3.2  Observation of antheridia and archegonia


Naoki Aono and Tomoaki Nishiyama



Antheridia and archegonia are formed at the shoot apex of a gametophore. They are covered with several young leaves, and it is necessary to remove the leaves for observation. Developmental processes at the cellular level are observed by differential interference contrast microscopy. 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.




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



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



Forceps with fine tips for surgery (e.g. FONTAX, INOX No5) are suitable for dissection. When it does not grip well, sharpen and adjust the tips using an oilstone.



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

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



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


DAPI staining



1. Cut the apex of the gametophore bearing gametangia away 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



DAPI Solution

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



CLSM observation of gametangia fixed with glutaraldehyde

See chapter 3.4.