3.1　 Observation of protonemata and gametophores

Yuji Hiwatashi

Introduction

Freshly inoculated and cultured protonemata are suitable for observation. When protonemata are cultured more than two weeks on a seat of cellophane on BCDATG or BCDAT media under continuous white light at 25˚C, protonemata start to change brownish and likely die. If you cultivate on the cellophane, it is better to observe protonemata in a week after inoculation especially on BCDATG.

Cell and organelle sizes change in different media. Chloroplasts become larger on BCDATG or BCDAT media than on BCD medium.

Chloronemata are easily observed in BCDATG or BCDAT media. When you inoculate protonemata on BCD medium, protonemata change to caulonemata earlier than on BCD.

For observation of gametophores, protonemata are inoculated in BCD medium rather than BCDATG and BCDAT media 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 a callus-like bud.

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 cover slip

3) Observe with stereomicroscope.　

2. To observe protonemata

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

2) Cover with a coverslip and observe immediately.　

Material

· Microscope

· Glass slide

· Coverslip

· Forceps

· Water

· Solid medium (ex. BCDATG plate)