In past iGEM seasons, there were relatively few teams dedicated to enhancing plant synthetic biology. This lack of plant-focused teams can be attributed to several factors. Firstly, the extended regeneration period required for plants poses a significant challenge, and the commonly used protocols for plant transformation involve the error-prone task of sterile plant regeneration. Additionally, there is limited access to pertinent information for iGEM teams interested in this field.

Nonetheless, it's essential to recognize the immense potential that plants hold within the realm of synthetic biology. As global temperatures continue to rise, the urgent need for rapid and effective crop improvement becomes paramount in ensuring food security for the world's growing population.

The most promising and most used procedure for introducing engineered DNA into the plant genome primarily relies on the utilization of Agrobacterium, a plant pathogen known for its capability to integrate DNA fragments into the plant's genome. Despite its widespread use in established protocols, Agrobacterium-dependent transformation is inefficient and only works reliably on a small number of model organisms.

As a part of our project, we conducted an analysis of certain regulatory components in Agrobacterium rhizogenes. This effort is aimed at providing a means for finely controlling gene expression, particularly in relation to the transfer of DNA to host plants over the long term. Despite Agrobacterium being a pivotal tool for plant transformation, there has been a notable absence of synthetic biology tools developed for this purpose. Also, the functionality and characterization of established genetic parts has not yet been verified to a large extent in important Agrobacterium strains.

To address this gap, we evaluated the functionality of regulatory parts from the iGEM community within Agrobacterium rhizogenes, including the important anderson promotors. Our findings underscore the significance of characterizing these well-known and extensively utilized parts when applied to new chassis. This characterization of parts in Agrobacterium now enables us and future iGEM teams to modulate the expression of genes, which are crucial for the transfer of DNA to plants, tailored to specific plant species.

Contribution to BBa_J23102

Anderson promoters play a vital role in synthetic biology and have been extensively studied in traditional chassis such as E. coli. These promotors have become the standard for constitutive expression in prokaryotic organisms, anchoring numerous iGEM projects. However, their performance in less traditional prokaryotic organisms has remained largely uncharted.

One of the first important experiments of our project represented the characterization of Anderson promotor collection in Agrobacterium rhizogenes strain ARqua1, the first choice for our plant transformations.

Figure 1: Comparison of the relative anderson promotor 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 promotors 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.

Contribution to low cost plant transformation protocol of non model organisms

Next to the characterization from important parts in Agrobacterium rhizogenes, we addressed several challenges common to working with plants. Conducting an iGEM project with native plant species presents several challenges to teams. The complexity associated with creating and implementing plant transformation protocols can be daunting. In addition, the lack of established transformation procedures for non-model species further complicates the task. In addition, growing and transforming plants takes a long time, which presents additional hurdles for iGEM teams working with local plant species. Most importantly, most transformation protocols are designed to work with expensive laboratory equipment, which is a huge barrier for many iGEM teams.

On of our contributions to future iGEM teams is centered on our revolutionary cut-dip-budding method, which has the potential to transform crop research. We prioritized agriculturally relevant plants, with a keen focus on Bambara Groundnut, a crucial legume in Africa renowned for its nutritional value and drought resistance. Our mission to provide accessible transformation methods led us to this innovative protocol, known for its simplicity - it doesn't require sterile cultures or antibiotics. It's a game-changer for iGEM teams with limited resources, as it entails growing plants in a non-sterile environment. This means that expensive equipment such as a sterile bench or growth chamber can be eliminated. Growing fully transgenic plants would also be possible with this method, provided that plants have the ability to induce new shoot tissue from root tissue. Many plant species show this useful property.

The results, including the successful transformation of Bambara Groundnut, mark a significant milestone in crop adaptation efforts. 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. We have thus laid the foundation for a bright future for iGEM projects worldwide and expanding opportunities in plant research, particularly in regions facing agricultural and nutritional challenges.

See below for more detail on our innovative 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.



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