Semi‐automated workflow for high‐throughput <i>Agrobacterium</i> ‐mediated plant transformation

Abstract

High-throughput experiments in plants are hindered by long generation times and high costs. To address these challenges, we present an optimized pipeline for Agrobacterium tumefaciens transformation and a simplified a protocol to obtain stable transgenic lines of the model liverwort Marchantia polymorpha, paving the way for efficient high-throughput experiments for plant synthetic biology and other applications. Our protocol involves a freeze-thaw Agrobacterium transformation method in six-well plates that can be adapted to robotic automation. Using the Opentrons open-source platform, we implemented a semi-automated protocol showing similar efficiency compared to manual manipulation. Additionally, we have streamlined and simplified the process of stable transformation and selection of M. polymorpha, reducing cost, time, and manual labor without compromising transformation efficiency. The addition of sucrose in the selection media significantly enhances the production of gemmae, accelerating the generation of isogenic plants. We believe these protocols have the potential to facilitate high-throughput screenings in diverse plant species and represent a significant step towards the full automation of plant transformation pipelines. This approach allows testing ~100 constructs per month, using conventional plant tissue culture facilities. We recently demonstrated the successful implementation of this protocol for screening hundreds of fluorescent reporters in Marchantia gemmae.