Plant Synthetic Biology

Key Achievement


Since the beginning of the iGEM competition there have been more than 4,300 iGEM teams, but only 48 teams worked on plant chassis in their projects. Moreover, most of them worked on the model species Arabidopsis thaliana and Nicotiana Benthamiana.
While our planet boasts about 400,000 plant species, humanity utilizes only around 31,000 for purposes ranging from food and medicine to building materials and decor. Yet, a striking 85% of our caloric intake comes from a mere 20 plant species, underscoring the limited diversity in our food crops.

Hence, we, the iGEM Marburg 2023 team, advocate for the utilization of the vast array of plant species by all future iGEM teams and the broader iGEM community. This approach empowers local teams to effectively address local challenges using the unique attributes of their regional plant species.

The Problems

There are a few challenges, which hinder iGEM teams starting their iGEM project with their local plant species:

We tried to dissect these problems and come up with solutions for the future iGEM community.

Our Solutions

Highly accessible plant transformation protocols

The first problem we wanted to address within our project was to lower the barrier of entry for the general plant transformation procedure. For that, we adapted, improved, and developed a low-cost/low-equipment plant transformation protocol, that appears to be easily adjustable for many plant species.

graphical abstract of the cut dip budding protocol. The figure shows the workflow for the CDB protocol using V. subterranea (Bambara groundnut as an example). Agrobacterium solution is centrifuged down and resuspended in fresh LB medium containing vanillin. The root is cut off and the shoot is incubated in the Agrobacterium solution for 30 minutes before planting. After two weeks, a plant with red roots is shown in the final picture.
Figure 1: workflow of our adapted CDB protocol.

We sought a versatile transformation method adaptable to various agricultural plants and found the cut-dip-budding protocol, a recent innovation (Cao et al., 2023). This protocol, notable for its simplicity, doesn't require sterile cultures or antibiotics, making it especially valuable for iGEM teams without a sterile bench. It involves growing plants in a non-sterile setting, cutting seedlings, inoculating with Agrobacterium rhizogenes, and then nurturing in vermiculite. In two weeks, hairy roots emerge, some of which are transgenic and develop into normal shoots. This technique creates new transgenic plants without extensive tissue culture. Bambara groundnut (Vigna subterranea) and (Fragaria x ananassa), both able to induce shoots from roots, are prime examples for this method.

After several rounds of optimization of the Cut-Dip-Budding protocol, Bambara groundnut showed signs of root development two weeks post-transformation. A part of the roots showed hairy cells. By the fourth week, distinct hairy root systems had formed, and two months post-transformation, a breakthrough occurred—the appearance of RUBY red root sections in two bambara groundnut plants. This phenotype marked the successful introduction of T-DNA into root cells, a pioneering feat as no transformation of bambara groundnut had ever been published before. We are deeply proud to announce that we were able to establish the first working method to transform the non-model organism bambara groundnut via Agrobacterium rhizogenes, marking a step forward for non-model crop transformation.

Bambara groundnut hold in two hands with gloves
Figure 2: bambara groundnut with new root system 8 weeks after transformation.
Red root photographed through binocular.
Figure 3: RUBY positive root tissue in bambara groundnut 8 weeks after transformation.

To further simplify the cut-dip-budding protocol, we sought an improvement in which the newly developed roots of the plants under study could be observed without disturbing the plant. We placed particular emphasis on ensuring that any improvement to the protocols remained simultaneously inexpensive and easy to implement. Looking for a creative solution to this problem, we found a novel method shared by plant biotechnology researcher Sebastian Cocioba via social media, which involved using rockwool saturated with bacterial culture. This innovation allowed us to observe root growth directly through a transparent tube, streamlining the process for coming iGEM-teams.

A bambara groundnut in the falcon with rockwool.
Figure 4: Construction of the adapted rock wool method with a bambara groundnut.

In conclusion, both the cut-dip-budding protocol and Sebastian's rockwool method proved to be invaluable additions to our toolkit. The complete protocols with all successful adaptations can be found below. Our successful transformation of bambara groundnut, a plant for which no established transformation protocol existed, stands as a proof to the effectiveness of our adaptations. These innovative methods not only advance our own project but also hold the potential to revolutionize the way we approach plant transformation. With this non-sterile protocol, free from the constraints of expensive labware, upcoming iGEM teams and researchers around the world can benefit greatly.

Here we summarized and documented our highly optimized plant transformation protocols for future iGEM teams. Click to open the full protocol:

graphical abstract of the protocol.



With this protocol we started to transform larger agricultural plants, such as Fragaria x ananassa, Taraxacum officinale, and Vigna subterrenea. We aimed to create a Agrobacterium rhizogenes based protocol that makes plant transformation more accessible to workgroups worldwide since it doesn't require a sterile environment but can be performed under non-sterile conditions.



Broadening the species range, which can be transformed by establishing new tools for engineering Agrobacterium

We aim to overcome the obstacles that hinder iGEM teams when initiating their projects with local plant species, including the scarcity of transformation protocols for non-model species and the absence of essential tools for engineering Agrobacterium. To tackle the second and the third challenges above we sought to improve plant transformation efficiency for non-model plant species.

Agrobacterium is the workhorse for the transformation of plants. However its natural host range is limited, and further improvements are needed to allow for a more broad range of plant species which can be transformed. Still the tools to rewire the regulation of the plant transformation machinery are lacking. That is why we set out to develop the basic synthetic biology tools to fine-tune gene expression in Agrobacterium.

First we tested the gene expression tools of the iGEM community for their functionality in Agrobacterium and could show significant differences when comparing to the results previously published for E. coli.

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Figure 5: Comparison of the relative anderson promoter strength between Agrobacterium and E. coli

These findings underscore the importance of characterizing fundamental components when establishing an organism as a synthetic biology platform. Our work uncovers a new dimension of Anderson promoters in Agrobacterium, igniting novel possibilities and solidifying our commitment to advancing plant transformation. Our findings demonstrate that well-established components can adapt effectively to new environments, providing opportunities for future plant engineers.

In order to control the virulence during the plant transformation procedure dynamically, we then moved on and characterized inducible promoter systems for Agrobacterium. We selected 9 promoters from the “Marionette Collection”, which contains a number of inducible systems highly optimized (in E. coli) for high dynamic range and low leakyness (Meyer et al., 2019). Additionally, Ptrc and Ptau were also included (Mostafavi et al., 2014; Stukenberg et al., 2021). Another consideration made when selecting the promoter systems to characterize was to include ones that use non-phenolic compounds as inducers (Ptau, IPTG, Pbetl, and Pbad), in the hope of minimizing cross talk with the native VirA/VirG two component system.

Measurement results of inducible promoter in A rhizogenes ARqua1
Figure 6: Measurement results of inducible promoter in A.rhizogenes ARqua1.

The results in Fig. 6 show relative luminescence (RLU) output from the negative control and maximum induction. This experiment demonstrated that most promoters did not respond significantly to induction in A. rhizogenes ARqua1, notable exceptions were Ptac, Pvan, PnahR and Ptau. With first two showing the highest overall induction strength and Ptau the widest dynamic range, in fact, the baseline expression of Ptau was as low as the dummy promoter, both at the threshold of detection for the plate reader used in the experiment, this demonstrates that the expression of Ptau is tightly regulated and has virtually zero leakiness.

Having developed these gene expression tools for Agrobacterium, we aspired to harness them to control the intricate plant transformation mechanisms of the bacterium. For that we went through 4 rounds of the DBTL cycle for our "Best composite part".

A rapid protocol for prototyping transformation efficiency

In order to tackle the 4th challenge for iGEM teams to work on a plant project, which is the time needed to get results, we successfully adapted a protocol that enables us and future iGEM teams to measure plant transformation efficiencies rapidly and straightforwardly using hairy root induction. This transformative method played a pivotal role in our project, allowing us to test composite parts, different bacterial strains, and the virulence enhancement.

For this protocol, we used the established model organism Arabidopsis thaliana to optimize the speed for generating valid transformation efficiency. Arabidopsis possesses several key attributes such as the short cultivation time proved to be advantageous for prototyping transformation protocols with speed and precision.

Graphical step-by-step of the Arabidopsis transformation protocol. step 1: harvest of Arabidopsis seeds. Step 2: seed surface desinfection. Step 3: germination in solid plant medium. Steps 3 and 5: germination over 7 days. Step 6: cutting of the roots and inoculation of the shoot with Agrobacterium culture. Step 7: transfer to new solid medium. Step 8: growth in solid medium over 3 days. Step 9: final inspection of the plantlets at 10 days of growth post infection.
Figure 7: workflow of the protocol for prototyping transformation efficiency

In total just 8 days are necessary from germination to obtain the first results of transformation efficiency. Another 7 days later the final results were gathered completing the evaluation of the transformation efficiency. Thus, this method is particularly suitable for upcoming iGEM teams to take further steps in A. rhizogenes mediated transformation. This protocol has been benchmarked by us with two different reporter constructs, demonstrating the versatility of this approach.

The first reporter we tested was the dsRed, and red fluorescence could be observed after 3 days. However, during our first test run, we faced difficulties in assessing and quantifying transformed areas using a fluorescent marker. Fluorescence microscopy took more time than normal microscopy, in which the experimental plants were particularly stressed under the coverslip and light. This noticeably reduced the survival rate of the plants after microscopy. Furthermore, it also poses a limitation for iGEM teams without dedicated microscopes.

To improve accessibility of our methods for future teams, we decided to come up with a method not relying on fluorescence microscopy. As we did research to find an alternative, we found a publication about a reporter for proteins which are converting tyrosine to betalain, called pRUBY (He et al., 2020). After cloning and transforming the plasmid 35S:RUBY into A. rhizogenes, we were excited to find that with this adaptation we could significantly speed up the evaluation with the microscope, reducing the stress on the plants. In fact, RUBY can be seen very well with the naked eye, which makes it suitable for protocols that do not require a microscope at all. This allowed us to make the work with A. rhizogenes more accessible and attractive for upcoming iGEM teams.

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Figure 8: A. thaliana on plate 3 days after transformation with Agrobacterium rhizogenes ARqua1.

Notably, the RUBY reporter could be detected after just 3 days after the transformation, while transgenic red hairy roots became visible by day 10. This breakthrough allowed us to draw initial conclusions about transformation efficiency a mere 8 days after seed plating.

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Figure 9: A. thaliana on plate 10 days after transformation with Agrobacterium rhizogenes ARqua1.

Our final protocol for fast prototyping of transformation efficiency can be found below

graphical abstract of the protocol.


30 minutes sterilization, 30 minutes put the seeds on plates, 1 h transformation, 1,5-2 h examination


As a model organism Arabidopsis Thaliana Col-0 is and has been well-known and researched and is therefore great to use for a quick estimation of the possible transformation efficiency. We used this organism to create a baseline working with Agrobacterium rhizogenes, to try out our constructs as well as to quickly quantify our transformation efficiency. While we first used dsRed as a marker we adapted to Ruby, with which we were able to omit the use of a fluorescence microscope and switch to using a light microscope and/or observing the transformation efficiency in our plants by the naked eye.


Put seeds on plates
Transformation day
Moving day


Put seeds on plates after stratification phase (minimum 3 days)
Transformation day
Moving day and first examination
You can see half of an open petri dish with bleached round filter paper on the medium. On the plate are 10 0,5 - 1 cm large plants with 2 small green leafs. One hypocotyl has a red color seeable with the bare eye.
Second examination 10 days after transformation
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Figure 10: A. thaliana on plate 4 weeks after transformation with Agrobacterium rhizogenes ARqua1.

In conclusion, we successfully adapted a protocol that enables us and future iGEM teams to measure plant transformation efficiencies rapidly and straightforwardly using hairy root induction. This transformative method played a pivotal role in our project, allowing us to test composite parts, different bacterial strains, and the virulence enhancement by vanillin as a cost-effective alternative to acetosyringone, all with a focus on optimizing plant transformation for a changing world.

Plant transformation protocols for monocots and woody species

Most of the world's vital agricultural crops belong to the monocots, making them important candidates for Agrobacterium-mediated transformation. Sadly, Agrobacterium-mediated transformation is particularly critical for monocots because they are not a natural host for the pathogen. Nevertheless, we were determined to achieve the transformation of monocots, and our focus turned to foxtail millet (Setaria viridis), an emerging model organism in plant physiology (Brutnell et al., 2010). Leveraging the success of our concise A. thaliana protocol, we sought to extend our efforts to transform Setaria using this approach. Therefore, we adapted our A. thaliana protocol to transform 35 germinated Setaria plants using Agrobacterium rhizogenes ARqua1, enhanced virulence with vanillin due to the absence of naturally occurring phenolic compounds in monocots.

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Figure 11: Foxtail millet during transformation process with Agrobacterium rhizogenes ARqua1.

Our transformation experiments yielded remarkable results. Surprisingly, 14 out of 35 plants exhibited red RUBY dye after 3 days, with 13 of them developing hairy roots within 10 days.

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Figure 12: Foxtail millet 3 days after transformation with Agrobacterium rhizogenes ARqua1.

In addition to our monocot efforts, we also got the chance to work with oak trees as a chassis, which are propagated in sterile culture at our university. Quercus robur is an important forestry species, producing long-lasting and durable timber. Also it is a particularly important tree in forest ecosystems, as it supports one of the highest biodiversity of insect herbivores (Kennedy & Southwood, 1984). Forests worldwide are already suffering from drought stress due to climate change. Accordingly, adaptation of tree species to the changing climate is also a pressing issue of today. In addition Prof. Dr. João Bespalhok mentioned in our human practices interview with him that having better transformation protocols for woody species would be a great chance to increase the number of used species.

Since the oak clones are cultivated sterile in the propagation, the development of a protocol adapted to sterile oaks was necessary. We devised a two-stage protocol - a "Transformation Day," where we exposed the oaks to the bacteria and placed them on a medium akin to the propagation protocol, followed by a "Moving Day" two days later.

For more information on our exciting journey to create this protocol, check out our oak diary!

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Figure 13: Oak plantlets in test tubes 2 weeks after transformation with Agrobacterium rhizogenes.

In our initial experimentation with 50 oak saplings, we observed the plants about 1-2 times a week. The evaluation revealed callus formation, but root observation was challenging due to the addition of activated charcoal, concealing the roots until they reached an appropriate length to be visible from below the sterile containers. Our knowledge from the propagation protocol indicated that oak rooting typically occurred between 3-6 weeks. At the 3-week mark, we closely examined the oaks, and to our astonishment, five of them had developed new roots, with two exhibiting hairy roots. This marked a significant success, confirming that our method had induced transformation events.

In summary, the development of our oak clone transformation protocol represents a significant milestone. We have developed a unique method for transforming these woody plants, and the observation of hairy transgenic roots in our initial attempt is truly remarkable. We hope this success serves as an inspiration for upcoming iGEM teams to encourage them to work with their local plant species

Outlook and Conclusion

Building on our refined CDB protocol, future iGEM teams now have a stepping stone to explore Agrobacterium-mediated transformation of non-model organisms, without the constraints of sterile environments and high-cost equipment. Our groundwork, which includes the detailed characterization of inducible promoters that stimulate virulence genes and their in-plant testing, paves the way for a more precise targeting of virulence. Our efficient A. thaliana protocol invites upcoming iGEM teams to delve deeper into the genetic intricacies of both Agrobacterium and plant hosts. Through the comprehensive suite of protocols, tools, and resources we've assembled, our vision is to empower subsequent teams, facilitating them to root new plant-centric projects in iGEM that resonate with the unique challenges and opportunities of their local ecosystems.

Looking ahead, our team is enthusiastic about refining the inducer concentration, a step that promises to fine-tune the plant transformation mechanisms in Agrobacterium using our inducible promoter constructs. Such advancements might reveal the optimal inducer concentration for gene activation. In addition, we're eager to broaden the species applicability of our CDB protocol, leveraging the inherent potential of plants to sprout shoot tissue from transgenic roots. This approach might revolutionize the regeneration of transgenic plants, sidestepping the complexities of traditional tissue culture methods.

In the next phases of our endeavor, we envision harmonizing these initiatives. This would involve optimizing the transformation machinery via our inducible promoter construct and directing its application in the transformation of non-model organisms through the cut-dip-budding technique.


  1. Brutnell, T. P., Wang, L., Swartwood, K., Goldschmidt, A., Jackson, D., Zhu, X.-G., Kellogg, E., & Van Eck, J. (2010). Setaria viridis: A Model for C4 Photosynthesis. The Plant Cell, 22(8), 2537–2544.
  2. Cao, X., Xie, H., Song, M., Lu, J., Ma, P., Huang, B., Wang, M., Tian, Y., Chen, F., Peng, J., Lang, Z., Li, G., & Zhu, J.-K. (2023). Cut–dip–budding delivery system enables genetic modifications in plants without tissue culture. The Innovation, 4(1), 100345.
  3. He, Y., Zhang, T., Sun, H., Zhan, H., & Zhao, Y. (2020). A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture Research, 7(1), Article 1.
  4. Kennedy, C. E. J., & Southwood, T. R. E. (1984). The Number of Species of Insects Associated with British Trees: A Re-Analysis. The Journal of Animal Ecology, 53(2), 455.
  5. Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196–204.
  6. Mostafavi, M., Lewis, J. C., Saini, T., Bustamante, J. A., Gao, I. T., Tran, T. T., King, S. N., Huang, Z., & Chen, J. C. (2014). Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression. BMC Microbiology, 14(1), 295.
  7. Stukenberg, D., Hensel, T., Hoff, J., Daniel, B., Inckemann, R., Tedeschi, J. N., Nousch, F., & Fritz, G. (2021). The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens. ACS Synthetic Biology, 10(8), 1904–1919.
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