Tissue culture of chrysanthemum, an elective knowledge subject in biology in senior high school
Basic process of plant tissue culture
Cell differentiation: A process in which cells show stability differences in morphology, structure and physiological functions during individual development.
In vitro plant tissues or cells, after a period of culture, will form callus through cell division. Callus cells are loosely and irregularly arranged, which is a kind of parenchyma cells, highly vacuolated and amorphous. The process of producing callus from highly differentiated plant tissues or cells is called dedifferentiation of plant cells, or dedifferentiation. Callus produced by dedifferentiation can continue to be cultured and can be redifferentiated into organs such as roots or buds. This process is called redifferentiation. The test-tube seedlings formed by redifferentiation can develop into complete plants when transplanted into the ground.
Plant cell engineering
Any living cell with a complete set of genetic information of an organism has the ability to develop into a complete individual, that is, every biological cell has totipotency. But it is not reflected in the growth and development of organisms, because different tissues and organs are formed through the selective expression of genes under specific time and space conditions.
The application of plant tissue culture technology includes: realizing rapid propagation of excellent varieties; Cultivate virus-free crops; Making artificial seeds; Cultivate new crop varieties and industrially produce cell products.
? Cell differentiation is a continuous change. What is its physiological significance?
The specialization of cell structure and function in multicellular organisms is beneficial to improve the efficiency of various physiological functions.
Compare the similarities and differences between apical meristem and callus.
Conditions affecting plant tissue culture
Materials: The difficulty of culture varies greatly with different plant tissues. The selection of plant materials is directly related to the success or failure of the experiment. The species of plants, the age of materials and the length of storage time will all affect the experimental results. In the tissue culture of chrysanthemum, the newly germinated lateral branches of non-flowering plants are generally selected as materials. Generally speaking, plants that are easy to reproduce asexually are easy to carry out tissue culture. Select buds with vigorous growth, good physiological state and easy to induce dedifferentiation and redifferentiation for tissue culture.
Nutrition: In vitro plant tissues and cells have relatively special requirements for nutrition, environment and other conditions, so it is necessary to prepare suitable culture media. The commonly used culture medium is MS culture medium, which contains a lot of elements such as nitrogen, phosphorus, sulfur, potassium, calcium and magnesium, trace elements such as iron, manganese, boron, zinc, copper, molybdenum, iodine and cobalt, and organic substances such as glycine, nicotinic acid, inositol, vitamins and sucrose.
Hormones: auxin and cytokinin are key hormones to start cell division, dedifferentiation and redifferentiation. In the presence of auxin, the role of cytokinin shows a trend of strengthening. Plant hormones such as auxin and cytokinin need to be added to the culture medium, and their concentration, application sequence and dosage ratio will affect the results.
4. Environmental conditions of operation process: PH, temperature, light and other environmental conditions.
Different plants often have different requirements for various conditions. Generally, the pH of chrysanthemum tissue culture is controlled at about 5.8, the temperature is controlled at 18~22℃, and the fluorescent lamp is irradiated at 12h.
Preparation of MS solid culture medium: preparation of various mother liquors: concentrated solution (culture medium mother liquor) prepared by various components according to the formula ratio.
? When in use, according to the concentration multiple of mother liquor, the dosage is calculated and diluted with distilled water.
? Preparation of culture medium: The added substances are agar, sucrose, mother liquor of macroelements, trace elements, organic substances and plant hormones, and the mixture is adjusted to 1000 ml with distilled water.
? In tissue culture of chrysanthemum, plant hormones may not be added.
The reason is that the tissue culture of chrysanthemum stem segment is relatively easy. ? Sterilization: The sterilization method adopted is high-pressure steam sterilization.
? What are the functions of various nutrients in MS medium? What are the characteristics of MS medium compared with broth medium?
Macroelements and trace elements provide inorganic salts necessary for plant cells; Sucrose provides carbon source and maintains cell osmotic pressure; Glycine, vitamins and other substances are mainly to meet the special nutritional needs of isolated plant cells after their normal metabolic pathways are affected to some extent.
Microbial culture medium is mainly organic nutrition, while MS culture medium needs to provide a lot of inorganic nutrition.
Aseptic explant: A plant organ or tissue fragment used for in vitro culture. When choosing the stem segments of chrysanthemum, we should take vigorous shoots. After washing the stems of chrysanthemum with running water, you can add a little washing powder, gently brush them with a soft brush, and then rinse them under running water for about 20min minutes. Absorb the water on the surface of the explant with sterile absorbent paper, put it into 70% alcohol by volume and shake it for 2~3 times, each time for 6 ~ 7 seconds, then immediately take out the explant and rinse it with sterile water. After taking out, the water on the surface of the explant was still absorbed by sterile absorbent paper and put into 0. 1% mercuric chloride solution 1 ~ 2 minutes. After taking it out, rinse it with sterile water for at least 3 times and rinse it with disinfectant.
Note: When disinfecting the surface of explants, the disinfection effect of chemicals and the tolerance of plants should be considered.
Inoculation: When explants are inserted in the inoculation process, the upper end of the shape is upward, and 7-8 explants are inoculated in each conical flask. Explant inoculation is similar to bacterial inoculation, and the operation steps are the same, both of which require aseptic operation.
Culture: it should be carried out in a sterile box, disinfected regularly, and kept at an appropriate temperature (18~22℃) and illumination (12h).
Transplanting: before planting, open the sealed membrane of the culture bottle, let it grow in the culture room for a few days, and then wash the root medium with running water. Then transplant the seedlings to sterilized vermiculite or perlite for a period of time to make the seedlings strong. Finally, open-air cultivation is carried out.
cultivate
The explants may be contaminated in the process of culture, because the disinfection of explants is not thorough; Incomplete sterilization of culture medium; Contaminated by miscellaneous bacteria during inoculation; The sealing performance of conical bottle is poor.
Pollen development of angiosperms in high January anther culture: The stamens of angiosperms are usually composed of filaments and anthers. Anthers are saccular structures with a lot of pollen in them. Pollen is formed by meiosis of pollen mother cells, so pollen is a haploid germ cell. The development of angiosperm pollen goes through four stages: microspore tetrad stage, mononuclear stage and binucleate stage. At the tetrad stage of microspore, four haploid cells are linked together and enter the mononuclear stage, and the four haploid cells of tetrad are separated from each other to form four mononuclear pollen grains. At this time, the cell contains dense protoplasm, and the nucleus is located in the center of the cell (monocytes are in the metaphase). With the growth of the cell, the nucleus moves from the center of the cell to one side (mononuclear side) and splits into 1 germ cell nucleus and 1 pollen tube cell nucleus, thus forming two cells, one is a germ cell and the other is a vegetative cell. Germ cells will divide again to form two sperm.
Note: ① There are two kinds of mature pollen grains. One is a binuclear pollen grain, which only contains pollen tube nucleus and reproductive nucleus, and the sperm of binuclear pollen grain is formed in pollen tube. The other is a trinuclear pollen grain. Before the pollen matures, the germ cells undergo mitosis to form two sperm. This pollen grain contains two sperm nuclei and one pollen tube nucleus (vegetative nucleus). The tetrad in pollen development is four connected haploid cells formed by meiosis of pollen mother cells; The tetrad in meiosis of animal cells is a pair of homologous chromosomes after synapsis, which is called tetrad because it contains four chromatids. ③ Two sperm formed by the same germ cell have the same genetic composition.
There are two ways to produce pollen plants (haploid plants) by anther culture. One way is that pollen develops into plants through embryoid stage, and the other way is that pollen first forms callus on induction medium and then induces it to differentiate into plants. There is no absolute boundary between these two pathways, which mainly depends on the types of hormones in the culture medium and their concentration ratio.
Note: ① No matter which production method, buds should be induced first, and then roots should be induced. ② embryoid: In the process of plant somatic tissue culture, the induced structure is very similar to the embryo developed by a fertilized egg, and its development is also similar to that of the embryo developed by a fertilized egg, with complete structures such as embryo, radicle and hypocotyl, just like a seed, also called cell embryo.
Factors affecting anther culture The success and success rate of pollen induction are affected by many factors, among which the selection of materials and the composition of culture medium are the main factors.
? Physiological status of parents: Early pollen anthers are more likely to produce pollen plants than late pollen anthers, so the early flowering period of roses is chosen.
? Suitable pollen development stage: Generally speaking, in the mononuclear stage, when the nucleus moves from the cell center to the cell side, the success rate of anther culture is the highest.
? Bud: Choose a completely unopened bud.
? The growth conditions of parent plants, low temperature pretreatment of materials and inoculation density all have certain effects on the induction success rate.
? Selection of materials: When selecting anthers, it is generally necessary to determine whether the pollen in them is in a suitable development stage through microscopic examination. The most commonly used method to determine the pollen development period is magenta acetate method. However, the pollen core of some plants is not easy to be colored, so the pollen core can be dyed blue-black by baking blue chrome alum.
? Disinfection of materials
? Inoculation and culture: Sepals and petals of sterile flower buds are removed under aseptic conditions, and anthers are immediately inoculated on the culture medium. When stripping the anther, try not to damage the anther (otherwise, callus will easily grow from the injured part after inoculation), and at the same time, completely remove the filament, because the anther connected with the filament is not conducive to the formation of callus or embryoid. Usually, each bottle is inoculated with 7 ~ 10 anthers, and the culture temperature is controlled at about 25℃ without illumination. Generally speaking, seedlings need light after 20 ~ 30 days of culture. The callus was transferred to the differentiation medium in time for further differentiation into regenerated plants. If the anther cracks and releases embryoids, a large number of young plants will be produced in one anther. After anther dehiscence, young plants must be separated as soon as possible and transplanted to new media, otherwise these plants will be difficult to separate. It is necessary to further identify and screen cultivated plants.
The similarity between plant tissue culture technology and anther culture technology is that the preparation method, aseptic technology and inoculation operation of culture medium are basically the same. The difference between them is that the selection of materials for anther culture is very important, and it is necessary to explore suitable buds in advance; Callus or embryoids released after anther cracking should also be replaced in time; Anther culture requires a stricter medium formula. All these make the difficulty of anther culture greatly increased.
The formula for calculating the moisture content (%) of the sample is as follows:
(container and sample mass before drying-container and sample mass after drying)/sample mass before drying
? Growth of Mucor: Conditions: Put the bean curd into a cage, control the temperature in the cage at 15 ~ 18℃ and keep a certain temperature.
Source: 1. Mucor spores in air, 2. Directly inoculate excellent Mucor species.
Time: 5 days
? Pickling: Put the tofu covered with Mucor into the bottle in layers and orderly, and add salt in layers. With the increase of layers, the amount of salt will increase, and the salt near the surface of the bottle mouth will be thicker. The curing time is about 8 days.
? When curing with salt, we should pay attention to controlling the amount of salt: the concentration of salt is too low to inhibit the growth of microorganisms, which may lead to tofu spoilage; Excessive salt concentration will affect the taste of sufu.
? Action of salt: 1. Inhibit the growth of microorganisms and avoid corruption. 2. Precipitation of moisture makes tofu hard and not brittle in the post-production process. 3. Seasoning to give sufu the necessary salty taste. 4. Extract the protease from the mycelium of Trichoenzyme.
? Preparation of marinated soup: marinated soup is directly related to the color, aroma and taste of sufu. Canned soup is made of wine and various spices. The content of wine in brine soup is generally controlled at about 12%.
? The function of wine: 1. Prevent contamination by miscellaneous bacteria and corrosion. 2. Combine with organic acid to form ester, which endows sufu with flavor. 3. The alcohol content in the later period of sufu is closely related to the length of fermentation time. The higher the alcohol content, the greater the inhibitory effect on protease and the longer the maturity time of sufu. If the alcohol content is too low, the protease activity is high, which will accelerate the hydrolysis of protein, and the mixed bacteria will multiply quickly. Tofu is perishable and hard to block.
? Function of spices: 1. Seasoning function II. Sterilization and disinfection function. Participate in and promote the fermentation process.
? Prevention of contamination by miscellaneous bacteria: ① The glass bottles used for curing sufu should be disinfected with boiling water after washing. (2) When bottling, the operation should be quick and careful. Tofu is placed neatly, and after adding the marinated soup, the bottle mouth should be sealed with tape. When sealing the bottle, it is best to pass the flame of alcohol lamp to prevent the bottle from being polluted.
trouble shooting
(1) Use the biological knowledge you have learned to explain how tofu grows white hair.
The white hairs of tofu are white hyphae of Mucor. Strictly speaking, it is upright hyphae, and there are creeping hyphae in tofu.
(2) Why do you want to sprinkle a lot of salt to pickle hairy tofu?
Salt can prevent the pollution of miscellaneous bacteria and prevent tofu from spoilage.
(3) What kind of tofu we usually eat is suitable for making sufu?
Tofu with a water content of about 70% is suitable for making sufu. Sufu made from tofu with high water content is not easy to form.
(4) When eating fermented bean curd, you will find that there is a dense layer of things outside the fermented bean curd? Skin? . This floor? Skin? How is it formed? Is it harmful to human body? What is its function?
? Skin? It is a mycelium (creeping mycelium) growing on the surface of tofu in the early stage of fermentation, which is harmless to human body. It can form sufu. Body? Shape fermented bean curd.
Senior one biology chromosome review handout nouns:
1. Chromosome variation: The variation of chromosome structure or chromosome number can be seen under the optical microscope.
2. Chromosome structural variation: refers to changes such as deletion (disappearance of one chromosome fragment), addition (addition of one chromosome fragment), inversion (inversion of one chromosome fragment 180o) or translocation (transfer of one chromosome fragment to another non-homologous chromosome) in a cell.
3. Chromosome number variation: refers to the change of chromosome number in cells.
4. Genome: A group of chromosomes with different shapes and sizes in germ cells is generally called genome. There are several chromosomes with the same morphology in the cell, that is to say, there are several chromosome groups.
5. Diploid: Any individual whose somatic cells contain two sets of chromosomes is called diploid. Such as human fruits, flies and corn. Most animals and higher plants are diploid.
6. Polyploid: Any individual with more than three chromosomes in a somatic cell is called ~. For example, potato contains four chromosome sets, called tetraploid, and common wheat contains six chromosome sets, called hexaploid (common wheat somatic cell 6n, 42 chromosomes, one chromosome set 3n, 2 1 chromosome). ),
7. Diploid: Any individual with a genome in a somatic cell is called ~.
8. Haploid refers to an individual whose somatic cells contain the chromosome number of gametes of this species.
9. Anther culture in vitro: Hybridize varieties with different advantages, culture flowers of F 1 in vitro to form haploid plants, double haploid chromosomes with colchicine, and select qualified individuals as seeds.
Statement:
1. Chromosome variation includes chromosome structure variation (the number and arrangement order of genes on chromosomes change) and chromosome number variation.
2. Polyploid breeding: a. Reason: During mitosis, after chromosomes are copied, cell division stops due to drastic changes in external conditions, and the number of chromosomes in cells doubles. When the spindle is destroyed in the late stage of cell mitosis, the cell can return to interphase without going through the late stage, thus doubling the number of chromosomes in the cell. ) b, characteristics: high nutrient content; However, the development is slow and the seed setting rate is low. C application of artificially induced polyploidy in breeding: common methods-treating germinated seeds or seedlings with colchicine; The function of colchicine-colchicine inhibits the formation of spindle; Example: Triploid seedless watermelon (tetraploid watermelon is obtained by treating diploid watermelon seedlings with colchicine; Triploid watermelon seeds were obtained by crossing diploid watermelon with tetraploid watermelon. Triploid watermelon has association disorder and cannot produce normal gametes. ), octoploid triticale.
3. Haploid breeding: Formation reason: It is formed by direct development of germ cells without fertilization. For example, drones in bees are haploid animals; Plants directly developed from corn pollen are haploid plants. Features: weak growth and development, high infertility rate. The common method of haploid breeding: anther culture in vitro. Significance: greatly shorten the breeding age. The advantage of haploid is that it greatly shortens the breeding cycle and is fast. Haploid plants are homozygous diploids after artificial doubling of chromosomes, and their offspring will not separate, and will soon become stable new varieties, and the cultivated seeds are absolutely pure.
4. Generally, several genomes are called ploidy. If an individual develops directly from the gametes of this species without fertilization, then no matter how many genomes it has, it is called. Haploid? .
5. The methods of biological breeding are summarized as follows: ① Mutation breeding: treating organisms with physical or chemical factors, inducing gene mutation, increasing mutation frequency and breeding excellent varieties. Example-Culture of Penicillin-producing Strain. (2) Cross breeding: the required fine varieties are cultivated by using the genetic recombination generated by biological hybridization and combining the fine characters of two parents. Example —— A new type of dwarf rust-resistant wheat was bred by crossing a long rust-resistant wheat with a short rust-resistant wheat. (3) Haploid breeding: haploids were obtained by anther culture in vitro, and then the number of chromosomes was doubled by artificial induction to obtain homozygotes quickly. Haploid breeding can greatly shorten the breeding cycle. (4) Polyploid breeding: a method of obtaining polyploid plants by artificial methods and then breeding new varieties by using their variation. Colchicine is usually used to treat germinated seeds or seedlings to obtain polyploid plants. ) example-cultivating triploid seedless watermelon and octoploid triticale (4n offspring are obtained by crossing 6n common wheat and 2n rye, and then the chromosome number is doubled to 8n by colchicine, which is octoploid triticale).