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Introduction to C-FERN®

Leslie G. Hickok, PhD and Thomas R. Warne, PhD
Department of Botany
University of Tennessee, Knoxville, TN 37996

Are plants alive? Do they do anything interesting? Ask these questions to any number of high school or college students and be prepared for a variety of answers! Even biology majors may give surprising responses. Teaching the basics of plant biology and capturing students' interest can be a daunting task. Nonetheless, plants are an integral and essential part of our living world, and learning about them is a necessary component of any liberal or specialized education; thus finding better ways to teach about plants is a challenge.

A Model Approach

Using model plant systems can be an effective way to develop compelling exercises and experiences for students. But for an organism to be useful in teaching plant biology, it must be interesting, simple to learn about and use, easy to grow and manipulate, and applicable to a broad range of disciplines and educational levels. C-Fern®, a specially derived cultivar of the tropical homosporous fern, Ceratopteris richardii, has all of these features and offers a dynamic new approach to teaching the basic aspects of plant biology.

A principal feature of C-Fern® is the rapid development of gametophytes over 2 to 3 weeks. Under optimum conditions, spores (Fig. 1) germinate 3 to 4 days from starting (DFS) the culture and develop to maturity by 10 to 12 DFS. The presence of 2 distinct sexes, males and hermaphrodites (Fig. 2), provides yet another interesting feature. Control of sexual differentiation depends on a chemical signal or pheromone. In the absence of the signal, early germinating gametophytes develop into hermaphrodites that synthesize and secrete the pheromone.

This then affects later germinating gametophytes that develop into males.

The presence of males with many sperm-containing antheridia (Fig. 3) allows students to visualize directly hundreds of swimming sperm that are released when water is added to the culture. Sperm are chemotactically attracted to eggs and form large frenetic swarms around mature archegonia on hermaphroditic gametophytes.

Students can view all phases of gametophyte growth and differentiation, fertilization, and early embryo development using a stereomicroscope and/or compound microscope. This demonstration of sex by a plant showing distinctive sexual types and actively swimming sperm that seek out and fertilize receptive eggs fascinates students. It provides a dynamic exposure to basic aspects of gametophyte development, sexual differentiation, pheromonal/chemical control of development, and plant sexual reproduction and alternation of generations.

Extended culture and observations of the sporophyte phase (Fig. 4) can also be carried out, culminating in the production of haploid spores to complete the life cycle.

Versatile and Easy

C-Fern® is both student and teacher friendly. Because they are free-living and autotrophic, both gametophytes and sporophytes can be cultured on a simple inorganic medium. Easy and foolproof sterile culture techniques are available for gametophyte cultures, and all stages can be conveniently cultured and manipulated with minimal training and equipment.

Getting started can be as easy as boiling water. Pour pre-made C-Fern® Basic Medium directly into sterile petri dishes after heating it in a boiling-water bath. C-Fern® spores are available in presterilized, premeasured units, and cultures can be inoculated by simply adding sterile water and dispensing the spores onto nutrient medium. After inoculation, the cultures are ready to go under the lights to initiate growth and development. Because the basic medium contains no carbon source (e.g., sugar), the likelihood of airborne contamination is greatly reduced.

Gametophyte cultures of C-Fern® are easy to maintain. Use a simple fluorescent lighting fixture (Fig. 5) to provide continuous illumination

By maintaining cultures in closed culture domes (plastic trays with clear covers), distance from the lighting fixture can be adjusted to achieve an optimum temperature within the culture dome of about 28º C (82º F). Culture domes also allow easy handling of a number of cultures, reduce the possibility of contamination, and buffer against changes in room temperature and humidity.

Population Studies and Large Data Sets

The rapid development and small size of gametophytes (0.5 mm to 3 mm at maturity) allows large populations to be cultured within small petri dishes (60 mm). Millions of gametophytes can be cultured in a very small space, allowing for the participation of large numbers of students.

Cultures of hundreds of gametophytes per petri dish allow students to work with populations as well as individuals.  This provides experience in sampling techniques and the statistical treatment of data.  Both quantitative and qualitative data sets (e.g., growth rate, germination rate, population sex ratio) can be obtained during the 2 to 3 weeks of culture in order to familiarize students with data acquisition, manipulation, and presentation. The importance of sample size can be demonstrated in exercises using both class and individual data. At the same time, it is possible to manipulate individual gametophytes so that controlled matings, both self and cross, are easy to accomplish.

Spores from a wide variety of stocks showing wild-type and mutant phenotypes are available. Mutant phenotypes range from striking visual types like polka dot to developmental mutants like maleless, dark germinator, and nonetiolated, to types resistant to environmental stresses from agents such as herbicides and salt. In addition to currently available stocks, additional visual and developmental mutants, including types showing altered sperm motility, are under development.

Class Kits and Independent Study

C-Fern® is a versatile teaching tool that can be used in both hands-on investigations and independent, student-initiated research. A number of separate C-Fern® kits have been developed for classroom use to highlight several types of investigations. Most kits are based on the rapid development of the gametophyte generation or include early sporophyte development so that the exercises can be completed within 2 to 4 weeks.

The rapid development and simple, inexpensive culture requirements also make C-Fern® an exceptionally useful organism for independent research projects. Students can use a variety of approaches to address many types of questions. The ability to work with large populations and to control experimental conditions enhances the ability to design and carry out meaningful projects.

Editor's Note:

The C-Fern® Manual and C-Fern® Laboratory Investigations have been developed jointly by Drs. Leslie G. Hickok and Thomas R. Warne with support from the National Science Foundation and The University of Tennessee. In addition, valuable advice and outstanding technical support were provided by Stephenie Baxter.

Further Resources

Banks, J. A. 1997. Sex determination in the fern Ceratopteris. Trends in Plant Science 2: 175.

Chasan, R. 1992. Ceratopteris: A model plant for the 90s. Plant Cell 4: 113.

Hickok, L. G., T. R. Warne, and R. Fribourg. 1995. The biology of the fern Ceratopteris and its use as a model system. International Journal of Plant Science 156: 332.

Raghavan, V. 1989. Developmental Biology of Fern Gametophytes. Cambridge University Press, NY.

Renzaglia, K. S., T. R. Warne, and L. G. Hickok. 1995. Plant development and the fern life cycle using Ceratopteris richardii. American Biology Teacher 57: 438.

Renzaglia, K. S. and T. R. Warne. 1995. Ceratopteris: An ideal model system for teaching plant biology. International Journal of Plant Science 156: 385.

Web Sites

The C-Fern® Web site: http://www.c-fern.org