Tradescantia Flowers Are Ism in Which to Study the Meiotic Be Purchased, Students Can Collect Borne in Clusters (Inflorescences) Process

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Tadescanzia:A Tool for Teaching Mejosis

Robert L. Hammersmith Thomas R. Mertens

A NYONE teaching about re- some, the location of the centro- derwort. Plants of the genus Tra- production, cytology and mere, and the presence of descantia are useful teaching tools genetics in eukaryotic or- chromomeres in early prophase I of for any biology classroom. Stu- ganisms is faced with getting stu- meiosis. An organism such as the dents can prepare root tip squashes dents to grasp the intricacies of grasshopper can be collected to demonstrate the stages of mito- Downloaded from http://online.ucpress.edu/abt/article-pdf/59/5/300/48038/4450313.pdf by guest on 02 October 2021 chromosome behavior in the mei- readily in the field, and many sis. Protoplasmic streaming can be otic process. As teachers of genetics higher plants can be cultivated eas- observed in the cells of the trichomes we have always believed that under- ily in the greenhouse, thus making (hairs) on the stamens of the plant's standing meiosis was essential to these organisms potentially useful flowers. Somatic mutations can be grasping the principles of Mendelian for study. detected in a gene regulating flower inheritance (Mertens 1971). That Over the years a number of or- color (MacKinnon & Mericle 1986) if teaching meiosis is a challenge to ganisms have been advocated for plants heterozygous for blue/red many who teach biology is reflected the classroom laboratory study of flower color are subjected to radia- in the science education literature meiosis. The roundworm, ascaris, tion. Finally, for our purposes, Tra- (Stewart et al. 1990; Smith 1991; is an old standby (Mertens & Ham- descantiais an ideal plant in which Mertens & Walker 1992; Kindfield mersmith 1995) and prepared to study the meiotic process by 1994). The literature cited suggests slides of ascaris oogenesis can be squashing the anthers of young strategies for teaching meiosis and purchased from biological supply flower buds. for improving instruction so as to companies. Likewise, grasshopper Plants of this genus have rela- avoid student misconceptions spermatogenesis may be studied in tively large chromosomes, and about the process. the laboratory (Mertens & Ham- some species have low chromo- The literature also reflects the mersmith 1995). While prepared some number (e.g. 2n = 12). In ongoing search for an ideal organ- slides of grasshopper meiosis can addition, Tradescantia flowers are ism in which to study the meiotic be purchased, students can collect borne in clusters (inflorescences) process. Among the characteristics male grasshoppers, dissect out with the most mature florets near that make an organism ideal for the their testes, and prepare their own the apex and the least mature near study of meiosis are the following: slides using acetocarmine or aceto- the base of the inflorescence. This low chromosome number, large orcein staining techniques. Many arrangement enables the student to chromosome size, and ease of ob- angiosperms (flowering plants) progress along the inflorescence, taining or culturing the organism. have been used in classroom stud- selecting flower buds of various Using an organism having low ies of microsporogenesis and mei- sizes until a bud at an appropriate number and chromosome large otic chromosome behavior. Among stage for revealing meiotic events chromosome size makes it possible those discussed in the literature are has been found. Each bud has six to count chromosomes at various maize or Indian corn (Rhoades stamens, the anthers of which con- stages in the meiotic process and to 1950), Rhoeo spathacea or Moses-in- tain microsporocytes in approxi- discern details of chromosome the-basket (Satterfield & Mertens mately the same stage of meiosis. structure, including the two chro- 1972), lily (Flagg et al. 1973), Vicia Since one microscope slide can be matids of a replicated chromo- faba or English broad bean (Bem- prepared from one or two anthers, pong 1973), Ornithogalum virens or if a student finds a bud at some Robert L. Hammersmith is Associate star of Bethlehem (auhar & Storey ideal stage (e.g. diakinesis of Professor and Thomas R. Mertens is 1982), and chives (Robinson 1982). prophase I or anaphase I), other Distinguished ProfessorEmeritus in the In this paper we wish to tout students can use the remaining an- Department of Biology at Ball State University,Muncie, IN 47306. another angiosperm for the study thers from the same bud to pro- of meiosis: Tradescantiaspp. or spi- duce slides of the same stage.

300 THEAMERICAN BIOLOGY TEACHER, VOLUME 59, NO. 5, MAY 1997 In this paper we will suggest a and are preserved in Carnoy's So- decant and filter the stain. Once procedure for making slides of mi- lution, a 6:3:1 mixture of absolute prepared, acetocarmine may be crosporogenesis in Tradescantiaand ethanol, chloroform, and glacial stored for reasonablylong periods, demonstrate with photographs acetic acid. This mixture must be but if a precipitate forms, refilter. that Tradescantiais, indeed, an ideal prepared at the time it is to be Alternatively, acetocarmine solu- organism for classroom use in used; 50 volumes of Camoy's are tion may be purchasedalready pre- studying meiosis. used to one volume of plant mate- pared from most biological and rial to produce the best preserva- chemical supply houses.] The an- Materials& Methods tion of chromosomes, nuclei and thers are macerated in a drop or spindles. After they are in this so- two of stain using dissecting nee- Sources of Tradescantia lution for two or three days, trans- dles and, perhaps, a scalpel or ra- Where can one obtain Trades- fer them to 70%ethanol and store zor blade. The stain interacts with cantia plants? Both Carolina Biolog- them in the freezercompartment of iron in the instruments to stain ical Supply Company and Wards a refrigeratoruntil they are to be chromosomes and nuclei a dark Natural Science Establishment list used. Material killed and fixed in reddish purple. After applying a Tradescantia in their catalogs. An- this way can be used for years to clean coverslip, heat (never boil!)

other mail-order source is Spring come. the slide over the flame of an alco- Downloaded from http://online.ucpress.edu/abt/article-pdf/59/5/300/48038/4450313.pdf by guest on 02 October 2021 Hill Nursery (6523 North Galena hol lamp. Heating improves stain Dissecting & Staining Anthers Rd., P.O. Box 1758, Peoria, IL uptake by the chromosomes.Next, 61656-1758). The plants we use in An inflorescence is a cluster of invert the slide on a paper towel on our classroom laboratory have flower buds. In Tradescantiathe the tabletop and apply pressure been identified as Tradescantia most mature buds are near the on the slide with your thumb to ohiensis. Our greenhouse staff apex of the inflorescence and the squash the microsporocytes, mak- maintains six or eight pots of the least mature near its base, where it ing it possible to see and count plants for our use. We like this is attachedto the plant. In studying chromosomes in cells in appropri- species because it has a low chro- meiosis, we suggest that students ate meiotic stages. Locate cells mosome number (2n - 12) and remove a bud from the middle of using 10OX magnification and large chromosome size. Other spe- the inflorescenceand from the bud study them more carefully at 430X cies have similar, large chromo- dissect out one or two of its six magnification.Should the slides re- somes but may have a higher chro- anthers. Since the inflorescences veal no microsporocytesin meiosis mosome number. have been killed and fixed in Car- (e.g. mature pollen grains only), noy's solution and stored in 70% return to the same inflorescence, Collecting & Preserving ethanol, they are totally devoid of use a smaller Inflorescences bud, and repeat the chlorophyll and white in color. An procedure using anthers from this As the number of daylight hours inflorescence should be placed in bud. If your first slide revealed begins to increase noticeably in several drops of 70% ethanol in a immature microsporocytes (prior March of each year, we find that watch glass. Position the watch to meiosis), start again but use a our greenhouse-grown Tradescantia glass directly on the stage of a slightly larger bud. Any one bud begins to produce inflorescences dissecting microscope and place a will usually contain a limited num- (flower clusters). Since we wish to black card under the dish to pro- ber of meiotic stages in its anthers. harvest inflorescences for class use vide good contrast for the white By preparing slides from several before any of the buds mature and inflorescence and flower parts. adjacentbuds one may obtain ear- flowers actually bloom, we watch Now, using 5x to lOx magnifica- lier or later stages as desired. the plants closely and remove im- tion, remove a bud from the inflo- mature inflorescences as they be- rescence and dissect out the an- Making Slides Permanent come evident each week. Immature thers from the bud. The anthers of Acetocarmine squashes pro- inflorescences are required because Tradescantiawill resemble a pair of duced as we have described may we want to "capture" microsporo- attached kidneys borne on a very be made permanentusing the sim- cytes going through the meiotic short stalk. ple freezing technique of Conger process. In mature buds and Transferthe anthers to a drop or and Fairchild(1953). Place the slide blooming flowers, meiosis has been two of acetocarmine stain on a on a block of dry ice for a mini- completed and only mature pollen clean microscope slide. [We pre- mum of one minute. While it is still grains are present in the flowers' pare the stain by dissolving dry on the dry ice, use a razor blade to anthers. carmine in boiling 45% acetic acid pry the coverslip off the slide. The The young inflorescences are (ig carmine: lOOml 45%0acetic frozen stain and the microsporo- taken to the laboratory immedi- acid). Stir the carminein the acid to cytes remain on the slide which is ately after they have been picked facilitate dissolving and then cool, then immediately transferredto a

TEACHINGMEIOSIS 301 Downloaded from http://online.ucpress.edu/abt/article-pdf/59/5/300/48038/4450313.pdf by guest on 02 October 2021

Figure 1. Meiosis in Tradescantia:a. zygonema, b. late pachynema, early diplonema, c. diakinesis, d. metaphase I, e. metaphase I going into anaphase I, f. anaphase I, g. late anaphase I, h. late telophase I, i. early prophase II, j. late prophase II, k. metaphase II, 1. late anaphase II. Photographs by authors of acetocarmine squash preparations. (Plate appears as Figure 7.5 in Mertens & Hammersmith 1995, ?CPrentice Hall.) coplin jar containing absolute eth- lection, and its cells can be photo- and along their lengths one can anol. Leave the slide in the alcohol graphed as we did for the illustra- discern bead-like chromomeres. By for 2.5 minutes and then transfer it tions in this article. pachytene of prophase I (Figure lb), to a second jar of absolute alcohol the chromosomes are fully syn- for 2.5 minutes. Finally, remove the Findings apsed and appear to be consider- slide from the second jar. Before ably thicker. We find diakinesis (Fig- the alcohol dries, place a drop of Meiotic Stages ure lc) to be especially spectacular. euparol mounting medium (avail- We believe that Figure 1 pro- Six tetrads (pairs of chromosomes) able from Carolina Biological Sup- vides convincing evidence of the can be counted and coils within the ply Co.) over the cells and apply a quality of the slides that can be chromosomes can be observed. Di- clean coverslip. (Euparol is used produced of meiosis in Trades- akinesis is typically an ideal time at because it is miscible with absolute cantia. Cells undergoing the major which to do a chromosome count ethanol.) The slide is best put aside stages and substages of the two since the two chromosomes of a for several days to permit the eup- meiotic divisions can be identified. pair are synapsed, extremely thick arol to dry. It can then become a In early prophase I (Figure la), and short, and widely spaced permanent part of your slide col- the chromosomes are very slender about the cell.

302 THE AMERICAN BIOLOGY TEACHER,VOLUME 59, NO. 5, MAY 1997 Metaphase I (Figures ld and le) can also be quite revealing with the six tetrads aligned across the equa- tor of the spindle. Anaphase I (Fig- ures lf and 1g) are especially note- worthy: One can count six dyads (duplicated chromosomes) migrat- ing to each spindle pole. That each chromosome consists of two chromatids is clearly evident and the location of each chromosome's cen- tromere is apparent as the site where the two chromatids of a chromosome are joined. We never cease to be amazed at the quality of anaphase I cells in Tradescantia!

Students often cannot grasp the Downloaded from http://online.ucpress.edu/abt/article-pdf/59/5/300/48038/4450313.pdf by guest on 02 October 2021 necessity of the second meiotic division. Seeing that each chromosome at anaphase I still consists of Figure 2. First mitotic division in a haploid microspore: a. early prophase, b. (note replicated chromosomes), d. metaphase, e. two joined chromatids makes it mid-prophase, c. late prophase anaphase, f. two-nucleate pollen grain. Magnification is identical to that in Figure 1. clear that only at anaphase II will these two chromatids be separated. While meiosis II (Figures li-il) is a single haploid nucleus produced generative nucleus will undergo less spectacular than meiosis I in by the second meiotic division another mitotic division to produce Tradescantia,it is still quite reveal- (Figure 2a). As the pollen grain two sperm nuclei. Special tech- ing. For example, one can detect matures, however, this nucleus un- niques can be used to reveal this (by the orientation of the chromo- dergoes a mitotic division (karyo- mitotic division, but they are not somes) that the spindles formed in kinesis) to produce two haploid addressed in this paper. cells in meiosis II are at nuclei-the spheroid tube nucleus adjacent Chromosome Aberrations right angles to the spindle of the and a nucleus that will eventually parent cell in meiosis I (compare become the elongate, worm-like During observation of freshly Figures lf and 11). At anaphase II generative nucleus. This mitotic di- prepared slides, an occasional an- (Figure 11) note that the chromo- vision can reveal chromosome ther may be found in which vari- somes are, indeed, single stranded number and morphology. Since the ous chromosome aberrations oc- (i.e. consist of only one chromatid). haploid number (n = 6) is present, cur. Typical types of chromosome The final product of the second mei- a chromosome count can be made aberrations found include chromo- otic division is four haploid micro- easily- especially at late prophase some bridges, acentric fragments, spores, each of which becomes a and metaphase (Figures 2c and d) lagging chromosomes, micronu- functional pollen grain. The four when the chromosomes are highly clei, and various combinations of chromatids of each tetrad have been contracted and separated from one these aberrations, depending on distributed to one of the four micro- another. Late prophase is also a the stage(s) of meiosis in the an- spores produced from an original good time to study chromosome ther. More rarely, changes in mother cell. morphology, identify the two chro- ploidy may also be observed. matids of each chromosome, and Slides with these types of chromo- Post-Meiotic Mitosis determine the site of the centro- some alterations provide not only If a student looking for the mei- mere. Usually the later stages- excitement for the student who otic stages finds a slide full of mi- anaphase and telophase- of this finds them, but also a wealth of crospores or pollen grains, all is not post-meiotic mitosis can also be observational information and dis- lost! Such a slide may be used to located. The completion of this first cussion material for the whole demonstrate the first post-meiotic post-meiotic mitosis results in two class. Exceptionally good slides can mitosis (Figures 2a-f), thus en- morphologically distinct nuclei- be made permanent for demonstra- abling one to learn a great deal the generative nucleus and the tion purposes. about the chromosomes of Trade- tube nucleus (Figure 2f). Figures 3a, b and c are photomi- scantia and angiosperm reproduc- After the pollen grains are shed crographs of typical chromosome tion in general. and when they germinate on the aberrations that we have observed Initially the pollen grain contains stigma of a pollinated flower, the in Tradescantia. These and other

TEACHINGMEIOSIS 303 References Bempong, M.A. (1973). Meiosis in Vicia faba. Carolina Tips, 36(14), 53-54. Burnham, C.R. (1962). Discussions in Cytogenetics. Minneapolis, MN: Bur- gess Publishing Co. Conger, A.D. & Fairchild, L.M. (1953). A quick-freeze method for making smear slides permanent. Stain Tech- nology, 28, 281-283. Flagg, R.O., Register, T.E. & West, W.R. (1973). Lily life cycle 1. Pollen. Caro- lina Tips, 36(10), 37-40. Jauhar, P.P. & Storey, W.B. (1982). Or- nithogalum virens: A useful organism for teaching cytology. Journal of He- Figure 3. Typical chromo- redity, 73(3), 243-244. some aberrations found in Kindfield, A.C.H. (1994). Assessing un- Tradescantia:a. anaphase I derstanding of biological processes: Downloaded from http://online.ucpress.edu/abt/article-pdf/59/5/300/48038/4450313.pdf by guest on 02 October 2021 where 2n = 24, b. late telo- Elucidating students' models of mei- phase I with a lagging osis. The American Biology Teacher, chromosome, c. late telo- 56(6), 367-371. phase showing a chromo- MacKinnon, C. & Mericle, L.W. (1986). some bridge, fragments The spiderwort: Nature's radiation and a micronucleus. The detector. Laboratory Activity from magnification is identical Wards. Rochester, NY: Wards Natu- to that in Figure 1; note ral Science Establishment, Inc. however, cell size is much McClintock, B. (1938). The fusion of larger due to the increase broken ends of sister half-chroma- in ploidy. tids following chromatid breakage at meiotic anaphases. Missouri Agricul- tural Experiment Station Bulletin, 290. Mertens, T.R. (1971). On teaching mei- types of chromosome aberrations rial may form micronuclei in the osis and Mendelism. The American provide a basis for a discussion of resulting cells (Figure 3c). Double Biology Teacher,33(10), 430-431. Mertens, T.R. & Hammersmith, R.L. and mechanisms leading to crossover events can result in more events (1995). Genetics LaboratoryInvestiga- chromosome alterations. For exam- complex chromosome configura- tions, 10th ed. Englewood Cliffs, NJ: ple, a chromosome bridge ob- tions, including double chromo- Prentice-Hall. served during anaphase I can be some bridges and fragments and Mertens, T.R. & Walker, J.O. (1992). A the result of a single recombination chromosome bridges in anaphase paper-&-pencil strategy for teaching event in a cell heterozygous for a II. (See McClintock 1938; Burnham mitosis & meiosis, diagnosing learn- ing problems & predicting examina- paracentric inversion. In this case, a 1962; and Swanson et al. 1967 for a tion performance. The American Biol- crossover involving an inverted further discussion of cytogenetics ogy Teacher,54(8), 470-474. and a noninverted chromatid and mechanisms for generating Rhoades, M.M. (1950). Meiosis in would result in a dicentric chroma- chromosome aberrations.) maize. Journal of Heredity, 41(3), 58- tid and an acentric fragment. Sep- More rarely, we have observed 67. A.D. meio- aration of the two centromeres of a changes in the ploidy of Trades- Robinson, (1982). Teaching sis with chives. Journal of Heredity, dicentric chromatid to opposite cantia, with the chromosome num- 73(5), 379-380. poles would result in a chromo- ber changing from n = 6 to n = 12 Satterfield, S.K. & Mertens, T.R. (1972). some bridge. An acentric fragment, (Figure 3a). Polyploid cells are Rhoeo spathacea: A tool for teaching on the other hand, would be ex- probably due to endomitosis in a meiosis and mitosis. Journal of Hered- pected to remain around the equa- cell or cells that ultimately form ity, 63(6), 375-378. Smith, M.U. (1991). Teaching cell divi- torial plate of the cell. Eventually, microspore mother cells. Chromo- sion: Student difficulties and teach- the bridge should break because of somes in these cells can behave ing recommendations. Journal of Col- the strain of the anaphase chromo- normally in meiosis with the ex- lege Science Teaching, 21(1), 28-33. some movement or be cut by cyto- ception that different combinations Stewart, J., Hafner, B. & Dale, M. kinesis, although occasionally the of multivalent and bivalent pairs (1990). Students' alternate views of The American bridge material may be suspended may occur. If polyploid cells are meiosis. Biology Teacher,52(4), 228-232. between two polar telophase nuclei observed, they can be used to in- Swanson, C.P., Merz, T. & Young, W.J. (Figure 3c). Large acentric frag- troduce the concepts of au- (1967). Cytogenetics. Englewood ments and suspended bridge mate- topolyploidy and allopolyploidy. Cliffs, NJ: Prentice-Hall.

304 THEAMERICAN BIOLOGY TEACHER, VOLUME 59, NO. 5, MAY 1997