Exercise 13.4 - Photomorphogenesis
- Pteridium aquilinum spores (Bracken fern)
- Agar plates of Knudson media 21
- Cellophane filters (red, blue and green 22)
- Dissecting microscope
- 1% (v/v) Tween 80 (Polyoxyethylene sorbitan mono-oleate)
- Place 10 ml of Tween 80 into a capped test tube and add
about 1-2 mg of dry fern spores. Cap the tube and shake the vial
to wet the spores.
- Obtain eight petri plates containing a minimum balanced salt
media. Pipette 1.0 ml of the spore suspension onto the surface of
each plate and swirl the plate to evenly distribute the spores.
Place the lids on the petri plates and seal the plates with tape
running completely around the edge. 23
- Wrap the plates in pairs of red, blue or green cellophane.
Leave two plates with no wrapping. Place the plates under a light
source (window sill or fluorescent "Gro-lux" with a 12/12 (12
hrs. light, 12 dark) light regime.
- Monitor the spores daily by observation with a dissecting
microscope. When they begin to germinate, monitor with the low
power of a regular microscope (or inverted, if the agar is not
The spores will extend a rhizoid in about 5 days and begin cell
division within 7-8 days. The first divisions are then crucial to
the development of the gametophyte shape. Continue to monitor the
division planes for a period of two to three weeks.
- Note the number of spores that germinate and calculate the
percent germination. As the prothalli grow, note the position and
direction of each cell division. Sketch each stage in the
development of the fern gametophyte.
Fix gametophytes periodically with acid-alcohol and prepare the
gametophytes for histological staining of the chromosomes. Note
the presence of mitotic figures and identify the poles of each
cell division. Based on the location of the division planes,
predict the direction for spindle fiber growth and hypothesize a
mechanism for controlling the direction of cell division (and
therefore the morphology of the gametophyte). Devise an
experiment to test your hypothesis.
One can do more advanced study of morphology by combining the
growth of the gametophyte with the presence of mitotic inhibitors
or chromosome damaging agents (Chapter Ten). Finally, the spores
can be subjected to x-ray exposure (40-60,000 roentgens) and the
morphology studied. As the fern "tumors" develop, the planes of
division will appear randomly, giving rise to a 3-dimensional growth
rather than the typical 2-dimensional gametophyte.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org