The looming threat of climate change poses an immediate risk to the global food supply. This makes the development of reliable tools for plant SynBio critical in order to engineer crops that are able to cope with extreme weather events and altered pest and disease dynamics. Despite their urgency, plant SynBio projects are rather scarce in iGEM due to the exceptionally demanding process of plant transformation. This year, the Marburg iGEM team tackled this problem by refining plant transformation protocols for non-model species, culminating in the first successful transformation of Bambara groundnut, a valuable legume for African countries. Furthermore, we thoroughly characterized novel genetic parts in Agrobacterium, and used them throughout several iterations of the DBTL cycle to create constructs that enhance plant transformation efficiency and broaden the range of genetically accessible plants. Ultimately, we hope to enable future teams to further extend the garden of plant projects in iGEM with new and diverse plant species.
Agrobacterium mediated transformation is one of the most prolific methods for plant engineering, by far the most common method for iGEM teams as well. However, working with plant SynBio is still far from a straightforward endeavor. This makes it especially hard for iGEM teams who might want to work with local plant species in their projects, often forcing them to engineer established model organisms such as Arabidopsis thaliana or Nicotiana benthamiana.
This year, we set our sights on creating tools that simplify and facilitate the process of achieving successful transformations in non-model plant species.
According to the UN Environment Program's "Emissions Gap Report 2022", current measures to curb global warming will fail to limit the rise in average global temperature to the 1.5 °C aim proposed in the Paris Agreement. Droughts, floods and heatwaves are predicted to undermine food security and nutrition across the globe, especially in economically vulnerable regions. Extensive changes in agricultural practices will be necessary to limit our contribution to climate change and to ensure we are able to feed the population in the future.
The current food supply chain is heavily reliant on a small group of staple crops. In fact, 60% of all calories consumed are derived from only four plant species: rice, wheat, maize, and potatoes (Sunderland, 2011). Consequently, threats to these cultivars, such as pests, diseases or environmental changes, have the potential to endanger the world’s food supply. Additionally, the unsustainable use of monocultures has led to the destruction of native ecosystems and to the displacement of local crops, which are vital to smallholder farmers and to the overall biodiversity.
The vulnerability of our agricultural practices is not a product of a small number of available plants though; in fact, the opposite is true: out of the 30,000 known edible plants, only 150 are actually consumed on a large scale (Shelef et al., 2017). Therefore, diversifying our food sources and identifying cultivars that are more resilient to the effects of climate change might deliver a significant contribution to ensure food security in the decades to come. In that regard, we believe an expansion in the range of species amenable to biotechnology would represent a huge leap towards that goal. On the one hand, it would lay the groundwork to improve existing local cultivars by shielding them against pests, climate change and ultimately facilitating their integration into the global market; on the other hand, completely new crops could be introduced through de novo domestication. Staple crops of today have gone through generations of artificial selection to enhance desirable traits and eliminate undesirable ones (Diamond, 2002). Synthetic biology offers a shortcuts to modify the genetic makeup of new domesticates, increasing their value as a source of nutrition in a matter of a few years.
Despite the tremendous advances in genetic engineering in the last few decades, less has been accomplished when it comes to building up new plant chassis. This delay is partially explained by the long growth time for each generation when compared to prokaryotic organisms. But it is also a product of the difficulties in introducing foreign genes to the plant cells. Currently, the most widely used method for plant transformation relies on the co-cultivation of plant tissue with the plant pathogen Agrobacterium tumefaciens. In the wild, this soil bacterium triggers the formation of tumors in plant tissue by inserting a DNA sequence with genes coding for the production of growth hormones. Scientists have taken advantage of this mechanism to replace the tumor causing genes by the desired genetic constructs. Despite that, the number of species where Agrobacterium-mediated transformation is well established is still rather limited, as most of the research is devoted to the crops with the largest market share (Chen et al., 2022). Local varieties, which hold immense value to smallholder farms as a source of subsistence, nutrition and cultural identity, have been so far overlooked by the GM revolution (Jacobsen et al., 2013). Part of the problem lies in the fact that specific strains of bacteria are needed to transform certain plants. As we found out, getting hold of a compatible Agrobacterium strain for a plant species (if one is even known) is extremely challenging, reaching up to hundreds of Euros and months of shipping time. This is made even worse by the confusing and often conflicting nomenclature of existing strains (De Saeger et al., 2021). While some work has been done to improve the existing strains, little has been done when it comes to approaching this issue with a synthetic biology framework, resulting in a meager collection of basic parts well characterized in Agrobacterium. This, in addition to the complex and expensive nature of the available protocols, puts many plant projects outside the reach of most iGEM teams.
This year, the Marburg iGEM team created the RhizoGene project with the main goal of addressing these issues. We believe the diversity of iGEM plant projects can be increased by lowering the barrier of entry for plant transformation, especially when it comes to non-model species. This will empower local teams to effectively address local challenges using the unique attributes of their regional plant species.
We achieved that by several iterations of optimizing existing transformation methods. For instance, we explored the cut-dip-budding protocol: known for its simplicity, it doesn't require elaborate sterile tissue cultures relying on expensive antibiotics or plant hormones, making it a game-changer for iGEM teams with limited resources (Cao et al., 2023). Through several rounds of tests and improvements, we were able to achieve the first recorded transformation of Bambara groundnut using Agrobacterium rhizogenes, proving that introducing completely new plant species to iGEM projects is a feasible endeavor.
The choice of working with Bambara groundnut was not arbitrary. It is a legume crucial for smallholder farmers in West Africa renowned for its nutritional value and drought resistance (Azman Halimi et al., 2019). Additionally, it presents opportunities for gender-focused innovation and commercial development, as it is primarily cultivated, sold, and prepared by women and serves as a valuable tool for elevating women's livelihoods (Lost Crops of Africa, 2006).
Another big part of our project focused on using synthetic biology approaches to improve Agrobacterium itself. Therefore, we created a composite part that leverages the function of the VirG transcription factor as a Master-Switch of plant transformation to improve the efficiency and host range of Agrobacterium. By incorporating the results of comprehensive part characterization experiments, we were able to iterate through several versions of our Master-Switch construct for optimal fine-tuning of the plant transformation machinery.
Based on our results, we could show that there is still plenty of room for improving current transformation methods, both on the side of the protocol itself, and by improving existing Agrobacterium strains. Additionally, we are confident that the true potential of Agrobacterium strain engineering is only beginning to be unlocked. With further part characterization and the development of even finer control of the vir genes, even higher transformation efficiencies could be achieved. For example, further experiments that titrate the level of virG induction might be able to find a sweet spot depending on the plant and Agrobacterium strain. The level of control over the virulence could also be more precisely controlled by not only inducing virG, but also the individual virulence genes. We are hopeful, that our findings will encourage future teams to work with plants from their local flora, further adapting the protocols we created to their own needs.