Chiyoda Shota, Ishizaki Kimitsune, Kataoka Hideo, Yamato Katsuyuki T, Kohchi Takayuki
Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan.
Plant Cell Rep. 2008 Sep;27(9):1467-73. doi: 10.1007/s00299-008-0570-5. Epub 2008 Jun 14.
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.
地钱（Marchantia polymorpha L.）属于基部陆地植物类群，是植物生物学中一种新兴的模式植物。我们建立了一种在实验室条件下制备多形地钱孢子囊的方法，通过远红光照射促进其向生殖发育转变。在此，我们报道了一种利用从孢子发育而来的未成熟叶状体改进的多形地钱直接转化系统。通过用携带赋予潮霉素抗性的潮霉素磷酸转移酶基因（hpt）的质粒进行转化，在4周内在选择培养基上获得了潮霉素抗性转化体。赋予壮观霉素抗性的氨基糖苷-3''-腺苷转移酶基因（aadA）也成功用作多形地钱核转化的另一个选择标记。除了hpt基因外，aadA基因的可用性应使多形地钱成为遗传操作的通用宿主。DNA凝胶印迹分析表明，转化的叶状体携带整合到基因组中的可变数量的转基因拷贝。尽管先前使用从芽孢生长而来的叶状体的系统需要在液体和固体培养基中进行两步选择，历时8周，但此处报道的使用从孢子发育而来的叶状体的系统通过在固体培养基上直接选择，能够在一半的时间内产生转化体，便于对这种模式植物进行遗传分析。
