Plant Material and Growth Conditions

The Physcomitrella patens Gransden strain was used throughout this study. Tissue maintenance and production were performed on BCDA medium as described by Cove et al. (2009). P. patens tissue and protoplasts were grown under long-day conditions (16 h of light [70–80 µmol m⁻² s⁻¹]/8 h of dark) at 25°C. Growth medium was supplemented with 20 µg L⁻¹ hygromycin B or 30 µg L⁻¹ G418 for selection of transformed cells. Tissue for phenotypic characterizations was grown on BCD medium unless stated otherwise. Cultures for bud counting were established as follows. A BCD-containing petri dish was inoculated with 16 equally spaced spot inoculums consisting of 10 µL of protonemal tissue suspension. After 14 d of growth, buds were counted on 100 filaments using a dissecting microscope, and the average number of buds per filament was calculated. Buds were counted on 15 cells from the tip of the filaments. The 100 bud-containing filaments were randomly chosen from different areas of the plate. Tissue for sporophyte production was grown on sterile Jiffy7 soil blocks placed in glass jars under short-day conditions (8 h of light [70–80 µmol m⁻² s⁻¹]/16 h of dark) at 15°C and manipulated as described (Perroud et al., 2011). Tissue for real-time quantitative RT-PCR was grown and harvested as follows. Protonemal tissue grown on BCDA under long-day conditions was collected, homogenized in sterile water, and inoculated on BCD medium overlaid with cellophane discs. The tissue was collected after 6 and 14 d of growth, snap frozen in liquid nitrogen, and stored at −80°C until further processing.