2.3 Induction of gametangia and sporophytes

Naoki Aono, Tomoaki Nishiyama, Mitsuyasu Hasebe

Introduction

Gametangia are induced at high frequency at 15˚C under short day conditions (8 hours light and 16 hours dark) as described in Hohe et al. (2002).

Hohe, A., S. A. Rensing, M. Mildner, D. Lang, and R. Reski. 2002. Day length and temperature strongly influence sexual reproduction and expression of a novel MADS-box gene in the moss Physcomitrella patens. Plant Biology 4:595-602.

Procedure

1. Use Jiffy7 (peat moss pot: Jiffy Products International AS, Kristansand, Norway) to grow healthy gametophores. There are 42 and 30 mm diameter peat pellets. Use 42 mm for regular culture. For crossing, put two 30 mm peat pots in a plastic box.

2. Put a dry Jiffy disc in a plastic box, and add water including 0.1% CaCO3 (saturated, 1 g/L) to immerse the dry Jiffy disc. Allow to swell for about 2 hours.

3. Remove excess water (otherwise, the peat pellet will be collapsed by autoclaving), then autoclave the wet Jiffy in a plastic box.

　Wet Jiffy in a plastic box after autoclaving

4. Add distilled water until 1 cm to the upper surface of the pot.

5. Inoculate with protonemata blended by polytron, glass beads or mortar and pestle.

6. Cultivate 1 to 1.5 months (until growth of gametophores with more than 10 leaves) at 25˚C under long day conditions (16 hours light and 8 hours dark) or under continuous light.

　

About a month cultivated at 25˚C with continuous light after inoculation.

7. Move to 15˚C under short day conditions (8 hours light and 16 hours dark). After a week, start observation of the gametophore apex.　 Antheridia are formed first, and then the archegonia are formed. No additional manipulation is necessary for fertilization in this method - just wait. Usually a single sporophyte grows at each apex. After a month, you can see a bunch of sporophytes. 2 months after the start of induction, matured spores are produced.

Development of sporophyte (days after moving to 16˚C culture)