pictures of hermaphrodites

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					                                   C-Fern Genetics
       Ceratopteris is a genus of homosporous (producing only one kind of spore) ferns
found in most tropical and subtropical areas of the world. Species grow as either
aquatics or sub-aquatics (growing partly on land and partly in water) and are limited in
habitat to ponds, rivers or other wet areas such as ditches, taro patches or rice paddies.
Although some require an aquatic habitat, most species can be successfully grown in
pot culture in the greenhouse under warm, humid conditions. Ceratopteris is eaten in
areas of Southeastern Asia and there was an early attempt to develop it as a crop in the
Philippines. Currently, commercial uses
are primarily limited to its widespread sale
as an aquarium plant in fish hobbyist
stores, where it is sold under the common
name of 'water sprite' and has even been
immortalized in plastic replicas.

        Ceratopteris is an excellent model
organism for genetic studies because of
its’ haploid-diploid life cycle (shown at
right). Haploid spores germinate under
moist conditions into autotrophic
gametophytes. Sexual differentiation is
regulated by the presence of the
pheromone ACe. If no ACe is present,
spores differentiate into hermaphrodites
that secrete ACe. Once hermaphtodites
are present, germinating spores will
produce males.

       Both sexes are autotrophic and
microscopic. Males are smaller than
hermaphrodites. Both sexes produce
haploid, motile sperm that swim down a
concentration gradient of pheremone
released by the hermaphrodite. This is
called positive taxis.

       After fertilization, a diploid
macroscopic sporophyte grows on top of
the haploid hermaphroditic gametophyte.
When mature, the sporophyte will produce
spores via meiosis reducing the nucleus
to haploid.

Adapted for classroom use by D. Maxwell in 2004 from information found at
The investigation:
Day 1:
       1. Obtain a Petri plate with C-fern agar and a sharpie.                         1
       2. On the bottom of the Petri plate, write your initials near the
          edge. Evenly space the numbers 1, 3, and 5 on the bottom of
          the plate (as shown at right).
                                                                                  3         5

       3. Obtain a spore suspension and sterile pipette.                           1

       4. Ensure the cap is on the spore suspension and invert 3 times
          to mix.
       5. Remove the cap and use the sterile pipette to transfer a single drop of spores onto the
          agar in your Petri plate on top of the 1.
       6. Allow the Petri plate to sit for a couple of minutes until the water drop soaks into the
          agar. This keeps your spores in a confined space.
       7. Seal the Petri plate with parafilm and incubate under the lights.
Day 3:
       1. Obtain your Petri plate from under the lights, spore suspension and a sterile pipette.
       2. Remove the parafilm from the Petri plate.
       3. Transfer a drop of spores to the top of number 3 as you did on day 1. Remember to
          allow the drop to soak into the agar before moving the plate.
       4. Seal with parafilm and return the plate to the lights.
Day 5:
       1. Inoculate the Petri plate with spores over the number 5 as you did on the previous two
       2. Remember to allow the drop to soak in before sealing the plate with parafilm and
          returning to the lights.
Day 10:
       1. Obtain a non-sterile Petri plate lid, double-sided clear tape, a microscope slide, and a
       2. Create a Petri plate cradle. Use the double-sided tape to attach the Petri plate lid to
          the microscope slide. Place the microscope slide in the clip on the microscope stage.
          You should now be able to move the Petri plate lid around on the stage using the
          knobs on the underside of the scope.
       3. Obtain your C-fern plate from under the lights, remove the parafilm and the lid.
          Place the lid face down on a clean surface to minimize the risk of contamination.
       4. Move the objective lenses as far away from the stage as your microscope permits.
          Place your C-fern plate in the cradle. Switch to the lowest magnification and move
          the objectives toward the agar surface until the surface is in focus. Have your partner
          watch as you do this to ensure that you do not touch the agar surface with the
          objective lens. Touching the surface with your lens causes two problems. It
          contaminates the agar, and it makes the lens dirty.
       5. Observe each of the areas where you placed your drops. Based on what you know
          about gametophyte development, predict which drop should contain more
          hermaphrodites, and which should contain more males.
       6. Count 20 randomly-chosen gametophytes in drop 1 and drop 5. Score them as male
          or hermaphrodite (see pictures on the following page).

Adapted for classroom use by D. Maxwell in 2004 from information found at
       7. Perform χ2 analysis of your sex ratio data for each drop. Do they differ significantly
          from a 1:1 ratio?
                                8. Observe the gametophytes.
                                    You will notice that
                                    gametophytes are either wild
                                    type or polka-dotted. In the
                                    picture at right, you will see
                                    males that are wild type (left)
                                    and polka-dotted. In the
                                    picture on the left, you will see hermaphrodites that are
                                    wild type (top) and polka-dotted (bottom). In the pictures
                                    below, you see a close-up of wild type (left) and polka-
                                    dotted cells (right).

        9. Determine the phenotype of at least 20 randomly chosen gametophytes. Given that
            the sporophyte parent was heterozygous, predict an expected ratio of haploid
            gametophytes. Perform χ2 analysis of your data.
        10. Obtain sterile sperm release buffer (SRB) and a sterile pipette. Transfer enough SRB
            to the Petri plate to cover the surface with a thin film of water.
        11. Observe your plate under the microscope. You should be able to find motile sperm.
        12. When you are finished observing, replace your lid on the Petri plate. Do NOT seal
            with parafilm. Return your plate to the lights.
Day 11:
        Obtain your Petri plate from under the lights. Seal with parafilm and return to the lights.
Day 17:
        1. Obtain your Petri plate from under the lights. Remove the parafilm and observe the
            developing sporophytes under the microscope.
        2. Count 20 randomly chosen sporophytes.
            Score them as either wild type (on the near
            right) or polka-dotted (far right).
        3. Based on a 1:1 ratio of wild type and polka-
            dotted gametophytes, and knowing that polka-
            dotted is recessive, predict the expected
            phenotype ratio.
        4. Perform χ2 analysis of your data.
If you would like to grow your sporophytes to maturity, you will need to thin your Petri plate to a
single sporophyte. There are not enough nutrients in the media to support more than one plant.
On day 24, you may transfer your sporophyte from the petri plate to a pot of wet potting soil.

Adapted for classroom use by D. Maxwell in 2004 from information found at