Micronucleus assay experiments on mutagenic substances
1. Understanding the methods and significance of micronucleus determination
2、Searching for new testing systems and new safer and more effective plant mutagens
Operation method
Micronucleus assay experiments on mutagenic substances
Principle
Micronuclei (micronuclei) abbreviated as (MCN) is an abnormal structure in eukaryotic cells, often produced by the cells through the action of radiation or chemicals. In the intercellular phase, the micronucleus is round, free from the main nucleus, and should be less than 1/3 the size of the main nucleus. The refractive index and cellular chemical reaction properties of the micronucleus are the same as those of the main nucleus, and it also has the ability of Woolman DNA. It is generally believed that the micronucleus is produced by the loss of mitotic fragmentation in late mitosis. Research work has confirmed that whole chromosomes or several of them can also form micronuclei. These fragments or chromosomes form a third nuclear mass when they are rejected by two daughter nuclei in prophase than in cytokinesis. It has been shown that the size of the micronucleus rate is positively correlated with the dose of the drug or the cumulative effect of radiation, as in the case of chromosomal aberrations. This is why many believe that a simple interphase micronucleus count can be used instead of the cumbersome intermediate aberration chromosome count. The synthesis of large numbers of new compounds, the use of atomic energy, and the discharge of a wide variety of industrial wastes have created the need for a highly sensitive, technically simple test system to monitor environmental changes. Only eukaryotic testing systems are more directly capable of hypothesizing the genetic hazards of mutagenic substances to humans or other higher organisms. In this regard, micronucleus testing is a more ideal method. At present, many departments at home and abroad have used "micronucleus test for radiation damage, radiation protection, chemical mutagens, new drug testing, chromosomal genetic diseases and pre-cancer diagnosis and other aspects. Most of the existing micronucleus test systems use mammalian bone marrow cells or peripheral blood cells, the disadvantage of which is that it requires certain culture conditions and time, and cell synchronization difficulties, low micronucleus rate, generally only about 0.2%. In recent years, some people use pollen spores of higher plants, using natural synchronization as micronucleus test material. Good results have been achieved. Among them, Madshew used a kind of duck metatarsal plant (Tradescantia paludosa), which is native to America, to establish a tetrad micro-nucleus rate counting (MCN-in-Tetrad) test system, which is generally recognized as one of the better systems in recent years. This system can achieve micronucleus rates of 10%-67% when treated with low doses of chemicals or radiation. In this experiment, we switched to a simple and easy to use tobacco seed for germination and treated it with a certain concentration of pinyonomycin hydrochloride to count the mutagenic rate of micronuclei.
Materials and Instruments
Tobacco seed Move I. Experimental materials Photo captions: 1, micronucleus,; 2 micronucleus,; 3, binucleus,; 4, nuclear outgrowth; 5, nuclear constriction; 6, nuclear auricle; For more product details, please visit Aladdin Scientific website.
Pindamycin hydrochloride Fixative
Triangular vials Scissors Tweezers Slides Coverslips
Tobacco seeds were soaked in warm water at 40°C for 40 minutes, and the seeds were gently handled by wrapping them in cloth, after which they were placed in Petri dishes with absorbent paper to be germinated at 25-28°C for about three days, and when the young roots grew to about 2-4 mm, the sprouted seeds were placed in a certain concentration of pingyromycin hydrochloride for mutagenicity for 5-8 hours, and then rinsed with water for three to five times, and then cultured overnight and fixed with fixative solution for use.
Experimental apparatus and drugs
1、Pingyangmycin hydrochloride, 20m g/ml, 40 m g/ml, 80 m g/ml concentration of the solution was prepared.
2、Fixing solution: 3 methanol: 1 glacial acetic acid
3、Staining solution
4、Laboratory tools: triangular flask, scissors, tweezers, slides, coverslips, etc.
Third, the experimental description
Cells in the division period are sensitive to the treatment of physical and chemical factors. Secondary and fine roots of tobacco are more sensitive to the treatment of paxillin from 17:00-24:00. The backward chromosomes or groups of backward chromosomes produced during the treatment can also be expressed in the middle chromosome.
IV. Experimental steps
1、Soak the tobacco seeds in warm water at 40℃ for 40 minutes, wrap the seeds with cloth and gently pinch them, after that, place them in petri dishes with absorbent paper to germinate at 25-28℃ (or place them in triangular flasks filled with distilled water to oscillate and cultivate them at 25-28℃) for about 2-3 days, and wait for the young roots to grow to about 2-4mm.
2. Put the germinated seeds into 20m g/ml, 40 m g/ml, 80 m g/ml concentration of pingyangmycin hydrochloride for mutagenesis for 5-8 hours, rinse with water for three to five times, and then incubate overnight, then fixed with fixative solution for use.
3,, Pick the appropriate young roots after mutagenesis, change to 70%, 50%, 30% alcohol for 10 minutes each, distilled water rinse twice.
4, 60 ℃ 1NHCL in 3-5 minutes, and then into the 1N cold HCL for 1 minute, take out with water slightly rinsed, placed on a slide, drop a small amount of staining solution, cover the coverslip, gently tapping, so that the root tip cells dispersed, and placed in the microscope to observe.
5、Compare the observed results with the following pictures.
7, bridge + backward chromosomes; 8, bridge + chromosome breaks; 9 double bridge; 10, multiple bridges; 11 free chromosomes, the
12 chromosome adhesion; Note: The above images are X 200