Agrobacterium-mediated Genetic Transformation of the Liverwort Marchantia polymorpha

Marchantia polymorpha is a liverwort and serves as an invaluable model organism for studying the evolution of land plants. It holds a unique position in the plant kingdom, representing a sister species to all other land plants.

Lifeasible has extensive research and service experience in plant genetic transformation. We offer Agrobacterium-mediated genetic transformation of the liverwort Marchantia polymorpha, which enables the precise introduction of specific genes into the plant genome, thereby facilitating molecular analysis and functional studies.

Transformation method

We offer an Agrobacterium-mediated genetic transformation method developed for Marchantia polymorpha, known as AgarTrap (agar-utilized transformation with pouring solutions). This technique promises to revolutionize research on Marchantia polymorpha and has the potential to be adapted for other plant species.

AgarTrap stands out among genetic transformation methods due to its simplicity and efficiency. Unlike traditional techniques, which involve multiple steps and the transfer of plant tissues between different media, AgarTrap streamlines the process by immobilizing Marchantia polymorpha tissues on a solid agar medium and allowing the necessary solutions to be absorbed. This results in a quicker and less labor-intensive procedure.

The process of transformation

The AgarTrap method can be divided into three major steps, including plating of Marchantia polymorpha tissue, pouring of the transformation buffer, and pouring of the selection buffer.

Step 1. Plating of Marchantia polymorpha tissue

• Spores (S-AgarTrap). The process begins by resuspending sporangia stored at -80°C in sterile ultrapure water. Spore suspension is then poured onto a solid medium, and glass beads are placed on the medium to facilitate even distribution. After incubation in a culture room, the spores are ready for transformation.
• Gemmae (G-AgarTrap). Gemmae are plated on a solid medium with the addition of sterile water. This step ensures that gemmae are evenly distributed and ready for subsequent transformation.
• Mature Thallus Pieces (T-AgarTrap). In this case, A. tumefaciens cells are streaked onto LB solid medium and cultured before plating the thallus pieces. These pieces are obtained from mature thalli and are cut into approximately 5x5mm sections. Unlike the previous two methods, thallus pieces do not require pre-culture.

Step 2. Pouring transformation buffer containing A. tumefaciens

• After culture, A. tumefaciens cells are skimmed from the LB solid medium and resuspended in a transformation buffer containing acetosyringone, MgCl2, and MES-NaOH. The density of A. tumefaciens cells in the transformation buffer is adjusted, and the buffer is poured onto the Marchantia polymorpha tissue on a solid medium. The Petri dish is then sealed and incubated at 22°C for 2-3 days, promoting the co-cultivation of the transformed cells.

Step 3. Pouring Selection Buffer Containing Antibiotics

• Following co-cultivation, A. tumefaciens cells covering the Marchantia polymorpha tissues and solid medium are removed by washing with sterile ultrapure water. After washing, a selection buffer containing a selective antibiotic is poured onto the solid medium. This buffer is absorbed by the medium, allowing transgenic cells to survive while non-transgenic cells perish. After 2-3 weeks of selection, the transformants are transferred to a new medium containing the selective antibiotic.

Advantages of our services

Lifeasible is dedicated to bryophyte transformation. Our Agrobacterium-mediated genetic transformation of Marchantia polymorpha using the AgarTrap method simplifies the transformation process. It improves efficiency also by eliminating labor-intensive steps and the need for liquid media. And it can effectively help researchers to deepen Marchantia polymorpha gene function study and promote further development of bryophyte transformation. If you are interested in us, please feel free to contact us.