The tissue culture of plants can not be done without the most basic conditions and items, when the rational use of some of the simplest appliances is not impossible to do.
One, the most necessary items
1, . Household pressure cooker 1 preferably larger, loaded with more things. For media, sterile water and other disinfection
2, inoculation box (sterile box) 1, you need to make their own, with some boards and a piece of glass and two feet of fabric. For inoculation and transfer.
3, pharmaceutical balance 1 frame preferably 500g weighing, smaller can also be used to weigh agar, sugar and other items.
4, stainless steel pot or aluminum pot (the kind of cooking soup, noodles) to buy. Used to cook the culture medium used.
5, mixing and dispensing medium with a spoon for cooking and dispensing medium.
6, a number of bottles for culture,
7, the cotton used to make cotton plugs, gauze and a number of threads, used to make bottle stoppers, it should be noted that the cotton to use ordinary cotton to do cotton clothing cotton, do not use hospitals to use skimmed cotton, it is easy to wear plugs contamination.
8, kraft paper, rubber bands a number of, used to wrap the bottle head and tie the head of the paper with.
9, 1 alcohol lamp, 1 scalpel, a number of blades, tweezers 2, 10ml, 100ml measuring cylinder each one, 2ml pipette 1, a number of cotton wool.
Two, how to solve the distilled water and basic medicines
The water used for the culture medium is always felt to be very important in the general mind, in fact, it is superfluous to worry about. Cold boiled water, clean river water are given to use, and does not affect the effect of culture. The pure water sold in the market is the ideal water for general culture medium. The most commonly used, the most necessary, the largest amount of just need to buy the following kinds of it.
1, MS medium of a large number of elements (home or the so-called pocket histoculture room culture generally use MS base on it.) ***5 kinds. You can also buy commercially available MS 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, bleach or bleaching agent
5, agar
6, sugar
7, formalin
8, potassium permanganate (you can go to the hospital or herbal store to buy)
9, trace elements, iron salts, vitamins, and hormone medicines are applied in very small quantities. For example, some hormones, vitamins and drugs and there are injections and tablets, they have a more accurate content, you can go to your needs to go to the proportion of preparation.
Hobbyists explore and suggest tissue culture in the home environmentThe technique of plant tissue culture, especially rapid propagation, is not something that can only be done by professionals and specialized laboratories. As a hobbyist with a strong interest in succulents and tissue culture, you can also prepare your own test tube seedlings by making full use of home conditions. In our cultivation of succulents, we have realized that some of the more valuable horticultural species, such as: Vientiane, Jade Fan, Shou, high striped eagle's claw and other dodecameric species, or colorful olive, Horehound petunia and other tubers, and even the planet, rock peony and so on, the conventional reproduction coefficient of these plants is very low, and they can be used to solve the problem of propagation of the organization of the culture, and can be obtained in considerable quantities. seedlings. Plants that are difficult to germinate from seeds, such as the whimsical sky, can also be aseptically sown with the help of tissue culture. The process of tissue culture not only provides a deeper understanding of the cultivated plant, but also has considerable significance for conventional cultivation. Many experts in tissue culture were not initially specialized in tissue culture, but achieved brilliant results based on their interest and spirit of exploration. The key is that hobbyists have strong interest, extraordinary observation and meticulous operation, which can make up for the defects of non-professionals, and can make full use of a variety of available resources in daily life, which is enough to allow hobbyists to complete the tissue culture in the home environment. At the same time this is not only to satisfy their own interests, but also can have a certain financial gain, and even can have innovation and invention.
I. How to solve the site and basic equipment:
To carry out tissue culture, without certain equipment can not be carried out. Tissue culture equipment in a formal laboratory is expensive and not suitable for home consumption, but it would be nice to have access to cheap laboratory equipment. If you don't have access to lab equipment, you may want to get your hands dirty and make some simple equipment yourself.
1. Aseptic operation of the main equipment - inoculation box and pressure cooker:
Inoculation box: the subject of plant tissue culture operations need to be carried out in a relatively closed sterile environment. So that we first want to create a closed environment, you can use some wood, plastic plates, plexiglass, aluminum alloy skeleton and other cheap materials, hands glued to a small inoculation box. Specific parameters can find a book "edible fungi cultivation", imitating the inoculation box of mushroom planting can be done. It should be noted that the inoculation box for tissue culture requires a high degree of airtightness, try to avoid rotten wood and rusty materials, and put two lights on the top of the box: 20w UV light and 20w fluorescent light. The periphery of the box try to use glass materials, because it is easy to observe, sometimes inoculation box can also be borrowed for the incubator, the periphery of the glass material is more convenient for observation and culture.
Autoclave: mainly used for sterilization of culture media and other materials. In the laboratory usually use a medical autoclave, but in the home to make full use of the kitchen to use the pressure cooker, pressure cooker pressure is lower than the sterilizer, but the appropriate extension of the sterilization time, you can still achieve better results. For example: the culture medium sterilization 40 minutes can basically achieve sterilization (sterilizer is 20 minutes); sterile water using intermittent sterilization method, the first pressure with a pressure cooker for 40 minutes, placed in 24 hours, and then press 20 minutes to achieve the effect.
2. Site:
This is easy for people, the overall requirement is to carry out tissue culture operations in the place to be as dust-free as possible, to reduce air flow, it is best to have a certain amount of sunlight. For example, a study or writing room can be used for aseptic operations.
The kitchen can be borrowed for media preparation, but the utensils used must be separate from the kitchen. Generally speaking, almost all chemicals in tissue culture are harmless to humans, except for a few hormones and sterilizing agents. For toxic substances, they need to be carefully stored to avoid danger.
3. Auxiliary equipment:
This mainly refers to the refrigerator, which is used to store chemicals and culture media. Balance, you can use a pallet balance with a sensitivity of 100 grams, or even use the medicine balance of traditional Chinese medicine can be replaced. Culture frame, this is more simple, using aluminum alloy - glass incubator can meet the requirements, if the daylight is insufficient, with fluorescent lamps to make up the light and so on.
4. Chemical reagents:
Chemical reagents are indispensable, because the core of tissue culture is the composition of the medium, and not simply aseptic operation. Some of the larger amount of chemicals is necessary to buy, such as large amounts 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 complexes do not need to be purchased because they are used in very small quantities and we can replace them with vegetable infusions or potato infusions. Hormones are essential, they need to be weighed with the help of an analytical balance, but this is not permitted by the family conditions, if you have the conditions you can use the relationship unit instead of formulating a masterbatch, and just take some to use each time. The best way is to go to an agricultural store (where pesticides and fertilizers are sold), which usually sells agricultural hormones, some of them in ampoules, which can be added to the medium in a certain proportion. In recent years, some chemical companies have begun to produce plant tissue culture medium powder, which can be easily added to the medium by weighing a certain amount and adding water. For example, the ms powder produced by Pansum Chemicals, only need to weigh 40 grams, add 1 liter of water, microwave heating for 5 minutes can be dispensed, quite simple.
Disinfectants are usually mercuric chloride (highly toxic), but if you can't get it easily, you can use bleach instead.
To adjust the pH, you can use soda ash and white vinegar, which are commonly used at home.
5. Cultivation containers and glass instruments:
The requirements for tissue culture containers are not high, as long as the glass color is transparent (sodium glass is not allowed).
We can just go to the dump and pick up some used jam bottles. A better one to use is the Aroma Granny Hot Sauce bottle, which is also used in our lab. The bottle sealing film using polypropylene plastic can be, if it is not good to find, you can use the microwave oven special tin foil or even the bag of instant noodles, folded into a double layer, fixed with a rubber band can be.
Other glass instruments, such as beakers, glass rods can be used in the family instead of cups and chopsticks; some measuring instruments, such as measuring cylinders and pipettes are best to buy at the glass instrument store, I'm afraid to spend no more than 20 dollars can be configured with a set of great glass instruments of the histoculture.
6. Other:
There are also some supplies that are necessary. For example, precision ph test paper (5.4 ~ 7.0), you need to buy a book, spend about 1.5 yuan can be done; alcohol lamps do not need to buy, with an ink bottle with a glass ring, plus a cotton wick, and finally use the cap of the agrocyanic spring mineral water to do the lamp, such a alcohol lamp is very small, suitable for inoculation box use. Knife scissors tweezers are necessary, to the flower market tool stalls, spend 10 yuan to get it all done, pay attention to choose more compact tools.
On the water, it is reasonable to say that we should use distilled water, but the pure water we drink at home is even purer than distilled water, so that borrowing some drinking water will be fine. We have used Kangshifu pure water for animal cell culture, the effect is very good, not to mention the plant cell culture.
The above are the basic equipment and reagents for tissue culture, mainly highlighting their related alternatives, because as a rapid propagation in tissue culture, its requirements are quite low. Our home culture is different from factory production, where high proliferation rates are sought for the production of histocultured seedlings, so its requirements are more delicate and standardized. Our home culture does not need to be so precise, as long as the purpose of successful culture can be achieved. Of course, if you want to improve the multiplication rate and the quality of in vitro seedlings, you must try to get as close as possible to the laboratory utensils and equipment, substitutes always have certain defects.
(A) the necessary facilities and items and substitutes
1, substitutes
Home use of the refrigerator, can be used to store the medium mother liquor (4 ℃) and need to be stored at low temperatures of the drugs (such as biological regulators).
Autoclave: it can be used for culture medium, sterile water, glassware and other histoculture utensils sterilization. If the hardness of the water used can be cool boiled water, disinfection and sterilization. Avoid being sterilized items surface scaling.
Stainless steel pot or aluminum pot: it can be used for culture medium, melting agar (as a water bath) and the sterilization of the histoculture utensils.
Brush clean the waste cooking oil barrel: it can be used to store distilled water.
Small white porcelain plate: it can be used for inoculation and holding disinfectant (if you put disinfectant, do not eat it to avoid poisoning).
Fluorescent lamp: it can be used for light source supplementation during the histoculture process.
Washed and discarded canning jars: it can be used for histoculture, instead of conical flasks and test tubes.
The site of histoculture can be done in your own room. When preparing the medium and inoculation, do not rush to do it when there are many family members, if the room has been installed with air conditioning, then it will be beneficial in the primary and secondary culture, and even the transition of seedlings.
2, homemade utensils
(1) the production of inoculation box: the ultra-clean bench in the process of histoculture is very expensive equipment. If we use homemade inoculation box instead, we can greatly save money, in favor of popularization.
Homemade inoculation box material, can be plywood, fiberboard, glass (3 mm thick), wood strips (decoration of houses with the keel), or even make cardboard boxes (the quality of the box to be slightly better, such as the box of the TV set), you can also use Plexiglas.
The size of the inoculation box can be made according to the conditions of their respective families. Make too small is not easy to operate, but relatively easy to disinfect, and less; make a larger easy to operate, but disinfection work does not seem easy, and covers an area of more. Generally speaking, 70 centimeters long, 45 centimeters wide. 50 centimeters high is more appropriate.
(2) the production of culture box: culture box can replace the culture room, can also be used for the transition of seedlings. It can be made of glass or utilize a rectangular fish tank. Therefore, it can be made with glass bonding. Its size can be decided according to the area of your family room and the amount of group culture.
3, need to purchase appliances
Ordinary balance (500 grams): used to weigh the preparation of media drugs, or weighing gold and silver, Chinese medicine arbitrage weighing, used to weigh the preparation of media drugs, trace elements and biological regulators; or buy trace elements and biological regulators when buying has been divided into packages. Alcohol lamp: used for inoculation, burning sterilization sterilization. Funnel (can also be used with the funnel supplied with the barrel of cooking oil purchased): used for dispensing culture medium. 2 long tweezers: for inoculation. 2 dissecting knives and several blades: for inoculation. 1 measuring cylinder each of 10, 50 and 100 ml: for preparing the culture medium. 2 long horn spoons: used for preparing culture medium to take medicine. 1, 2, 5 ml pipette each: used for preparing culture medium.
High-temperature plastic film or kraft paper: used to wrap the mouth of the culture bottle and table paste homemade inoculation box internal corners. Rubber ring: used to tie the culture bottle mouth. Degreased cotton wool: used for operators and histoculture utensils alcohol cotton ball disinfection. pH (5 ~ 7) test paper 1: when used can be cut into small strips to detect the pH value of the culture medium. Alcohol 1 bottle: for alcohol lamp and sterilization. Bleach 1 bottle: for sterilization. 1 bottle of formalin: for sterilizing the inoculation box (pour 10-20 ml of formalin into a small white ceramic dish 2-10 hours before each operation, and take it out before the operation).Drugs needed for MS medium; for preparing the medium. Hydrochloric acid and sodium hydroxide: for pH adjustment. It would be ideal to purchase a UV lamp for indoor and inoculation box disinfection.
(2) Precautions during operation
Home operation is different from the unit, so we should pay attention to the following matters: pay attention to safety, and keep the drugs properly. Especially for the elderly and children; the operation should be carefully and strictly disinfection, inoculation operators wear a good mask, work cap, avoid electric fans and sandy weather, family members to avoid operation; before and after the disinfection to avoid family pets into the workplace; drug weighing should be accurate; if the family does not have the temperature control (such as temperature control air conditioning), should be avoided in the height of the summer, cold winter; crocus group culture should be step by step to make, can first make easy to group culture flowers. You can start with the flowers that are easy to cultivate, and then do the flowers that are difficult to cultivate after you have gained experience.
1. Preparation of culture containers and glass instruments:
The first thing to do is to prepare culture bottles and glass instruments for the preparation of culture medium. Culture bottles generally use a variety of jam bottles, to soak the bottle with detergent for 4 to 8 hours, and then rinse with running water several times. Other glass instruments need to be cleaned in the same way, until the glass walls do not hang water droplets but become a film of water. Cleaned glass containers should be inverted, empty the bottle of water.
The cleaned containers should be protected from dust and placed in a glass cabinet if possible. It is simpler to cover the container with a clean towel.
Pipettes should be cleaned with a suction ball, repeatedly blowing on it with distilled water until it is clean, placing the pipette in a long tube for ease of use. General pipettes can only be used once, that is, to be cleaned, so there is more than necessary to prepare a few pipettes, it is recommended that: 10 ml / 2, 5 ml / 2, 1 ml / 5.
2. Preparation of the medium:
The classic medium for tissue culture is the ms formula, which is basically divided into: macroelements, microelements, iron salts, organic complexes, phytohormones, sugars and supports. The dosage of these components are in milligrams, which is difficult to weigh with an ordinary balance. In order to solve this problem, we can proportionally enlarge the dosage of each component to formulate a masterbatch, and just add it according to a certain proportion when using.
The following will be a ms culture medium mother liquor preparation program is announced as follows:
Massive elements: ammonium nitrate 66.0 grams;
Potassium nitrate 76.0 grams;
Anhydrous calcium chloride 13.3 grams;
Magnesium sulfate heptahydrate 14.8 grams;
Potassium dihydrogen phosphate 6.8 grams.
The above were dissolved separately and combined and fixed to 1 liter, and 25 ml were taken for every 1 liter of standard ms medium prepared.
Iron salt: ferrous sulfate heptahydrate 5.56 g;
Disodium ethylenediaminetetraacetic acid (edtana) 7.46 g.
The above two were dissolved by heating, mixed, and fixed to 1 liter, adjusted ph to below 5.5, and 5 ml was taken for each liter of ms prepared.
The quantities of trace elements and organic complexes are too minute, for simplicity we can replace them with vegetable extracts or potato extracts. Usually we use 100 grams of spinach with 100 ml of water and boil for 10 minutes, filter and leave the filtrate, and add 20-50 ml of extract for every 1 liter of ms medium. Similarly, you can use 100 grams of potato with skin plus 200 ml of water and boil for 15 minutes, filter and leave the filtrate, add 50~100 ml per liter of ms can be.
After adding the above components, 30 grams of sugar and 7 grams of agar should be added per liter of ms medium.
It is important to note here that both sugar and agar are variable amounts and should be adjusted as needed. Also carrageenan for jelly can be used instead of agar for better clarity.
After the above ingredients are added, the volume is set to about 800 ml and heated in a microwave oven until all the agar is dissolved. At this point, add the desired plant hormone (usually formulated as a 0.1% solution, so that every 1 ml taken, converted to the medium is 1 ppm). Water is then added to finalize the volume to 1100 ml (1.1 liters), the reason for the extra 0.1 liters is to offset losses in dispensing and sterilizing. Finally, adjust the ph to 5.8~6.0 with acid and alkali.
3. Preparation of culture medium using ms powder:
At present, some domestic chemical companies have developed simple ms medium powder, which greatly simplifies the preparation of culture medium, and you can choose the type of medium according to the instructions of different companies. The following is an example of ms original powder: ms culture medium original powder, including massive elements, trace elements, iron salts, organic complexes, sugar and agar, generally weighed according to the use of the method, microwave heating to dissolve, add the hormone, volume to 1100 ml (1.1 liters), adjust the ph value of 5.8 ~ 6.0.
4.
Dispense the prepared medium into culture containers, be careful to dispense it while it is still hot, because agar will solidify below 40 degrees Celsius. Each bottle should contain about 1/6~1/5 of the volume of the bottle, and be careful not to hang the medium on the outer wall of the bottle, which may cause contamination. After dispensing, use polypropylene film or an instant noodle bag to seal the bottle, and secure it with an adhesive ring (try to choose the inner tube of a bicycle, which is more heat-resistant).
5. Sterilization:
To sterilize the media in a home pressure cooker, it is recommended to buy some "sterilizing effect test strips" from a medical equipment store, which can indicate the effect of sterilization, and will change color when the sterilizing effect is achieved. Put the test paper and media into the pot at the same time, heat until there is a lot of steam out, and then add pressure to cut, maintain a low heat for 30 ~ 40 minutes.
After the sterilization is completed, cool naturally, remove the medium and place it in a cool, dust-free place.
6. Preparation of inoculation box:
The newly made inoculation box must be clean and clean, as far as possible without dead corners. 2 hours before each use, the operation of all the items needed into the inoculation box, and then a small beaker to add 3 ~ 5 grams of potassium permanganate into the inoculation box, in the box to the beaker poured into 2 ~ 3 ml of formaldehyde solution, about a few seconds after the formaldehyde steam will be seen to appear, so that you can be all the items in the inoculation box for the first time to disinfect; at the same time, turn on the ultraviolet lamp irradiation. Ultraviolet light is a high-voltage penetration excitation of mercury vapor generated, ultraviolet light for the second sterilization, in the ultraviolet radiation about 15 minutes, will stimulate the formation of ozone oxygen, ozone as the third line of defense sterilization, so after three sterilization, inoculation box can basically reach the standard of sterility.
15 minutes before the operation, just evaporate formaldehyde with a beaker poured into 5 ml of concentrated ammonia, because formaldehyde has a toxic effect on the human body, ammonia and formaldehyde and the formation of a solid substance called "hexamethylene tetramine", which neutralizes the formaldehyde irritating steam. At this time, the UV lamp should not be turned off, but continue to irradiate until 5 minutes before the operation is turned off to maintain darkness for 5 minutes, and then turn on the fluorescent lamp for operation. The reason for maintaining darkness for 5 minutes is that the killing of microorganisms by ultraviolet light is to act on the thymine of dna to form tetramers, but microorganisms have a kind of photorevival protein, which can restore the thymine tetramers under the environment of visible light and revive microorganisms, which is called "photorevival", so after turning off the ultraviolet light, it is necessary to maintain darkness for a few minutes to avoid the photorevival. Therefore, after turning off the UV light, you must maintain darkness for a few minutes to avoid the emergence of photoreactivation.
How to carry out the work of family culture
Preparation
Family culture because of the limitations of the conditions, it is impossible to configure a large number of nutrient preparation base, generally 1 liter each time to configure the appropriate. After boiling and measuring with PH test paper, divided into 35-40 bottles, stuffed with cotton plugs and wrapped with wrapping paper. (Cotton plugs must be used as cotton rolled cotton, can not use skimmed cotton, and then wrapped tightly with gauze, tied with cotton thread. Cotton cork loosening and tightening to hand-held cotton cork bottle does not slip off as appropriate.) Then put into the autoclave autoclave sterilization, when the nozzle of the autoclave spray steam, buckle the pressure valve, from the pressure valve when the jet timing, continuous maintenance of 15-20 minutes after turning off the fire, the pressure disappeared, open the lid of the pot, take out the matching nutrient base, put into the inoculation box to be used. For the first experiment, the sterilized culture medium should be placed for three or five days, and be used only when no mold growth is observed. If there is mold growth, this indicates that the sterilization time is not enough, and need to increase the sterilization time.
Inoculation
Put the sterilized medium, sterile water, disinfectant and equipment into the inoculation box, because the inoculation box is relatively small in size, so all the items used to be placed in an orderly manner in the appropriate location, can not be placed randomly, the box is more humid, the point of the alcohol lamp must be if you want to use a lighter, do not use matches. Special attention should be paid to the preparation of sewage, dirt containers, as large as possible, because the operation is not allowed to open the box to take things. After placing all the items, place a magnetic cup containing 10-20 ml of formalin into the box (it is better not to use a glass cup, as the temperature is high during the reaction and it is easy to explode). Pour in 2-5g of potassium permanganate (PP powder) so that its vapor fills the box to achieve the effect of sterilization. At this time, the inoculation box ventilation holes and operating holes closed to avoid formaldehyde vapor quickly dissipated. To be in the box after the vapor dissipation, can start inoculation work, this time about 5-10 hours.
Inoculation, sealed in the inoculation box ventilation holes and operating holes on the seal, remove the fumigation of the magnetic cup, will be inoculated with the material into the inoculation box, open the ultraviolet lamp, fifteen minutes after the closure of ultraviolet lamps, turn on the lighting, you can start working. Operation must establish the concept of asepsis, to see all the items as germ-carrying, should always pay attention to the precaution. Work is due to the higher temperature in the box, humidity, hands are easy to sweat, so after each inoculation of a bottle should use alcohol to wipe your fingers, you can also wear finger cots. In addition, the operation should pay attention to fire prevention, due to the small space, a carelessness is easy to burn fingers or cause alcohol fire. Open the cork, the mouth of the bottle should be placed above the alcohol lamp, with the ring finger and little finger of the right hand to clamp the end of the cotton plug, gently pull out the cotton plug, the cotton plug can not be placed on the items in the box, the application of finger clips, after inoculation, the mouth of the bottle in the alcohol lamp to burn a little, the cotton plug is also burned, and then gently stuffed above the flame of the alcohol lamp, tied the head wrapping paper, with a pencil labeled with the species, date of inoculation, numbering, etc., and then repeat the next bottle.
In fact, the inoculation box inoculation and ultra-clean bench compared to the work of people to feel much more comfortable. The rate of contamination is also very low, and there is almost no contamination after skillful operation. After inoculation, healing tissue differentiation, seedling differentiation, growth status and inoculation on the ultra-clean bench is no different.
Cultivation
Cultivation of culture in the amateur conditions of the family is not possible to get special control, so to fully utilize the natural conditions, after inoculation of culture bottles can be placed in a place with a strong diffused light, such as bookcases against the window, writing desks, but to avoid direct daylight, the general room temperature of 22-28 degrees Celsius can be a normal growth, not Special varieties do not need special care. If the room temperature is too low, you can use cardboard, wooden strips, plastic film to make a simple incubator, which is equipped with one or two 15-25 watt white woven lamps, which can both subsidize the light can also improve the temperature of the culture. When the temperature is too high in summer, those who have the conditions can be placed in an air-conditioned room, and those who do not have an air-conditioned room, the cooling is a little more difficult, but most of the test-tube seedlings can be tolerated, not to die. After the seedlings grow, placed in the bookcase or workbench, feel their own cultivation of vibrant little life, undoubtedly a kind of enjoyment.
Cultivation
When the seedling grows to a certain point, you can configure some rooting with nutrient base to transfer the seedling to cultivate roots, and when it grows a certain amount of roots, it can be planted out of the bottle. Seedlings out of the bottle planting must pay attention to the following aspects, one is to reduce the temperature appropriately; the second is to increase humidity; the third is to strictly control the harm of fungi; four is to ensure that the planting medium is loose and breathable; five is to ensure that the appropriate light. I usually use metatarsal stone, wood chips, rice husk mix for substrate, plant the seedlings with Chlorothalonil spray to water the roots, covered with plastic film, a small number of seedlings simply covered with a glass, about 15 days after the cover can be removed, you can carry out normal management.
Family to carry out group cultivation in fact is not a very difficult thing, as long as more brainstorming, think of some ways to do it is also very easy. The beginning of everything is difficult, the solution is always more than the difficulty. I was in a county in 1974 when the director of the Tobacco Research Institute, the original conditions do not allow, research funds are very tight, at the beginning of the time, the year's scientific research funds only 3,000 yuan, that year I did the anther haploid of tobacco, rice anther culture, citrus endosperm culture, black fruit cane stem tip tissue culture, field Panax quinquefolium guaiacum tissue culture, black knapweed (Dendrobium officinale) seeds of the aseptic culture, somatic fusion, have been very good results. At that time, a lot of research units in Beijing and Shanghai came to see and were very surprised, they thought that the precious seedlings of histoculture were thrown everywhere by us, and found it unbelievable that what they couldn't do could be done in a very closed mountainous area of ours. I say this is not to brag about myself, but to tell friends who want to engage in this industry, do not look down on their own energy, and do not be superstitious authority, as long as they have confidence, in strict accordance with the laws of science to do, there is nothing that can not be done, there is nothing that can not be done.
Selection and effect evaluation of home culture medium
Selection of suitable medium is the basis for the success of plant tissue culture.
The selection of suitable culture medium is mainly considered from the following two aspects: first, the basic medium; second, the concentration and relative proportion of various hormones.
MS medium is suitable for most dicotyledonous plants, B5 and N6 medium are suitable with many monocotyledonous plants, especially N6 medium is very effective for graminaceous plants such as wheat, rice, etc. White medium is suitable for root culture.
Trying these media first for preliminary experiments can be less detour and greatly; reduce the consumption of time, labor and material resources. When through a series of initial trials, can then be based on the actual situation of some of the ingredients to make small adjustments.
When making adjustments, the following cases can be referred to. One is that when a compound is used as a source of nitrogen, nitrate works better than ammonium, but nitrate alone causes the pH of the medium to drift toward alkalinity, which is overcome if nitrate and a small amount of ammonium are added at the same time. Secondly, when the supply of certain elements is insufficient, the cultured plants will show some symptoms, which can be adjusted according to the symptoms, such as nitrogen deficiency, the cultured tissues often show the color of anthocyanin (red, purplish-red), and it is difficult to see the differentiation of conduit molecules inside the healing tissues; when there is a shortage of nitrogen, potassium, or phosphorus, the cells will be obviously overgrown, forming some very fluffy, or even hyaline, healing tissues; the lack of iron and sulfur When iron, sulfur lack of tissue will lose green, cell division stagnation, healing tissue brown senescence symptoms; boron deficiency when the trend of cell division is slow, excessive elongation; lack of manganese or molybdenum when the cell growth is affected.
The role of exogenous hormones in the culture medium will also make the culture appear some of the above similar situations, so it should be carefully analyzed and not easily concluded.