Tissue culture of plants is impossible without the most basic conditions and articles, and it was not impossible to make rational use of some of the simplest appliances at that time.

First, the most necessary items

1,. Household pressure cooker 1 It's better to have a bigger one with more things in it. Used to disinfect culture medium, sterile water, etc.

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

3. The medicinal balance 1 rack is preferably 500g, but it can be smaller. It is mainly used to weigh agar, sugar and other items.

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

5. A spoon for stirring and repackaging the culture medium is used for cooking and repackaging the culture medium.

6, a number of bottles for culture,

7. A number of cotton, gauze and thread are used to make cotton stoppers, which are used to make bottle stoppers. It should be noted that cotton should be made of ordinary cotton-padded clothes, not absorbent cotton used in hospitals, so it is easy to wear plugs and pollute.

8. Some kraft paper and rubber bands are used for wrapping bottle heads and wrapping paper.

9. Alcohol lamp 1 lamp, scalpel 1 handle, several blades, two tweezers, one measuring cylinder for 10ml, one measuring cylinder for 100ml, 2ml pipette 1 piece, and several medicinal cotton.

Second, how to solve distilled water and basic 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 cultivation effect. Pure water sold in the market is an ideal water for general culture media. The most commonly used, necessary and used ones only need to buy the following ones.

1, a large number of elements in MS culture medium (family or so-called pocket tissue culture room culture generally uses MS base. ) ***5 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. White sugar

7. Formalin

8, potassium permanganate (you can go to the hospital or medicine store to buy)

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

Discussion and suggestion on tissue culture of enthusiasts in family environment Plant tissue culture technology, especially rapid propagation, can not be completed only by professionals and professional laboratories. As a lover who has a strong interest in succulents and tissue culture, he can also prepare his own test-tube seedlings on the basis of making full use of family conditions. During the cultivation of succulents, we realized that some rare horticultural varieties, such as Vientiane, Jade Fan, Shouyi, Gaowen Eagle's Claw, etc., or tuber plants such as Colorful Olive and He Lu Petunia, even Planet and Lithocarpus, etc., have low conventional propagation coefficient, and they can all adopt tissue culture to solve their propagation problems and get a considerable number of seedlings. Tissue culture can also be used for aseptic seeding of plants with difficult seed germination, such as Fantasia Tianwai. In the process of tissue culture, we can not only have a deeper understanding of the cultivated plants, but also have great significance for conventional cultivation. Many experts engaged in tissue culture did not specialize in tissue culture at first, but made brilliant achievements with interest and exploration spirit. The key point is that fans have strong interest, extraordinary observation and meticulous operation, which can make up for non-professional defects and make full use of various available resources in daily life, which is enough for fans to complete organizational training in a family environment. At the same time, it not only satisfies one's own interests, but also has certain economic benefits, and even can make innovations and inventions.

A, how to solve the site and basic equipment:

Tissue culture cannot be carried out without certain equipment. 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 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 subject operation of plant tissue culture needs to be carried out in a relatively closed aseptic environment. Then we must first create a closed environment, and we can use some cheap materials such as wood, plastic board, plexiglass and aluminum alloy skeleton to glue a small inoculation box. The specific parameters can be found in a book on "Edible Mushroom Cultivation", and one can be made by imitating the inoculation box for growing mushrooms. It should be noted that the inoculation box for tissue culture requires high tightness, so as to avoid decaying wood and rusty materials as much as possible. Two lamps are placed at the top of the box: a 20w ultraviolet lamp and a 20w fluorescent lamp. The periphery of the box should be made of glass material as far as possible, because it is easy to observe, and sometimes the inoculation box can be borrowed as an incubator, and the periphery is made of glass material, which is more convenient for observation and culture.

Pressure cooker: mainly used to sterilize culture medium and other materials. Medical autoclaves are usually 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 pressure cooker is lower than that of the sterilizer, but we can still achieve good results by extending the sterilization time appropriately. For example, the sterilization effect can be basically achieved by sterilizing the culture medium for 40 minutes (the sterilizer is 20 minutes); Sterile water is made of fractional sterilization, which can be pressed in a pressure cooker for 40 minutes, left for 24 hours and then pressed for 20 minutes.

2. Venue:

This is easy for people. The general requirement is that the place where tissue culture is performed should be as dust-free 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 kitchen to prepare the culture medium, 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 kept 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, or even a traditional Chinese medicine scale can be used instead. Culture rack, this is even simpler. The aluminum alloy-glass incubator can meet the requirements. If the sunlight is insufficient, it can be supplemented with fluorescent lamps.

4. Chemical reagents:

Chemical reagents are indispensable, because the core of tissue culture is the composition of culture medium, not simple aseptic operation. It is necessary 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, white sugar, agar and so on. Trace elements and organic compounds do not need to be purchased, because their dosage is very small, and we can use vegetable juice or potato juice instead. Hormones are essential, and their weighing needs the help of analytical balance, but this is not allowed by family conditions. If possible, you can use relational units instead of making mother liquor, and you can take some at a time. The best way is to go to agricultural stores (where pesticides and fertilizers are sold). They generally sell hormones for agriculture, some of which are packed in ampoules and added to the culture medium according to a certain proportion. In recent years, some chemical companies have started to produce the original powder of plant tissue culture medium, which is convenient only by weighing a certain amount of water. For example, ms raw powder produced by Pantech Chemical Company only needs to weigh 40 grams, add 1 liter of water, and heat it in microwave oven for 5 minutes, which is quite simple.

The disinfectant is usually mercuric chloride (highly toxic). If it is not easy to get, it can be replaced by bleaching powder.

When adjusting pH, soda ash and white vinegar commonly used in families 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 and pick up some old jam bottles. The better bottle is the Axiangpo Chili sauce, which is also used in our laboratory. The sealing film of the bottle can be made of polypropylene plastic. If it is difficult to find it, you can use tin foil paper or even instant noodle bags specially used for microwave ovens, fold them into two layers and fix them with rubber bands.

Other glass instruments, such as beakers and glass rods, can be replaced by family cups and chopsticks; Some measuring instruments, such as measuring cylinders and pipets, are best bought in glass instrument stores. I'm afraid you can configure a set of excellent tissue culture glass instruments without spending 20 yuan money.

6. Others:

There are also some supplies that are necessary. For example, you need to buy a precision ph test paper (5.4~7.0), which costs about 1.5 yuan. You don't need to buy an alcohol lamp. Use an ink bottle with a glass ring, plus a cotton core. Finally, use the bottle cap of Nongfu Spring mineral water as a light bulb. This alcohol lamp is very small and suitable for inoculation boxes. Knives, scissors and tweezers are necessary. Go to the tool booth in the flower market and spend all the money 10 yuan. Pay attention to choosing smaller tools.

As for water use, distilled water is supposed to 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 used Master Kong purified water to cultivate animal cells, and the results were 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 the factory production of tissue culture seedlings should pursue high proliferation rate, so its requirements are more precise and standard. Our family training does not need to be so accurate, as long as we achieve the goal of successful training. Of course, if you want to improve the multiplication rate and the quality of test-tube seedlings, you must try to be close to the laboratory utensils and equipment, and the substitutes always have certain defects.

(a) the necessary facilities and substitutes

1 substitute

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

Pressure cooker: it can be used for disinfection and sterilization of culture medium, sterile water, glassware and other tissue culture utensils. If the hardness of the water used is high, cold boiled water can be used for disinfection and sterilization. Avoid scaling on the surface of sterilized 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-cleaned waste oil barrel: it can be used to store distilled water.

Small white porcelain dish: it can be used to inoculate and hold disinfectant (if disinfectant is put, don't eat it again to avoid poisoning).

Fluorescent lamp: it can be used to supplement the light source in tissue culture.

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

Tissue culture sites can be carried out in their own rooms. 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 appliances

(1) Production of inoculation box: clean bench is very valuable equipment in the process of tissue culture. If we use self-made inoculation boxes instead, we can greatly save money and be conducive to popularization.

The materials of the self-made inoculation box can be plywood, fiberboard, glass (3 mm thick), wooden strips (keel for decorating houses), even cardboard boxes (the quality of the boxes is slightly better, such as the boxes of televisions), or plexiglass.

The size of the inoculation box can be made according to their family conditions. It is too small to operate, but it is relatively easy to disinfect and occupies less land; It is easy to operate to make a large 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 high 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 a rectangular fish tank. Therefore, it can be made by glass bonding. Its size can be determined according to the area of your family 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 medicine; Or when buying trace elements and biological regulators, buy packaged ones. Alcohol lamp 1 lamp: used for inoculation, burning, disinfection and sterilization. Funnel (can also be used to buy bottled edible oil and distribute it): used for sub-packaging culture medium. 2 long tweezers: used for inoculation. 2 scalpels and several blades: used for inoculation. 10, 50, 100 ml measuring cylinders 1 each: used for preparing culture medium. 2 long horn spoons: used to prepare culture medium and take medicine. 1, 2 and 5 ml pipettes 1 each: used for preparing culture medium.

High-temperature resistant plastic film or kraft paper: used to wrap the corners of the culture bottle mouth and the self-made inoculation box. Rubber ring: used to tie the culture bottle mouth. Absorbent cotton: used for disinfection of alcohol cotton balls for operators and tissue culture equipment. PH (5 ~ 7) test paper 1 book: 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, pour10 ~ 20 ml of 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 1 UV lamp is purchased, indoor and inoculation box disinfection is more ideal.

(II) Matters needing attention in operation

Family operation is different from that of the unit, so we should pay attention to the following matters: pay attention to safety and keep drugs properly. Especially for the elderly and children; Disinfect carefully and strictly during operation. When inoculating, operators should wear masks and working caps to avoid electric fans and windy and dusty weather, and family members should avoid operation for a long time. Avoid family pets 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 done step by step. Flowers that are easy to be cultured can be made first, and flowers that are difficult to be made after gaining 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 used for preparation. Culture bottles generally use various jam bottles. First, soak the bottles with detergent for 4-8 hours, and then rinse them 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 inverted, and the water in the bottle should be emptied.

Clean containers should be dust-proof and placed in glass cabinets as far as possible. It is simpler 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 easy use. Generally, pipettes can only be used once, that is, they need to be cleaned, so it is necessary to prepare several pipettes. Suggestions are: 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 components 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 a mother liquor of ms culture medium is announced as follows:

Macroelements: 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 materials respectively, then combine them to constant volume to 1 liter, and take 25ml of standard ms medium for each preparation 1 liter.

Iron salt: 5.56g of ferrous sulfate heptahydrate;

7.46 grams of disodium EDTA.

Dissolve the above two kinds by heating respectively, mix them, adjust the volume to 1 liter, adjust the ph to below 5.5, and take 5 ml for each liter of MS.

The amount of trace elements and organic compounds is too small, so we can use vegetable extract or potato extract instead for simplicity. Usually, we are used to boiling spinach with 1 00g and water with 100ml for10min, filtering to keep the filtrate, and adding 20 ~ 50ml of extract for each preparation of1l ms medium. Similarly, potato with skin100g and 200ml of water can be used to boil15min, and the filtrate can be filtered, and 50 ~100ml can be added per liter of MS..

After adding the above components, 30 grams of white 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 the above ingredients are added, the volume is about 800 ml, and it is heated by microwave oven until all agar is dissolved. At this time, add the required plant hormones (usually prepared into 0. 1% solution, so that every 1 ml is converted into 1ppm in the culture medium). Then add water to constant volume to 1 100 ml (1. 1 liter). The reason for adding 0. 1 liter is to offset the loss in sub-packaging and disinfection. Finally, adjust the ph to 5.8~6.0 with acid and alkali.

3. Using ms powder to prepare culture medium:

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, added with hormones, and the volume is fixed to 1 100 ml (1. 1 liter).

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 will easily cause pollution. After repackaging, seal it with polypropylene film or a bag with instant noodles, and fix it with a rubber ring (try to choose the inner tube of a bicycle as the rubber ring is heat-resistant).

5. Sterilization:

It is recommended to buy some "sterilization effect test paper" from medical equipment stores for disinfection in 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, add pressure cutting, and keep the fire low 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 try to have no dead ends. 2 hours before each use, put all the articles needed for operation into the inoculation box, then add 3-5g potassium permanganate into a small beaker and put it into the inoculation box. Pour 2-3ml formaldehyde solution into the beaker in the box, and formaldehyde vapor will appear after a few seconds, so that all the articles 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 generated by high voltage penetrating mercury vapor, and ultraviolet rays are sterilized for the second time. After ultraviolet rays are irradiated for about 15 minutes, oxygen will be excited to form ozone, and ozone will be used as the third line of sterilization. After three times of sterilization, the inoculation box can basically reach the aseptic standard.

Before the operation15min, pour 5ml of concentrated ammonia water into the beaker used to evaporate formaldehyde just now. 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, the ultraviolet light should not be turned off, and it should continue to irradiate until it is turned off 5 minutes before operation, and it should be kept dark for 5 minutes, and then the fluorescent lamp should be turned on for operation. The reason for maintaining the darkness for 5 minutes is that ultraviolet light kills microorganisms by acting on the thymine of dna to form tetramers, but microorganisms have a light-reactivating protein, which can reduce the thymine tetramers in the presence of visible light and revive microorganisms. This effect is called "light-reactivation", so it is necessary to maintain the darkness for several minutes after turning off the ultraviolet light to avoid the emergence of light-reactivation.

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 in family tissue culture, and it is generally appropriate to configure 1 liter each time. After boiling, measure it with PH test paper, pack it into 35-40 bottles, plug it with cotton plugs and wrap it with wrapping paper. (The tampon must be used to roll cotton for cotton-padded clothes, and absorbent cotton cannot be used, and then it is wrapped tightly with gauze and tied with cotton thread. The tightness of the tampon should be that the portable tampon bottle does not slip. ) and then put it into an autoclave for high-pressure sterilization. When steam is sprayed from the nozzle of the autoclave, the pressure valve is buckled, and the time is counted from the time when the pressure valve sprays air, and it is continuously maintained for15-20 minutes, then the fire is turned off. After the pressure disappears, the pot cover is opened, the nutrient medium is taken out, and it is put into an inoculation box for later use. In the first experiment, the sterilized culture medium should be left for three to five days before it can be used when no mold growth is observed. If mold grows, it means that the sterilization time is not enough, and it is necessary to increase the sterilization time appropriately.

inoculate

Put the sterilized nutrient medium, sterile water, disinfectant and used equipment into the inoculation box. Because the volume in the inoculation box is relatively small, all the items used should be placed in the corresponding position in an orderly manner, and they should not be placed at will. The humidity in the box is high, so you must use a lighter 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 putting all the articles in place, put a magnetic cup filled with10-20 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 of potassium permanganate (PP powder) to make its steam fill the box to achieve the sterilization effect. At this time, the vent hole and operation hole of the inoculation box should be sealed to avoid the formaldehyde vapor from dispersing quickly. Inoculation can only be started after the steam in the box is exhausted, which takes about 5-10 hours.

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 work. When operating, we must establish the concept of sterility, treat everything as carrying bacteria, and always pay attention to prevention. Work is due to the high temperature and humidity in the box, and hands are easy to sweat, so you should wipe your fingers with alcohol after each bottle of inoculation, and you can also wear finger covers. In addition, pay attention to fire prevention when operating, because the space is small, it is easy to burn your fingers or cause alcohol fire if you are not careful. When opening the bottle stopper, place the bottle mouth above the alcohol lamp, hold the cotton plug tail with the ring finger and little finger of the right hand, and gently pull out the cotton plug. The cotton plug cannot be placed on the articles in the box. After inoculation, burn the bottle mouth on the alcohol lamp and the cotton plug, then gently plug it above the flame of the alcohol lamp, tie the wrapping paper, mark the variety, inoculation date and number with a 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 almost no pollution occurs after skilled operation. After inoculation, the callus differentiation, seedling differentiation and growth state are no different from those inoculated on the 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 natural conditions. The inoculated culture bottles can be placed in places with strong scattered light, such as bookcases and desks near the window, but direct sunlight should be avoided. Generally, they can grow normally at room temperature of 22-28 degrees Celsius, and they are not special varieties and do not need special care. If the room temperature is too low, a simple incubator can be made of cardboard, wood strips and plastic film, and one or two15-25 watt white woven lamps can be installed in it, which can not only supplement the illumination, but also increase the culture temperature. When the temperature is too high in summer, it can be placed in an air-conditioned room if possible. If there is no air-conditioned room, it is difficult to cool down, but most of the test-tube seedlings can be tolerated and will not die. After the seedlings grow up, it is undoubtedly a kind of enjoyment 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 to transfer and cultivate the seedlings to take root, and when a certain root system grows, they can be planted out of the bottle. When planting seedlings out of bottles, we must pay attention to the following aspects: first, we must appropriately reduce the temperature; The second is to increase the 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 husks as a mixed substrate. After planting the seedlings, I spray the roots with chlorothalonil and cover them with plastic film. A small number of seedlings are simply covered with glass. After about 15 days, the mulch can be removed and normal management can be carried out.

In fact, it is not a very difficult thing to carry out tissue culture at home. As long as you use your brains and think of some ways, it is easy to do it. Everything is difficult at the beginning, and the method is always more difficult than it is. I/KLOC-0 was the director of a county tobacco research institute in 1974, but the original conditions did not allow me, and the research funding was very tight. At the beginning, the research funding for one year was only 3,000 yuan. In that year, I did anther haploid of tobacco, anther culture of rice, endosperm culture of citrus, stem tip tissue culture of black-peel sugarcane, callus culture of Panax notoginseng, sterile culture of black-knot grass (Dendrobium) 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 the precious tissue culture seedlings were thrown everywhere by us. It was incredible that they could not do it, but they could do it in a very isolated mountainous area. What I mean by saying this is not to brag about myself, but to tell my friends who want to engage in this industry not to underestimate their own energy, nor to be superstitious about authority. As long as they have confidence, they will do it in strict accordance with the laws of science. Nothing is impossible.

Selection and effect evaluation of home 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 is 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 for preliminary experiments, which can avoid detours and greatly; Reduce the consumption of time, manpower and material resources. After passing a series of preliminary tests, some components can be adjusted in a small range according to the actual situation.

When making adjustments, the following conditions can be used for reference. 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. If nitrate and a small amount of ammonium salt are added at the same time, this drift will be overcome. Second, when some elements are in short supply, the cultured plants will show some symptoms, which can be adjusted according to the symptoms. For example, when nitrogen is insufficient, the cultured tissues often show the colors of anthocyanins (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 lacking, the tissue will lose its green color, the cell division will stagnate, and the callus will appear brown aging symptoms; When boron is deficient, the cell division tends to be slow and excessively elongated; Cell growth is affected when manganese or molybdenum is lacking.

The effect of exogenous hormones in the culture medium will also cause some similar situations in the culture, so it should be carefully analyzed and it is not easy to draw conclusions.