Experiments on the meiotic preparation of plant pollen mother cells
Experiments on the meiotic preparation of plant pollen mother cells
Meiosis is a specialized type of mitosis that occurs in organisms during the maturation of the gametes formed by the gametocytes, and it consists of two consecutive cell divisions, the first of which is meiotic and the second of which is isochronous. The first division has a longer prophase with more complex chromosome changes, which can be subdivided into five periods, namely, the fine lineage, the even lineage, the thick lineage, the double lineage, and the terminal phase. The behavior of chromosomes in meiosis has a major impact on the distribution and recombination of genetic material.
Operation method
Experiments on the meiotic preparation of plant pollen mother cells
Principle
Meiosis is a specialized type of mitosis that occurs in organisms during the maturation of the gametes formed by the gametocytes, and it consists of two consecutive cell divisions, the first of which is meiotic and the second is isochronous. The first division has a longer prophase with more complex chromosome changes, which can be subdivided into five periods, namely, the fine lineage, the even lineage, the thick lineage, the double lineage, and the terminal phase. The behavior of chromosomes in meiosis has a major impact on the distribution and recombination of genetic material. In higher plants, during the formation of male gametes, the microspore mother cell (2n) within the anther, undergoes meiosis to eventually produce four microspores. The number of chromosomes within each microspore has been halved to n. Each microspore later develops further into a pollen grain. The anthers of plants are generally used as meiotic preparation materials because they are easy to obtain and convenient to handle. The flower buds of the plant are collected at a suitable period, and are killed and fixed by a fixative solution so that the cells maintain their morphology when they are alive. Then press staining and other treatments, made of meiosis slide specimens, under the microscope for observation, you can see the meiosis process of the microspore mother cell (that is, the process of chromosomes from double to haploid), the study of chromosomes in the morphology and the number of dynamic changes in the characteristics.
Materials and Instruments
Corn male inflorescences Move 1. Sampling: Selection of appropriate sized flower buds is a critical step in the observation of meiosis in pollen mother cells. Different plants take different periods. For more product details, please visit Aladdin Scientific website.
Anhydrous ethanol 95% ethanol Potassium dichromate Concentrated hydrochloric acid Concentrated sulfuric acid Glacial acetic acid Xylene Canada gum Magenta
Microscope Dissecting needle Slide Coverslip Tweezers Petri dish Alcohol lamp Absorbent paper Gauze Blade Filter paper Match
Corn: about two weeks before the male pumping (trumpet stage), with a finger from the trumpet downward squeeze leaf sheaths, touch a soft feeling, that is, where the male inflorescences. In the place with a razor blade to make a longitudinal cut, with tweezers to take out the inflorescence branches. At this time, the apex of the male inflorescence spikelet glume length of about 4mm, anther length of 2 ~ 3mm, the upper part of each branch spikelet development is the earliest.
Taking time is generally in the morning at 9:00 ~ 10:00, with slight variations among different materials.
2. Fixation: The material should be immediately put into the fixative to kill the fixation. Under the condition of 4～15℃, the general fixation time is 24 hours. After fixation, the material is first rinsed with 95% alcohol to no acetic acid odor, and then moved to 70% alcohol in the refrigerator for preservation. Commonly used fixing solution is Fahrenheit fixing solution, in the use of this fixing solution should pay attention to the ready-to-use, fixed not to be exposed to the sun.
3. Staining and pressing: First, place the fixed material in a petri dish and pour a little preservation solution. Use tweezers to pick an appropriate size of the spikelet or bud on absorbent paper to absorb excess alcohol, and then placed in the center of the slide. Two to three anthers were picked out with a dissecting needle and drops of magenta acetate or ferroalum hematoxylin acetate stain were immediately placed on the anthers. The anthers were cut transversely with a dissecting needle and gently squeezed from one end toward the incision to allow the pollen mother cells to escape from the incision. The anther wall and other debris were then removed with forceps. This is one of the key steps in the excellence of the slide pressing. If the debris is not removed, it will not be easy to flatten the dividing cells so that the chromosomes are not easily dispersed, and the cells will be dislodged from the slide in large numbers during the process of making the permanent slides. After removing the debris, a preliminary microscopic examination is performed under low magnification, and if the pollen mother cell is in meiosis, a coverslip can be added. When adding the coverslip, one side of the coverslip was placed on the slide, and when the staining solution covered the whole edge, the left hand held the forceps against the coverslip, and the right hand held the dissecting needle to hold the coverslip to put it down gently. After adding the coverslip, if there is excess staining solution, absorbent paper can be used to absorb it; if the staining solution can not cover the coverslip, add a little staining solution on one side of the coverslip, and then bake the slides on the alcohol lamp. When baking the film, hold the film flat in the hand above the alcohol lamp to move back and forth, and often put the film on the back of the hand to test the temperature, in order to the film is not hot for the appropriate, can be repeated for many times. Baking is a very important step in the preparation process. By baking the slides, the cytoplasm becomes lighter in color, and the chromosomes can be fully and distinctly colored. After baking the film can be added to a small piece of absorbent paper on the coverslip, with a thumb or pencil with an eraser tip to gently press the coverslip. Care should be taken not to move the coverslip. If the microscopic examination found that the chromosome staining is too light, can be slightly added around the coverslip staining solution and then baked; if the staining is too deep, can be added from one side of the coverslip drops of 45% glacial acetic acid, on the other side of the absorbent paper suction, so that the glacial acetic acid from the underside of the coverslip flow through the cytoplasm until the color of the lighter chromosomes and chromosome coloring is obviously clear analysis.
4. Production of permanent slices: To make the slices can be preserved for a long time, they can be made into permanent slices. The production of permanent film mainly includes three steps, including slice, dehydration and sealing time. First of all, with a petri dish (built-in U-shaped glass tube or a short glass rod) a number of sets of numbers, the preparation of multi-stage dehydration agent (commonly used dehydration agent see appendix II).
(1) Segmentation: Prepare to make a permanent slice by first turning it over so that the coverslip is immersed face down into the first stage of the petri dish to be segmented. Let one end of the slide rest on a short glass rod, and the other end and the bottom of the dish contact, so that the coverslip naturally fall off, and mark the original direction and position of the slide and coverslip corresponding.
(2) Dewatering transparency: Hold the coverslip with a dephosphorylated matchstick split at one end (orientation unchanged, material facing up) and place it into the second-stage Petri dish. And the slide should be turned over so that the side with material is facing up. Then dehydrate in turn, one stage at a time, usually about 1 to 2 minutes per stage. If the third dehydration is used about 20 to 30 seconds per stage. In order to prevent the cells from falling off the slides during the dehydration process, a layer of protein glycerin glue can be applied to the coverslip during the preparation, baked on an alcohol lamp until light smoke is emitted, and then cooled for a few moments and covered on the slide with material.
(3) Sealing: After the slides are removed from the last petri dish and cooled slightly, place a drop of complete Canada gum in the center of the slide where the coverslip was originally placed, then turn the coverslip (so that the material side is facing downward) and gently cover it with the gum in the same direction and position as before. It is important to allow the coverslip to sink naturally as the gum expands; do not apply pressure or move the coverslip. Then lay it flat in a cool place to dry before microscopic examination. For microscopic examination of compliant slices, label the right end of the slide with the name of the specimen and the date of preparation.
[Attachment] Frozen slice method: this method is fast and simple. For the slides ready to make permanent slices, first freeze them with liquid carbon dioxide dry ice, or freeze them with a cryogenic cooler, and when they are completely frozen, use a razor blade to separate the coverslips from the slides, and dry the slides and coverslips in a warm box at about 37°C. The slides should be removed and placed in xylene to soak them in water. After removal, the slides and coverslips were soaked in xylene for 10-20 minutes, and the slides were sealed with drops of gum.