How-To-Do-It Plant Development& the Fern Life Cycle Using Ceratopterisrichardii KarenS. Renzaglia Thomas R.Warne Leslie G. Hickok Plants can make importantcontribu- gametophytic phase of the life cycle. studies (Hickok 1985; Hickok et al. tions in demonstratingbasic biological In particular,phenomena such as ger- 1987). Ceratopterisis an ideal experi- Downloaded from http://online.ucpress.edu/abt/article-pdf/57/7/438/47354/4450034.pdf by guest on 27 September 2021 principles of development, genetics, mination, organogenesis, interactions mental organism because of several physiology and cytology because of between individuals, control of devel- unique features that distinguish it the ease with which they can be cul- opment, fertilization,and embryo de- from other ferns; these include a rapid tured, manipulated and observed. velopment can be investigated using life cycle, the ease of culture and ma- Presently, two flowering plant model very simple techniques. nipulation of both sporophytes and systems, Fast Plants (rapid cycling In this paper, we describe a labora- gametophytes, and the availabilityof a Brassica)and Arabidopsis,are used ex- tory exercise for high school students large variety of specific mutant lines tensively in research and teaching or undergraduatesin General Biology (Hickoket al. 1987). (Williams& Hill 1986; Goldberg 1988; using C. richardii.This exercise ex- Perhaps the most attractivefeature Patrusky 1991). However, the exclu- tends over four weeks, with potential of Ceratopterisis that it can complete its sive focus on angiosperm models con- for continued observation, and fo- life cycle from spore to spore in less tributesto a limited appreciationof the cuses on the development of sexually than 90 days, thus permitting semes- diversity of plant development, mor- mature gametophytes from single- ter use. Both gametophytes and sporo- phology and life cycles. In fact, many celled spores. We have successfully phytes are easily cultured on a simple aspects of plant biology are more used this inquiry-based exercise for mineral medium, and can be manipu- readily explored in less specialized three years in a one-semester course lated, bred and propagated asexually. plants such as ferns and bryophytes, with 25 laboratorysections, each with The entire sexual phase of the life cycle in which there is a striking alternation about 20-25 students. We also present may be completed under axenic (ster- of generations or life cycle phases de- techniques for culture and manipula- ile) conditions. In addition to culture fined by meiosis and syngamy. A tion of gametophyte development on an agar-solidifiedmedium, game- model system based on the homo- from spores that are applicable for tophytes can be grown by a variety of sporous fern, Ceratopterisrichardii has hands-on activities for students at all means, including suspension culture, received relatively widespread appli- levels. The guiding text poses many on soil or on sand (Warne & Lloyd cation in research but has not been questions that promote interaction 1981). exploited to its full potential as an with the instructor. In addition, this Under standard culture conditions, instructionaltool (Chasan 1992). exercise can be used to reinforce or spores (Figure 1) initially germinateat Like all ferns, C. richardiiis charac- integrate with other biological princi- 3-6 days following spore imbibition terized by an alternationbetween mor- ples, such as genetics (homozygosity, and maximum germination occurs by phologically and physiologically inde- heterozygosity), evolution (mating 6-10 days. Gametophytes (prothalli) pendent haploid gametophyte and systems, sexual reproduction)and de- reach sexual maturity in 8-20 days, diploid sporophyte phases which de- velopment (mitosis, meiosis, cellular depending on culture conditions (Fig- velop from single-celled spores and differentiation),depending on the dis- ures 24). At sexual maturity two ga- zygotes, respectively. The relative cretion of the instructor. metophytic types are evident, males structural simplicity of these gen- and cordates. Male gametophytes are erations allows for direct illustration Organism small, lack a defined meristematicre- of fundamental principles of plant gion and bear only male sex organs or growth, differentiationand population Ceratopterisis a fern genus with one antheridia (Figures 4 & 5). The large dynamics, especially in the sexual or tetraploid and three diploid taxa cordate gametophytes exhibit a char- (Lloyd 1974). Plants are found rooted acteristicwell-defined notch meristem in swampy areas or floating in open and eventually bear both female sex Karen S. Renzaglia is Professor of Biolog- water throughout the tropics and sub- organs (archegonia) and antheridia ical Sciences at East Tennessee State tropics, and commonly exhibit a (Figures3 & 6). Sexual dimorphism is University,Johnson City, TN 37614. Tho- weedy habit, e.g. in roadside ditches mediated by a species-specific phero- mas R. Wame is a Research Associate in and Hawaiian the Department of Botany and Lesie G. taro patches (Lloyd mone, antheridiogen, which is se- Hickok Ph.D., is Professor of Botany at 1973). A strain of the diploid C. richar- creted by gametophytes into the the Universityof Tennessee, Knoxville,TN dii, Hn-n, is well-characterizedand medium and causes sensitive gameto- 37996. has been used for a variety of genetic, phytes to become male. Sexual dimor- developmental and physiological phism occurs even among populations 438 THEAMERICAN BIOLOGY TEACHER, VOLUME 57, NO. 7, OCTOBER1995 of genetically identical gametophytes (Warneet al. 1988). Fertilizationof the egg by sperm is effected by water- induced release of motile sperm which swim to and down the archegonial neck (Figure 7). Within 3-7 days fol- lowing fertilization,swollen regions at the base of the archegonia manifest development of young embryos. Un- like in seed plants, sex organs, sperm, egg cells, developing embryos and the process of fertilization may be ob- served in whole mounts of Ceratopteris gametophytes without extensive effort in dissection. Sporophytes develop over several weeks, producing a het- eroblasticsequence of sterile and then fertile leaves (sporophylls) that bear A,~~~~~~~~~~ sporangia (Figures 8-10). Downloaded from http://online.ucpress.edu/abt/article-pdf/57/7/438/47354/4450034.pdf by guest on 27 September 2021 Preparation for Laboratory Exercise For this exercise, students sow spores and culture gametophytes in 60 x 20 mm plastic petri dishes con- taining an agar-solidified nutrient medium. The composition of stock solutions and the working nutrient medium (modified Parker/Thompson's culture medium, Klekowski 1969) is given in Table 1. Not all commercially available media are appropriate for fern culturing, but a half-strength modified Murashige and Skoog me- cordate gametophyte showing numerous/ arhgoi (AR. Br=5 Fgr .Hg dium with 1/2 x NH4NO3 and 1/2 x KNO3 (Sigma M8900) yields results comparable to the modified Parker/ Thompson medium. We usually pour mgifcto of~anaceoimwt-ueossemcls(P tepigt wmdw h about 100 petri dishes per liter of me- dium. Petri dishes can be poured on a nec an fetilze he gg.Bar= 1 ym clean bench without the use of a lam- inar flow hood. Culture dishes from each laboratory Mm~~ section are placed in a tray under grow lights (ideally four tubes, 4 inches apart at 20 cm from the cultures) at room temperature (ca. 25? C). For ex- perimental purposes, optimal condi- tions for gametophytic growth and development are a constant thermo- Plate 1, Figures 1-7. Gametophyte development in Ceratopteris richardii photoperiod of 28 ? 1? C and 80-85 Figure 1. Ungerminated spore showing wall ornamentation. Bar = 50 ALm.Figure 2. Young, ,umolesPAR per m2 per sec. However, developing gametophytes. The upper gametophyte is slightly younger than the lower gameto- can be successfully cul- phyte, which has begun to develop a notch meristem (M). Cell divisions in this meristem result in gametophytes a mitten-shaped or heart-shaped mature gametophyte such as that in Figure 3. Note rhizoids (R) tured under a wide variety of temper- at the base of the gametophyte near the ruptured spore walls (arrows). Bar = 50 ,um. Figutre ature and light regimens, including 3. Nearly mature cordate (hermaphrodite) gametophyte. Archegonia (AR) are visible near the ambient light conditions. Precise tim- notch meristem (M) and a few antheridia (AN) are developing along the wings. Rhizoids (R) are ing of development will depend on numerous around the base of the gametophyte, near the spore wall (arrow). Bar = 100 ,um. Figure environmental regimen, including 4. Male gametophyte of an age and magnification comparable to the hermaphrodite in Figure 3. type of culture medium, light inten- Growth of this form is limited to the production of numerous antheridia that cover the surface. Abundant rhizoids (R) are located at the base of the gametophyte near the spore watl (arrow). Bar sity, photoperiod and temperature. = 50 ,um. Figure S. Higher magnification of the surface of a male gametophyte similar to that in On the window sill or at room temper- Figure 4. Each antheridium contains 16 or 32 sperm cells (SP). The distinctly cofled sperm are atures below 240 C, spore germination mature and ready to be released. Bar = 50 ,am. Figure 6. Details of the notch meristem (M) of a and growth will be slowed consider- ably. A trial run under specific class- room conditions is recommended to determine timing of developmental FERNUFE CYCLE 439 events. Generally, at warm room For the following three weeks, we temperature and under constant light, prompt observations with questions spores germinate in one week and about specific aspects of development motile sperm are visible in three and other issues. Discussion between weeks. instructor and student may be simple, p1P Spores of Ceratopterisare relatively or more complex and detailed, de- easy to manipulate as they are atypi- pending on the background of the cally large (60-100 ,um diameter); there student. Thus, this exercise is applica- are about 1.25 x 106 spores per gram. ble at a variety of educational levels. Spores can be stored indefinitely at Spores are currently available from room temperature.