Experiments on the mutagenic effect of radiation on plant chromosomes
Experiments on the mutagenic effect of radiation on plant chromosomes
Understand the mutagenic effects of physical factors on genetic material through radiation on plants.
Operation method
Experiments on the mutagenic effect of radiation on plant chromosomes
Principle
Physical radio Ionizing radiation, with very short wavelengths and correspondingly large frequencies, has a large penetrating power, γ-rays in the air, with a range of up to a few hundred meters and a speed of 300,000 km/sec. Therefore, giving a certain dose of irradiation to plants can easily cause genetic mutations and chromosomal aberrations, commonly found in cytological phenomena such as chromosome adhesion, breaks in the region of the node, disruption of spindle filament formation, and breaks in chromosomes or chromatid monomers. This phenomenon can be observed through cell division. In practice, it is important to choose the right dose. The general requirement is to minimize physiological damage to the plant and to improve the genetic aspects of the effect in order to effectively select superior variant types and individuals.
Materials and Instruments
Onion Garlic Move I. Experimental materials and supplies 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 Multimedia System
1. Materials: round onion, garlic,.
Supplies: microscope, thermostat, refrigerator, water bath, analytical balance, scissors, tweezers, razor blades, slides, coverslips, filter paper, measuring cylinders, Petri dishes, beakers, dropping bottles, alcohol lamps, section boxes, labels and so on. Multimedia system (with microscopic demonstration).
2. Drugs: anhydrous ethanol, 95% ethanol, glacial acetic acid, distilled water, alkaline magenta, hematoxylin, colchicine, saturated p-dichlorobenzene solution, 0.002 mol/L 8-hydroxyquinoline, 1 mol/L hydrochloric acid, 4% aqueous ferric alum solution, 2% magenta acetate staining solution, 2.5% cellulase and 2.5% pectinase mixture.
II. Experimental content and experimental steps
1. Treatment and rooting: Garden onion and garlic bulbs were treated with 1000 roentgen, 2000 roentgen and 3000 roentgen respectively.
After treatment of garden onions placed in the water on the mouth of a small beaker, so that the rhizome part of the contact with water, or the round onion half buried in wet sand. Cultivated in 20 ~ 25 ℃ light conditions for 2 ~ 3 days, when the root length of 1 ~ 2cm, select the robust root from the tip of about 1cm cut for pre-treatment. After treatment of garlic cloves peeled, strung 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. Pre-treatment: In order to stop the spindle activity, obtain more intermediate division phases, shorten the chromosomes, and facilitate the dispersion and counting of chromosomes, the root tip needs to be pre-treated. If it is only to observe the chromosome dynamics in the mitotic phases, no pretreatment can be done. There are two methods of pretreatment: freezing method and drug method.
(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, which is effective, simple and easy to implement, and 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 pretreated 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 (Experiment 3). The action of these drugs shortened and thickened the chromosomes, which facilitated the observation of the chromosomes.
3. Fixation or anterior hypotonic: In order to maintain the intrinsic morphology and structure of the chromosomes, the pre-treated root tips should be fixed promptly after rinsing twice with distilled water (about 5 minutes). The fixation solution is Carnot's fixative, using anhydrous alcohol: glacial acetic acid = 3:1, or 95% ethanol instead of anhydrous ethanol. The fixative should be prepared and used now. Observation can be made after 20 to 24 hours of fixation. If long-term storage is needed, it can be rinsed twice with 70% alcohol and then transferred to 70% alcohol for storage.
4. Dissociation: The methods of dissociation are:
(1) Rinse the root tips twice with distilled water and then put them into 1 mol/L hydrochloric acid that has been preheated in a 60℃ water bath. Process at a constant temperature of 60°C for 10 to 15 minutes and remove the root tip when it is 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.
5. Post-hypotrichosis: The dissociated root tips were placed in distilled water and rinsed 2 to 3 times (about 10 minutes) and placed in 70% alcohol for spare time.
6. Staining and pressing: there are several methods for staining and pressing:
(1) Magenta acetate staining method: take a root tip and put it on absorbent paper to absorb the excess preservation liquid, then put it in the center of a clean slide. Use a razor blade to 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 slides. 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. 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. Microscopy: 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 and the image is clear, draw the chromosome types of the aberrant chromosomes under the microscope and count them.