that the plant contains when growing, so we can track better the spread of the genetic material in the environment and the GM seed in the food chain. The marker can be introduced either in the host cell or to the nitroplast organelle itself. The uTP sequence we came up with and tried to test during our project could a marker that indicates the spread of the transgenes introduced into the crop.</p>
<divclass="h2">Ownership</div>
<p>Now we can again return to our question "Is our idea a techno-fix?" and how we can respond to the second practical criticism which states that techno-fixes only create more problems. By the critical thinking about environmental consequences and by implementing the said ideas could greatly help prevent or at least see clearer the future uncertain environmental effects of our GM crop.Now we can again return to our question ‘Is our idea a techno-fix?’ and how we can respond to the second practical criticism which states that techno-fixes only create more problems. By the critical thinking about environmental consequences and by implementing the said ideas could greatly help prevent or at least see clearer the future uncertain environmental effects of our GM crop.</p>
<divclass="h2">Ownership</div>
<divclass="h3">Giving access to non-profit partners</div>
<divclass="h2">Choosing a Suitable Crop</div>
<divclass="h2">Alternative Approaches</div>
<p>Learning from our HP work, our final idea on how we would handle ownership is to make sure that our developed seed with our proposed technology is owned by multiple actors and all owners act responsibly when using the seed. Such as during the development by scientist, distribution by companies or organisations, planting by farmers. This requires that knowledge is available (technical and about safety as well) to all actors and they collaborate and share the information they learn upon the usage of the seed. </p>
<p>This could be possibly done if the development of the GM seed and information on the technology is not owned by a commercial private company but a local research Institute or university. Examples we could learn from are the cases of the pest resistant Bt eggplant in Bangladesh mentioned before, or the rainbow papaya case in Hawaii where the local university developed a Papaya ringspot virus resistant strain for local farmers. </p>
<p>So to reply to the third practical criticism that techno-fixes are conservative, by rethinking the ownership model, we are truly stepping out of the current industrial agriculture system and hope to implement a more inclusive and accessible technology.</p>
<p>However, this could be difficult if the development and risk assessments are too costly to develop a nitrogen-fixing crop with our method since it requires the insertion of multiple genes and an organelle. Alongside with the entrepreneurship part of our project we thought of a compromise. If we have the final product as a seed incorporating the nitroplast, a patent on our developed seed or on our method (information) would be filed for return on investment. To make the technology more accessible we would provide the seeds for (free) or share the information on engineering with relevant non-profit partners like local institutes or NGOs in Sub-Saharan African regions or in South America where our technology good help the most. </p>
<divclass="h2">Choosing a Suitable Crop</div>
<p>It is relevant what target crop we choose because it limits where it can be grown and defines the environment which is relevant for safety. This is also important because Europe doesn’t allow the cultivation of GM crops. Amrit Nanda advised us that we should focus outside of Europe like USA or Brazil if we want to implement our idea. Potential crops are ones with high nitrogen need and that are mass-produced in the world like maize or sugar beet. If these species are native outside of Europe than it is something to think about from safety perspective. On the other hand a design requirement we thought of is that our technology can be applied easily to different varieties so local varieties could also have the nitrogen-fixing trait. As Elhai J mentions in his article the need of a technology to transplant nitroplasts not only available for the big corporations. He suggests two methods that might work. The regeneration of mature plants after protoplast fusion or transferring the organelle by grafting. Nicotiana benthamiana could be used for this idea which is known to be a super grafter, able to form grafts with 87% of a wide variety of plant species. [19]</p>
<p>As a first step in the project when working with plants, the approach could be to do experiments first on plant cell cultures or try to modify the germplasm. Max van Hooren advised to choose a model plant organism first like tobacco plant with shorter reproduction time. Or maybe it would be easier to start with a plant that has already symbiosis with n-fixing bacteria so it would incorporate easier the nitroplast like soybean. It could be also a good test if a nitrogen fixing soybean with our approach would have higher yield than a one inoculated with bacteria.</p>
<divclass="h2">Alternative Approaches</div>
<p>We were aware that the nitrogen pollution problem can be approached in many ways from a synthetic biology perspective therefore we were thinking critically of our idea. During our discussion with KWS seeds the concern was raised that it could be <strong>too energy costly for the plant</strong> in many situations to have the organelle incorporated into its cell and to maintain it with proteins and nutrients constantly. For example, <strong> if there is abundant nitrogen in the soil, then it might not be beneficial for the crop and yields would get lower</strong>. Considering this the researchers at KWS suggested that an <strong>alternative approach</strong> is to <strong>not use UCYN-A as an organelle</strong> but rather <strong>focus on a less host dependent cell</strong> or endosymbiont as nitroplast that could provide usable nitrogen for the plant cell.<strong> Elhai J</strong> in his elaborate article on the potential use of nitroplasts concluded the same.[19] This way mostly only metabolites would be exchanged between host and organelle and less genes need to be inserted into the host cell making the engineering process easier.</p>
<p>One possibility is to use cyanobacteria or rhizobacteria that already live in close symbiotic relationship with many plants as nitroplast candidates. This way we think that creating artificial endosymbiosis based on auxotrpohies might be an easier task then with UCYN-A. The work by Cournoyer, J.E., Altman, S.D., Gao, Yl. et al [20]creating yeast cyanobacteria chimeras could serve as an example. Their method could be applied for nitrogen fixation and not CO2 fixation and eventually moving to plant cells and using appropriate bacteria strains.</p>
<p>Another candidate for a nitroplast could be the endosymbiont of the Rhopalodia gibberula microalgae called RgSB. that has a genome small for a Cyanothece (close cyanobacteria relative) but 10- to 20-times larger than a chloroplast genome. [19] RgSB is only lacking the genes for photosynthesis, it has purely metabolic dependency on the host algae so there is no protein dependency. Roughly an approach could be to try to incorporate encapsulated RgSB’s into an eukaryotic cell (not C. reinhardtii because the size difference is too small) and select cells incorporating the encapsulated bacteria by reducing the ammonia supply in the medium.</p>
<p>Additional approaches that the researchers of KWS suggested are stopping at the level of transforming and incorporating nitroplast into an algae cell that can be used industrially as protein source for example or as organic fertilizer in the field. Then we don’t have to totally reinvent our target crop plant's nutritioning. This is an approach we thought of before, but we think it has less impact, firstly because what we learned during other stakeholder interviews is that for industrial algae fermentation the carbon source in sustainability aspect is more relevant than the nitrogen source. Second this approach would have many safety issues as well. None the less, using our approach in alternative microbial food protein or artificial meet production to cut down on supplying a nitrogen source could be something to consider in the future. </p>
