Plant Root Tip Pressing Experiment
Plant Root Tip Pressing Experiment
The root tip compression technique can be used to: study chromosome number and morphology in mid-stage plants.
Operation method
Plant Root Tip Pressing Experiment
Principle
Mitosis is the main way of plant cell division and proliferation of somatic cells. During mitosis, the chromosomes in the cell nucleus are accurately replicated and regularly and evenly distributed to the two daughter cells, so that the daughter cells and the mother cell have the same number of chromosomes with the same morphology and structure, which ensures the consistency of the hereditary traits of the plant cells. In various plant tissues with vigorous growth, such as root tip tissue, stem tip tissue, intermediate meristematic tissue, healing tissue, etc., the cells often undergo vigorous mitosis. By taking the material at the appropriate period of cell division and by treating the test material in a certain way, it is possible to observe the characteristics of chromosome changes and the morphological features of the chromosomes under the microscope, and to count the chromosomes. Since the chromosomes in the middle of mitosis have typical morphological features and are easy to count, in order to obtain more images of the chromosomes in the middle of mitosis, drug treatment or freezing treatment can be used to prevent the formation of the spindle and stop the cell division in the middle of the process. The treatment can also shorten the chromosomes and make them easy to disperse for observation and study. In addition, acid hydrolysis or enzyme treatment of cell tissues removes the pectin layer between the cells and softens the cell wall, making the cells easy to disperse from each other, which is conducive to pressing and staining.
Materials and Instruments
Onion Garlic Rye Corn Wheat Broad Bean Move 1. Taking materials: can be cultivated indoors at the root tip, can also be dug directly from the field of freshly grown young root tip. The method of indoor culture is: Caveat The length of the plant cell division cycle varies, usually between ten and tens of hours, and temperature significantly affects the division cycle; for a less familiar experimental material, it is best to grow roots at a specific temperature to get a handle on the peak of mitosis in order to get more mitotic cells. Common Problems Root tips are the most commonly used material for observing plant chromosomes due to the ease of obtaining the material, and some plant seeds that are difficult to germinate, or only have plants without seeds, can also use stem tips as material. For more product details, please visit Aladdin Scientific website.
Anhydrous ethanol 95% ethanol Glacial acetic acid Distilled water Basic magenta Hematoxylin Colchicine Saturated p-dichlorobenzene solution 8-hydroxyquinoline Hydrochloric acid Ferroalum aqueous solution Magenta acetate staining solution Cellulase Pectinase mixture
Microscope Thermostat Refrigerator Water Bath Analytical Balance Scissors Tweezers Blades Slides Coverslips Filter Paper Measuring Cylinder Petri Dish Beaker Drip Bottle Alcohol Lamp Slicing Case Labeling
(1) round onion and garlic root tips: rooting before the round onion to the root up in the light for two days, and then placed in the mouth of a small beaker of water, so that the rhizome contact with water, or the round onion half buried in wet sand. In 20 ~ 25 ℃ under the light conditions of culture 2 ~ 3 days, to be root length 1 ~ 2cm, select the strong root from the tip of about 1cm cut for pretreatment. Garlic as a material can be peeled off the garlic cloves, and then strung up with fine wire in a petri dish of water culture, other requirements as above. Cut the root tip of the time to about 10:00 in the morning is good.
(2) corn (or rye, wheat, broad bean) roots: first soak the seeds for a day, to be absorbed and swollen and then transferred to the petri dish with several layers of filter paper (or blotting paper), moistened with water, and then evenly placed in the seeds on the filter paper, covered with a lid. Put in the dark conditions of 18 ~ 20 ℃ cultivation, when the root grows to 1 ~ 2 cm, select the robust root tip at about 10:00 a.m. to take off the spare.
The method of taking roots directly from plants in the field is applicable to most graminaceous plants, for example, a large number of adventitious roots grown during the growing season of wheat crops are more robust than those sprouted from seeds, and are good materials for observing mitosis.
2. Pre-treatment: In order to stop the activity of the spindle and obtain more intermediate division phases, and at the same time to make the chromosomes relatively shorter, so as to facilitate chromosome dispersion and counting, the root tip can be pre-treated. If it is only to observe the chromosome dynamics in all phases of mitosis, no pretreatment can be done.
Two methods of pretreatment are freezing pretreatment and drug pretreatment.
(1) Freezing pretreatment: Soak the root tips in distilled water and place them in a refrigerator at 1～4℃ or in a thermos flask filled with ice for 24 hours. This method has no destructive effect on the chromosomes, and the chromosomes are shortened uniformly with good effect, simple and easy to implement, and is applicable to all kinds of crops.
(2) Drug pretreatment: Commonly used drugs are 0.05-0.2% colchicine solution, saturated p-dichlorobenzene solution, 0.002mol/L 8-hydroxyquinoline, etc. The root tip is then pre-treated with 0.002mol/L 8-hydroxyquinoline. For pretreatment, the root tips were directly immersed in the drug solution and treated for a certain period of time at a suitable temperature (Table 1). All of these drugs can shorten the chromosomes, but at the same time, they also have a certain destructive effect on the chromosomes. Care should be taken when using them, with wheat, rye, barley, onion and garlic being treated for about 4 hours, and cotton and rice for about 2 hours. The duration of treatment is highly dependent on the temperature. 
3. Fixation or former hypotonic: the purpose of fixation is to use chemicals to kill the living cells quickly, and make the composition of the chromosome nucleoprotein denaturation and precipitation, in order to maintain the intrinsic morphology and structure of the chromosome. Fixation solution is generally used Carnot's fixation solution, the preparation method is anhydrous alcohol: glacial acetic acid = 3:1, can also be used 95% ethanol instead of anhydrous ethanol.
The pre-treated root tips were rinsed twice with distilled water (about 5 minutes) and then transferred to Carnot's fixative. The root tips were fixed at 4-15℃ for 20-24 hours, and then they were ready for observation. If long-term preservation is required, the root tips can be rinsed twice with 70% alcohol then transferred to 70% alcohol for preservation.
4. Dissociation: The purpose of dissociation is to decompose the pectin substances in the middle layer between the cell walls as well as part of the cytoplasm, so that the cells can be easily dispersed. At the same time can also make the cell wall moderately soft and easy to press the film. Dissociation methods are mainly the following.
(1) The root tips were rinsed twice with distilled water and then placed in 1 mol/L hydrochloric acid that had been preheated in a 60℃ water bath. The root tip was treated at a constant temperature of 60°C for 10 to 15 minutes and removed when the root tip was transparent and beige in color.
(2) The root tips were treated in an equal mixture of 95% alcohol and concentrated hydrochloric acid for 2 to 10 min. Or, the root tips were treated within 5ol/L hydrochloric acid for 5 to 10 minutes.
(3) The root tips were placed in an equal mixture of 2.5% pectinase and 2.5% cellulase (PH 5 to 5.5) and treated for 2 to 3 hours at room temperature of 18 to 28°C.
5. Post-hypolysis: the root tips were rinsed in distilled water 2 to 3 times (about 10 minutes). Enzymatically digested root tips may be soaked for 10 minutes and then rinsed once more. Root tips must be rinsed well or the staining will be affected. After hypotonicity, the root tips are placed in 70% alcohol and set aside.
6. Staining and pressing: The following methods are commonly used for staining and pressing.
(1) Magenta acetate staining method: take a root tip and put it on absorbent paper to absorb excess preservation liquid, then put it in the center of a clean slide. With a razor blade, cut off the root tip meristematic tissue, cut it into thin slices (the thinner the better), drop 1 drop of 2% magenta acetate staining solution, cover the coverslip, and press the slice. The coverslip was covered with 1 drop of 2% acetic acid magenta staining solution and pressed. The coverslip was held in place with one hand, while the other hand was used to tap the coverslip with the tip of a forceps or the handle of a dissecting needle to disperse the material. The back of the slide was lightly roasted with fire, and then continued to tap, until the material was cloudy to be examined microscopically. The purpose of heating is to fully stain and soften the chromosomes as well as to disrupt cytoplasmic staining. If a more desirable split phase is found, the slides can be lightly baked again, then cover the coverslip with a piece of absorbent paper, press the slides firmly with the thumb (do not move the coverslip), and then microscopically examine.
You can also use the cross-pressing method. That is, the root tip is taken and placed on an absorbent paper to absorb the excess preservation fluid, and then placed in the center of a clean slide. Cut off the meristematic tissue with a razor blade and cut it into thin slices. Take another clean slide and cross it in a cross shape over the slide with the material on it. Cover the slide with a piece of absorbent paper and press the piece with your thumb so that the material is pressed into a thin layer. The two slides were then separated and stained by adding 1 drop of staining solution to each. After a short pause a coverslip was added, the slides were lightly toasted on an alcohol lamp, the coverslip was held in place with one hand, and with the other hand the material was gently tapped with the eraser tip of a pencil to disperse the root tip cells evenly.
(2) Modified Carbol Magenta Staining Pressing Method: Place the root tip in the center of a clean slide, cut off the root tip meristematic tissue, cut it into thin slices, drop a drop of Modified Carbol Magenta Staining Solution, and cover the coverslip and press the slices.
(3) Ferroalum hematoxylin staining and pressing method: first, put the root tip into 4% ferroalum aqueous solution for 2~4 hours. Then rinse with running water for 20 minutes, and then put the root tip into 0.5% hematoxylin staining solution to avoid light for 40-120 minutes, and then put it into distilled water for spare. For its preparation, a stained root tip was taken and placed in the center of a clean slide. Cut off the root tip meristematic tissue with a razor blade, cut it into thin slices (the thinner the better), drop 1 drop of 45% glacial acetic acid, add a coverslip, and press the slides in the same way as above.
7. Microscopic examination: the pressed piece first in the low magnification microscopy, find the split cells after conversion to high magnification observation. If the chromosomes are well dispersed, the image is clear, it can be dehydrated and sealed to make a permanent film.