Highly efficient G-AgarTrap-mediated transformation of the Marchantia polymorpha model strains Tak-1 and Tak-2

The liverwort Marchantia polymorpha L. is an important model species for investigating land plant evolution. Effective genetic transformation techniques are crucial for plant molecular biology and simplified or improved techniques for specific cultivars or strains can accelerate research. Over the past several years, we developed a simple Agrobacterium-mediated transformation technique for M. polymorpha named AgarTrap (Agar-utilized transformation with pouring solutions). AgarTrap is an easy technique that involves pouring the appropriate solutions onto plant materials on a single solid plate of medium. We recently improved AgarTrap using gemmalings (G-AgarTrap) of the M. polymorpha female model strain BC3-38 and achieved a transformation efficiency of nearly 100%. Based on this improved technique, in the current study, we adopted two factors (sealing the Petri dish with Parafihn and dark treatment during co-cultivation) and optimized two factors (Agrobacterium strain and pre-culture period) of the improved G-AgarTrap for other model strains of M. polymorpha, the male strain Takaragaike-1 (Tak-1) and the female strain Takaragaike-2 (Tak-2). After optimization, the transformation efficiency of Tak-1 using G-AgarTrap was as high as 55% compared to approximately 30% using the previous protocol. Furthermore, using Tak-2, we achieved a transformation efficiency of nearly 100%. Our improved G-AgarTrap technique for Tak-1 and Tak-2 represents a promising tool for promoting the study of Marchantia.