How to carry out tissue culture of family plants?

It is impossible to plant tissue culture without the most basic conditions and articles, and it is not impossible to make rational use of some of the simplest instruments at that time.

First, the most necessary items

1, 1 domestic pressure cooker, preferably larger and loaded with more things. Used for disinfection of culture medium, sterile water, etc.

2. There is a 1 inoculation box (sterile box), which needs to be made by yourself, using some boards, a piece of glass and two feet of cloth. Used for inoculation and transfer.

3. 1 The medical balance is 500g or smaller, which is mainly used for weighing agar, sugar and other items.

4. Buy stainless steel pot or aluminum pot (the kind that cooks soup and noodles). Used for cooking medium.

5. Spoons for stirring and repackaging the culture medium are used for cooking and repackaging the culture medium.

6. Several culture bottles,

7. Many cotton, gauze and thread are used to make cotton plugs, and cotton plugs are used to make bottle stoppers. It should be noted that ordinary cotton-padded clothes should be used for cotton instead of absorbent cotton used in hospitals, which is easy to wear, block and pollute.

8. Some kraft paper and rubber bands are used to wrap bottle caps and wrapping paper.

9.65,438+0 alcohol lamp, 65,438+0 scalpel, several blades, two tweezers, one measuring cylinder with 65,438+00 ml and 65,438+000 ml respectively, 65,438+02 ml pipette and some absorbent cotton.

Second, how to solve distilled water and alkaline drugs

The water used in the culture medium is always very important in the minds of ordinary people, but it is actually an unnecessary worry. Cold boiled water and clean river water are used, which does not affect the breeding effect. Pure water sold in the market is the ideal water for general culture medium. The most commonly used, necessary, used, just buy the following.

1, a large number of elements in MS medium (home or so-called pocket tissue culture room culture generally adopts MS basis. ) There are five kinds. You can also buy commercially available MS culture medium.

(1), ammonium nitrate

(2) Potassium nitrate

(3), calcium chloride

(4) Magnesium nitrate

(5), potassium dihydrogen phosphate

2. Alcohol

3. Precision test paper (PH 5.4-7.0)

4, bleaching powder or bleaching essence

5. Agar

6. refined sugar

7. Formalin

8, potassium permanganate (can go to the hospital or drugstore to buy)

9. Trace elements, iron salts, vitamins and hormones are rarely used. For example, some hormones, vitamins, drugs, injections, tablets, the content is relatively accurate, you can make the proportion according to your own needs.

Discussion and Suggestions on Tissue Culture by Family Environment Enthusiasts Plant tissue culture technology, especially rapid propagation, can not be completed by professionals and professional laboratories alone. As lovers who are interested in succulents and tissue culture, they can also prepare their own test-tube seedlings on the basis of making full use of family conditions. During the cultivation of succulents, we have recognized some rare horticultural varieties, such as Vientiane, Jade Fan, Shroud and Gao Wen Eagle Claw. Or tuberous plants, such as colored olives and Helu petunias, and even planets and stone oak. The conventional propagation coefficient is low, and tissue culture can be used to solve the propagation problem and obtain a considerable number of seedlings. Tissue culture can also be used for aseptic sowing of plants with difficult seed germination, such as the flower-like year. In the process of tissue culture, we can not only have a deeper understanding of the cultivated plants, but also have great significance for routine culture. Many experts engaged in organizational training did not specialize in organizational training at first, but made brilliant achievements with interest and exploration spirit. The key is that fans are interested, observant and meticulous in operation, which can make up for non-professional defects and make full use of various available resources in daily life, enough for fans to complete organizational training in a family environment. At the same time, it not only satisfies its own interests, but also has certain economic benefits, and can even make innovations and inventions.

A, how to solve the site and basic equipment:

Without certain equipment, tissue culture is impossible. Tissue culture equipment in regular laboratories is expensive and not suitable for family consumption, but it would be great if there is a way to buy cheap laboratory equipment. If you can't get the laboratory equipment, you might as well make some simple equipment yourself.

1. Main equipment for aseptic operation-inoculation box and pressure cooker:

Inoculation box: The main operation of plant tissue culture needs to be carried out in a relatively closed sterile environment. Then we must first create a closed environment, and we can stick a small inoculation box with some cheap materials such as wood, plastic board, plexiglass and aluminum alloy skeleton. Specific parameters can be found in a book "Edible Mushroom Cultivation", and it is enough to imitate the inoculation box for growing mushrooms. It should be noted that the inoculation box for tissue culture requires high sealing, and decaying wood and rusty materials should be avoided as much as possible. Two lamps are placed on the top of the box: a 20w ultraviolet lamp and a 20w fluorescent lamp. The periphery of the box is made of glass as far as possible, because it is easy to observe, and sometimes you can borrow the inoculation box as an incubator, and the periphery is made of glass, which is more convenient for observation and cultivation.

Pressure cooker: mainly used for sterilization of culture medium and other materials. Medical autoclaves are generally used in the laboratory, but in the family, we should make full use of the pressure cooker used in the kitchen. The pressure of the autoclave is lower than that of the sterilizer, but we can still achieve good results by extending the sterilization time appropriately. For example, sterilizing the culture medium for 40 minutes (sterilizer for 20 minutes) can basically achieve the sterilization effect; Sterile water is made by sterilization in several stages. It can be pressed in an autoclave for 40 minutes, and then pressed for 20 minutes after standing for 24 hours.

2. Location:

It's easy for people. The general requirement is that the place for tissue culture should be as clean as possible, reduce air flow, and preferably have some sunshine. For example, study and writing room can be used for aseptic operation.

You can borrow the culture medium prepared by the kitchen, but the utensils used must be separated from the kitchen utensils. Generally speaking, almost all chemicals in tissue culture are harmless to human body, except a few hormones and disinfectants. Toxic substances need to be carefully preserved to avoid danger.

3. Auxiliary equipment:

Mainly refers to the refrigerator, which is used to store chemicals and culture media. The balance can be a tray balance with a sensitivity of100g, and can even be replaced by a traditional Chinese medicine scale. Cultural shelf, this is simpler. Aluminum alloy glass incubator can meet the requirements. If the sunlight is insufficient, it can be supplemented by fluorescent lamps.

4. Chemical reagents:

Chemical reagents are essential, because the core of tissue culture is the composition of culture medium, not simple aseptic operation. Need to buy some chemicals with large dosage, such as a large number of elements (ammonium nitrate, potassium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, etc. ), potassium permanganate, formaldehyde, alcohol, sugar, agar and so on. Trace elements and organic compounds don't need to be purchased, because their dosage is very small. We can use vegetable juice or potato juice instead. Hormone is essential, and its weighing needs the help of analytical balance, but this is not allowed by family conditions. If possible, you can use relational units instead of mother liquor, and you can take some at a time. The best way is to go to an agricultural store (selling pesticides and fertilizers). They generally sell agricultural hormones, some of which are packed in ampoules and added to the culture medium according to a certain proportion. In recent years, some chemical companies began to produce plant tissue culture medium powder, which is convenient only by weighing a certain amount of water. For example, the ms powder produced by Pantech Chemical Company only needs to weigh 40 grams, add 1 liter of water and heat it in the microwave oven for 5 minutes, which is quite simple.

The disinfectant is usually mercuric chloride (highly toxic). If it is not easy to get, you can use bleach instead.

When adjusting the pH value, soda ash and white vinegar, which are commonly used at home, can be used.

5. Culture containers and glassware:

The container for tissue culture is not very demanding, as long as the glass color is transparent (sodium glass is not allowed).

We can go to the garbage station to pick up some old jam bottles. The better bottle is Axiangpo Chili sauce, which is also used in our laboratory. The sealing film of the bottle can be made of polypropylene plastic. If it's hard to find, you can use tin foil or even an instant noodle bag special for microwave ovens, fold it into two layers and fix it with rubber bands.

Other glassware, such as beakers and glass sticks, can be replaced by household cups and chopsticks; Some measuring instruments, such as measuring cylinders and pipettes, are best bought in glass instrument stores. I'm afraid it doesn't cost 20 yuan to configure an excellent tissue culture glass instrument.

6. Others:

There are also some supplies that are necessary. For example, if you need to buy a precise ph test paper (5.4~7.0), you probably need 1.5 yuan. You don't need to buy alcohol lamps. Use an ink bottle with a glass ring and a cotton core. Finally, use the bottle cap of Nongfu Spring mineral water as a light bulb. This alcohol lamp is small and suitable for inoculation box. Knives, scissors and tweezers are necessary. All the tools for the flower market 10 yuan are done. Pay attention to choosing smaller tools.

As for water, distilled water should be used, but the pure water we drink at home is purer than distilled water, so it is enough to borrow some drinking water. We use Master Kong purified water to cultivate animal cells, and the results are very good, not to mention plant cell culture.

The above are the basic equipment and reagents for tissue culture, mainly highlighting their related substitutes, because as a rapid propagation in tissue culture, its requirements are quite low. Our family culture is different from factory culture, and factory production of tissue culture seedlings should pursue high proliferation rate, so its requirements are more accurate and standard. Our family training doesn't need to be so precise, as long as we can achieve the goal of successful training. Of course, in order to improve the proliferation rate and the quality of test-tube seedlings, we must be as close as possible to the instruments and equipment in the laboratory, and the substitutes always have certain defects.

(a) Necessary facilities and substitutes

1, substitution

Household refrigerator can be used to store the mother liquor of culture medium (4℃) and drugs (such as biological regulators) that need to be preserved at low temperature.

Pressure cooker: it can be used for disinfection and sterilization of tissue culture vessels such as culture medium, sterile water and glassware. If the hardness of the water used is high, it can be disinfected with cold boiled water. Avoid scaling on the surface of disinfected articles.

Stainless steel pot or aluminum pot: It can be used for disinfection and sterilization of culture medium, dissolved agar (as a water bath pot) and tissue culture equipment.

Brush waste oil drum: it can be used to store distilled water.

Small white porcelain plate: it can be used for inoculation and disinfection solution (if disinfection solution is put, don't eat it again to avoid poisoning).

Fluorescent lamp: it can be used as a supplementary light source in tissue culture.

Wash away discarded cans: It can be used for tissue culture instead of conical bottles and test tubes.

Tissue culture can be done in your own room. When preparing culture medium and inoculation, don't do it when there are many family members. If air conditioning has been installed indoors, it will be beneficial to primary culture, secondary culture and even seedling transition.

2. Homemade household appliances

(1) Production of inoculation box: The clean workbench is very valuable equipment in the process of tissue culture. If the self-made inoculation box is used instead, it can greatly save money and be beneficial to popularization.

The materials of the self-made inoculation box can be plywood, fiberboard, glass (3 mm thick), battens (keels for decorating houses), even cartons (boxes with slightly better quality, such as TV boxes), or plexiglass.

The size of the inoculation box can be determined according to their family conditions. Too small, inconvenient to operate, but relatively easy to disinfect, occupying less space; It is easy to make a big one, but the disinfection work is not easy, and it covers a large area. Generally speaking, it is 70 cm long and 45 cm wide. 50 cm height is more suitable.

(2) Production of incubator: the incubator can replace the culture room and can also be used for transitional seedlings. It can be made of glass or rectangular fish tanks. So it can be made by glass bonding. Its size can be determined according to the area of your room and the amount of tissue culture.

3. Need to buy equipment

Ordinary balance (500g): used for weighing and preparing culture medium drugs, or weighing and preparing culture medium drugs, trace elements and biological regulators by weighing gold, silver and traditional Chinese medicines; Or when buying trace elements and biological regulators, buy packaged ones. Alcohol lamp 1: used for burning, disinfection and sterilization during inoculation. Funnel (can also be used to buy bottled edible oil for distribution): used for sub-packaging culture medium. 2 long tweezers: used for inoculation. Two scalpels and several blades: for inoculation. 10,50, 100 ml measuring cylinder: used for preparing culture medium. Long horn spoon: used for preparing culture medium and taking medicine. 1, 2,5 ml pipette: 1 used to prepare culture medium.

High temperature resistant plastic film or kraft paper: used to wrap the corners of culture bottle mouth and self-made inoculation box. Rubber ring: used to tie the bottle mouth of culture. Absorbent cotton: used for disinfection of alcohol cotton balls for operators and tissue culture equipment. PH (5 ~ 7) test paper 1: When used, it can be cut into small strips to detect the pH value of the culture medium. Alcohol 1 bottle: used for alcohol lamp and disinfection. Bleaching powder 1 bottle: used for disinfection. Formalin 1 bottle: used to disinfect the inoculation box (2 ~ 10 hours before each operation, pour 10 ~ 20 ml formalin into a small white porcelain dish and take it out before operation). Drugs needed for MS culture medium; Used to prepare culture medium. Hydrochloric acid and sodium hydroxide: used to adjust pH value. If you buy 1 ultraviolet lamp, indoor and inoculation box disinfection is ideal.

(II) Operational considerations

Family surgery is different from unit surgery, so we should pay attention to the following matters: pay attention to safety and keep drugs properly. Especially the elderly and children; Carefully and strictly disinfect during the operation. During inoculation, operators should wear masks and working caps to avoid electric fans and windy and dusty weather, and family members should avoid long-term operation. Avoid domestic pets from entering the workplace before and after disinfection; Drug weighing should be accurate; If the family does not have temperature control (such as temperature regulation and air conditioning), it should be avoided in midsummer and winter; Tissue culture of Anthurium andraeanum should be gradual. Flowers that are easy to cultivate can be made first, and flowers that are difficult to make can be made after experience.

1. Preparation of culture containers and glass instruments:

To prepare the culture medium, we must first prepare the culture bottle and the glass instrument for preparation. Culture bottles generally use various jam bottles. First, soak the bottle with detergent for 4-8 hours, and then rinse it with running water several times. Other glass instruments also need to be cleaned in this way until the glass wall becomes a water film instead of water droplets. The cleaned glass container should be turned upside down, and the water in the bottle should be emptied.

Clean containers should be dust-proof and put in glass cabinets as far as possible. It is easier to cover the container with a clean towel.

The pipette should be cleaned by sucking the ball and repeatedly sucked with distilled water until it is clean. Put the pipette in a long tube for use. Generally, pipettes can only be used once, that is, they need to be cleaned, so several pipettes need to be prepared. Suggestions: 10ml/2, 5ml/2, 1ml/5.

2. Preparation of culture medium:

The classic culture medium for tissue culture is ms formula, which is basically divided into: macroelements, microelements, iron salts, organic compounds, phytohormones, sugars and supports. The dosage of these ingredients is in the milligram level, which is difficult to weigh with ordinary balance. In order to solve this problem, we can enlarge the dosage of each component in proportion to make mother liquor, and add it in a certain proportion when using it.

The preparation scheme of ms culture medium mother liquor is as follows:

Macroelement: ammonium nitrate 66.0g;

76.0g of potassium nitrate;

Anhydrous calcium chloride13.3g;

Magnesium sulfate heptahydrate14.8g;

6.8 grams of potassium dihydrogen phosphate.

Dissolve the above substances respectively, then combine them to constant volume to 1l, and take 25ml of standard ms medium for each preparation1l.

Iron salt: 5.56 grams of ferrous sulfate heptahydrate;

7.46 grams of disodium edetate.

Heat and dissolve the above two kinds respectively, mix them, adjust the volume to 65438 0 liters, adjust the ph to below 5.5, and take 5 ml per liter of MS.

The amount of trace elements and organic compounds is too small. For simplicity, we can use vegetable extract or potato extract instead. Usually, we are used to boiling 100g spinach with 100ml water for 1 0min, filtering to retain the filtrate, and adding 20~50 ml of extract per1liter of ms medium. Similarly, add 200ml water to100g potato with skin to boil for15min, and filter the filtrate, and 50 ~100ml can be added per liter of MS. ..

After adding the above ingredients, 30 grams of sugar and 7 grams of agar are needed for each liter of ms medium.

It should be noted here that both sugar and agar are variable quantities and should be adjusted as needed. In addition, carrageenan used to make jelly can also replace agar, which has better transparency.

After adding the above ingredients, the volume is about 800 ml, and the agar is heated by microwave oven until it is completely dissolved. At this time, the required plant hormones are added (usually 0. 1% solution is prepared, so every 1 ml in the culture medium is converted into 1ppm). Then add water to the volume of 1 100 ml (1. 1 liter). The reason for adding 0. 1 liter is to offset the loss in repackaging and disinfection. Finally, adjust the ph value to 5.8~6.0 with acid and alkali.

3. Prepare culture medium with ms powder:

At present, some domestic chemical companies have developed simple ms culture medium powder, which greatly simplifies the preparation of culture medium, and selects the type of culture medium according to the instructions of different companies. Let's take the raw powder of ms as an example: the raw powder of ms culture medium, including a large number of elements, trace elements, iron salts, organic compounds, sugar and agar, is generally weighed according to the use method, heated and dissolved in a microwave oven, and added with hormones to a constant volume of1100 ml (1.1)

4. Sub-packaging:

Sub-package the prepared culture medium in a culture container, and pay attention to sub-package while it is hot, because agar will solidify below 40 degrees. The culture medium in each culture bottle is about 1/6~ 1/5 of the bottle volume, so be careful not to hang the culture medium on the outer wall of the bottle, which is easy to cause pollution. After repackaging, seal it with polypropylene film or bag filled with instant noodles and fix it with rubber ring (rubber ring is heat-resistant, try to choose bicycle inner tube).

5. Disinfection:

It is suggested to buy some "sterilization effect test paper" from medical device stores for disinfection of domestic pressure cookers, which can indicate the sterilization effect and will change color when the sterilization effect is achieved. Put the test paper and culture medium into the pot at the same time, heat until a large amount of steam comes out, cut under pressure, and keep it on low fire for 30~40 minutes.

After sterilization, naturally cool, take out the culture medium and put it in a cool and dust-free place for later use.

6. Preparation of inoculation box:

The newly made inoculation box must be clean, and there should be no dead angle as far as possible. 2 hours before each use, put all the items needed for operation into the inoculation box, then add 3-5g potassium permanganate into the small beaker and put it into the inoculation box. Pour 2-3ml of formaldehyde solution into the beaker in the box, and formaldehyde vapor will appear after a few seconds, so that all items in the inoculation box can be disinfected for the first time. At the same time, turn on the ultraviolet lamp for irradiation. The ultraviolet lamp is produced by high pressure penetrating mercury vapor, and the ultraviolet ray is sterilized twice. After ultraviolet irradiation for about 15 minutes, oxygen will be excited to form ozone, which is the third line of defense for sterilization. After three times of sterilization, the inoculation box can basically meet the aseptic standard.

Before the operation 15 minutes, pour 5 ml of concentrated ammonia water into the beaker just used to evaporate formaldehyde. Because formaldehyde is toxic to human body, ammonia water can form a solid substance called "hexamethylenetetramine" with formaldehyde, thus neutralizing the irritating steam of formaldehyde. At this time, do not turn off the ultraviolet lamp, continue to irradiate until it is turned off 5 minutes before the operation, keep it dark for 5 minutes, and then turn on the fluorescent lamp for the operation. The reason for keeping dark for 5 minutes is that ultraviolet light kills microorganisms by acting on thymine of dna to form tetramer, but microorganisms have a light-reactivating protein, which can reduce thymine tetramer in the presence of visible light and revive microorganisms. This effect is called "light resurrection", so it is necessary to keep it dark for a few minutes after turning off ultraviolet light to avoid light resurrection.

How to carry out tissue culture at home

prepare

Due to the limitation of conditions, it is impossible to configure a large number of nutrient media for family tissue culture, and it is generally appropriate to configure 1 liter each time. After boiling, measure with PH test paper, subpackage into 35-40 bottles, plug them with cotton plugs and wrap them with wrapping paper. (Cotton slivers must be wrapped with cotton, not absorbent cotton, and then wrapped with gauze and tied with cotton thread. The tightness of the tampon should be such that the portable tampon bottle will not slide. ) and then put it into an autoclave for high pressure sterilization. When steam is ejected from the nozzle of the autoclave, the pressure valve is locked, and the air ejected from the pressure valve starts to count, and it is maintained at 15-20 minutes, and then the fire is turned off. After the pressure disappears, open the pot cover, take out the nutrient medium and put it in the inoculation box for later use. In the first experiment, when no mold growth was observed, the sterilized culture medium should be left for three to five days before use. If mold grows, it means that the sterilization time is not enough, and the sterilization time needs to be increased appropriately.

vaccination

Put the sterilized culture medium, sterile water, disinfectant and used equipment into the inoculation box. Because the volume in the inoculation box is relatively small, all the used items should be placed in the corresponding position in an orderly manner, and can not be placed at will. The humidity in the box is high, so a lighter must be used instead of a match to light the alcohol lamp. Special attention should be paid to preparing containers for sewage and dirt, as large as possible, because it is not allowed to take things out of the box during operation. After all the articles are put away, put the magnetic cup with 10-20 ml formalin into the box (it is best not to use a glass, because the reaction temperature is high and it is easy to burst. ), pour 2-5g potassium permanganate (PP powder) to make its steam fill the box, so as to achieve sterilization effect. At this time, the vent hole and operation hole of the inoculation box should be closed to avoid the rapid spread of formaldehyde vapor. It takes about 5- 10 hours to start inoculation after the steam in the box is exhausted.

During inoculation, remove the seals sealed on the ventilation holes and operation holes of the inoculation box, take out the magnetic cup for fumigation, send the inoculation materials into the inoculation box, turn on the ultraviolet lamp, turn off the ultraviolet lamp and turn on the illuminating lamp after 15 minutes, and then start working. During the operation, we must establish the concept of sterility, treat everything as bacteria, and always pay attention to prevention. Work is due to the high temperature and humidity in the box, so hands are easy to sweat, so after each bottle of inoculation, you should wipe your fingers with alcohol, and you can also wear finger covers. In addition, pay attention to fire prevention when operating, because the space is small, and it is easy to burn your fingers or cause alcohol fire if you are not careful. When opening the cork, place the bottle mouth above the alcohol lamp, hold the tail of the tampon with the ring finger and little finger of the right hand, and gently pull out the tampon. The tampon can't be put on the items in the box. After inoculation, burn the bottle mouth on the alcohol lamp and cotton plug, then gently put it above the flame of the alcohol lamp, tie the wrapping paper, mark the variety, inoculation date and number with pencil, and then repeat.

In fact, compared with clean bench, people feel much more comfortable at work. The pollution rate is also very low, and there is almost no pollution after skilled operation. After inoculation, there was no difference in callus differentiation, seedling differentiation and growth status between inoculation and ultra-clean platform.

cultivate

It is impossible to control the cultivation conditions under the amateur conditions at home, so we should make full use of the natural conditions. Inoculated culture bottles can be placed in places with strong scattered light, such as bookcases and desks by the window, but direct sunlight should be avoided. Generally, it can grow normally at room temperature of 22-28 degrees Celsius. It is not a special variety and does not need special care. If the room temperature is too low, you can make a simple incubator with cardboard, wood strips and plastic film, and put one or two 15-25 watt white woven lamps in it, which can not only supplement the light, but also increase the culture temperature. When the temperature is too high in summer, you can put it in an air-conditioned room if possible. If there is no air-conditioned room, it is difficult to cool down, but most test-tube seedlings can tolerate it and will not die. When the seedlings grow up, it is undoubtedly a pleasure to put them on the bookcase or workbench and feel the vibrant little creatures they have cultivated.

cultivate

When the seedlings grow to a certain extent, some rooting media can be configured, and the seedlings can be transferred and cultivated to take root. When a root system grows, it can be planted out of the bottle. When raising seedlings outside the bottle, we must pay attention to the following aspects: first, we must appropriately reduce the temperature; The second is to increase humidity; Third, we must strictly control the harm of miscellaneous bacteria; The fourth is to ensure that the planting medium is loose and breathable; The fifth is to ensure proper lighting. I usually use metatarsals, sawdust and rice husk as mixed substrates. After planting the seedlings, I sprayed the roots with chlorothalonil and covered them with plastic film. A few seedlings are simply covered with glass. After about 15 days, the mulch can be removed for normal management.

In fact, it is not a very difficult thing to organize training at home. It's easy to do as long as you use your head and think of something. Everything is difficult at the beginning, and the method is always more difficult than it is. From 65438 to 0974, I was the director of a county tobacco research institute, but the original conditions did not allow, and the research funds were very tight. At the beginning, the research funding was only 3000 yuan a year. In that year, I made anther haploid of tobacco, anther culture of rice, endosperm culture of citrus, shoot tip tissue culture of black-skinned sugarcane, callus culture of Panax notoginseng, sterile culture of black knot grass (Dendrobium nobile) seeds and somatic cell fusion. We did it in this way. At that time, many research institutes in Beijing and Shanghai came to see it and were very surprised. They thought that precious tissue culture seedlings were thrown everywhere by us. Incredibly, they can't do it, but they can do it in a very remote mountain area. I don't mean to brag about myself, but to tell my friends who want to engage in this industry not to underestimate their energy and not to be superstitious about authority. As long as they have confidence, they will do it in strict accordance with scientific laws. Nothing is impossible.

Selection and effect evaluation of domestic tissue culture medium

Choosing suitable culture medium is the basis of successful plant tissue culture.

The selection of suitable culture medium is mainly considered from the following two aspects: first, the basic culture medium; The second is the concentration and relative proportion of various hormones.

MS medium is suitable for most dicotyledonous plants, B5 and N6 medium are suitable for many monocotyledonous plants, especially N6 medium is very effective for gramineous plants such as wheat and rice, and white medium is suitable for root culture.

First of all, try these media to carry out preliminary experiments, which can avoid detours and greatly; Reduce the consumption of time, manpower and material resources. After a series of preliminary tests, some components can be adjusted in a small range according to the actual situation.

When making adjustments, you can refer to the following situations. First, when a compound is used as nitrogen source, nitrate is better than ammonium salt, but using nitrate alone will make the PH of the culture medium drift to alkaline direction. This drift will be overcome if nitrate and a small amount of ammonium salt are added at the same time. Second, when the supply of some elements is insufficient, the cultivated plants will have some symptoms, which can be adjusted according to the symptoms. For example, when nitrogen is insufficient, the cultured tissues often show anthocyanin colors (red and purple), and it is difficult to see the differentiation of vessel molecules inside the callus; When nitrogen, potassium or phosphorus are insufficient, cells will obviously overgrow and form some very fluffy and even transparent callus; When iron and sulfur are deficient, the tissue will lose its green color, cell division will stagnate, and callus will appear brown aging symptoms; When boron is deficient, cell division tends to be slow and excessively elongated; When manganese or molybdenum is deficient, cell growth is affected.

The effect of exogenous hormones in culture medium will also cause some similar situations in culture, so we should analyze it carefully and not draw conclusions easily.