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BSCI 442, 2011 Plant Physiol Lab. 1 Expt. 7. Sexual Plant Reproduction: Testing conditions required for pollen germination and tube growth ____________________________________________________________________________ File: pollen Modified from E. Moctezuma & others for BSCI 442 (Sze) Objectives: Get familiar with the parts of flowers: male and female organs Observe pollen germination and tube growth in real time. Test the conditions that stimulate pollen germination and tube growth. Observe cytoplasmic streaming (due to vesicle traffic). Introduction Flowering plants, or angiosperms, are the largest and most successful plant group in the world. From giant eucalyptus trees to minuscule duckweeds, from desert cacti to water lilies, these familiar plants dominate the world. They play a significant role in providing food and shelter for animals and other organisms. There are over 250,000 species of flowering plants and they are all vascular seed plants that have flowers and fruits. Angiosperms derive their name from the Greek words angion (vessel) and sperma (seed). The flower contains the vessel that houses the seed. It is also the organ for sexual reproduction. A typical flower is composed of modified leaves arranged in four whorls (circles) on the end of an enlarged portion of the stem called the receptacle. The calyx is the outer (and lowest) whorl of floral parts and often functions to enclose and protect the flower bud before it opens. Individual members of the calyx, usually green, are called sepals. Moving inward, the second whorl of floral parts is the corolla. Individual members of the corolla are called petals, which are often conspicuously colored to attract insects or birds that serve as pollinators. Plants that are wind-pollinated or that selfpollinated do not have colorful petals, such as the model plant Arabidopsis. Fig. 1. Arabidopsis plant and flower (Taiz Fig 16.1) Sepals and petals are considered the sterile parts of a flower because they are not directly involved in meiosis and sexual reproduction. The third whorl consists of the male portion of the flower, the stamens. Each stamen is composed of two parts, an anther and a filament. The anther is a four-chambered structure where pollen is produced. The male gametes are contained within pollen. The filament is the threadlike stalk that supports the anther. The female part of the flower contains one or more pistils (also referred to as carpels) and occupies the innermost whorl. Each pistil is composed of three parts: a stigma, the receptive tip where the pollen lands; the style, which is BSCI 442, 2011 Plant Physiol Lab. 2 an elongated tubular structure through which the pollen tube grows after pollen germination; and at the base is the ovary. Arabidopsis had multiple ovules within two fused carpels (Fig. 1). The production of flowers and seeds within fruits are two evolutionary advancements that only flowering plants possess. A number of other advancements can be summarized as part of their overall adaptability. That is, flowering plants have evolved numerous adaptations in structure and function in order to survive in the many different environments found on earth. This adaptability can be demonstrated in their form and structure, their physiology, and their biochemistry. Alternation of Generations in Flowering Plants Flowering plants reproduce sexually and have an alternation of generations. The life cycle of flowering plants involves the alternation of a diploid sporophyte stage and haploid gametophyte stage. The haploid gametophyte generation in flowering plants is very reduced and is enclosed within and dependent on the sporophyte. Flowering plants, like the gymnosperms, are heterosporous (produce two kinds of spores). The two types of spores are called microspores and megaspores. Fig. 2 Life cycle of plants. Plants have an alternation of generations: the diploid sporeproducing plant (sporophyte) alternates with the haploid gamete-producing plant (gametophyte). Pollen, the male gametophyte, has 3 cells. The female gametophyte has 8 cells. Animal life cycles have meiosis followed immediately by gametogenesis. (Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman) Pollen Development The diploid male sex organ of the flower, the anther, encloses four microsporangia (Figure 2). Within the microsporangia are microsporocytes, or microspore mother cells. Each one of these cells undergoes meiosis to produce haploid microspores. The haploid microspores grow and mature into haploid male gametophytes, known as pollen grains, which are released and travel to the female. The pollen houses the sperm gamete. Embryo Sac Development The female ovary contains the diploid female sex organs of the flower called the ovules. Specific cells in the ovules undergo meiosis to produce haploid megaspores. One of these megaspores grows and develops into the female gametophyte or embryo sac within which develops an egg cell. Angiosperm Pollination Pollination, the transfer of pollen from the male anther to the female stigma, must occur before seed plants can reproduce sexually. Pollen grains have a limited life span. Depending on the species, pollen may be viable for only a few hours up to several weeks after it is released from the anther. Many plants, including gymnosperms and some flowering plants, rely on wind to aid in pollination. However, most flowering plants use animal agents to transfer pollen. BSCI 442, 2011 Plant Physiol Lab. 3 A primary function of flowers is to attract pollinators with colorful petals, scent, nectar, and pollen. In exchange for moving their pollen around, flowers have evolved many rewards to attract pollinators. The most common attraction is food, either nectar (a sugar solution) or pollen, which is high in protein. In many plants, nectar is produced in special glands called nectaries. Nectaries are most commonly found in the flowers, but can be located on leaves or other parts of the plant (extra-floral nectaries). Regardless of where nectar is produced, it is usually protected from evaporation into the atmosphere and from dilution by rainwater. The nectar is also protected from being robbed by other animals that do not aid in pollination. Fertilization The pollen grain germinates when it lands on the stigma. Once the pollen grain is on the stigma, fertilization can begin. The sperm in the pollen grain must get to the egg, located at the base of the pistil in the ovary. When the pollen grain germinates, its tube nucleus grows a tube from the stigma, through the style, and down to the ovary. The two sperm from the pollen travel down this tube to the ovary. One sperm fertilizes the egg in the embryo sac to produce a diploid zygote, which grows into an embryo enclosed in a seed. The other sperm fuses with the two polar nuclei to form the 3n endosperm that provides food for the developing embryo. This event is called double fertilization and is unique to flowering plants. Thus, the seeds are the mature ovules that contain the embryos and the ovary develops into the fruit, a structure for seed dispersal. When the seed germinates in the soil, the young embryo uses the energy stored in the cotyledons to start growth. One of the reasons that flowering plants are so successful today is that their seeds and fruits are adapted for dispersal in many different ways. Materials Spiderwort (Tradescantia) – are not flowering yet Pansies Snapdragon Alyssum Part A. Observe the structure of the flowers 1. Obtain two different flowers per group. With each flower and examine its structure with the dissecting microscope 2. Identify and count the number of sepals, petals, stamens, and pistils. Use a dissecting microscope to help you see stamens and pistils. 3. Now, use tweezers to remove the sepals and petals. Look at the stamen and pistil using the dissecting microscope. Draw and label the stamen, anther, filament, pistil, stigma, style, and ovary. PART B. POLLEN GERMINATION Successful fertilization results if the pollen grains that are sticking to the stigma germinate. This results in the production of a pollen tube that grows through the style into the ovary and down to the ovule. The male gamete will travel down this tube to fertilize an egg. For pollen germination to take place, several requirements must be met. Pollen grains are able to germinate and to extend a tube in the absence of the female organ, if a suitable nutrient media is provided. Here you will test and determine which are the required nutrients, and propose why. BSCI 442, 2011 Plant Physiol Lab. 4 We will use a pollination solution containing sucrose. Boric acid and calcium nitrate have been added to the solution to speed up the germination. Day 1: Make several solutions to test the effect of various nutrients. Discuss what purpose each nutrient serves. Complete medium for pollen germination and tube growth in vitro Medium content sucrose Boric Acid(H3BO3) Calcium Nitrate.4H2O MW 342 61.83 236.15 amount 20 g 0.02 g 0.06 g % 10% 0.01% 0.03 KCl Conc 294 mM 1.5 mM 1.28 mM 1 mM 200 ml DI water These solutions lack one nutrient. Absent nutrient a. No suc b. No NO3 c. No Ca d. No B Substitute MW Mannitol (sugar substitute) 182 Amt(gm)/ 200 ml ? Ca chloride KCl (same) KNO3 KCl (same) No substitute 111 (anhy) ? 101.1 Final Conc 295 mM 1.5 mM 1 mM 2.5 mM 1 mM Tests i). Test and compare pollen from two flowers for their ability to germinate and extend a tube. Choose 2 different flowers. ii) Nutrient requirement: Each group tests the effect of one nutrient on in vitro germination and tube growth, and compare results with control. E.g. a) sugar, b) Nitrate, c) calcium, and d) Boron iii) Other tests (optional): Stage of pollen maturation. Immature versus mature pollen. Nutrients: 1. Prepare and obtain the test solutions 10 ml/each. 2. Take a tiny petri-plate. Dust pollen over the bottom of the plate. 3. Add drops of the solution onto the pollen. Look at the slide using the scanning (4X or 10 x) objective and find a spot where there is a lot of pollen. 4. Change to the low power objective (10X) and watch the pollen. After about 15 minutes, you should see pollen tubes starting to form. The first evidence of this is a small bump on the side of a pollen grain. 5. When germination starts, take out a sample and place on a slide with a coverslip so you can look at it with 40x. If you see a pollen grain germinating, center it in your field of vision and change to the high-power objective (40X) to watch the tube grow. You can probably see the cytoplasmic streaming within the tube. [Many pollen tubes will break apart (lyse) because the tube membrane is too thin to hold the cell together in this sucrose solution. However, it would not lyse if it were growing in a flower’s style.] BSCI 442, 2011 Plant Physiol Lab. 5 6. Analyse results a. Determine % germination. (Time at 30 min). In a field, count the number of total pollen grains, and the number of those showing emerging tube. (total count >20) Express results as % grains germinated at __ specified time. b. Tube length of pollen tubes. (Time at 1- 1.5 h) Measure the relative length of tubes (count 20 tubes for controls, and 20 for a treatment). *Hint: Take pictures. Save and measure according to pixels with Image J. c. Express data as averages with SD. Put in a table showing % Germination, and average tube length. 7. Share results of all groups with the lab section. Report Germination Control for a. a. no sucrose Control for b b. No nitrate Control for c c. no calcium Control d. no B % Germination Ave tube length e.g. 14/20 Discuss & include in your report: 1. What do results show? How does the absence of sucrose, nitrate, calcium or B affect pollen germination and tube growth. How do you interpret the results? 2. What purpose(s) does sucrose serve? Give at least 2 distinct examples. In the plants, where does the sucrose in the flower come from? 3. What is a likely reason for NO3- requirement? Propose a likely fate of nitrate from its uptake to its incorporation into a macromolecule. Show specific reactions. 4. Pollen tube growth is fairly fast, and reaches the ovule in a few h or less. Why? 5. Actinomycin D, an inhibitor of transcription, blocks pollen germination by 15% only relative to control. What does this result mean? Ref. Taiz & Zeiger 2002, 2006. chap. 16 Fig. 2. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates and WH Freeman Moctezuma E (2008). Manual for Plant Biology Lab (U. Maryland, College Park)