Plastid transformation of sporelings and suspension-cultured cells from the liverwort Marchantia polymorpha L.

We describe simple and efficient plastid transformation methods for suspension-cultured cells and sporelings of the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as suspension-cultured cells and sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the methods described here, a few plastid transformants are obtained from a single bombardment of sporelings, while more efficient plastid transformation is expected in suspension-cultured cells, ~60 transformants from a single bombardment. Homoplasmic transformants of thalli are obtained immediately after primary selection, whereas homoplasmic transformants from suspension-cultured cells are obtained after 12-16 weeks of repeated subculture.