Plant Development & the Fern Life Cycle Using Ceratopteris Richardii

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 development (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 following 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 observed 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 containing 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 medium. 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 experimental purposes, optimal conditions 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. Dr. Leslie Hickok, Botany Depart- Additional materials needed for this ment, University of Tennessee, Knox- exercise include cotton-tipped applica- ville, TN 37996. Individuals adopting s f~~~~~~~~~~~~~~~~~~Itors (at most, one per student), inex- Ceratopterisin large enrollment courses pensive plastic sandwich bags (one are encouraged to generate and har- per culture dish), and 60 x 20 mm vest spores from plants grown in ter- m~~~~~~ plastic petri dishes. Cultures sown by raria or greenhouses. 8 students usually contain some fungal, bacterial or algal contamination. Since the culture medium lacks a carbon Downloaded from http://online.ucpress.edu/abt/article-pdf/57/7/438/47354/4450034.pdf by guest on 27 September 2021 source, contamination is usually rela- Laboratory Exercise Al, tively minor and its appearance is in- Week 1 tegrated into this exercise. For axenic cultures, spores may be Goal: To observe development of easily disinfected by surface steriliza- the gametophytic generation of a fern. tion for three minutes with a 1:5 solu- Observations will be made on spore tion of commercial bleach to water, germination, gametophyte ontogeny, followed by three washes with dis- sexual maturation, fertilization, and tilled water (Wame et al. 1986). During the development of the young sporo- the process, spores are maintained in a phyte. You should be able to relate all conical centrifuge tube. Solutions are of these events to the complete life removed by releasing air from a bulb cycle of a fem. connected to a Pasteur pipet while Background: The life cycle of land placing the pipet tip securely against plants is characterized by an alterna- the bottom of the tube. If the tip of the tion between two phases or genera- pipet is flat (pipets must be carefully tions that are morphologically distinct screened prior to the process) and and perform different functions. The rests snugly against the tube bottom, gametophyte generation is the sexual only liquid will be drawn into the phase of the life cycle and produces pipet when the bulb is released; spores gametes (egg and sperm), and houses will aggregate around the outside of and nourishes the embryo. Gameto- the pipet. This technique should be phytes may be extremely small as in practiced prior to spore culturing to seed plants or they may be the domi- determine the time required to draw nant phase of the life cycle as in bryo- solutions from the tube, and this time phytes. The fern gametophyte is quite it is free-living and indepen- Plate 2, Figures 8-10. Sporophyte de- should be included in the three- small, but more familiar velopment in Ceratopterisrichardii. minute sterilization period. dent from the large, The Ceratopteris exercise is an on- spore-producing fern plant. In vascu- such as fems and seed Figure 8. Two gametophytes or proth- going laboratory activity that requires lar plants, sporophyte is the domi- alli (P) with developing sporophytes observation, data collection and syn- plants, the nant (S). The sporophyte emerging from thesis over several weeks. Initial estab- generation. herbs, ferns and all the gametophyte on the right has a lishment of spore cultures and sub- Trees, vines, spores by meiosis primary (L) and a secondary leaf (L'). sequent observations are not time land plants produce division. This is different Note numerous rhizoids (arrows) at- consuming and, therefore, we inte- or reduction most animals, in which meiosis taching the prothalli to the agar me- grate this exercise with other topics from This means that dium. Bar = 2 mm. Figure 9. Numer- dealing with botany or developmental yields gametes. cells and that the ous young sporophytes with primary biology. Simple experimental treat- spores are haploid which spores, the leaves emerging from underlying pro- ments (e.g. spore density, tempera- plant produces must be diploid. After thalli. Prothalli (P) are visible on the ture or light conditions) may be incor- sporophyte, and they germi- left side of the micrograph. Bar = porated readily into this exercise. In spores are dispersed what do they grow? You will 5 mm. Figure 10. Mature plant (sporo- the first week, we present the overall nate, into this (if you do not phyte) with fertile leaves only. Spores goal of the exercise and provide suffi- answer question know the answer!) by first- are produced all along the inrolled cient background on the plant life cy- already of developing fern margins of the highly dissected leaves. cle. If sporophytes are available, it is hand observations culture A young, uncoiling fiddlehead (arrow) impressive to place a vial of spores spores. You will sow your own sharpest pow- is at the base of the plant. Pot diameter next to a mature plant. Enthusiasm of spores and use your to see how fern is 6 inches. can be generated about observing the ers of observation development of one from the other. spores develop.

440 THEAMERICAN BIOLOGY TEACHER, VOLUME 57, NO. 7, OCTOBER1995 Exercise:A spore is a single cell that germinatesunder the right conditions, Table 1. Modified Parker's Macronutrient/Thompson'sMacronutrient Basal grows by mitotic divisions, and differ- Medium Stock Solutions (Klekowski1969). entiates into a multicellularplant that produces gametes. Look at the vial of Stock Final fern spores on the front table. These Solution MediummL are spores of an unusual tropicalfern NutrientComponents gILa Stock/Lb called Ceratopterisor the water sprite; 1 NH4NO3 25 5 you may have seen this fern sold as an 2 K2HPO4 20 25 aquariumplant. How many spores do 3 MgSO4 7H20 10 12 you think are in this vial? Are these 4 CaCl2* 2H20 13 2 spores dry? If they are dry, how can 5 Micronutrients 10 they survive?What do you thinkwould MnSO4* H20 0.025 be necessaryfor them to germinate? CuSO4 5H20 0.037 Take a cotton-tipped applicatorand ZnSO4*7H20 0.052 carefullyput it into the spores so that H3BO3 0.186 some spores adhere to the cotton. Do (NH4)6Mo7024* 4 H20 0.0037 not take many spores! Since spores are 6 Chelated Iron Solutionc 10 very small, only a slight brown color- FeS04 * 7H20 2.78 Downloaded from http://online.ucpress.edu/abt/article-pdf/57/7/438/47354/4450034.pdf by guest on 27 September 2021 ation on the cotton end is plenty. Raise Disodium EDTA* 2H20 3.73 the lid of a petri dish containing the nutrient agar just enough to position a Prepare each stock solution by dissolving listed quantities of component(s) in the cotton tip over the agar and tap off distilled water brought to 1 L volume. Stock solutions will keep for over 6 months some of the spores. After distributing and should be stored at 4? C. the spores as sparsely and as evenly as b Add appropriate volume of each stock solution and bring to 1 L volume. possible, replace the petri dish lid. It is Transfernutrient solution to 2 L Erlenmeyer,add 10 g (1%w/v) agar (Bactoagar). important to work quickly while the Titratenutrient solution to pH 6.0 prior to autoclaving. lid is up so that you do not introduce c Dissolve each component separatelyinto c. 450 ml water. Heat EDTAsolution to many foreign airborne contaminants boiling, add hot EDTA solution to iron solution. Boil combined solutions for 1 into your culture. Write your initials hour. Cool completely, then bring to 1 L volume. and date on the lid and place the dish into a plastic sandwich bag. Sprinklea few of the spores that are next several laboratory sessions. You can identify the developing ferns When still adhering to the applicator on a you arrive for your lab period, because they will always be growing small drop of water on a microscope you should immediately check your out of spores. What is happening fern slide and place a cover slip over them. spore cultures! Make good notes when spores germinate?Have all the Observe this wet mount of spores un- and drawings of what you see. We will spores germinated?What could be the der the compound microscope at high give you additional instructions and reasons for germination of some magnification. Can you see any pat- questions to aid in your observations. spores and not others? Are there fun- terns on the spore walls? What varia- Week 2 gal, bacterialor algal contaminantson tions in the spore wall ornamentation the plate? Where do these contami- occur on different surfaces of the Yourfirst activitytoday should be to nants come from? spores? Draw a few spores showing observe the spores you sowed last Carefully lift the lid of the culture the wall pattern or ornamentation. week. Many spores should have ger- and remove a few germinated spores Observe the spores on your dish minated. First observe the size, color using a small spatula or forceps. Pre- through the dissecting microscope. and distributionof germinated spores pare a wet mount of the germinated Note and record the general size and through the lid of the dish with the spores and observe them under the color and distributionof spores on the dissecting microscope and compare compound microscope. Locate the dish. It is your job to observe carefully the appearance of your spores with original spore wall and note how what happens to these spores over the the observationsyou made last week. the developing gametophyte broke

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FERNLIFE CYCLE 441 through this wall. The minute plants from? When you see movement, in- (to KSR),and NRI Competitive Grant poking out of the spore wall should be crease the magnificationand observe Program/ USDA Grant 92-3700-7673 green. Locatethe rhizoids. What is the these small, flagellated gametes. Be (to LGH and TRW).We would like to function of rhizoids in a plant that is very patient-give it some time. The thank Cassandra A. Holloway and composed of only a few cells? Locate sperm cells can move very quicklyand Douglas L. Bernhardfor excellenttech- cells that contain chloroplasts. Draw are hard to examine unless they are nical assistance,and DarrellMoore and several of these young plants and label caught on something or slow down. Ann Rushing for comments on the the spore wall, rhizoids, and photo- Draw and describe the shape of the manuscript.Many of the micrographs synthetic cells with chloroplasts.What sperm cells and try to locate flagella. included in the paper were taken by differences are there, if any, among Once you have observed swimming undergraduatestudents. We would es- the young plants that you observe? sperm cells, locate the archegonia on pecially like to thank Michelle Treece, Replace your dish into the sandwich another gametophyte. If you are pa- StevenTester and BillHamilton for their bag and put it back in the culture area. tient and lucky, you will see the sperm contributions. cells swimming down the neck of the Week 3 archegonium on their way to fertilize the egg inside. How can the spermcells References As in the beginning of the last labo- find theirway to the egg cell?How long ratory, you should start today's lab by and how far can one sperm cell swim? Chasan, R. (1992). Ceratopteris:A observing your fern gametophyte cul- Before you put your dish back into model plant for the 90s. The Plant tures. As before, observe the fern its plastic bag, sprinklethe plants with Cell, 2, 113-115. Downloaded from http://online.ucpress.edu/abt/article-pdf/57/7/438/47354/4450034.pdf by guest on 27 September 2021 plants on the dish and record your distilled water from the squirt bottles. Goldberg, R.B. (1988). Plants: Novel observations. Identify the two mor- This water will allow the sperm cells to developmental processes. Science, phologically distinct kinds of gameto- swim easily to the eggs in the archego- 240, 1460-1467. phytes (prothalli, sing. prothallus). nia and fertilizethem. The sporophyte Hickok, L.G. (1985).Genetics of game- The larger mitten-shaped gameto- is the productof fertilizationand devel- tophyte development.Proceedings of phytes produce both male and female ops from the zygote inside the archego- the RoyalSociety Edinburgh, 86B, 21- sex organs. The male sex organs (an- nium. By next week, the next genera- 28. theridia, sing. antheridium)are found tion-the diploid sporophyte-will be Hickok, L.G., Warne, T.R. & Slocum, around the margins of the plant and very small but it should be visible. M.K. (1987). Ceratopterisrichardii: near the rhizoids at the lower part of Applications for experimentalplant the plant. The female sex organs Week 4 biology. AmericanJournal of Botany, (archegonia, sing. archegonium) are 74, 1304-1316. aggregated near the growing notch, As in the beginning of the last labo- Klekowski, E.J., Jr. (1969). Reproduc- usually on the underside of the plant. ratory,you should continue observing tive biology of the Pteridophyta.III. Among these larger gametophytes your fern gametophyte cultures. Gen- A study of the Blechnaceae.Botanical should be numerous small, exclusively tly remove a few of the mitten-shaped Journalof LinneanSociety, 62, 361-377. male plants. Each male gametophyte gametophytes from the dish and pre- Lloyd, R.M. (1973). Systematicsof the is covered with numerous antheridia pare a wet mount. Carefully observe genus Ceratopteris(Parkeriaceae). I. that give a "bubbly"appearance to the the region close to the growing notch. Sexual and vegetative reproduction plant. These entirely male prothalli If fertilization was successful last in Hawaiian Ceratopteristhalictroides. develop in response to the presence of week, you may be able to see bumps at AmericanFern Journal, 63, 12-18. a special chemical (antheridiogen) in the base of older archegonia!What do Lloyd, R.M. (1974). Systematicsof the the medium that is secreted by older you think these bumps will grow into? genus CeratopterisBrongn. (Parkeri- gametophytes. Can you think of any Where have these bumps come from? aceae). II. Taxonomy. Brittonia,26, ecological or genetic advantages for By next week, the young sporo- 139-160. such a condition in nature? phytes should be easily visible. Some Patrusky, B. (1991). Drosophilabotanica Make a wet mount that includes a sporophytes may be large enough to (the fruit fly of plant biology). Mo- few gametophytes of both types and transplant from the petri dish to a saic, 2(2), 32-42. observeyour slide under the compound terrarium containing potting soil. A Warne, T.R. & Lloyd, R.M. (1981). microscope.Draw one of each type of 2-literclear plastic beverage bottle can Inbreedingand homozygosity in the gametophyte. Indude and label the make a small, cheap terrarium.When fern Ceratopterispteridoides (Hooker) spore wall, rhizoids, photosynthetic you transfer the young sporophytes, Hieronymus (Parkeriaceae).Botani- cells, sex organs and growing notch. try not to take a lot of agar, and make cal Journalof the LinneanSociety, 83, What is differentabout these two types sure that you separate out individual 1-13. of gametophytes?How have the game- plants. If you watch your fern grow Warne, T.R., Walker, G.L. & Hickok, tophytes changed from last week? long enough (30 or more days), you L.G. (1986). A novel method for In addition, this week you should should find sporangiathat develop on surface-sterilizingand sowing fern see the male gametes in action-the the underside of specializedleaves. At spores. AmericanFern Journal,76, swimming sperm cells. Preparea new that point, you may wish to grow 187-188. wet mount of several of the older male spores produced in these sporangia Warne, T.R., Hickok, L.G. & Scott, gametophytes so that you can care- and begin another cycle of the life R.J. (1988). Characterization and fully observe their antheridia. Include history of Ceratopteris. genetic analysis of antheridiogen- one or two of the largermitten-shaped insensitive mutants in the fern gametophytes that have 1-3 archego- Acknowledgments Ceratopteris.Botanical Journal of the nia. Patiently observe the antheridia LinneanSociety, 96, 371-379. with the compound microscope. What This work was supported by NSF Williams, P.H. & Hill, C.B. (1986). happens to antheridia?What does wa- grants DEB 9207646,Research Experi- Rapid-cycling populations of Bras- ter do? Where do sperm cells come ences for UndergraduatesSupplement sica. Science,232, 1385-1389.

442 THEAMERICAN BIOLOGY TEACHER, VOWME 57, NO. 7, OCTOBER1995