Direct transformation of the liverwort Marchantia polymorpha L. by particle bombardment using immature thalli developing from spores

The liverwort, Marchantia polymorpha L., belongs to a group of basal land plants and is an emerging model for plant biology. We established a procedure to prepare sporangia of M. polymorpha under laboratory conditions by promoting its transition to reproductive development by far-red light irradiation. Here we report an improved direct transformation system of M. polymorpha using immature thalli developing from spores. Hygromycin-resistant transformants were obtained on selective media by transformation with a plasmid carrying the hygromycin-phosphotransferase gene (hpt) conferring hygromycin resistance in 4 weeks. The aminoglycoside-3 ''-adenyltransferase gene (aadA) conferring spectinomycin resistance was also successfully used as an additional selectable marker for nuclear transformation of M. polymorpha. The availability of the aadA gene in addition to the hpt gene should make M. polymorpha a versatile host for genetic manipulation. DNA gel-blot analyses indicated that transformed thalli carried a variable number of copies of the transgene integrated into the genome. Although the previous system using thalli grown from gemmae required a two-step selection in liquid and solid media for 8 weeks, the system reported here using thalli developing from spores allows generation of transformants in half the time by direct selection on solid media, facilitating genetic analyses in this model plant.