Plant Pollen Morphology Observation Research Experiment
Source: Experiments in Botany
Operation method
basic program
Principle
The science of observing the fine morphology of plant surfaces through light microscopy and scanning electron microscopy is called micromorphology. The study of micromorphology has a wide range of contents, from the shape and arrangement of epidermal cells of stem and leaf of plant body, the protrusion of outer cell wall to the structure and morphology of epidermal hairs, stomatal apparatus and glands of leaf blade, as well as the withered and fine structure of the surface of fruits, seeds and pollen, among which the study of the morphology and structure of plant pollen is called Sporangiology. The development of sporology has had a great impact on modern plant taxonomy, providing a large amount of information for taxonomy, and at the same time, it has also played a positive role in promoting the development of geology and occupational botany. The morphology and structure of pollen reveal the phylogenetic course of plants and reflect the adaptive characteristics of plants to different media of pollination. By observing the morphological features of pollen, we can understand the diversity of pollen morphology and structure and its adaptation to pollination, learn to correctly describe the morphological features of pollen, and master the method of pollen preparation, on the basis of which we can recognize the role of pollen morphology and features in plant taxonomy, occupational botany, geology and plant systematics.
Materials and Instruments
Pollen Move 1 Collection of pollen material Pollen of plant material from different families can be taken from fresh plants or waxy leaf specimens. Collect as many flowers as possible that are about to open and record the details of the sample taken at the time of collection. Generally, if the flower is large and has many stamens, the stamens or anthers can be removed directly with forceps and placed in a small glass tube and numbered. If the chemical is very small, such as Caiaceae, Kuanaceae, Luoaceae and other plants, the stamens of the flowers are not easy to identify, you can remove a few of their small flowers and put them into a glass tube. 2 Decomposition of pollen Decompose pollen by acetic anhydride decomposition method. (1) Add 2mL of glacial acetic acid into the small glass tube with anthers, and break the anthers with a glass rod after the anthers are softened. The pollen was filtered through a fine copper mesh into the numbered corresponding centrifuge tube, and after precipitation by centrifugation, the glacial acetic acid was poured off. (2) Add acetic anhydride-concentrated sulfuric acid mixture (9:1) ( prepared temporarily before use to avoid failure), and heat the centrifuge tube in a water bath (or a beaker with water) until it boils. During the decomposition process, a glass rod can be used to stir gently once or twice to make it homogeneous. At the same time, the glass rod can be used at any time to take out a few tears of pollen on the slide, observed under the microscope, to see whether the inner wall of the spore powder and the protoplasmic body has been completely dissolved if the inside of the protoplasmic body is not completely dissolved, you can continue to carry out the fraction of shaos, until it is completely dissolved. Generally about 2-3min. (3) Decomposition is good, and then put the centrifuge tube into the centrifuge centrifugation precipitation, pour off the mixture Add distilled water, and then centrifugation, repeat 3 times. Add 50% glycerol, pour the glycerol and pollen into a small glass tube and add a little preservative (carbolic acid or muscimol). 3 Preparation of pollen (1) will be kept in a glass tube of pollen with a glass rod or pipette to take out a few instruments, placed on a slide, such as debris, can be removed with tweezers; and then put into a small piece of glycerol glue, slightly heated to melt or add a drop of glycerol glue has been dissolved in hot water, gently stirred with a regent; and then the coverslip and then a little baked on the alcohol lamp or a miniature hot plate, quickly covered (baked coverslip can reduce the occurrence of bubbles, the thickness is generally more than a little more than the thickness of the coverslip. The thickness of the coverslip is usually less than 17µm.) Seal and label the coverslip. (2) After the glycerol gel has completely solidified, seal the edges around the coverslip with Canada Tree Gum to make a permanent preparation, and put it into the specimen box for storage. 4 Preparation of pollen SEM samples Take a little anther placed on the slide, then drop 50% alcohol a drop, with a small camera to crush the anther, the pollen particles will be eluted by natural drying, placed under the optical microscope to check the number of pollen particles, when the pollen to reach a full address, and then use the tip of a small brush quasi-pollen particles to the double-sided transparent tape paper on the sample table, containing pollen particles Xiang Pin the sample table moved to the mirror film machine, spraying the gold town of the film can be stored after 3min. Scanning Electron Microscope (SEM) 5 Pollen morphology analysis Put the permanent preparation made by acetic anhydride decomposition method under the light microscope, pay attention to the observation of the shape of pollen grains, the type of germination holes, etc., and use the eyepiece microscope ruler to measure the pollen grains into the small, each type of pollen grains measured 20 grains, the minimum value, maximum value and the average value, but in the SEM to observe the shape of the pollen grains, type of germination holes (furrows), especially the outer wall ornamentation, use the scanning electron microscope photographs to measure the size of germination furrows, the measured value is based on the magnified photographs of the pollen grains, and the measured value is based on the magnified photographs of the pollen grain size. size, and the measured values were recalculated accurately based on the scale on the enlarged photographs, the actual magnification estimation of the photographs, and then found by dividing the length of the actual size on the photographs by the magnification. (1) Types of pollen Mature pollen can be categorized into several types: ① Single-grain pollen: Pollen grains that stand alone at maturity are called single-grain pollen. The pollen of most plants belongs to this type. ② Compound Pollen: If two or more pollen grains are gathered together, it is called compound pollen. In addition, there are many pollen grains gathered together to form pollen masses, such as the pollen of Orchidaceae and Lauraceae. (2) Symmetry and polarity of pollen grains Most pollen grains are symmetrical and very few are asymmetrical. Pollen grains have two different kinds of symmetry, i.e. radial symmetry and left-right symmetry. The polarity of pollen is determined by the position of the pollen in the tetrads. (3) Emergence pore Pollen grains can be categorized into two types: (1) without a pore, in which pollen grains do not have a pore; and (2) with a pore, to which most pollen grains belong. The shape, structure, location, number and size of the pore often vary greatly depending on the family, even in the same genus and different species of pollen, there can be cross fertilization; on the other hand, some families of the genera of pollen is very consistent, such as Gramineae, Ricaceae, Umbelliferae and so on. The germinal pores are generally divided into two types: (1) grooves are long germinal pores, whose long axis is more than twice as long as the short axis; (2) pores are short germinal pores, whose long axis is two times as long as the short axis or smaller, or are rounded. (4) Outer wall ornamentation After the pollen grains are treated with acid or alkali, the living material inside the pollen and the soft inner wall are dissolved, leaving only the outer wall of the pollen. The outer wall of pollen is usually subdivided into the (outer) outer layer and the (outer) inner layer. Pollen surface is smooth, or wavy, in some pollen also has a variety of sculptured molecules, such as small spines, tumors, particles, etc., to form a variety of sculpture. The various sculptures on the surface of pollen are categorized as: ① granular sculpture. The surface of the pollen has particles, and the size of the particles can vary. ② Tumor-like pattern. The maximum width is greater than the height. ③ Stripe-like sculpture. The carvings become parallel to each other. ④ Rod-shaped carving. The carvings have a rounded head, and the height is greater than the maximum width. ⑤ Rasping carving. Spiny or small spines, pointed or blunt at the unterminal end, but much wider at the base than at the unterminal end. ⑥ Brain-like carving. The carvings form curved lines that resemble brain wrinkles. (vii) Cavate sculpture. The surface of the pollen has concave cavities. ⑧ Reticulated sculpture. The net consists of ridges and mesh. Ridges can be wide or narrow, and the size and shape of the mesh can vary greatly. ⑨ Negative mesh carving. Equivalent to the part of the mesh ridge concave, and equivalent to the part of the mesh convex. (5) Shape and size of pollen The three-dimensional shape of pollen can be derived from observing the shape of pollen grains in different positions and at different focal points (under high magnification). The common shapes of pollen are: super-elongated spherical, oblong spherical, sub-spherical, spherical, oblate spherical, and super-oblate spherical. The size of pollen varies greatly, with the smallest pollen grains having a maximum diameter of less than 10µm, and the largest ones reaching more than 250µm. Measurement of pollen size: For general pollen, the polar axis (the line extending outward from the center of the tetrads through the center of the pollen grain is the polar axis of the pollen) and the equatorial axis (the line perpendicular to the polar axis is the equatorial axis) were measured; if there were two different equatorial axes, a total of three measurements were made; if the pollen was nearly spherical, only its diameter was measured. Each measurement was standardized to 20 pollen and averaged; if the number of pollen was less than 20 or more than 20, this was noted in parentheses. General measurements were made under a 40x objective lens, and figures were generally taken to one decimal place and derived from calculations, as the accuracy of the micrometer in general is small enough to reach decimals of less than 1µm. The thickness of the outer wall or the diameter of the holes, etc., are measured with a precision micrometer. For more product details, please visit Aladdin Scientific website.
Ice acetic acid Acetic anhydride Concentrated sulfuric acid Glycerol Carbonic acid Canadian gum Distilled water Alcohol, etc.
Optical microscope, scanning electron microscope, water bath, centrifuge, small test tubes, tweezers, dissecting needles, fine copper mesh, slides, coverslips, double-sided transparent tape, alcohol lamp, eyepiece, micrometer, etc.