"pictures of hermaphrodites"
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 http://cfern.bio.utk.edu/ 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 1 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 days. 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 microscope. 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 http://cfern.bio.utk.edu/ 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 http://cfern.bio.utk.edu/