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BIOL 153L General Biology II Lab Black Hills State University Lab 8: “Advanced Land Plants" Seed Plants: The "primitive land plants" described in last week's lab have vascular and reproductive systems that restrict them to reasonably wet locations. All bryophyte stages and pteridophyte gametophytes are non-vascular, and both groups have motile sperm that require water to swim. However, the development of complex vascular systems and reproductive processes that don't require water has allowed gymnosperms and angiosperms to survive in both wet and dry habitats on land. Seeds have given gymnosperms and angiosperms an added advantage. A seed is composed of a sporophyte embryo and a food source that are enclosed in a protective coating. The coating protects against harsh conditions—such as desiccation—and may allow the embryo to remain dormant until conditions are adequate for growth. Sometimes seeds remain viable for centuries! Seed plants follow the same life cycle as primitive land plants—sporophyte, spores (via meiosis), gametophyte, and gametes (via mitosis) are all present. However, for both gymnosperms and angiosperms, sporophytes are dominant and gametophytes are tiny. (The female gametophyte actually forms inside, and is supported by, the sporophyte.) Because of the reduced size of the gametophyte, life-cycles of gymnosperms and angiosperms superficially resembles that of animals. I. Gymnosperms Gymnosperms are a major group of vascular plants (see textbook, Chapter 18). Conifers are familiar members of this group, but it also includes Gingkoes (which resemble deciduous trees), Cycads (which resemble palm trees), Gnetophytes (including Ephedra, a locally common shrub in western North America) and other groups. Gymnosperms are characterized by “naked” seeds— they develop on the surface rather than being enclosed in an ovary. Conifers occur in temperate areas worldwide, with greatest diversity in the northern hemisphere. Western North America is especially rich in conifers. The world’s oldest trees (Bristlecone Pines), largest trees (Giant Sequoias), and tallest trees (Coastal Redwoods) are conifers found in California. Conifers are often adapted to fire: some have fire-resistant bark that enable them to survive fires, while others are in fact adapted to burn! Fire-adapted conifer species may not release their seeds from cones, or be able to germinate, until fire blazes through the environments where they live (textbook, Chapter 18, pp. 437-448). Conifers of local importance in western South Dakota include Ponderosa Pine (Pinus ponderosa, ubiquitous in the Black Hills ecoregion and the adjoining foothills), White Spruce (the Black Hills Spruce, Picea glauca var. densata, is endemic to our region), and Rocky Mt. Juniper. Pine life cycle In pines—and also spruces and firs—spores, gametophytes, and gametes are produced in male or female cones. Both cone types are generally found on the same tree in conifers (Chapter 18, Fig. 18-19, pp. 442-443 in textbook). Before lab, watch this video that provides an excellent overview of the pine life cycle. https://www.youtube.com/watch?v=WqGhmkYXcdM Page 1 The male cone (=microsporangiate cone) is small and flexible. Within it, microsporangia give rise to microspores via meiosis, and the microspores develop into microgametophyes (pollen). Germinating pollen produce nonmotile sperm and pollen tubes— the sperm are delivered to the egg through the pollen tube (Figs. 18-16 thru 18-18, 18-22). The female cone (=ovulate cone) is larger and woody. Each ovuliferous scale has two ovules. Each ovule contains a megasporangium (=nucellus) in which meiosis occurs to produce four megaspores (1x). Only one megaspore survives, and it grows into a megagametophyte (1x) that develops two archegonia; each archegonium produces an egg via mitosis (Figs. 18-20 thru 18-23). Pine pollen is transported by the wind, and if it lands on the scales of an ovulate cone, pollination drops may draw it through the micropyle. The pollen will germinate and the pollen tube will bring the nonmotile sperm to the egg. Pollination occurs when pollen is captured in the micropyle; fertilization occurs when the sperm fuses with the egg. In pines, pollination occurs ~15 months before fertilization, which means reproduction is a multi-year process! The fertilized egg develops into the embryo, which is enclosed within the seed. These seeds will appear as visible bumps on the ovuliferous scale and have wings that allow them to be dispersed by the wind. The conifer seed is complex and consists of both sporophytic and gametophytic tissue. The embryo (2x) is the baby sporophyte that will grow into a new plant. The seed coat (2x), which provides protection, originated from the parental sporophyte, and the food reserve (1x) is the remaining megagametophyte (Figs. 18-23 thru 18-24). 1. Take a quick look at the male (microsporangiate) and female (ovulate/megasporangiate) cones of spruce. For now, just be able to recognize the two cone types (Fig. 18-22, p. 845). You will look at these cones more thoroughly later in lab. Page 2 2. Go outside to examine the spruce (Picea) trees on either side of the entrance to Jonas Science. Note where the cones are located on the tree—you may need to stand back to view the entire tree and/or look at several trees. Locate both pollen-producing (microsporangiate) cones and ovule-producing (ovulate/megasporangiate) cones. Please do not remove cones from tree! Find a female cone on the ground and bring it back inside to your lab bench. Sketch a spruce tree that shows where male and female cones are located. Sketch a male and female cone as they grow on a branch. Needles are leaves. Lightly touch a spruce branch with needles. Are they sharp (could make you say “ouch!”) or are they soft? Look at areas of the branch that are missing needles. You should see stout pegs, or bumps, where the needles were attached. Draw a spruce branch that shows pegs and needles. 3. Return to classroom with your female cone. Leave it at your lab bench for a few minutes. 4. Examine the female cones. Note the diversity of sizes and shapes. Examine one of the large cones thoroughly. The ovuliferous scales are the most obvious scales that you can see. Ovules form on the top of ovuliferous scales, and sterile bracts form underneath. Sketch this cone. Identify an ovuliferous scale and sterile bract. Look to see if any seeds (or ovules) are present—if not, where would they would be? (Figs. 18-20 thru 18-21, p. 444). Page 3 5. Return to lab table, and examine the spruce cone you collected from outside. Feel free to remove ovuliferous scales to get a better look! Look for ovules or seeds and sterile bracts. Sketch the cone and an ovuliferous scale. Label the location of the ovules. a. What is the ploidy of the spruce cone (i.e., the ovuliferous scales)? __________________ b. How many ovules—and ultimately seeds—develop on each scale? __________________ 6. Take a male cone back to your lab table, and examine it under a dissecting scope. Be sure to put the cone in a petri plate rather than directly on the microscope stage! The main structures you see on the male cone are microsphorphylls; there are two microsporangia under each. Tear off a few microsporophylls and look at them under the dissecting scope. Sketch the entire male cone and label a microsporophyll and microsporangia. Sketch a magnified view of a microsporangia. 7. Inspect and then sketch a conifer seed. 8. Examine dried sample and fresh needles of pine (Pinus). Notice that the needles occur in bundles. These bundles are called fascicles—needles are attached at the bottom and will be shed together. Fascicles are characteristic of pines, and species can be identified by the number of needles in the bundle. Draw a branch of pine and a fascicle of pine needles. Page 4 9. Examine the dried samples and cones of juniper (Juniperus). Note the berry-like objects— these are the cones of juniper and are used to flavor gin. Multiple species of Juniperus occur in South Dakota and are on display here. Draw a branch of juniper with cones. 10. Although conifers are the most familiar of the gymnosperms, some gymnosperms lack needles and/or cones. Some of these other extant gymnosperms are described below. Ginkgoes (pp. 450-453) have broad fan-shaped leaves and fleshy (and stinky) yellow cones. Although once found worldwide, their natural range is now limited to China. In North America, they are commonly grown as attractive landscape trees—but in South Dakota they do not thrive in the harsh environment and unpredictable climate. There is one small (and rather unhappy!) Ginkgo biloba tree growing in Jorgensen Park in Spearfish. Cycads (pp. 448-451) have palmlike leaves and grow in tropical and subtropical areas. Some species are grown as large potted plants. Gnetophytes (pp. 453-455) consist of three genera--all of them are weird gymnosperms. Gnetum is a tropical plant with leathery broad leaves, Welwitschia, found only in the Namib Desert, has two straplike leaves, and Ephedra is an arid-land plant with very small leaves and jointed stems—it looks a lot like Equisetum (see lab 7). Ephedra is the only Gnetophyte found in the United States. Ephedra, which contains the alkaloid ephedrine (chemically similar to "speed") and many other medicinally active chemicals, has been used for thousands of years for allergies, bronchial issues, weight loss, and energy. Unfortunately, ephedrine can raise blood pressure and cause cardiac problems—many people have died from Ephedra supplements, including dozens of U.S. soldiers, a Minnesota Viking, and a Baltimore Oriole. These deaths were linked to concentrated extracts rather than teas/tinctures made from the whole plant. In 2006, the FDA banned ephedrine for use in dietary supplements in the U.S.; Ephedra extracts that do not contain ephedrine are still legal. The North American Ephedra species, “ Mormon Tea,” contain very low concentrations of ephedrine and are still consumed by many people; its safety is unknown. Watch this short video to learn a bit more about Mormon Tea: https://www.youtube.com/watch?v=QfHpHQ8nFM0 1. According to the video, what are medical conditions that have been treated by Ephedra? 2. How have the Chinese traditionally served, and used, Ephedra? 3. Why are the species of Ephedra in the U.S. generically called Mormon or Brigham Tea? Page 5 Examine dried samples of Ginkgo, Cycads, and Ephedra. Sketch and label them. PINE SLIDES 1. Examine prepared slide number 162 (Pine staminate cone l.s.). Calling these “staminate cones” is a misnomer given that they do not contain stamens, only pollen. The bract-like structures on the male, pollen-bearing cones are microsporophylls. Microsporangia grow on the bottom of microsporopylls. Microspores—which are the spores!—form via meiosis in the microsporangia. Microspores grow into pollen via mitosis. (Textbook, Chapter 18, Fig. 18-19, pp. 442-443). Draw a pine microsporangiate cone and label a microsporophyll, microsporangia, and pollen grains. (Microspores are not present.) (Textbook, Chapter 18, Fig. 18-17, p. 440). a. What is the ploidy level of the microsporophyll? __________________________ b. What is the ploidy level of the microsporangia? __________________________ c. What is the ploidy level of the microspore (= spore)? __________________________ d. Did the microspore form via meiosis or mitosis? __________________________ e. What is the ploidy level of pollen (= male gametophye)? __________________________ f. Did pollen form via meiosis or mitosis? __________________________ 2. Examine prepared slide number 231 (Pine pollen). Gymnosperm pollen is dispersed by wind, and has wings that look like Mickey Mouse ears to catch the breeze. Pollen grains are the male gametophyte and are called microgametophytes. A mature pollen grain will produce a sperm nucleus, which is the male gamete—these sperm do not swim. (Chapter 18, Fig. 18-18, p. 441). Sketch a pollen grain. Page 6 3. Examine prepared slide number 258 (Pine ovulate cone l.s.). The bract-like or scale-like structures on female cones are more complex than the male microsporophylls. Each ovuliferous scale bears two ovules on its upper surface and has a sterile bract beneath it. Each ovule has a megasporangium (=nucellus) that gives rise to one megaspore (=spore) via meiosis (four megaspores are made but only one survives). The megaspore grows into the megagametophyte via mitosis and develops the archegonia that produce the eggs via mitosis. Integuments surround the ovule, and the micropyle is an opening in the integument. (Chapter 18, Fig. 18-21, p. 444). Draw an ovuliferous scale and label the ovuliferous scale, sterile bract, ovule, integuments, and micropyle. What is the micropyle for? What do the integuments become? a. What is the ploidy level of the ovuliferous scale? _______________________ b. What is the ploidy level of the megaspore (= spore)? _______________________ c. Did the megaspore form via meiosis or mitosis? _______________________ d. What is the ploidy level of the megagametophye? _______________________ e. Did megagametophye form via meiosis or mitosis? _______________________ f. What is the ploidy level of the archegonia? _______________________ g. What is the ploidy level of the egg? _______________________ h. Did the egg form via meiosis or mitosis? _______________________ 4. Examine prepared slide number 235 (Pine Ovule l.s.). The available slides do not include the act of fertilization. These slides show the pair of egg cells, which are the female gametes, within the archegonia of a megagametophyte, on an ovuliferous scale. The megagametophyte is the female gametophyte. Draw and label an ovuliferous scale, megasporangium (=nucellus), megagametophyte, the two archegonia with a single egg cell each, and integument. Page 7 5. On p. 443 of textbook, find the image of ovule and seed. Sketch these images below, making notes that show how parts of the ovule are incorporated into the seed. Be sure to show how the seed contains diploid material from mom, diploid material from “baby,” and haploid material from mom. (Note, the textbook image does not show that the integument becomes the hard seed coat.) II. Angiosperms (flowering plants). Angiosperms have flowers and fruits, a distinctive life cycle, and have been the dominant phylum of land plants for over 100 million years (see textbook, Chapter 19). Angiosperms range from tiny duckweeds (1 mm across) to towering Eucalyptus trees (100 m tall) and are found in all but the most extreme environments. Almost all food plants are angiosperms. Some have recognizable flowers—sunflowers, peas and beans, apples. Some have obscure flowers—grasses such as corn and rice. Others are eaten before they flower—carrots, celery, broccoli, spinach. Most broadleaf trees and shrubs are angiosperms—maple, oak, birch, aspen, palm trees. The flower often consists of four modified leaves: sepals, petals, stamens (microsporophylls), and carpels (megasporophylls). There are many groups of flowering plants that have flowers that lack petals or sepals, such as the oaks and birches. Grasses have reduced and highly specialized flowers adapted to wind pollination: their flowers typically have only stamens and carpels. (In the case of the grasses, it is clear that their flowers represent a reduction in complexity from ancestors that had petals and sepals.) (Textbook, Chapter 19, pp. 460-465). Angiosperm sperm seeds are covered by fleshy or dry fruit. Their seeds develop within the ovary, and fruit includes the ovary wall. Gymnosperms are called “naked seeds” because their seeds develop directly on the cone scale rather than in an ovary—gymnosperm seeds are protected by a seed coat but not surrounded by fruit. Angiosperm life cycle The male reproductive structure of a flower is the stamen. It is composed of the narrow filament that is topped by the two-parted anther. Pollen forms in the anthers. The female reproductive structure is the pistil (composed of one or more carpels). The rounded base is the ovary; it contains one to several ovules that produce eggs. The lobed, and often sticky, top is the stigma (Fig. 19-6, p. 461). Pollination occurs when pollen lands on the stigma. The style is the narrow part that connects stigma to ovary. Pollen tubes grow down the style on their journey to the egg. In angiosperms, spores, gametophytes, and gametes are produced in flowers. The ovary contains one to several ovules in which the megagametophyte develops. The megagametophyte develops an embryo sac with 8 nuclei and 7 cells (i.e., one cell has two nuclei). One of those cells is the egg. Pollen, the male gametophyte, forms in the anthers. When pollen lands on the stigma, a pollen tube grows into the ovule. Each pollen grain produces two sperm. One pollen sperm fertilizes the egg, which will become the sporophytic embryo (2x). The other pollen sperm nuclei fuses with the 2-nuclei cell and becomes the endosperm (3x). The endosperm will be the food source for the developing plant, and it is what we consume when we eat grains. This process is called double fertilization. (Textbook, Chapter 19, Fig. 19-22, pp. 472-473). Page 8 Before lab, watch this video that provides an overview of the angiosperm life cycle and compare with the image below. https://www.youtube.com/watch?v=AykzPemLs7Q ALSTROMERIA FLOWER Terminology Floral whorls = sepals, petals, stamens, pistil(s) Sepals (collectively calyx): the outermost whorl; often green and leafy Petals (collectively corolla): the second whorl; often showy and brightly colored Stamens (collectively androecium): the male reproductive structure: filaments and anthers Pistil(s) (collectively gynoecium): the female reproductive structure: ovary, stigma, and style; may be referred to as carpel(s) Tepals: when the petals and sepals look the same Perfect flower: contains stamens and pistil(s) Imperfect flower: contains stamens or pistil(s), but not both Complete flower: contains all four floral whorls—sepals, petals, stamens, pistil(s) Incomplete flower: missing one or more of the four whorls—sepals, petals, stamens, and pistil(s) Page 9 Take an Alstroemeria flower, razor blade, and petri dish to your lab bench (one per group). As with many monocot flowers, the sepals and petals of Alstroemeria look alike, and they are therefore called tepals. Alstroemeria flowers are three-merous, meaning that flower parts are in multiples of three. (Some other angiosperm flowers are four-merous or five-merous.) Examine the Alstroemeria flower. Draw and label sepals, petals (or tepals), corolla, calyx, stamens, filaments, anthers, pistil(s), ovary, style, stigma, androecium, and gynoecium. The calyx and corolla can attach above or below the ovary. If the ovary is above the flower parts, the plant has a superior ovary. If the ovary is below the flower parts, the plant has an inferior ovary. Carefully pull the flower parts away from the ovary. Draw the ovary showing flower part attachment. Does this plant have a superior or inferior ovary? Dissect the Alstroemeria flower—remove and sort all the floral whorls. Count the number of sepals, petals, stamens, and pistils. Record those numbers below. No. of sepals = No. of petals = No. of stamens = No. of pistils = ______ ______ ______ ______ Is the Alstroemeria flower perfect or imperfect? Explain. Is the Alstroemeria flower complete or incomplete? Explain. Look at an anther with a dissecting scope or hand lens. How many lobes are present on each anther? Draw the anther below and indicate the number of lobes. Page 10 Look at the stigma with a dissecting scope or hand lens. Is the surface sticky or dry? How many stigmatic lobes are present? Draw the stigma below and indicate the number of lobes. Place the pistil in a petri dish and cut the ovary in cross section—not a longitudinal section! The ovary wall encloses everything; locules are the spaces within the ovary; ovules are the round balls; carpels are the “walls” within the ovary. Draw the ovary cross section. Label the ovary wall, locules, carpels, and ovules. Show where the ovules are attached. Take a Petri dish from the bench that has a longitudinal section of an ovary. Draw the ovary longitudinal section and label the parts that you see. Discard the Alstroemeria flower parts and return the Petri dish and razor blade. LILIUM SLIDES Note, the textbook does not have examples illustrating most of these slides. While viewing them, compare what you are seeing on the slide to what you saw on the Alstroemeria. 1. Examine prepared slide 108 (“LILIUM Flower Bud, c.s.”) which shows what a flower looks like scrunched up in the bud. Draw and label the sepals, petals, anthers, filaments, pollen grains, pistil, and ovules. Page 11 2. Examine prepared slide 201 (Lilium ovary), a cross section from farther down on the flower than slide #108. Each of the three little notches on the outside of the ovary wall represents part of the three carpels. The carpels are modified leaves that are curled in at their edges and have ovules attached along their margins. Draw and label the ovary wall, ovules, locules, and placentation (point of attachment of the ovules), and carpel walls. 3. Examine prepared slide number 207 (Lily anther pollen tetrads), which is a cross section from farther up on the Lilium flower than on slide number 108. (Fig. 19-14, p. 466). Draw and label the style, anthers, and pollen. Page 12