3.6　 Observation of antheridia and archegonia

　　　　　　　　　　　　　　 Naoki Aono and Tomoaki Nishiyama

Introduction

Antheridia and archegonia are formed at the shoot apex of gametophore. They are covered with several young leaves, and it is necessary to remove the leaves for observation. Developmental processes at cellular level are observed by differential interference cotrast microscope. The fluorescence of DAPI (4’,6-diamidino-2-phenylindole) for nuclei and the autofluorescence emitted after glutaraldehyde fixation for both cytoplasm and nuclei will be helpful for detailed observation.

Instruments

・ Stereomicroscope

Stereomicroscope with magnification from 100x (for removing to leaves) to 400x (for dissecting gametangia) (e.g. Leica MZ125 and Leica MZ APO ) and transmitted light. An objective lens with long working distance (e.g. Leica Plan Apo 1x) is recommended for comfortable dissection.

・ Microscope

Microscope with differential interference contrast and 20x, 40x and 100x objective lenses (e.g Leica DMLB). If you observe DAPI stained antheridia, fluorescence microscope equipped with a UV excitation filter is required.

・ Forceps

Forceps with fine tips for surgery (e.g. FONTAX, INOX No5) is suited for dissection. When it does not bite well, sharpen and adjust the tips using oilstone.

Procedure

1. Place a gametophore into water on a plastic plate or a slide glass. When matured antheridia are produced, brown antheridia will be also found further inside of leaves (Arrowhead).

2. Carefully remove leaves around gametangia using forceps under stereomicroscope. Be careful not to separate gametangia from gametophore.

　

Fig. Antheridia and archegonia on the shoot apex of a gametophore after removal of leaves

　

DAPI staining

Procedure

1. Cut away the apex of gametophore with gametangia from the stem.

2. Place the tissue into DAPI solution. And then incubate at 37˚C for 2 hours.

3. Wash the tissue with 50 mM NaH2PO4 (pH 7.0)

4. Observe the gametangia under UV illumination

Solution

DAPI Solution

50 mM NaH2PO4 (pH 7.0), 1 mM EDTA, 0.2% TirtonX-100, and 1 mg/ml DAPI

CLSM observation of gametangia fixed with glutaraldehyde

See chapter 3.4.