By: Jennifer Harper and Valerie Friedmann

2. Perplexing Polka Dots A Study of C-fern Genetics By: Jennifer Harper and Valerie Friedmann

3. C-ferns are a hybrid of species found in Cuba and Nicaragua. The strain of DNA that causes the ferns to have a short life cycle was taken from one fern and introduced into the genes of the other fern by means of repeated cross breeding. The abbreviated name C-fern comes from Ceratopteris richardii. Found in tropical areas of the world. Semi-Aquatic C-Ferns

4. C-ferns • Exhibit both haploid and diploid phases.

5. Problem • The researchers wished to learn more about genetics and the way traits are passed from one generation to the next. • F1 generation polka dot C-ferns will be cross bred to create an F2 generation of plants. Wild Type Hermaphrodite C-fern

6. The growth from spore to gametophyte takes only 2 weeks. From fertilization, it takes another 2 weeks to produce a sporophyte. In 90 days, the fern is fully mature. C-Ferns

7. What is a polka dot C-fern? • The polka dot characteristic is a mutation that was induced using an EMS mutagen. • The chloroplasts clump together in the fern cells to create the look of polka dots. • When mature, polka dot C-ferns will appear more pale than wild type C-ferns, but will still grow and reproduce the same.

8. Polka Dot Wild Type

9. Hypothesis It is expected that, because the polka dot trait is believed to be recessive, the crossing of two mutant forms of polka dot C-fern plants will produce only polka dot offspring.

10. The first attempt at producing Polka dot C-ferns was made by using spores that had been subjected to an EMS mutagen. EMS causes many types of mutations in C-ferns, not only polka dots. Some other mutations are pale, lethal, dwarf, ruffled, and bubbles. The plates were sown and the gametophytes were searched through only to find that not enough polka dot C-ferns had been produced to continue the experiment and acquire accurate results. Procedure

12. Because the ferns sewn at UT could not be used in the project, they were used to provide the researchers with basic knowledge of C-fern maintenance. • Temperature control 28ºC • Medium Production solidification • Sterilization contamination prevention

13. After the first unsuccessful attempt to produce polka dot C-ferns, spores were purchased from Carolina Biological Supply. • These spores were an F1 hybrid of polka dot and wild type C-ferns in a 1:1 ratio.

14. Growth of the C-fern spores was accelerated using the “Dark Start” method. The spore vial was inoculated with 5 mL of distilled water and covered completely with aluminum foil.

15. Because hermaphrodites mature more rapidly than males, 32 plates were sown and labeled “male” and two days later 32 more plates were sown and labeled “female.”

16. Once gender was visible under the stereoscope, male ferns were removed from the hermaphrodite plates. Hermaphrodites were removed from the male plates. All wild type ferns were removed from both male and female plates. This was done to prevent unwanted fertilization from occurring. All ferns were counted and recorded for comparison.

17. C-ferns are a homosporous plant, meaning that a single spore can become male or hermaphrodite depending on climate and the pheromone, antheridiogen. Because Antheridiogen is secreted by developing gametophytes, it affects only the gametophytes that develop later. C-ferns grown in the presence of the Ace pheromone develop antheridia, which are the male reproductive organs.

18. Once the ferns were about two weeks old and had reached sexual maturity, sperm release buffer (SRB) was added to the male plates to induce the release of sperm in male C-ferns. Water can also be used to release sperm. This sperm was taken from the males using a sterile pipette and placed at the archegonia of the hermaphrodite ferns.

20. Archegonia, the female reproductive organs, contain one egg each that lies at the base of a small neck. The neck, which protrudes out from the surface of the gametophyte, consists of four rows of cells and neck canal cells in the middle. When archegonia are mature, water may be added to the ferns to cause the neck canal cells to burst open forming a small open canal leading to the egg. The contents of the neck canal cells are deposited at the neck as an archegonial discharge that attracts the sperm to the top of the open neck.

21. Egg Neck Canal

22. Archegonia

23. Once fertilized, the archegonia swell and by mitotic cell division, a diploid embryo is formed. • After one to two weeks, the embryo has developed into a sporophyte, identified by the rhizome, a short upright stem with roots and leaves. • Sporophytes produce an unlimited number of genotypically identical spores.

24. Results

25. Results

26. Because the F2 generation was a 1:2 ratio of polka dot to wild type C-ferns, it was believed that human error occurred and that wild type sperm was present during fertilization. With further research, it was discovered that the polka dot trait could be present on two different genes. This would mean that the Polka dot trait is not a simple trait. Conclusion

27. The hermaphrodites would be removed from their original plates and isolated in a new plate as soon as gender and phenotype is evident. This would prevent any wild type sperm from being present during fertilization. Temperature would be regulated in the growth pods before ferns were placed in them to ensure that maximum growth potential can be achieved. Researchers must be extremely patient in order to find the polka dot ferns amongst the wild type ferns. Research Improvements

28. Further studies are being done to increase knowledge of the location of the polka dot trait. Two types of polka dot ferns will be cross bred. One type of fern has more condensed chloroplasts than the other. If in crossing these two kinds of polka dot ferns we receive all polka dot ferns, we can conclude that the trait is on the same gene. If we receive wild type ferns, assuming that human error has not occurred, we can conclude that the trait is on at least two different genes. Further Studies

29. Acknowledgements • We would like to thank the Tennessee Junior Academy of Science for generously providing us with the finances needed to perform this experiment. • We would also like to express our gratitude to Dr. Les Hickock and Stephenie Baxter for mentoring us and for making the UT facilities available to us, as well as allowing us to use the pictures on his website. • Additionally, we would like to thank Mrs. Jan Coley for motivating us, providing us with constant guidance, and teaching us to dig deeper into the world of science.