Exercise 13.4 - Photomorphogenesis

LEVEL I

Materials

Procedure

  1. 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.

  2. 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

  3. 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.

  4. 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 too thick).
    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.

  5. 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.

Optional

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 -- cellab@gac.edu