Laboratory 29 - Magnoliophyta: Reproductive Morphology II

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1 58 Laboratory 29 - Magnoliophyta: Reproductive Morphology II I. Microsporogenesis and microgametogenesis A. Pollen tube growth Growing pollen tubes is simple if you are using bicellular pollen. Physiologically, it is possible to grow many pollen grains on a fairly simple Brewbaker-Kwack medium (0.01% H 3 BO 3, 0.03% Ca(NO 3 ) 2 4H 2 O, 0.02% MgSO 4 7H 2 O and 0.01% KNO 3 ) with from 0% to 20% sucrose added. Obtain a slide and place a small drop of pollen growth solution on it. Then sprinkle some fresh pollen on the drop. Wait for a minute or two for the pollen to settle into the solution and hydrate. Then, VERY CAREFULLY add a cover slip. (Hydrated pollen grains break easily!) Now, to prevent the slide from drying, either put it in a humid chamber or seal the edges of the cover slip by applying liquid Vaseline to the outside edges of the cover slip. (A warm paper clip is an ideal applicator, if used carefully.) Pollen tubes should form within minutes. These will be visible using a compound microscope or high magnification with a dissecting microscope. For showing rapid growth, pollen of Tradescantia and Impatiens is excellent. To show the movement of the generative cell down the pollen tube, Amaryllis is ideal. As these elongate, can you see the vegetative nucleus? the generative cell? a linkage between these structures? When are the sperm cells formed? B. Pollen grain formation A developmental sequence of light and electron micrographs is available on demonstration. Each of the following developmental stages in the formation of the mature pollen, typified by Plumbago zeylanica. The following stages are represented: 1. Microsporocyte in prophase I. Notice the number of microsporocytes, their organization, and their coenocytic organization during early microsporogenesis. The fine structural organization of the meiotic cytoplasm and organelles appears nearly as drastic as those events occurring in the nucleus. 2. Cytokinesis occurs simultaneously in all four of the microspores in the dicotyledonous pattern of microsporogenesis, shown here. 3. Cytokinesis is followed by enclosure of the microspores within a callosic special wall which is presumed to regulate the entry of certain external metabolites, including nucleic acids. This is also the stage at which the carbohydrate skeleton of the exine is laid down. 4. Expansion and vacuolization of the microspores accompanies the formation and differentiation of the pollen wall. At this stage, the nucleus has a small nucleolus and is centrally located in the cytoplasm. The tapetum actively secretes sporopollenin precursors at this stage. Microspores have not completed inflation. 5. Post-meiotic mitosis results in the formation of a separate vegetative cell with a vegetative nucleus and a lenticular generative cell attached to the intine wall of the pollen. Vacuolization continues until full volume of the pollen grains is reached. The tapetum begins degeneration. 6. The generative cell next separates from the intine to become a free cell. At this stage, the pollen reaches full inflation, morphogenesis of the generative

2 59 cell into a spindle-shaped cell occurs, and it comes to lie next to the vegetative nucleus. 7. At the transition to maturity, the two sperm cells are formed. Vacuoles are replaced by cytoplasm at this stage. The cytoplasm contains many is secretory vesicles (polysaccharide vesicles used in pollen tube growth), numerous mitochondria, plastids, and either starch or lipid bodies as a storage material. The storage material in pollen grains appears to be consistent with pollination strategy. Insect-borne pollen tends to contain lipid bodies; wind-borne pollen tends to contain starch grains. In angiosperms, the mature pollen may be either bicellular, containing a generative cell, or tricellular, containing fully formed sperm cells. This characteristic varies by genus and family in the angiosperms. (In Populus species, including cottonwood, there is the transitional condition-- sometimes both bicellular and tricellular pollen may occur in the same anther. The formation of sperm cells prior to germination is considered to be the derived characteristic. Compatibility systems and pollen physiology relate to the time of sperm cells formation relative to anthesis. See table. C. Pollen wall micromorphology Pollen wall morphology consists of two layers: the intine, which is primary cell wall, and the exine, which contains the extremely resistant compound sporopollenin. The exine provides protection against desiccation and physical deformation; however, the pollen tube emerges from and is continuous with the intine. Therefore, one or more apertures are present. Observe scanning electron micrographs of selected pollen grains, observing apertures, sculpturing and diversity. These are extras, so you may take some souvenirs to keep, if you wish. Functionally, the pollen must be protected by a resistant cell wall, but must respond as a naked cell to recognition stimuli. One response to this evolutionary pressure has been the diversification of pollen wall sculpturing to retain superficial glycoproteins and lipids on the grain. These proteins and lipids form a sort of pseudomembrane on the pollen that activate imbibition when deposited on the appropriate stigma. Remarkably different sculpturing patterns are present on different pollen grains. Presumably, these have occurred in response to evolutionary pressures from a variety of directions-- most notably for efficient dispersal, germination and recognition. Cell wall organization and cellular condition of the pollen is systematically significant in some plant groups. D. Internal organization of the male germ unit The male germ cells (either the sperm or generative cells, depending on the pollen grain and stage of development) are typically associated with the vegetative nucleus prior to germination and during pollen tube growth. This association has been termed the male germ unit. Examples are in the lab. Although the exact form of the MGU varies from one species to the next, this association may be important in delivering the two gametes into the embryo sac at the same time, preventing heterofertilization (a condition in which sperm cells from different pollen tubes fertilize the egg and central cell.) The two sperm cells are linked by retaining a common cell wall, as a result of generative cell cytokinesis and both are contained within an internal vegetative cell plasma membrane. A second physical association occurs between at least one of the sperm cells and the vegetative nucleus. This forms a more or less intricate arrangement of complementary surfaces that is maintained up to the time of fertilization. The grasses are exceptionally interesting in this regard, because their association is severed early in development, but re-established just prior to fertilization. This argues strongly for the importance of the MGU. This association appears to occur in the vast majority of angiosperms.

3 60 II. Megasporogenesis and megagametogenesis A. Megagametophyte development The vast majority of flowering plants (over 70% of the families) have monosporic development, in which the embryo sac is genetically homogeneous and all cells within are derived from a single megaspore, the others aborting (see Fig ). On demonstration are slides representing these events from initiation of the megasporocyte to maturity in both Zea mays, corn, and Cucurbita, the cucumber. The captions explain the events in greater detail and, at least in corn, illustrate how callose wall patterns may determine the pattern or abortion between the four megaspores. Note that in corn, the micropylar dyad cell may begin to abort prior to the completion of meiosis, so that in this case, sometimes only three cells are formed as a result. The other types of embryo sac formation require the participation of either two megaspores (bisporic development) or four megaspores (tetrasporic development), resulting in genetically heterogeneous nuclei within the megagametophyte (see Fig , p. 584, for the diversity of different types). The majority of these variant forms are tetrasporic. One form of tetrasporic embryo sac development that is particularly striking is that of lily, which displays Fritillaria- type embryo sac development. The events of that type of embryo sac development are discussed in the text (p. 587). B. Organization and variability in the mature megagametophyte A small display shows the general organization of the mature megagametophyte and a major variant of this type in the synergid-lacking megagametophyte of Plumbago. III. Embryogenesis A. Fertilization The function of the mature embryo sac is a complex process that can be divided into four stages: 1. Progamic phase - A preparative stage in which the male and female gametophytes are slowly modified prior to arrival of the pollen tube. This includes, for example, the degeneration of the receptive synergid 2. Gamete discharge - Arrival and discharge of the pollen tube, constituting sperm deposition 3. Gametic fusion - fusion of plasma membranes of the gametes, transmitting the nuclei of the male gametes into the egg cell and central cell 4. Nuclear fusion - fusion of the nuclear envelopes of the male and female gametic nuclei Demonstrations are available on each facet of the process. The exhibit centers on results using Plumbago zeylanica and Populus deltoides, two plants in which these processes have been clearly elucidated. Several alternative models are also shown in another figure on display. B. Embryogenesis After the formation of the zygote and primary endosperm nucleus, considerable development is usually required to organize the mature seed. Endosperm development, which creates the nutritive material for the embryo, usually precedes the division of the zygote and further embryogenesis. The

4 61 processes of embryogenesis and endosperm development are illustrated in Capsella bursa-pastoris, shepard's purse, which is the classical plant for this research (and is similar to Arabidopsis in its pattern of developement). Slides are available illustrating the organization of the young embryo, including: 1. globular embryo stage - the first stage in the formation of a three-dimensional cell division system producing tissue thickness in the embryo. Note the long suspensor and unusual basal cell, which is found in many members of the Brassicaceae. What is the function of the suspensor? 2. heart-shaped stage - differentiation of the early cotyledons. This stage also shows the first conspicuous evidence of a protoderm. What is the developmental potential of that layer? 3. torpedo stage - elongation of the embryo. The embryo becomes more complicated at this stage and the apices are becoming organized. 4. maturational stage - growth of the embryo is completed within the sclerified ovular integuments, forming the seed. One caveat--capsella has a curved megagametophyte (ana-amphitropous), which is a highly restricted characteristic in angiosperms. The events are given in detail for Capsella on pages of Gifford and Foster. A remarkable reduction of the embryo is shown in the embryo of Monotropa uniflora, the parasitic Indian pipe. Embryos of this plant are only two cells at maturity. In angiosperms, there is wide variation in the organization of the embryo in different groups. As a last exercise on the organization of embryos, examine the mature embryo of Zea mays, as a representative of the monocots. The embryo of corn is particularly precocious compared to most flowering plants. Observe a prepared slide and identify the scutella, shoot apex, root apex, epiblast, coleoptile, coleorhiza and young foliage leaves. What constitutes the cotyledon? Is there any evidence that a second cotyledon was ever present in monocots?

5 62 COMPARISON OF BICELLULAR AND TRICELLULAR POLLEN GRAINS Those released with generative cells at anthesis are known as bicellular pollen grains, while those containing sperm cells at anthesis are tricellular. Characteristic: Tricellular Bicellular Sperm formed: Yes No Duration of growth: Short Long (< 10 hr) (12 hr or longer) Growth rate: Rapid at stigma Slower growth initially slower near end of faster after sperm growth formation Mitochondria: Extremely active Sluggish Mitochondrial longevity: Short Normal Incompatibility system: Sporophytic Gametophytic Rejection site: On stigma (lack of In upper, mid and hydration) or early lower style usually in stylar growth prior to sperm formation Means of rejection: Inhibition at stigma Callose walls thicken imbibition fails growth slowly ceases tube aborts/bursts. tube dies in situ. Specific recognition gene regulation Growth inhibition / starvation