Team:Imperial College London/Human Implementation
We consulted numerous experts in various fields to ensure that the design of the AuxIn system respects all relevant social, ethical and legal issues. One module of our system, Gene Guard, is a direct result of brainstorming around the issues involved in the release of genetically modified organisms (GMOs). Although we have only reached the proof of concept stage, we have put a lot of thought into how AuxIn may be implemented as a product and the legal issues that would be involved.
Although our project is only at the proof-of-concept stage, we have done extensive research to be able propose a future implementation plan for AuxIn. With the advice of several experts, we decided to develop our product in a pre-coated seed package. Although proof-of-concept has only been tested with E. coli on Arabidopsis thaliana roots, we hope that future work will enable the production of a general seed coat that can be applied to any seed type to tailor to the needs of different ecosystems.
AuxIn also has potential as a platform technology in that we can deliver virtually any compound to plant roots via our Phyto-Route module. The advantage of this approach, over direct genetic modification of plants, is that the same product can be applied (in theory) to any plant type, including those already established in the soil.
Because soil erosion and desertification pose a great threat all over the world, we plan to sell AuxIn to industrialised countries such as the USA and Australia, to protect barren land or restore depleted crop lands. With this revenue we can provide AuxIn to pre-established local organisations in developing areas free of charge.
Figure 1 shows an implementation pipeline diagram for our project. It will need to go through extensive safety testing and optimisation before being implemented.
Figure. 1: Predicted developmental stages of the AuxIn project. Source of biology, toxicology, and environment data: CropLife International, as supplied by Dr Stuart Dunbar.
Trip to Syngenta
We wanted to get advice from some people in the field of plant biotechnology to review our project and discuss the feasibility of implementing such an engineered system to augment plant roots.
We had the pleasure of meeting three scientists who work at Syngenta, a company specialising in agricultural products and research that employs some leading plant scientists - Professor Stuart Dunbar, Dr Torquil Fraser and Dr John Paul Evans. Professor Dunbar leads a team that investigates the functionality of agrochemicals. Dr Fraser is an expert in soil science and Dr Evans specialises in auxins. Together we discussed the strengths and weaknesses of our project in regard to a whole variety of issues. (To read about what we learned on ecology during this visit, please click here).
We were made aware of seed coat technology, which we were told can be used to supply our bacteria to plants in the soil. It is possible to tailor-design the contents of the seed coat as well as the localisation (it can stay attached to the seed or disperse).
Depending on the seed type, it is possible to determine how long you want it to last in the soil by what you put in the coat. For example, we would be able to have nutrients for the bacteria inside and pesticides for the plant inside the coat. We were informed that it would cost $10-20 million US dollars to develop a new seed coat and there would be extra costs associated with proving its safety.
Overall, the Syngenta team gave us very positive feedback and could foresee potential for our system as a tool to aid in preventing soil erosion as well as remediating degraded land. They underscored the importance of no-till farming is essential for sustainability and future production in addition to protecting land from erosion by planting vegetation. We were also urged to market our project as a platform system of natural biochemical delivery to roots of different plant types.
Figure 3: Prof. Dunbar showed us the intricate robot that could take leaf cuttings without the veins by mapping out the vein structure. (Picture by Imperial College London iGEM team 2011).
Figure 2: In the conference room at Jeallot's Hill research centre with Prof Dunbar and his team. (Picture by Imperial College London iGEM team 2011).
Seed coat design
We needed to find a method suitable for applying our bacteria to the soil or seeds to improve root growth of germinating plants. The method had to be amenable to planting seeds by hand without the need for machinery. We were aware of the seed coat technology from the meeting with Syngenta, but not on the specifics in implementing it.
We were put in touch with Mathijs Wuts, a seed coat expert from Syngenta, for more information on types of seed coats and which would best suit our project. He suggested a Phyto-Drip seed treatment as the best option for survival of our bacteria.
Phyto-Drip is a high precision application technology for crop protection agents or other beneficials for young plant production. A droplet (0.2 to 0.5 ml) with the compounds you want to apply is positioned on top of the seed directly after sowing. Due to the liquid application, a part of the droplet will enter the soil and awaits the growth of the young plant to guarantee further protection in the root zone. On a professional level this technology is exclusively available for transplanted vegetable crops but the principle is easily copied in a lab using a (multi-)pipette.
A huge advantage of the technology is that the survival chances of bacteria are significantly increased. The precision of the application is better than a seed coat due to the fact that it is applied in exactly the same amount of compound to each seed. A seed coat covers the surface of the seed and thus will inevitably result in a certain variation of the compound loading for each individual seed due to the variability of the seed surface.
Although this technology is not yet commercially available for direct sown field crops, the pipette application method may be useful for small scale trials and practical proof of concept.
Figure 4: A schematic diagram of what a coated seed looks like. The seed coat is cracked and melted when exposed to the rain and the bacteria could be released. (Picture by Imperial College London iGEM team 2011).
Although the Phyto-Drip would be better for keeping bacteria alive, a pre-applied seed coat would be more practical for simple application by hand in local areas. Mr Wuts informed us of Incotec and their recent partnership with TJ Technologies Inc. They have developed QuickRoots, a seed supplement containing natural microbes such as B. subtilis, which has proven to increase root mass as well as improve crop yields. Incotec is developing a seed coat to apply QuickRoots to seeds so that the product can be provided as pre-treated seeds. Such a seed coat would be ideal for implementing AuxIn so that pre-coated seeds can be sown without a second step of adding liquid medium.
We then contacted Dr Frans Tetteroo from the Incotec Group to gain insight into the specifications behind production of microbe-containing seed coats. Incotec develops and commercialises special powder and liquid formulations that promote seed germination, provide beneficial nutrients to microorganisms, and often contain active ingredients to protect plants from pathogens (insecticides and fungicides). Shelf life is an important parameter for the bacteria and the seed and therefore coating formulas must pass very stringent quality tests on the survival of both. Storage temperature and humidity also play an important role in optimising shelf life. However the shelf life of microorganisms will depend mostly on the species' dessication tolerance. If engineering bacteria with a dessication tolerance such as Bacillus subtilis there will be no problem, however Rhizobia have no dessication tolerance. The coating materials developed by Incotec are inert and should not affect the survival of engineered bacteria nor the shelf life of the seed.
Figure 5: Scanning electron micrograph images of a lettuce seed (left) and the surface of a lettuce seed coated with beneficial bacteria (right). (Images provided by Dr Frans Tetteroo from Incotec).
Figure 6: Images of a coated lettuce seed. (Provided by Dr Frans Tetteroo from Incotec).
Data fitting was used to determine the IAA concentration for optimal root growth (0.1 nM) and this value was used to model the number of our engineered bacteria that would be needed in a seed coat, based on modelling of chemotaxis towards the root and bacterial IAA expression (Figure 7). The number of bacteria calculated to be needed in the seed coat to induce optimal root growth is 4.97 x 106(see more on Auxin Xpress modelling) and from the modelling of phyto-route, the bacteria should be positioned at a distance of less than 0.012 m from the seed.
Figure 7 (a): The evolution of IAM vs. time. Figure 2 (b): The evolution of IAA vs. time. IAA expression level is 72.35 µM, therefore each bacterium produces 7.24×10-14 µM at steady state with bacterial cell volume equal to 10-15 dm3. From wet lab experiments we know that the optimal concentration of IAA to promote root growth is 0.1 nM, and the volume of an Arabidopsis seed coat approximately equals 3.6×10-9 m3. Therefore the number of bacteria required to be present in seed coat to maximally increase root growth of roots is 4.97 x 1024. (Modelling by Imperial College London iGEM team 2011).
Although soil erosion is a world-wide problem, we wanted to focus on one area to propose in our implementation pipeline. Originally we were looking into the Sahel area of Africa where large-scale initiatives such as the Great Green Wall project (link) are taking place. Yet we didn’t know how a seed technology could be sustainably integrated into such a project and if it would be realistic.
Figure 8: Rebekka and Nikki had the pleasure of meeting Louise and Rodney (two founders of BRT) over lunch to discuss the implementation of tree planting programs. (Picture by Imperial College London iGEM team 2011).
In order to gain some more understanding of how small scale re-vegetative organisations work, we contacted and met with Louise Cooke and Rodney Portman from the Berkely Reafforestation Trust (BRT). They are part of a four-person organisation that form partnerships with local organisations to initiate holistic tree planting programs in areas such as the Sahel and Himalayas. The main points we took away with us about implementing tree-planting programs in developing areas were:
- Local involvement and initiative is essential for sustainability. Large scale tree-planting programs may physically be affective but are not sustainable in the long run because the locals of that area do not have an interest or benefit from caring for the vegetation. Additionally, international organisations often come and go, leaving projects unfinished or discontinued, therefore those that are benefiting from the program must be integral to its development.
- It is vital to use native plant/tree species. There will be much less risk of disaster because indigenous species will already be adapted to that climate and ecosystem.
- There can be no added costs for locals and application must be VERY simple. In the BRT programs, seeds are collected from existing trees and then planted rather than purchasing from seed companies in order to promote sustainability. Generally seeds are sown in small nurseries until they reach seedling height and are then planted in larger spaces because seeds are often very difficult to establish in harsh environments.
- There is a growing program already well established in India and East Africa to promote tree planting for cash return from carbon offset. TIST is a large scale organisation with the resources to track and monitor tree planting at local levels so that locals can qualify to access their carbon credits. A few people in each local area are trained to map the geographic location of trees that have been planted and take several measurements. This information is entered directly into a palm computer and then transferred to a large database where such information from several areas is massed together. They then fill out all the paper work required to access cash credits and filter the money back down to locals. The organisation receives early investment capital from carbon trading companies that can buy “future carbon” at a heavily discounted price. This sophisticated hierarchy provides the infrastructure needed for locals to obtain carbon offset credits which otherwise would be impossible to monitor.
From Louise and Rodney’s advice on simplicity of application, we decided to go for the seed coat rather than the Phyto-Drip which would require an additional step after sowing. We also decided it would be best to supply of pre-coated seeds to established re-vegetation initiatives such as BRT. This was the best choice because these organisations will already have built strong relationships and established trust with the locals of an area. They are the ones who will be planting the seeds, tending to them and benefiting from them so it is essential that they are integral to the program. We would aim to fund this by deriving profit from commercial markes such as the agricultural sector. IAA has been shown to improve the quality and yield of cotton crops, providing potential application of our system in agriculture. Additionally, our experiment results showing that Arabidopsis watered with IAA increases plant biomass which could have a significant impact on enhancing crop yields.
 Chen J and Guan X (2011) Auxin boost for cotton. Nature Biotechnology 29: 407–409.