A Simple and Scalable Chopped-Thallus Transformation Method for Marchantia polymorpha

Abstract The liverwort Marchantia polymorpha has emerged as a valuable model for studying fundamental biological processes and the evolutionary history of land plants. Agrobacterium-mediated transformation is widely used for genetic modification of M. polymorpha using spores, thalli, and gemmae. While spores offer high transformation efficiency, they result in diverse genetic backgrounds due to sexual reproduction. Conversely, thallus- and gemma-based methods maintain genetic consistency but are impractical for large-scale applications. To address these limitations, we developed a novel chopped-thallus transformation method. This technique involves generating numerous plant fragments by chopping thalli, eliminating the need for complex preprocessing. The method demonstrated superior transformation efficiency compared to traditional approaches and achieved sufficient numbers of transformants using simplified Gamborg’s B5 medium, previously considered suboptimal. This scalable and straightforward method enables the generation of large numbers of genetically consistent transformants, facilitating high-throughput experiments, including mutant screening and other large-scale applications. Competing Interest Statement The authors have declared no competing interest. Footnotes Abbreviations - ANOVA - Analysis of variance - EF1 - Elongation factor 1-α - GUS - β-glucuronidase - HSD - Honestly significant difference - LB - Left border - mALS - mutated acetolactate synthase Marchantia Marchantia polymorpha - RB - Right border - Tak - Takaragaike - TE - Transformation efficiency