A model plant for a biology curriculum: Spider Flower (Cleome hasslerana L.).
Article Type: Report
Subject: Annuals (Plants) (Research)
Authors: Marquard, Robert D.
Steinback, Rebecca
Pub Date: 04/01/2009
Publication: Name: The American Biology Teacher Publisher: National Association of Biology Teachers Audience: Academic; Professional Format: Magazine/Journal Subject: Biological sciences; Education Copyright: COPYRIGHT 2009 National Association of Biology Teachers ISSN: 0002-7685
Issue: Date: April, 2009 Source Volume: 71 Source Issue: 4
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 246348874
Full Text: [ILLUSTRATION OMITTED]

* Rationale

Major advances in fundamental science are developed using model systems. Classic examples of model systems include Mendel's work with the common garden pea (Pisium sativa), classic inheritance work by Morgan with the fruit fly (Drosophila), developmental studies with the nematode (C. elegans), and transposable elements in maize (Zea maize). Other model species include mouse-ear cress (Arabidopsis thaliana), poplar trees (Populus spp), and mouse (Mus musculus). Each of these organisms has become a model system to study because it possesses attributes that allow for intimate study and manipulation, such as rapid generation cycles or a small genome size.

While these systems are not tractable for study in secondary education, the concept of a model species for study remains appealing. A model species for study in a biology curriculum provides a foundation and a constant for study and an appreciation of wholeness of an organism. To this end, there is an annual landscape plant that is easy to culture and can serve as a model species: Spider Flower or Cleome (Cleome hasslerana L.). Four teaching modules have been developed, with discussion questions and additional experiments where appropriate. The four major teaching modules center on: reproductive biology, inheritance of flower color, biochemistry, and seed germination/ experimental design. It is hoped that Cleome will provide a teaching platform for instructors of biology and a starting point for industrious students considering independent study.

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* Cleome as a Model Species

Cleome is a tough annual plant that produces perfect flowers over a long season. The plant is tractable with many physical attributes that make study quite easy. For example, the (typically) perfect flowers have large parts for ease of study. Anthers produce abundant pollen that can be readily collected and germinated. Plants quickly begin to flower in 10-12 weeks from seed; the fruit develops rapidly, and seeds mature in copious amounts about seven to eight weeks after flowers open. In fact, Cleome regularly reseeds itself and will voluntarily return to one's garden with little effort.

Flower color of Spider Flower ranges from white through red to violet, and three genes were identified that control coloration (Laddet al., 1984). True-breeding lines of Cleome have been developed that produce uniform flower color. Seeds of "White Queen," "Violet Queen," "Cherry Queen," and "Rose Queen" are readily available both from mail-order companies (e.g., Stokes Seed Inc., Buffalo, NY, and Pinetree Seed Company, New Gloucester, ME) and local display racks. As an alternative to growing plants from seed, plants can be bought from some garden centers in the spring.

* Basic Biology

Spider Flower (Cleome hasslerana L.) is an annual plant native to South America and is a member of the Capparidaceae family. The plant is a diploid, with 10 or perhaps 12 pairs of chromosomes (Darlington & Wylie, 1955). Plants are started from comma-shaped seeds, which are small (~13,000/oz) and black in color (Figure 1). Conditioned seeds readily germinate in three to five days and rapidly produce vigorous seedlings under favorable conditions. The central growing point of the plant and the lateral branches will eventually terminate in an inflorescence. This inflorescence is a collection of flowers that are produced in a whorl (Figure 2). The most immature flowers are at the center of the inflorescence, while more mature flowers are on the outside. New flowers open daily, and the inflorescence will continue to bloom for weeks.

Initially, flowers are perfect, and each has a complement of four petals, six stamens, and a pistil (Figure 3). As flowers open, mature, and senesce, fruit begins to develop below the growing stem that was formerly the inflorescence (Figure 4). Later, after considerable fruit set, newly opened flowers lack the ability to produce fruit. That is, the pistil becomes vestigial, while the petals and stamens remain normal. Seemingly, the production of subtending fruit signals the termination of pistil production in subsequent flowers. Why and how these developing fruit regulate future flower morphology is a question that students could ask, research, and speculate about the answer.

While lax horticultural practices are not encouraged, Cleome is not finicky and is quite tolerant of cultural errors. For example, plants that wilt from lack of water will rebound surprisingly well after watering. Plants that are grown in a confined pot (10-15 cm diameter) with a restricted root volume will produce a small and single-stemmed plant that is easy to manage. These plants will produce a terminal inflorescence of flowers with few, if any, lateral branches. At the other extreme, garden-grown or pot-grown plants (in 3-5 gallon pots) that are given ample space, water, and nutrition can become quite large (~ 1 m tall and 1 m wide!). It is recommended that plants be grown either in the garden or in 1-2 gallon pots.

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While many plant species are self-incompatible, Cleome is self-fertile. Fruits mature in six to eight weeks, and the long, slender silique will eventually split longitudinally (Figure 5). Seeds are black when fully mature, and about 50 are produced from a typical fruit (Figure 6). As with many plant species, Cleome seeds will not immediately germinate after harvesting and require a cold period. To promote germination, store seeds dry in a refrigerator for several weeks. Seeds will remain viable for at least two years and perhaps longer.

* Overall Objectives

** Build teacher familiarity with Cleome as a model teaching species.

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** The primary science objectives are embedded in aspects of floral anatomy, pollen germination, Mendelian genetics, plant breeding methods, extraction and elucidation of plant pigments, and experimental design.

** The primary objectives are expanded with questions and activities appropriate for independent student activity.

* Reproductive Biology

Lesson 1.1. Examination of Flower Anatomy

Cleome flower parts are showy, large, easy to manipulate, and ideal for instructional purposes to examine a perfect flower. While flower size varies based on plant culture, stamen length typically is 50-65 mm, including the slender anther, which is about 8-10 mm in length. The flower pistil is normally observed in two lengths. A fully functional pistil includes a 55-75 mm stalk with a 5-10 mm ovary, style, and stigma. The pistil terminates in an obvious globose stigma, which is sticky for receiving pollen. Nonfunctional (vestigial) pistils are also commonly found, and they are less than 20 mm in length. The four flower petals are each 35-45 mm in length and can be white, pink, red, lilac, or violet in color. At the base of the petals on a newly opened flower is a small, sticky droplet of nectar to attract pollenizers.

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Questions

1. Does Cleome produce perfect or imperfect flowers? Explain.

2. Are Cleome flowers insect- or wind-pollinated? Explain.

3. Do all flowers (from all plants) have petals? Explain.

4. Find five examples of plants that are insect-pollinated and five plants that are wind-pollinated.

5. What are the advantages of wind-pollination? Disadvantages?

6. What happens inside the ovary, and what does the ovary produce for the plant?

Lesson 1.2. Pollen Germination

Individual flowers are fully functional for only one day. That is, within a flower cluster, new flowers open daily, while one-day-old flowers lack pollen, and petals have already begun to wither. Ovaries, with stigmas that had been pollinated, begin to visibly swell in 24-48 hours post-pollination. An advantage of using this plant for teaching reproductive plant biology is that new flowers open daily, and ovary development and seed production are very rapid. In addition, the stamens are large with a long filament, which serves as a convenient handle. Pollen can be easily collected by wiping the opened anthers (pollen is visibly orange in color) into a small microfuge tube commonly used in biotechnology labs. Pollen can also be stored for several days (4[degrees]C), if necessary, and used as needed.

Surprisingly, pollen germinates like a seed germinates. Individual pollen grains germinate by absorbing water and pushing out a pollen tube from a pore or groove in the individual pollen grain. The function of the pollen is to provide the male nucleus during fertilization, and the tube is simply the conduit to deliver the nucleus to the egg cell located in the ovary. While natural pollen germination is an in vivo system, it also can be accomplished in vitro on a simple gelatin medium.

Medium Preparation

Pollen is best germinated on a semi-solid medium made from gelatin (Knox gelatin), simple household cane sugar, boric acid, and water. Pollen germination is often improved in slightly acidic medium (hence the boric acid), and boron and calcium in very small concentrations are helpful. The following medium has been made and successfully tested with Cleome pollen, with excellent results. Medium is made by combining gelatin (~3.0%), sucrose (10%), plus about 10 grains of boric acid per 50 ml of tap water. The medium is heated and stirred in a beaker on a combination hot-stir plate. The medium is heated to near the boiling point (95[degrees]C) and then carefully poured into small Petri dishes (50 x 9 mm diameter). Petri dishes are about half-filled with hot medium, which is allowed to cool and solidify. The medium can be refrigerated to promote solidification.

Hints & Precautions

* Do not boil the medium during preparation.

* Medium should be taken from the refrigerator and warmed to room temperature prior to use. Medium will keep for one to two weeks if refrigerated. One problem that may arise is if the medium is warmed significantly above room temperature; it then melts, and the pollen experiment will fail. The medium can be damaged if warmed either during transport in a vehicle or if left on a microscope stage with the light on longer than five to 10 minutes.

* Pollen can be streaked directly on the medium by holding the Cleome stamen by the slender filament and streaking the anther across the medium. When plating pollen, less is better, because clumped and heavy pollen density makes observations much more difficult. Also, one Petri dish can be "divided" by using an indelible marker on the bottom of the plate to create two, three, or four sections for students to plate their individual pollen.

* Camel hair brushes are frequently recommended for manipulating pollen. We find such brushes impractical and ineffective. Alternatively, the torn end of a paper match works well to move pollen in small quantities. This technique is suggested if it is necessary to move pollen from a storage container to the medium or stigma.

* Medium will become contaminated three to five days after pollen has been introduced onto the medium, and plates can be discarded or washed.

* Cleome produces six stamens per flower, which are ladened with pollen. Flowers should be collected early in the morning (7:30am-8:30am) to ensure that pollen has not been stripped by insects in the field. Flowers can be brought to the lab for later use, and the pedicels should be kept in water. A file card with a small hole punched in the center can be used to help support the flowers over a small beaker of water. Wet anthers (from dew) can be quickly dried with a hair dryer, if necessary.

* Cleome pollen germinates quickly. Within one hour of plating pollen, some grains will have germinated, and the tubes will have extended 1-3X the diameter of the pollen grains. After four hours, the pollen tubes are 3-5X; after 24 hrs, they are 5-10X; and after 48 hrs, tubes are 10-30X. Typical germinating Cleome pollen is shown in Figure 7.

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Questions

1. When pollen germinates, how long must tubes grow in vivo for Cleome? What about for corn?

Pollen tubes must grow from the stigma to the egg cells. Therefore, pollen tubes must grow several mm for Cleome but several cm for corn because the "silk" is the stigma.

2. What is the likely reason that boric acid was used in the medium?

It lowers the pH and also provides some boron ions that enhance germination.

3. Why was sugar added to the medium?

It provides an energy source for the germinating pollen.

4. What would happen if only 10 pollen grains landed on the stigma?

A maximum of 10 seeds would be produced. One pollen grain is needed for every seed produced. The instructor may expand this thinking into an eat of corn or a watermelon, where many seeds are produced.

5. What is the function of the pollen tube?

It serves as a conduit for male nuclei to travel.

6. What happens during fertilization?

In simple terms, egg and pollen nuclei fuse to form a diploid zygote, which divides to become the embryo that eventually becomes a part of the seed.

7. Did pollen density on the plate influence germination?

Normally, very, very low pollen density can result in lower germination rates.

Expansion of the Concepts: Testable Experiments

1. What variables could influence the percent germination of Cleome pollen?

Percent sugar, pH, temperature, addition of other ions such as calcium.

2. What happens when no pollen lands on the stigma of Cleome?

The stigma remains receptive for a longer period of time.

3. What happens when foreign pollen (a species other than Cleome) lands on a Cleome stigma?

Foreign pollen will not germinate or will grow erratically and never grow into the egg cells.

4. How long will Cleome pollen remain viable?

It depends on storage temperature and plant species. Frozen pollen of some species (at -20[degrees]C) can remain viable for five or more years. The longevity of Cleome pollen was not tested here but could be a student project. (Pollen can be stored in small gel caps, then placed in a Ziploc bag with desiccant and frozen at -20[degrees]C.)

* Inheritance of Flower Color in Cleome

Lesson 2.1. A Three-Gene System

Students need to be familiar with the basic parlance of genetics. That is, a basic understanding of genotype, phenotype, dominance, diploid, co-dominance, recessive, alleles, locus, and segregation of alleles is essential. Construction of a Punnett square is also very helpful and should be studied to better understand segregation of alleles.

Five different flower forms are possible in Cleome, including pink, rose, lilac, violet, and white (Figure 8). The inheritance of flower color in Cleome has been documented and was shown to be controlled by three genes (Ladd et al., 1984). In simple terms, one gene determines whether the flowers are white or pigmented. A second gene controls whether the plants are in one color class (pink/rose) or another (lilac/violet). The third gene controls whether pigmented flowers are dilute (pink or lilac) or intense (rose or violet). According to Ladd et al. (1984), each of the three genes has two possible alleles.

Let us first consider white or pigmented flowers. Using Ladd's nomenclature, the W allele is dominant to the w allele, and homozygous ww plants will produce white flowers. Plants with either the WW or Ww genotype will have pigmented flowers. The second gene controls the type of anthocyanin pigmentation. That is, the R allele is dominant to the r allele, and plants that carry an R will produce violet- or lilac-colored flowers. Plants that carry the homozygous rr condition will produce pink or red flowers. Lastly, a third gene controls the intensity of the pigmentation. Dilute flower color is dominant over intense pigmentation. Therefore, plants that carry the I allele will be more dilute (e.g., pink and lilac), while the recessive condition (ii) will produce intense flower color (e.g., red, violet).

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Determining the flower color from a given genotype requires a three-step process. First, consider whether the plant can produce pigment. If the plant genotype includes ww, the alleles carried by the other two genes are immaterial because the plant cannot produce petal pigments (anthocyanins). Plants that carry at least one W allele can produce anthocyanin pigments. The two pigment classes are the violet class (R allele present) and the red class (rr genotype). Finally, the third gene determines the quantity of pigment that is present, as described above. All possible genotypes and corresponding phenotypes are shown in Table 1.

Questions

1. Genetically, how are white flowering plants different? Explain.

2. Specifically, how could one determine the genotype of a red flowering plant?

Answer: Conduct a "test cross." Cross the red plant with a white plant and analyze the flower color of the progeny. There are two possible genotypes for red flowering plants, and their genotypes differ by the alleles carried for pigmentation production (Table 1). One type of red plant will produce all red flowering progeny. The second genotype for red will produce both red and white flowering progeny in an approximate ratio of 3:1.

Lesson 2.2. Hand-Pollinations To Produce an [F.sub.1] Generation

Hand-pollinations with this species are very easy. Anthers are subtended by a long filament that can serve as a handle. Flower pistols terminate with a stigma that is visible to the naked eye. Pollination is a simple matter of moving pollen from the desired male parent and placing it on the stigma of the desired female parent. In addition, Cleome is a self-fertile plant in that seed will be produced when pollen is applied to stigmas of the same plant (self-pollination).

While easy to pollinate, there are some challenges in doing this as a classroom assignment. First, bees are tenacious and will remove all pollen from the flowers before mid-morning An ideal situation would be if flowering plants were potted (1 gal) and could be moved into a classroom or other space where bees can be excluded. Another consideration is that pollinations should be made with virgin stigmas that have not been pollinated. Insects are an obvious concern, but wind can also buffet the plants and cause pollen to be transferred to virgin stigmas. It is recommended that plants be moved indoors and that flowers which will open in one day be emasculated and labeled. That is, flowers that are one day from opening have stamens that are enclosed within the rolled-up petals. Those virgin stigmas (encased in petals with unopened anthers) can remain unpollinated by cutting away the stamens (using fine scissors), and petals can also be removed to minimize interest by insects. These flowers that are prepared will then be ready for pollination the next day. The instructor must decide how this preparation can best be achieved

Pollen can also be collected from anthers that are bristling with pollen. Holding the filaments, the anthers can be wiped into a gel capsule or small microfuge tube, such as those used in DNA amplification for the polymerase chain reaction technique. To make effective pollinations using stored pollen, a torn paper match is best for moving collected pollen to a receptive stigma. If available, fresh pollen can also be used for pollinations.

Controlled crosses in breeding programs often use bags to exclude insects and promiscuous pollination. While isolation bags could be used, students will do well with a simple removal of stamens and petals. Student pollinations are best labeled with colored tape close to the stem but around the pedicel of the floater. Alternatively, small jewelry tags could also be used. We recommend that the students' initials be used, followed by a number that represents a cross number. For example, "RDM 2008.01" could be used as a cross number. The initials "RDM" would indicate who made the cross, "2008" indicates the year, and "01" is used to indicate the parental phenotypes. A notebook should be used to compile a record of all crosses that were made. Multiple pollinations can be made to represent a specific cross if sufficient flowers are available. In fact, there is always attrition in hand-pollinations, and duplicate pollinations are advantageous for obvious reasons. Because multiple plants of one color may be used, it would be advantageous if parent plants are uniquely numbered.

We recommend one to three plants per student group, depending on plant size. Flowers can be prepared the day before, either by the instructor or by students, and set aside for 24 hours. Pollinations can be done very quickly, and the instructor must plan not to emasculate all flowers that will open when pollinations are to be made, because pollen will clearly be needed, unless it has been stored (see above).

The example below could represent a line of information in a notebook corresponding to a specific pollination combination:

* Biochemistry of Cleome

Chromatography, in theory, is a simple method to separate compounds and then to detect those compounds. Paper chromatography (PC) begins by spotting a small sample on a strip of paper (Figure 9). Next, the paper is placed in a solvent that normally ascends the paper by capillarity, and the solvent travels up the paper. Chemical differences in the sample mixture cause compounds to rise at different rates and separate from each other. Hence, PC results in one or several colored spots on the paper. Improvements in chromatography beyond PC include thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC).

PC is very manageable in high school biology, while TLC is more demanding. HPLC is outside the reach of high school capacity without a connection to a well-equipped college or neighboring lab. Several references may help the teacher become more familiar with PC (Halborne, 1967) and TLC (Sherma & Fried, 1996).

Lesson 3.1. Separation of Leaf Pigments by Paper Chromatography

Leaves typically contain a mixture of pigments that are associated with photosynthesis, including two types of chlorophylls (chl-A and chl-B) plus some less-important carotenoids. A classic experiment is to use PC to separate chlorophyll types in spinach. Chlorophyll and carotenoid separation is quite easy and relatively fast when Cleome is used.

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Lesson 3.2. Separation of Floral Pigments by Paper Chromatography

Flower color is the most conspicuous attribute of most ornamental plants. Two major classes of plant pigments include the carotenoids and flavonoids. The carotenoids are typically yellow or orange and fat-soluble. The flavonoids are a complex group of compounds, and more than 2000 are thought to occur (Anderson & Francis, 1996). Tissue color associated with flavonoids typically includes pink, red, lavender, violet, and blue hues. Flavonoids are what give fruits such as strawberries, blueberries, cranberries, and eggplant their color. The chemistry of these plant pigments is complex and outside the scope of this article. However, it is valuable to know that the colors of these compounds are influenced by pH, co-factors present in the tissue, and molar concentration. Second, these compounds are three-ringed carbon structures with one or more sugars attached. The number and type of sugar will influence the resultant color.

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Lesson 3.3. Floral Pigments by Thin-Layer Chromatography

Thin-layer chromatography (TLC) is a refinement of PC and results in better (clearer) separation of the pigments. Typically, a small amount of sample is spotted onto commercially purchased plates (Figure 12). While plates are rather expensive, they have a long shelf life (three to six years). Suggested plates for this TLC work are silica gel 60 F254 (10 X 20 cm and 250 [micro]m) from EM Science, Gibbstown, NJ. The glass plates come as 10 X 20 cm plates. These can be cut with a glasscutter available at hardware stores, and 6 X 10 cm plates seem to work best, with a 600 ml beaker to hold the solvent.

Like PC, the pigment is extracted as described previously, the sample of interest is spotted onto the plate (Figure 12), and the plate is then placed in an appropriate solvent. The TLC work can be done in a simple 600 ml beaker with about 20 ml of solvent (Figure 13). While many solvents are possible, we suggest a solvent mixture of ethyl acetate: formic acid: acetic acid: water in a 100:11:11:27 ratio by volume. Chemicals are available from Flynn Scientific and Sigma Chemical. The flower pigments will be carried through the silica gel matrix on the plate, and separation should be quite good after about 45-90 minutes.

As a reminder and precaution, the spots should be placed about 1 cm from the bottom edge of the plate and must not be submerged into the solvent mixture when the TLC is started. The acidic solvent used in this technique should be handled with care, and the procedure is best done in a fume hood, if available. TLC plates need less starting pigment than PC, so do not overload the plate with pigment sample. If there is room on the plate to load more samples, try two series of spots. In the second series, spot on about a quarter of what was used initially. Overloaded samples will not resolve well. Also, the beaker and small TLC plates should be carefully covered in foil to keep the pigments dark and to minimize solvent loss while the pigments elute. Expected results are shown in Figure 14.

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Questions

1. What differences are there between red and violet flowers?

2. Did you note any qualitative differences between red and pink flowers?

3. How might flower pigments relate to genotype?

* Experimental Design & Seed Germination in Cleome

Lesson 4.1. Seed Germination

Science, by its nature, requires experimentation that itself requires a plan and a design to conduct the experiments. A correct design of an experiment allows the data to be analyzed and a meaningful conclusion to be drawn, while a flawed design is useless. An example of a flawed design is a new diet. Consider if 10 individuals were placed into Diet Group A and another 10 were placed into Diet Group B. After six months, if we simply determined the percentage of each diet group that lost weight, we would have a flawed design! One cannot do a statistical analysis comparing just one percentage to another. A valid design would include pairing up the individuals by sex, body mass, and lifestyle. Then, each of the pairs is randomly assigned to one of the diets. After six months, one could make a paired comparison, and a design could be statistically evaluated in a valid way.

Good experimental design usually has the following elements:

* Treatments must be assigned randomly, to individuals or groups.

* At least two groups or individuals must receive the same treatment to create replication in the experiment.

* When possible, similar groups (or blocks) are identified, and they are then assigned to specific treatments. There are college courses and textbooks on experimental design (Hoshmand, 1988).

* Finally, it is imperative that the experiment includes a control group that represents the status quo.

* In summary, replication, randomizing treatment assignments, blocking, and ensuring that one has a control are important considerations when setting up an experiment.

Freshly-collected Cleome seeds will not germinate in the fall. Why? Because, if those seeds germinated immediately, they would not have enough time before frost to mature another seed crop. Most seeds from plants in the temperate regions require a cold treatment before they will readily germinate. This cold treatment (or chilling requirement, which is necessary to break seed dormancy) is either received naturally or artificially. Seeds that over-winter in the soil receive a natural period of cold that helps break that dormancy. The breaking of dormancy is thought to be associated with a decline in the class of plant hormones called cytokinens.

Consider the following data collected from seed that had been chilled in moist paper towels for various lengths of time (Table 2). With no chilling treatment, less than 1% of the seed germinated. In contrast, more than 65% of the seed germinated when a moist chilling period of nine weeks was received (Table 2). Conditioned (chilled) seeds readily germinate in three to five days and rapidly produce vigorous seedlings (Figure 15).

Possible Discussion Lessons

1. Design an experiment to determine the chilling treatment required to germinate Cleome seed. Cold treatments are normally achieved by placing seed in a refrigerator (4 [degrees]C) for a number of days. The cold treatment can be conducted dry, or sometimes the cold treatment is better in damp paper towels. With this as an introduction, ask students how they would design an experiment with 300 Cleome seeds to determine the chilling requirement.

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Answer:

* You must first determine how many different treatments you want to test. Say you wanted to test five treatments: 0, 25, 50, 75, and 100 days of cold treatment.

* Next, we suggest at least four replicates of each treatment, which requires 20 tests.

* If you have 300 seeds, examine the seeds and eliminate any that look small or broken to ensure that all remaining seeds are high quality. Try to have 250 seeds for the experiment.

* Divide the seeds into five groups of 50 seeds each.

* We would plan the experiment so that all testing of seed germination begins on the same day. Look at the calendar and assume that you are going to start the germination part of the experiment on Feb. 1. Now, back up 100, 75, 50, and 25 days from Feb. 1 and mark those dates on the calendar. On those dates, 50 seeds are moved together in a small envelope into the refrigerator to begin the chilling of the seeds. Make sure the envelope is labeled!

* On Feb. 1, start the seeds. That is, for each envelope of 50 seeds, choose four groups of 10 seeds. Each group is plated between wet filter paper in a Petri dish. Discard the remaining seeds, since they were prepared in case of a spill or other problem.

* Repeat with the other packets, and don't forget the control, which is seed without any cold treatment.

* Keep the Petri dishes in close proximity at room temperature, and monitor for seed germination in two to seven days.

* Seeds are considered germinated if they have produced a root (radical) that has elongated to at least the diameter of the seed. Record data!

* An appropriate statistical analysis is needed to validate the treatment differences.

2. Some seeds require light to germinate. Why would the adaptive strategy be to require light to germinate? Does Cleome seed require light to germinate? Once you determine the chilling requirement of the seed (Activity #1), have the students think about how they would accomplish this experiment with 120 good-quality seeds.

Answer:

* Chill all 120 seeds to ensure good germination potential for the prescribed length of time.

* Divide the seeds into two groups of 60 seeds. The seeds are selected at random because they were already well mixed in the envelope.

* Place 10 seeds in moist filter paper in a Petri dish, as described above. Create five replicates that will remain in a lighted area, and repeat for the second treatment. These dishes will be wrapped in foil and kept near the other Petri dishes.

* Allow to remain untouched for seven days, and then determine the percent germination of the 10 dishes.

Additional Questions

1. What temperature will kill Cleome seed?

2. Is the chilling requirement enhanced when seeds are kept moist?

3. How long will seed remain viable at 4[degrees]C?

4. Does freezing seed (-20[degrees]C) reduce germination?

References

Anderson, O.M. & Francis, G.W. (1996). Natural pigments. In J. Sherma & B. Fried (Eds.), Handbook of Thin-Layer Chromatography. New York, NY: Marcel Dekker, Inc.

Darlington, C.D. & Wylie, A.P. (1955). Chromosome Atlas of Flowering Plants. London, UK: George Allen & Unwin, Ltd.

Halborne, J.B. (1967). The Comparative Biochemistry of Flavonoids. New York, NY: Academic Press.

Hoshmand, A.R. (1988). Statistical Methods for Agricultural Sciences. Portland, OR: Timber Press.

Ladd, D.L., Albrecht, Wewnes M. & Clayberg, C.D. (1984). Genetics of flower color in Spider flower. Journal of the American Society for Horticultural Science, 109, 759-61.

Sherma, J. & Fried, B. (1996). Handbook of Thin-Layer Chromatography. New York, NY: Marcel Dekker, Inc.

BIO

ROBERT D. MARQUARD (RMarquard@us.edu) is Middle School Science Chairman, University School, Shaker Heights, OH 44122. REBECCA STEINBACK is Academic Dean (retired), Andrews Osborne Academy (retired), Willoughby, OH 44094.
Materials

* binocular microscope

* student light microscope

* fresh Cleome flowers (collected
the morning of lab)

* expanding fruit (ovary) from
Cleome flowers that are two to
three days post-pollination

* prepared slides of a pollen mixture
(Carolina Biological)

* store-bought flowers (optional)

* microscope slides and cover
slips

* disposable pipets

* small beakers of water

* dissecting probes, scalpel

* Petri dishes (100 mm)

Activities

1. Macroscopically examine the
flowers to identify the flower
parts, including the petals, style,
stigma, ovary, anthers, and filaments.
Draw the flowers from a
macroscopic view. Does the flower
you observe have a functional
pistil or a short vestigial pistil that
is non-functional?

2. Compare Cleome to store-bought
flowers and identify
the differences.

3. Place flowers in half of a
Petri dish; examine the flowers
under a dissecting microscope
(20-40X). Identify
whether the stigma appears
wet and clumpy.

4. Look at the stigma. Is there
any visible pollen on the
stigma? Carefully remove a
stamen from the flower that
has pollen on the anther.
Wipe the stigma of the flower
against the anther and again
observe the stigma under a
dissecting microscope.

5. Draw all the flower parts
under low magnification.

6. Take an expanding ovary from
below the stigma that is three
to six days post-pollination and
carefully cut open the ovary longitudinally.
Observe the developing
embryos, and draw the
structure.

7. Place a small drop of water in
the center of a microscope slide.
Hold a stamen by the filament.
Dip the anther into the drop of
water to release the pollen. Apply
the cover slip and observe the
pollen under a light microscope
at 100-400X. Draw the pollen.
With a light microscope, estimate
the diameter of a pollen grain by
comparing it to a human head hair,
which has a diameter of about 100 [micro]m.


Materials

* light microscope

* combination hot-stir plate

* stir bar

* thermometer (optional)

* 50 X 9 mm Petri dishes

* Knox or other gelatin

* sucrose

* boric acid

* prepared microscope
slides of germinating
pollen

* fresh Cleome flowers
for pollen, or
stored pollen

* microfuge tube
or gel capsule
(optional)

* paper matches
(optional)

* dissecting forceps
(optional)

Activities

1. Students (in small groups) can make pollen medium, or
it can be prepared in advance by the teacher. The medium
will take at least 30 minutes to solidify after it is poured into
plates and is available for use in the next class.

2. Using the stamen's filament as a handle, lightly streak pollen
on the gelatin medium. Strive for a very light streaking
of pollen, because moderate or heavy pollen streaks will
make observations difficult. Check pollen 1-48 hours later
for germination.

3. Draw the germinated pollen.

4. Estimate percent germination and estimate the length of
the pollen grains. Normally, pollen tube length is estimated
as a multiple of the diameter of the pollen grain, so 2X, 10X,
or 15X are examples of that estimate.

5. Observe prepared slides of germinated pollen. Compare
those germinating pollen grains to Cleome pollen. What
are the obvious differences in pollen anatomy?


Materials

* potted and flowering
Cleome plants of each possible
color as an example for
the classroom (optional)

Activities

1. Paper exercise: Students are
asked to write all possible
genotypes for each phenotype (provided in Table 1).

2. Ask students to explain why some specific genotypes
have the corresponding phenotype. For example, why is
WWRrII lilac and Wwrrii red in color?

3. The teacher should prepare a set of crossing schemes
between various genotype combinations. Students should
work out the flower color and frequency of the progeny.
Four examples are provided below:

a. What are the possible phenotypes and frequency of the
progeny from a cross between Wwrrii X Wwrrii parents?

3:1, red:white

Also, what is the phenotype of the parents?

Both red

b. What are the possible phenotypes and frequency of the
progeny from a cross between wwrrii X wwRrii parents?

100% white

Also, what is the phenotype of the parents?

White and violet

c. What are the possible phenotypes and frequency of the
progeny from a cross between WWRrii X WWRrii parents?

3:1, violet:red

Also, what is the phenotype of the parents?

Violet and violet

d. What are the possible phenotypes and frequency of the
progeny from a cross between WwRrii X
WwRrii parents?

9:4:3, violet:white:red

Also, what is the phenotype of the
parents?

Violet and violet


Color      Date     Who   Female     Male       Number of
Cross No.   of tape                   parent     parent      crosses
                                                              made

2008.01       Red     July 29   RDM   Red #3    White #5        4

Materials

* colored tape and indelible marker

* dissecting scissors

* flowering Cleome plants

* notebook

Activities

1. Practice collecting pollen into gel capsules or microfuge
tubes.

2. Prepare the flowers for future pollinations. Remove petals
and emasculate the flowers.

3. Select one or several parent combinations from the population
of plants, and make pollinations. Label the pedicel
with tape for identification purposes.

a. From the parent combinations that you chose, what will
the progeny probably look like for flower color?

b. Don't forget to collect fruit/seed later in the season.

4. Controlled crosses and subsequent seed could be tested for
correct segregation by subsequent classes.


Materials

* roll of Whatmann chromatograph paper (2 cm wide)

* test tubes 25 X 200 mm

* test tube rack

* mortar and pestle (small)

* acetone, or fingernail polish remover as a substitute

* disposable pipets

* Cleome leaves

Activities

1. Separate leaf pigments of Cleome by
PC:

a. Use the mortar and pestle to grind a small amount of
leaf tissue (0.3-0.5 g) in about 3 ml of acetone. The solution
should be a dark green when finished.

b. With a disposable pipet, apply very small droplets of
the extract to a strip of chromatography paper that is
cut to a point (Figure 9). Let
dry and repeat with another
droplet until adequate pigment
has been applied. Repeat again
if necessary.

c. With pencil, label the top of
the paper with time, date, and
name.

d. Slightly crease the paper
lengthwise to create some
rigidity to the paper.

e. Place the pointed end of the
paper into the test tube that
contains 5 ml of 80% acetone.
Do not allow the spot to submerge
into the solvent. Rather,
let capillarity ascend through
the spot. Allow to stand for
about 30-40 minutes to separate
the pigments.

f. Examine the paper within 24
hours to see whether there are
leaf pigments that were separated
by PC. An example of the
results is shown in Figure 10.


Materials

* roll of Whatmann chromatograph paper
(2 cm wide)

* test tubes 25 X 200 mm

* test tube rack

* crucibles (as a small mortar)

* glass rods as pestle

* acidified methanol (1 drop HCl to 12 ml
methanol)

* water

* disposable pipets (or capillary tubes) for
spotting

* Cleome petals (violet and red)

Activities

1. Separate petal pigments of Cleome
by PC:

a. Use the rod and crucible to grind
(mash) a small amount of petal
tissue in 1.0 ml acidified MeOH
to extract petal pigments. With a
pipet, spot the extract onto chromatography
paper, and place the strip
into a solvent of a 1:1 isopropanol:
water solution. It will take 2-2.5
hours to separate the petal pigments.

b. Expected results: Red flowering
Cleome appear to have a single
flower pigment, while violet flowers
appear to have at least two
(Figure 11).


Materials

* acidified methanol
(1 drop HCl to 12
ml methanol)

* silica gel 60 TLC
plates

* solvent, 20 ml per
plate, of ethyl acetate:
formic acid:
acetic acid: water
(100:11:11:27)

* formic acid

* acetic acid (glacial)

* ethyl acetate

* 600 ml beakers

* glass rods and crucible

* pipets (or capillary tubes)

Activities

1. Separate floral pigments of Cleome
by PC:

a. Use the rod and crucible to
grind (mash) a small amount
of petal tissue in 1.0 ml acidified
MeOH to extract petal pigments.

b. Cut plates to the appropriate
size (6 X 10 cm). With a disposable
pipet, apply very small
droplets of the extract to a plate.
Let dry and repeat with another
droplet until adequate pigment
has been applied.

c. Place about 20 ml of solvent
(see above) in a 600 ml beaker.

d. Carefully place the spotted plate
in the beaker, making sure the
spots are not submerged in the
solvent.

e. Place in fume hood, if available, and allow the solvent
front to travel about 5-8 cm before the plate is removed
from the solvent (60-120 minutes). Place the plate on a
paper towel in the fume hood and allow to dry. Observe
plates within 36 hours, because the pigments will likely
fade after 36 hours.


Materials

* Cleome seed

* Petri dishes

* moist filter paper

* refrigerator


Table 1. All possible genotypes for each flower color in Cleome.

Phenotype (flower color)   Possible genotype of the plant

White                      wwRRII, wwRRIi, wwRRii
                           wwRrII, wwRrIi, wwrrii
                           wwrrII, wwrrIi, wwrrii

Pink                       WWrrII, WWrrIi
                           WWrrII, WWrrIi

Red                        WWrii, Wwrrii

Lilac                      WWRRII, WWRRIi, WWRrII, WWRrIi
                           WwRRII, WwRRIi, WwRRII, WwRrIi

Violet                     WWRRii, WWRrii, WwRRii, WwRrii

Table 2. Percent germination of Cleome seed
after chilling (6[degrees]C) in moist paper towels for
various lengths of time.

Chilling period (wks)   Germination (%)

0                             0.5
2                            2.30
3.50                          10
5                             48
9                             66
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