There are 400.000 plant species on our planet and humanity is using roughly 31.000 for food, medicine, animal feed, building material and decoration. However, plant transformation protocols are limited to a few species and often proves costly and time-consuming, our iGEM team has taken significant strides forward. By refining protocols, particularly the cut-dip budding method, we provide the iGEM community with more accessible tools. This broadens the spectrum of transformable species, emphasizing non-model and agriculturally vital plants.
For the first time, we have successfully transformed bambara groundnut, a non-model plant from Africa, with the soil bacterium Agrobacterium rhizogenes, marking an important milestone in our protocol. In addition, our dedicated efforts in developing a transformation protocol for woody species and a rapid protocol for monocotyledons with the ability to test improvements within a shorter timeframe underscore our team's relentless commitment to improving the accessibility and adaptability of plant transformation in the face of climate change and global agricultural challenges.
In the following text, we will look at our journey through the development of transformation protocols. We worked with model organisms like Arabidopsis thaliana and foxtail grass (Setaria sp.), as well as non-model organisms such as bambara groundnut (Vigna subterranea), strawberry (Fragaria x ananassa) and dandelion (Taraxacum officinale). In addition, we worked with the woody species oak (Quercus robur).
In March 2023, the Intergovernmental Panel on Climate Change (IPCC) released a sobering report that once again underscored the undeniable truth: our planet is warming at an alarming rate due to man-made climate change. This unprecedented temperature rise has triggered a cascade of consequences, including a surge in extreme weather events, increasing number of plant pathogens and a steady decline in agricultural yields (Singh et al., 2023). The urgent need for adaptation to these changing climatic conditions has never been more apparent (Calvin et al., 2023).
Traditionally, the process of adapting crops to new environmental challenges through conventional breeding methods is a time-consuming endeavor, often spanning several years. However, in the face of the climate crisis, we find ourselves in a race against time to secure our world's food supply. This urgency demands innovative and accelerated solutions.
Within the realm of plant biotechnology, various transformation techniques have been established. These include the gene gun, which is used to deliver DNA into tissue culture, or the floral dip method for the model organism Arabidopsis thaliana. These techniques have shown promise in introducing desirable traits into plants. However, they are not without their challenges.
Three key issues loom large in the realm of plant transformation: the methods are particularly time-consuming and a long cultivation period is required before results are obtained. Established protocols are limited to model organisms only and are not applicable to non-model species. In addition, the setups of the methods are often very complicated and cost-intensive. These major challenges are especially an obstacle for interested iGEM teams that want to tackle the big tasks of plant synthetic biology.
In response to these constraints, our iGEM team embarked on a mission to streamline and optimize accessible transformation protocols. We focused on Agrobacterium rhizogenes mediated plant transformations because it allows us to use the natural hairy root induction of the soil bacteria to induce transgenic roots. This method has been used for successful transformation since the 1970s, but most work focused on the expansion of transformed species rather than the optimisation of the method itself. Our research spans across a diverse spectrum of plant species, including both dicots and monocots.
Why did we choose to work with Arabidopsis thaliana in our mission to optimize accessible transformation protocols? The answer lies in the need for a method that can deliver transformation efficiency information to us quickly and easily - a crucial factor for the success of any iGEM project. At the beginning of our project, we soon realized the importance of a method that would allow us to run multiple transformation experiments in parallel to quickly quantify transformation efficiency. This is where the proven model organism Arabidopsis thaliana came into play.
Arabidopsis possesses several key attributes that made it an ideal candidate for our research. Its small size allowed us to study numerous plants within the confines of a small growth chamber space, maximizing our research efficiency. Additionally, the short cultivation time and relatively small genome of Arabidopsis proved to be advantageous for prototyping transformation protocols with speed and precision.
In the realm of Agrobacterium rhizogenes-mediated transformation, we were specifically searching for a fast and straightforward protocol. Our quest led us to a method (Mai et al., 2016) which outlined a procedure for obtaining transgenic hairy roots capable of producing heterologous proteins. In this method, Arabidopsis seeds undergo sterilization and are then spread on petri dishes, followed by stratification for four days. The plates were then moved to a 21 °C environment in darkness for germination for three days. After that, the roots were cut from the seedlings, and the hypocotyls were exposed to a Agrobacterium suspension for a short duration.
In our pursuit of efficiency, we made modifications to this protocol. Initially, we stratified the seeds in sterile MilliQ water after sterilization at 4 °C, effectively shortening this phase from four to three days. After that, we took a closer look at the germination phase. Early on, we encountered challenges in handling the tiny and delicate A. thaliana seedlings after only three days of germination, which led us to extend the germination period to five days. This adaptation significantly increased the survival rate of seedlings during transformation, making the process more manageable.
To assess transformation efficiency, we initially employed Agrobacterium rhizogenes ARqua1 with the fluorescent reporter
gene dsRed, provided by our PI Anke Becker. 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 gene encoding for
proteins that convert tyrosine to betalain, called pRUBY
(He et al., 2020). After cloning 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, after a few days, 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.
Our quest for efficiency didn't stop there. We aimed to determine the earliest time for assessing transformation efficiency, streamlining the protocol for initial evaluations. Through a series of experiments, we monitored plants for red dye starting on day 2 and continuing every day for 14 days. This comprehensive assessment revealed that plants could be best examined twice — 3 days and 10 days after transformation. Notably, the RUBY reporter could be detected 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.
With our goal to make this method usable for many iGEM teams, we wanted to specifically reduce the cost per transformation round. Therefore, we optimized the biggest cost drivers in this protocol. First, we revised the selection cassette on the T-DNA of the RUBY plasmid. It utilizes a hygromycin resistance, which we replaced with a kanamycin resistance, as kanamycin is significantly cheaper. In a second step, we found that the "Whatman paper type 5" used in the original protocol is a very expensive material. Therefore, we looked for a cheaper alternative. The solution was round, bleached coffee filter paper, which can be purchased in supermarkets. After autoclaving it in an aluminum foil bag, it was equally functional for our method. If you want to read this protocol with all adaptations, see the experiments page.
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.
Our mission extended beyond the confines of laboratory research, reaching out to agriculturally relevant plants, especially those with the potential to address emerging challenges in global nutrition, particularly in regions with hot climate. After exhaustive research, we identified two such candidates — the legume Bambara Groundnut (Vigna subterranea) and the beloved strawberry (Fragaria x ananassa). Each had its unique significance in our project.
Bambara Groundnut, one of the most important legumes cultivated in Africa, boasts exceptional nutritional value and
exceptional drought resistance. Its potential to thrive in regions susceptible to crop losses due to global warming
piqued our interest, aligning perfectly with our mission to facilitate crop adaptation in the face of climate change
(Tan et al., 2020).
Meanwhile, the strawberry, a cultural icon in our home-country Germany (over 130 000 t strawberry
yield per year), presented an opportunity to address the water-intensive nature of strawberry cultivation and explore
varieties resistant to drought stress (Betriebe, Anbauflächen, Erträge und Erntemengen von Gemüse, o. J.).
Our quest for a transformation method that would be accessible to many laboratories and adaptable to various
agricultural plants led us to the cut-dip-budding protocol, a recently published advance in the world of plant
transformation (Cao et al., 2023). What sets this protocol apart is its minimalistic approach — it doesn't demand sterile
cultures or antibiotics. Especially for iGEM teams that do not have access to a sterile bench, this protocol offers an
outstanding opportunity to explore ways in plant synthetic biology. The method relies on a straightforward principle:
growing plants in a non-sterile environment, cutting seedlings near the shoot-root junction, inoculating the upper parts
with Agrobacterium rhizogenes at the cutting site by dipping into the bacterial solution, and nurturing them in
vermiculite. Thanks to the root-inducing plasmid carried by A. rhizogenes, hairy roots sprouted approximately two weeks
later. The authors identified a subset of these roots as transgenic. These transgenic roots continued to grow over 8-10
weeks, eventually resembling the secondary roots of wild-type plants. The authors then cut these transgenic roots into
3-cm-long segments and placed them on non-sterile soil for budding. They observed the emergence of green buds that
developed into normal-appearing shoots, which were also transgenic.
This method allowed us to generate entirely new transgenic plants without the need for extensive tissue culture. Both
Vigna subterranea and Fragaria x ananassa are able to induce shoots out of roots by themselves, making them ideal
candidates for this transformative technique. Many plants have this property, which makes the protocol particularly
attractive for other iGEM teams to work on local agroplants. A protocol that enables the transformation and subsequent
regeneration of transgenic plants without sterile tissue culture is outstanding. Therefore, we were particularly excited
to work with this method, test it on exciting and important crops, and make it as applicable as possible for upcoming
iGEM teams.
Notably, Taraxacum kok-saghyz, a dandelion species, had been successfully transformed using this method in previous research, prompting us to include a close relative, Taraxacum officinale, in our project (Cao et al., 2023).
Our initial experiments followed the protocol of growing the plants in the greenhouse, removing their roots, incubating
them in Agrobacterium rhizogenes ARqua1 for 15 minutes, followed by placement in vermiculite. While strawberry and
dandelion plants readily formed new roots, bambara groundnut did not exhibit this response during the first trials.
Furthermore, all roots in strawberry and dandelion were non-hairy and non-red, resulting in a negative outcome. In the
subsequent experiment, we extended the incubation time to 30 minutes, but the results remained negative. Thus, we
further optimized the transformation step in the third round of experiments by increasing the bacterial concentration
through centrifugation and adding vanillin to the Agrobacterium dipping solution in order to enhance the virulence genes
of the bacteria.
For Agrobacterium-mediated gene transfer into plant hosts, the substance acetosyringone is often
favored, which induces the natural inducible promoter of Agrobacterium virulence genes. However, according to a paper,
as an alternative to acetosyringone, other phenolic compounds can also induce the virulence genes (Cha et al., 2011).
For this reason, we decided to test vanillin in Agrobacterium culture to see if it can increase the transformation
efficiency. This innovation holds particular significance for upcoming iGEM projects, offering a cost-effective way to
boost virulence.
This time, the experiment bore fruit. Hairy roots appeared on the strawberry plants for the first time, although no red dye from the reporter RUBY could be seen, marking a significant milestone as evidence of transformation events. 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 so 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 big step in non model crop transformation.
In parallel with these experiments, we explored the use of a second bacterial strain, Agrobacterium rhizogenes K599, known for its higher virulence (Foti & Pavli, 2020). The original CDB paper also used Agrobacterium rhizogenes strain K599. Accordingly, we hope for a better transformation result. As of the time of writing, the results of this experiment are still pending.
Throughout our experimental runs, we realized that removing and counting newly rooted plants from the vermiculite multiple times inflicted significant stress on them. 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. However, we encountered a challenge—desiccation risk, especially in bambara groundnut, due to its large leaves and thus significant transpiration. To address this issue, we devised a solution: small greenhouses, made from autoclave bags and metal rods, were placed over the plants for two days after transformation, which improved survival rates greatly.
Furthermore, we optimized the timing of transfection, as we explored the possibility that significantly younger plants could better withstand the stress of transformation, given their smaller leaf area and thus reduced water loss. Indeed, this adjustment resulted in significantly fewer plant fatalities post-transformation.
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 here. 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. This accessibility promises to expand the horizons of plant research, offering new ways to address global challenges in agriculture and nutrition.
Since the beginning of the year, Philipps-University Marburg is coordinator of the DFG research project PhytOakmeter. This project works on climate stress monitoring using the Quercus robur clone DF159, whereby the clone DF159 is propagated at our university. Thankfully, we got the chance to work with these special oaks. 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. The collaboration with PhytOakmeter thus provided a suitable model to work on woody species. Since the oak clones are cultivated sterile in the propagation, the development of a protocol adapted to sterile oaks was necessary.
The critical question was at what stage of oak propagation we could successfully introduce transformation. In the oak propagation process, buds are nurtured in a medium infused with phytohormones. After approximately 8 weeks, sprouts emerge, only with callus growth and no roots. Given that we needed to remove the roots for A. rhizogenes-mediated transformation, this stage of development proved to be the ideal entry point. However, the oaks, accustomed to sterile conditions, are highly susceptible to stress.
To address this, 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. On this day, the
oaks were dipped in cefotaxime and then transferred to a medium containing cefotaxime, to decrease bacterial numbers and
thus reduce biotic stress on the plants. We conducted this work in a laminar flow cabinet, albeit with the added
precaution of providing a plastic hood to shield the oaks from the airflow. For more information on our exciting journey
to create this protocol, check out our oak diary!
In our initial experimentation with 50 oak saplings, the results varied. While the hood didn't offer adequate
protection, resulting in leaf loss and contamination in some cases, the resilient oaks managed to recover. They
displayed new branch and leaf growth from lateral buds after 3-4 weeks.
Regular observation, about 1-2 times a week, 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 .
Armed with this promising evidence, we were eager to fine-tune our protocol to enhance transformation efficiency. We also explored the possibility of testing the bacterial strain K599 instead of ARqua1 in a second round, with the generous support of Lars Opgenoorth, who provided us with oak saplingsonce again. This time, we opted for a simpler approach. By turning off the sterile bench during incubation and closing the shutter to prevent contamination, we reduced water-loss by evaporation. We also increased the bacterial concentration through centrifugation and resuspension, and introduced vanillin to the culture to increase bacterial virulence.
As of the publication of this wiki, the second round has been transformed 2 weeks ago. Encouragingly, withered leaves are scarce, and contaminations have significantly reduced. Presently, 23 out of the 50 oaks have developed callus. In our quest to better evaluate the oaks, we positioned the shoots at an angle in the medium during the second round. This strategic adjustment allows us to observe roots much earlier than before, with small roots already visible on three plants after 2 weeks. We eagerly anticipate the results we can showcase before the Grand Jamboree.
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 contribute to this vital project, securing our agriculture future in the face of climate change. With this protocol, the possibilities for accessible plant research are boundless, offering a pathway to address pressing global challenges.
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 monocots are not a natural host for the pathogen. Nevertheless, we were determined to achieve the transformation of monocots, and our focus turned to 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.
Our journey with Setaria viridis commenced with the challenge of seed sterilization with sodium hypochlorite. To effectively work with these seeds, it was necessary to completely remove the husks surrounding them to prevent contamination. Initially, we developed a technique involving the removal of these layers by eroding the seed husk via manual grinding against adhesive strips and individually breaking open each seed with a thin metal pin. While effective, this method proved time-consuming. However, optimization efforts led to a more efficient destemming technique, offering a promising solution for future iGEM teams. By gently crushing the seed husks with a mortar and pestle until the glumes naturally separated, we streamlined the process significantly. The complete protocol with all successful adaptations can be found here.
With contamination concerns addressed, our next hurdle was germinating Setaria viridis seeds. Extensive research led us to a method involving overnight incubation in gibberellic acid at 28°C before plating them out, greatly improving germination rates (Sebastian et al., 2014). Encouraged by these developments, 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.
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. As our initial batch of Setaria seed was exhausted, leading us to acquire a new batch. However, germination proved elusive, despite our efforts with the previously successful protocol. After many negative trials, our instructor came up with the creative idea of ordering bird food instead of regular seeds because bird food is being dried out to store it longer and that could increase the germination rate. The new Setaria species, Setaria italica, was chosen for the experiment for several key reasons. First, Setaria italica and Setaria viridis are closely related genetically, as they both belong to the same genus, which makes Setaria italica a suitable candidate for studying certain genetic traits. Second, Setaria italica seeds are more readily available commercially, simplifying the acquisition of materials for researchers. Lastly, Setaria italica is a cultivated plant with a longer history of domestication compared to Setaria viridis. This domestication history results in more uniform and quicker germination, which is essential for the experiment, as it exhibits a typical domestication syndrome trait of loss of seed dormancy. So, we bought foxtail millet. We treated the new seed in a large experimental set with different variants of our previously developed protocol: we varied the sterilization time, tried it with and without gibberellic acid. Our results showed that the new seed germinated significantly better without the gibberellic acid.
Having acquired Setaria seedlings once more, we endeavored to build upon our earlier successes in monocot transformation. Our investigations spanned both Agrobacterium strains, ARqua1 and K599, as well as variations in incubation time and the testing of our promising VirG constructs in Setaria sp. Ultimately, transformation was not successful using the new variety, as no more transgenic tissue could be generated.
Nevertheless, this journey underscores the challenges inherent in developing feasible and reliable transformation protocols for monocots. Yet, it also illustrates the power of creative problem-solving and the application of an engineering cycle, ultimately leading to success and innovative advancements in this vital field of research.