Chapter 10 Laboratory Investigations With C-Fern™ (Ceratopteris richardii ) Leslie G. Hickok and Thomas R. Warne Department of Botany University of Tennessee Knoxville, TN 37996 (423) 974-2256; [email protected] Leslie Hickok received his B.S. in Biology from Murray State University, his M.S. in Botany from Ohio University, Athens, and his Ph.D. in Botany from the University of Massachusetts, Amherst. Dr. Hickok is a Professor at the University of Tennessee, Knoxville. He has studied extensively the biology and use of Ceratopteris in basic research ranging from cytotaxonomic to genetic, physiological and molecular genetic studies. Thomas Warne received his B.S. in Botany from the University of Georgia, Athens and his M.S. and Ph.D. in Botany from Ohio University, Athens. Dr. Warne is a Research Associate at the University of Tennessee, Knoxville and has a research background in the evolutionary and population biology of Ceratopteris. Jointly, Drs. Hickok and Warne have pursued basic research on the physiology and genetics of salt tolerance in plants using Ceratopteris and are currently developing educational materials for university-level and grade 7-12 biology courses using this unique organism. Reprinted From: Hickok, L. G. and T. R. Warne. 1998. Laboratory investigations with C-Fern™ (Ceratopteris richardii). Pages 143-176, in Tested studies for laboratory teaching, Volume 19 (S. J. Karcher, th Editor). Proceedings of the 19 Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 365 pages. - Copyright policy: http://www.zoo.utoronto.ca/able/volumes/copyright.htm Although the laboratory exercises in ABLE proceedings volumes have been tested and due consideration has been given to safety, individuals performing these exercises must assume all responsibility for risk. The Association for Biology Laboratory Education (ABLE)© disclaims any liability with regards to safety in connection with the use of the exercises in its proceedings volumes. © 1998 Leslie G. Hickok and Thomas R. Warne Association for Biology Laboratory Education (ABLE) ~ http://www.zoo.utoronto.ca/able 143 144 C-Fern™ Contents Introduction ....................................................................................................................................... 144 Overview of Investigations and Notes for the Instructor .................................................................. 147 Investigation 1. Plant Development and C-Fern Life Cycle............................................................ 150 Investigation 2. Quantitative Measurement -- Serial Dilution And The Mass Of An Individual C- Fern Spore........................................................................................................................................154 Investigation 3. Environmental Controls On Sex Determination - Gametophytic Density And Sex Expression in C-Fern ........................................................................................................................ 158 Investigation 4. Ecology in a dish - Environmental Controls On Sporophyte Development - Population Density And Growth of C-Fern Sporophytes................................................................. 162 Investigation 5. Genetics In Action! Visualizing Mendelian Inheritance Using C-Fern ................ 165 Acknowledgments............................................................................................................................. 171 Literature Cited ................................................................................................................................. 171 Appendix A. Surface Sterilization and Sowing of C-Fern Spores .................................................. 171 Appendix B. C-Fern Nutrient Medium - Composition And Preparation ........................................ 173 Appendix C. General Culture Instructions....................................................................................... 175 Appendix D. Sources of Supplies and Materials.............................................................................. 176 Introduction Laboratory-based education can serve many functions, such as, the reinforcement and illustration of lecture and text material and the exposure of students to the uses of sophisticated contemporary equipment and methodologies. However, an equally important and often neglected function is the use of the biological laboratory to promote general understanding of the scientific method. Laboratory-based education can teach students how the process of science works, what constitutes scientific information and how scientific information is acquired. In other words, this approach can teach students how to DO science. Comprehension of the scientific method involves understanding and applying a large set of interrelated skills -– problem identification, hypothesis formulation, experimental design, implementation, data collection, analysis and synthesis—that necessitate a different approach to teaching. However, normal constraints on time and equipment can make it difficult to implement such an approach. One option is the introduction of exercises in experimental science using amenable and flexible model organisms relevant to a broad range of disciplines and subject areas. In plants, only a few model systems have attributes that meet the demands of a wide range of situations and levels. Here, we describe a broadly useful model system, C-Fern, which is based on the homosporous fern, Ceratopteris richardii. C-Fern possesses a number of characteristics and developmental features that make it ideal for use in the classroom and in student-initiated research. Why Use C-Fern? The advantages of C-Fern as a model plant system derive from the unique properties of its development and life cycle (Chasan, 1992; Hickok et al., 1995; Hickok et al., 1997). Both haploid (gametophyte) and diploid (sporophyte) phases exist independently and, thus, experimental studies are possible at the cellular and whole plant level without artificial manipulations of the life cycle. Development from spores to sexually mature gametophytes to young sporophytes can be observed within a 2-3 week period and the complete, spore to spore, life cycle takes less than 12 weeks. The rapid growth and small size of gametophytes permit the ‘miniaturization’ of experiments, such that large numbers of individuals and treatments can be used in a small space and short time. The haploid / diploid genetic system is very simple. C-Fern combines features of both 'higher' and C-Fern™ 145 'lower' plant systems. The developmental simplicity and haploid status of the gametophytic phase provides opportunities that are not available in angiosperm models. At the same time, the ability to study any genotype or process within the complex vascular sporophyte phase allows direct comparisons with higher plant systems (e.g., Wisconsin Fast PlantsTM, Arabidopsis) that are not possible with developmentally simpler systems such as mosses (e.g., Funaria, Physcomitrella) or algae (e.g., Chlamydomonas, Euglena). We present here the protocols necessary to culture and use C-Fern™ for educational settings. Some investigations that capitalize on the unique attributes of this model plant system are given. Although these investigations are formatted in the ‘standard approach’, they are easily modified to a ‘student-guided’ or inquiry-based approach. What Is Ceratopteris? Ceratopteris is a genus of homosporous ferns found in most tropical and subtropical areas of the world (Lloyd, 1973). Species grow as either aquatics or sub-aquatics and are restricted in habitat to ponds, rivers or other intermittent wet areas such as ditches, rice paddies or taro patches. Although some require an aquatic habitat, most species can be successfully grown in standard greenhouse pot culture under warm, humid conditions. Currently, commercial applications are primarily limited to its use as an aquarium plant, where it is sold under the common name of 'water sprite' and has even been immortalized in plastic replicas. Although a number of homozygous diploid and tetraploid accessions of Ceratopteris are available, the Hn-n strain has been used in most experimental studies (Hickok, 1987; Hickok et al., 1995; Hickok et al., 1997). Hn-n is a diploid (n = 39) derived from a collection of C. richardii from Cuba (Killip 44595 GH). During the past several years, an improved diploid strain, RN, has been developed from a series of crosses and backcrosses. All of the exercises presented here are based on the use of the RN strain. The C-Fern Life Cycle (See Figure 10.I.1) Like all homosporous ferns, C-Fern has two independent, autotrophic phases: a developmentally simple haploid gametophyte and a vascular diploid sporophyte. The gametophytic phase, which develops mitotically after germination of the single-celled spore, can be cultured axenically on a simple inorganic nutrient medium. Development of this haploid phase is very rapid. Germination occurs three to four days following inoculation and full sexual maturity is attained within six days after germination. At maturity, the gametophyte consists of a small (2 mm), simple essentially two-dimensional (flat) thallus with rhizoids, vegetative cells and sexual organs (archegonia and antheridia). Archegonia (singular-archegonium) are female organs that contain one egg each that lies at the base of a small neck that sticks out from the surface of the gametophyte. The neck consists of four rows of cells and a few ‘central canal cells’