A Simple Protocol for Thallus Culture-Based Genetic Transformation of the Liverwort Marchantia polymorpha

The liverwort Marchantia polymorpha, as the earliest land plant, is an important model system for studies of molecular evolution. Liverworts, which undergo a haplo-diplontic life cycle, spend most of their life as haploid gametophyte bodies, called thalli. In addition, the thalli can produce vegetative propagules, termed gemmae, conferring asexual reproduction in liverworts. Thus, to obtain sufficient isogenic tissues for genetic manipulation, the thallus is a suitable material. Here, we found that 23- to 25-d-old fully expanded thalli can be used for the genetic transformation with the pre-cultivation process, setting up the mature thalli pre-cultivation method. Next, we elucidated that 10- to 12-d-old young thalli can be used for the tissue-culture-based transformation in the absence of the pre-cultivation step, establishing the young thalli direct co-culture method without significant changes of transformation efficiency compared with the mature thalli pre-culture method. Overall, we set up the protocol using various stages of thalli of M. polymorpha with stable transformation efficiency. Mainly, we focused on reducing the hands-on labor time for performing the genetic transformation by widening the available stage of thalli for transformation or excluding the regenerating process of the thallus plantlets. In this report, we describe the detailed procedures employed under experimental conditions.