Introduction

Freshly inoculated and cultured protonemata are suitable for observation. When protonemata are cultured for more than two weeks on cellophane on BCDATG or BCDAT medium under continuous white light at 25˚C, the protonemata start to turn brownish and die. If you cultivate tissue on cellophane, it is better to observe protonemata at about one week after inoculation, especially on BCDATG.

The size of cells and organelles changes in different media. Chloroplasts become larger on BCDATG or BCDAT medium than on BCD medium.

Chloronemata are easily observed on BCDATG or BCDAT medium. When you inoculate protonemata on BCD medium, protonemata differentiate into caulonemata earlier than on BCDAT. For observation of gametophores, protonemata are inoculated in BCD medium rather than BCDATG and BCDAT medium and the plate is sealed with surgical tape to prevent evaporation of water. For observation of bud formation, BCD medium is appropriate because addition of ammonium in the medium enhances growth of buds that are callus-like, rather than normal.

Protocol

1. To observe a gametophore with more than 10 leaves.

1) Place a gametophore on solid medium.　

2) Drop water on the gametophore and cover with a coverslip

3) Observe using a stereomicroscope.　

2. To observe protonemata

1) Drop water on a glass slide and place a few protonemata in the water. For living cells, do not use glycerol solution.

2) Cover with a coverslip and observe immediately.　

Material

· Stereomicroscope

· Glass slide

· Coverslip

· Forceps

· Water

· Solid medium (eg. BCDATG plate)