GENETIC TRANSFORMATION AND HYBRIDIZATION Direct transformation of the liverwort Marchantia polymorpha L. by particle bombardment using immature thalli developing from spores Shota Chiyoda Æ Kimitsune Ishizaki Æ Hideo Kataoka Æ Katsuyuki T. Yamato Æ Takayuki Kohchi Received: 7 March 2008 / Revised: 30 May 2008 / Accepted: 30 May 2008 / Published online: 14 June 2008 Ó Springer-Verlag 2008 Abstract 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. Hygromy- cin-resistant transformants were obtained on selective media by transformation with a plasmid carrying the hygromycin-phosphotransferase gene (hpt) conferring hy- gromycin resistance in 4 weeks. The aminoglycoside-3 00 - adenyltransferase gene (aadA) conferring spectinomycin resistance was also successfully used as an additional selectable marker for nuclear transformation of M. poly- morpha. 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. Keywords aadA Á Bryophyte Á Immature thalli Á Liverwort Á Marchantia polymorpha Á Particle bombardment Á Spore Á Transformation Introduction Liverworts, mosses, and hornworts are the earliest- diverging extant lineages of land plants, and the liverworts in particular are considered the first land plants to evolve and colonize the ancient landscape (Qiu et al. 1998, 2006; Dombrovska and Qiu 2004). The morphology of liverworts is less complex than that of vascular plants, and they exhibit a number of characteristics of primitive land plants. Liverworts have been studied for their unique characteristics of regeneration by developmental biologists since the early years of the 20th century (Hohe and Reski 2005). The liverwort, Marchantia polymorpha L., is a geographically widespread species that is dioecious. The dominant stage of its life cycle is a haploid thallus, pro- viding advantages over diploid vascular plants for genetic analysis. M. polymorpha can propagate not only sexually, but also by asexual bud-like structures called gemmae that arise from single initial cells in gemma cups on thalli (Barnes and Land 1908; Hughes 1971). M. polymorpha is expected to provide insight into the nature of ancestral land plants from comparative genomic analyses (Bowman et al. 2007). In addition to previous intensive studies on organ- ellar genomes (Ohyama et al. 1986; Oda et al. 1992), and the Y chromosome (Yamato et al. 2007), the nuclear genome of M. polymorpha is being sequenced by the Community Sequencing Program of Joint Genome Shota Chiyoda and Kimitsune Ishizaki contributed equally to this work. Communicated by R. Reski. S. Chiyoda Á K. Ishizaki Á H. Kataoka Á K. T. Yamato Á T. Kohchi (&) Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan e-mail: tkohchi@lif.kyoto-u.ac.jp 123 Plant Cell Rep (2008) 27:1467–1473 DOI 10.1007/s00299-008-0570-5