C-Fern - A Model Plant for Teaching
Leslie G. Hickok, Professor of Botany, University of Tennessee

Ferns have been traditionally used to illustrate a unique plant life cycle and alternation of generations. However, their use in the biology curriculum has generally been restricted by the limited ability to provide students the opportunity to directly observe the sexual phase of the life cycle - the free-living haploid gametophyte. With the development of C-FernTM, this limitation no longer exists and many features of the C-Fern life cycle can now be integrated into the curriculum in ways that go well beyond traditional applications. Because of the ease of C-Fern culture and manipulation, a wide variety of fundamental biological principles can be readily observed and explored. With its large, single-celled haploid spores and their rapid and predictable germination and development into haploid gametophytes, C-Fern provides a wealth of developmental stages and events that can be directly observed and manipulated. Simple, inexpensive and dependable culture techniques using a basic mineral nutrient agar medium and Petri dishes provide an introduction to sterile culture techniques without the contamination difficulties commonly encountered in using enriched nutrient medium. Using a simple and compact culture container (i.e., the C-Fern Growth Pod), gametophytes can be grown to sexual maturity in less than two weeks and developing diploid sporophyte embryos can be observed in as little as three days following fertilization. Maintenance of sporophytes in a simple bottle terrarium for eight weeks or longer provides opportunities to observe vegetative growth and development, asexual reproduction from marginal leaf buds and, ultimately, the production of haploid spores via meiosis.

Gametophyte development within the first two weeks clearly illustrates basic aspects of tissue and organ differentiation in a two-dimensional, single-cell-thick plant that reaches 3 mm in length at maturity. This includes differentiation of a distinct meristem and sex organs. Pheromone-based control of sexual type among developing gametophytes results in the formation of distinct male and hermaphrodite gametophytes. Manipulation of culture conditions can directly affect population sex ratio and reveals how the environment can play a role in breeding strategies. The addition of plant stress agents or growth regulators (e.g., salt, herbicides, ABA) to the culture medium affects both growth and differentiation in the wild type strain, but a variety of resistant mutants show distinctly different responses. Male gametophytes with numerous antheridia provide a highly predictable and nearly limitless source of swimming sperm. Sperm can be seen easily at magnifications of 20X or higher, allowing students to observe directly key processes in sexual reproduction. In addition to observing the natural chemotactic behavior of sperm as they search for the egg, students can experiment directly with sperm suspensions by testing various chemicals for their ability to attract sperm. Students can even discover that sperm can readily differentiate between certain stereoisomers (e.g., L- and D-malic acid).

In genetics applications, the availability of a wide variety of distinct mutant phenotypes, along with the free-living haploid gametophyte and diploid sporophyte stages, provides many advantages. Students can observe and quantify segregation ratios in F1-produced spore populations, providing a direct illustration of the consequences of meiotic segregation and independent assortment. Analysis of ratios in gametophyte populations (equivalent to gametic ratios) allows hypothesis formation concerning the subsequent F2 sporophyte generation. After adding water to segregating populations of gametophytes, students can directly observe random fertilizations and, within 2-3 weeks, collect F2 data to test their hypotheses. Because each Petri dish culture contains hundreds of individuals, students analyze large populations, providing ample opportunities for the development of quantitative skills. Whether used in a traditional setting, in inquiry-based laboratories or for independent student research projects, C-Fern provides a simple, predictable and reliable organism with a wide variety of curricular applications.