General method of plant tissue culture?

Home tissue culture\x0d\\plant tissue culture without the most basic conditions and items can not be engaged in, when the reasonable use of some of the simplest appliances is not impossible to do. \x0d\ I. The most necessary items \x0d\ 1. Household pressure cooker 1 preferably a larger one that holds more. Used for sterilizing media, sterile water, etc.\x0d\2, inoculation box (aseptic box) 1 , you need to make your own, with some wooden boards and a piece of glass and two feet of fabric. For inoculation and transfer. \x0d\3, medicinal balance 1 rack Preferably 500g weighing, smaller can be, mainly used to weigh agar, sugar and other items. \x0d\4, stainless steel pot or aluminum pot (the kind used to cook soup and noodles) to buy. Used for cooking culture medium with. \x0d\5, One spoon for mixing and dispensing culture medium for cooking and dispensing culture medium. \x0d\6, a number of bottles for culture, \x0d\7, a number of cotton, gauze and thread for making cotton stoppers, used to make stoppers for the mouth of bottles, it is important to note that the cotton should be used to use ordinary cotton for making cotton clothes, do not use the skimmed cotton used in hospitals so that it is easy to wear the stopper contamination. \x0d\8, kraft paper, rubber bands a number of, used to wrap the bottle head and tie the head paper with. \x0d\9, 1 alcohol lamp, 1 dissecting knife, a number of blades, 2 forceps, 10ml, 100ml measuring cylinder each, 2ml pipette 1, a number of cotton wool. Cold boiled water, clean river water are given to use, does not affect the effect of culture. The pure water sold in the market is even more ideal for general culture medium. The most commonly used, necessary, and used in the largest quantities is simply to purchase the following. \x0d\1, MS medium of large amounts of elements (home or so-called pocket histoculture room culture generally use MS base can be.) ***5 kinds. You can also buy commercially available MS medium\x0d\(1), ammonium nitrate \x0d\(2), potassium nitrate \x0d\(3), calcium chloride \x0d\(4), magnesium nitrate \x0d\(5), potassium dihydrogen phosphate \x0d\2, alcohol \x0d\3, precision test strips (PH 5.4-7.0)\x0d\4, bleach powder or bleach Essence\x0d\5, agar\x0d\6, sugar\x0d\7, formalin\x0d\8, potassium permanganate (can be bought at hospitals or herbal stores)\x0d\9, trace elements, iron salts, vitamins and hormone drugs are used in very small quantities. For example, some hormones, vitamins drugs and there are injections and also tablets, they have a more accurate content, you can go to the proportion according to your needs to prepare. \x0d\ \x0d\ Hobbyist Discussion and Suggestions on Tissue Culture in the Home Environment Plant tissue culture techniques, especially rapid propagation, are 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 your home conditions. In our cultivation of succulents, we have realized that some of the more valuable horticultural varieties, such as: Vientiane, Jade Fan, Shou, High-grained Eagle's Claw and other dodecameric species, or Colorful Olive, Hollu Petunia and other tubers, and even Planetaria, Rock Peony and so on, the conventional reproduction coefficients of these plants are very low, and they can be used to solve the problem of propagation by using tissue 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 the various resources available 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 amount of financial gain, and can even have innovation and invention. \x0d\ I. How to solve the site and basic equipment: \x0d\ To carry out tissue culture, it is impossible to carry out without certain equipment. Tissue culture equipment in a formal laboratory is expensive and not suitable for home consumption, but it would be great if there is a way to get cheap laboratory equipment. If there is no way to get laboratory equipment, it is good to get your hands dirty and make some simple equipment yourself. So that we first need to create a closed environment, you can use some wood, plastic plates, plexiglass, aluminum alloy skeleton and other cheap materials, hands glued a small inoculation box. Specific parameters can find a book "edible fungi cultivation", imitating the inoculation box of mushroom planting to do a can. 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 perimeter of the box try to use glass material, because it is easy to observe, sometimes inoculation box can also be borrowed as an incubator, the perimeter of the glass material is more convenient for observation and cultivation. \x0d\ autoclave: mainly used for sterilization of culture media and other materials. In the laboratory usually use a medical autoclave, however, in the home to make full use of the kitchen to use the pressure cooker, pressure cooker is lower than the sterilizer, but the appropriate extension of the sterilization time, you can still achieve better results. For example: on the culture medium sterilization 40 minutes can basically achieve the sterilization effect (sterilizer is 20 minutes); sterile water using intermittent sterilization method, the first pressure with the autoclave 40 minutes, placed in 24 hours, and then pressure 20 minutes to achieve the effect. \x0d\2.venue: \x0d\this is easy depending on the person, the overall requirement is that the place where the tissue culture operation is carried out should be as dust-free as possible, with less air movement, and preferably with a certain amount of sunlight. For example, study rooms and writing rooms can be used for aseptic operations. \x0d\ Conducting the preparation of culture media can be done by borrowing the kitchen, but the utensils used must be separate from the kitchen utensils. Generally speaking, almost all chemicals in tissue culture are harmless to humans, except for a few hormone substances and sterilizing agents. Substances that are toxic need to be carefully stored to avoid danger. \x0d\3.Auxiliary equipment:\x0d\ Mainly refers to the refrigerator, which is used to store chemicals and culture media. Balance, you can use a pallet balance with a sensibility of 100 grams, or even use the medicine balance of traditional Chinese medicine can be used instead. Culture frame, this is more simple, the use of aluminum alloy - glass incubator can meet the requirements, if the daylight is insufficient, with fluorescent lamps to make up the light and so on. \x0d\4. Chemical reagents: \x0d\ Chemical reagents are indispensable, because the core of tissue culture is the composition of the medium, and not simply aseptic operation. Some chemicals in large quantities are 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 Chemical Company only requires 40 grams of weighing and 1 liter of water, and can be dispensed by heating in a microwave oven for 5 minutes, which is quite easy. \x0d\ Disinfectant is usually mercuric chloride (highly toxic), which can be replaced by bleach if it is not readily available. \x0d\ When adjusting the pH level, you can use soda ash and white vinegar, which are commonly used at home. \x0d\5. Culture Containers and Glass Instruments: \x0d\ The requirements for containers for tissue culture are not high, as long as the glass color is transparent (sodium glass is not acceptable). \x0d\\ We can just go to the dump and retrieve some used jam bottles. A better bottle to use is the Aroma Poo Hot Sauce bottle, which is also used in our lab. Just use polypropylene plastic for the bottle's sealing film. If you can't find it well, you can use microwave-safe tin foil or even an instant noodle bag, fold it into a double layer and fix it with a rubber band. \x0d\ Other glass instruments, such as beakers, glass rods can be replaced by family cups and chopsticks; some measuring instruments, such as measuring cylinders and pipettes are best purchased at a glass instrument store, I'm afraid that for less than 20 dollars you can configure a set of quite great glass instruments for histoculture. \x0d\6.Other:\x0d\ There are also some supplies that are necessary. For example, precision ph test paper (5.4~7.0), you need to buy a book, you can spend about 1.5 yuan to get it 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 Nongfusanquan mineral water for the lamp, so that alcohol lamps are very small, suitable for use in the inoculation box. Knife clippers tweezers are necessary, to the flower market tool stall, spend 10 yuan all done, pay attention to choose more compact tools. \x0d\ About water, it is reasonable to say that distilled water should be used, but the pure water we drink at home is purer than distilled water, so it is fine to say that we borrow some drinking water. We once used Kangshifu pure water for animal cell culture, the result is very good, not to mention in plant cell culture. \x0d\ Above are the basic equipment and reagents for tissue culture, mainly highlighting their related alternatives, because as a rapid propagation in tissue culture, it is quite less demanding. Our home culture is different from factory production, which is looking for high proliferation rate in producing tissue culture 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 paraphernalia and equipment, and substitutes always have certain defects. \x0d\\\\\x0d\(I) Necessary facility items and substitutes \x0d\1, substitutes \x0d\ home-use refrigerator, can be used to store the culture medium master batch (4 ℃) and need to be stored at low temperatures of drugs (such as biological regulators). \x0d\\ autoclave: it can be used for culture medium, sterile water, glassware and other histoculture utensils sterilization sterilization. If the hardness of the water used can be cool white water, disinfection and sterilization. Avoid scaling on the surface of sterilized items. \x0d\ stainless steel pot or aluminum pot: it can be used for culture medium, melting agar (to play the role of water bath) and histoculture utensils sterilization sterilization. \x0d\\ Brushed discarded cooking oil drum: it can be used to store distilled water. \x0d\ small white porcelain plate: it can be used for inoculation and holding disinfectant (if you put disinfectant, do not eat it again to avoid poisoning). \x0d\ fluorescent lamp: it can be used for light source supplementation during histoculture. \x0d\\ Wash discarded canning jars: it can be used for histoculture, instead of conical flasks, test tubes. \x0d\\ 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 more family members. If air-conditioning has been installed in the room, it will be beneficial in the primary and secondary cultures, and even in the transition of seedlings. \x0d\2, homemade utensils \x0d\(1) the production of inoculation box: the ultra-clean bench in the process of histoculture is very expensive equipment. If we replace it with homemade inoculation boxes, we can save a lot of money and help popularize it. \x0d\\ homemade inoculation box of materials, can be plywood, fiberboard, glass (3 mm thick), wood strips (decoration of the house with the keel), and can even be used to make cardboard boxes (the quality of the box to be slightly better, such as the box of the TV set), can also be used in plexiglass. \x0d\\ Their inoculation box size can be made according to the conditions of their respective homes. Make too small is not easy to operate, but relatively easy to disinfect, and occupies less space; make a larger easy to operate, but disinfection work does not seem easy, and occupies more space. Generally speaking, 70 centimeters long, 45 centimeters wide. 50 centimeters high is more appropriate. \x0d\(2) the production of culture box: culture box can replace the culture room, can also be used for the transition of seedlings to use. 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 one's family room and the amount of tissue culture. \x0d\3, need to purchase paraphernalia \x0d\ Ordinary balance (500 grams): used for weighing the preparation of medium drugs, or weighing with arbitrage weighing gold, silver and Chinese medicine, used for weighing the preparation of medium drugs, microelements and bioregulators; or buying microelements and bioregulators when they are purchased in already divided 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 medicines. 1 each of 1, 2, 5 ml pipette: used for preparing culture medium. \x0d\ high temperature plastic film or kraft paper: used to wrap the mouth of culture bottles and table paste homemade inoculation box internal corners. Rubber ring: used to tie the mouth of the culture bottle. Degreased cotton wool: used for operators and histoculture utensils alcohol cotton ball disinfection. pH (5 ~ 7) test paper 1 book: 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 more desirable to acquire 1 UV lamp for indoor and inoculation box disinfection. \x0d\(ii) Precautions during operation \x0d\ Home operation is different from that of the unit and therefore the following should be noted: Be safe and keep the medicines properly. Especially for the elderly and children; operation to disinfection carefully and strictly, inoculation operators wear masks, 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 to 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, winter; Anthurium group culture to be step by step to make, can first make easy to group cultivation of 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. Culture bottles generally use a variety of jam bottles, to first soak the bottle with detergent for 4 to 8 hours, and then rinse several times with running water. 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. The cleaned glass container should be inverted and emptied of water. \x0d\ 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. \x0d\ Pipettes should be cleaned with a suction bulb, repeatedly blowing on it with distilled water until it is clean, placing the pipette in a long tube for ease of use. Generally pipettes can only be used once, that is, to clean, so there is more than necessary to prepare a few pipettes, recommended: 10ml / 2, 5ml / 2, 1ml / 5. \x0d\ 2. Preparation of medium: \x0d\ The classic medium for tissue culture is the ms formulation, which is basically divided into: macroelements, trace elements, iron salts, organic complexes, plant hormones, sugars and supports. The dosage of all these components is in milligrams, which is difficult to weigh with an ordinary balance. In order to solve this problem, we can proportionally scale up the dosage of each component to formulate a masterbatch, and just add it according to a certain ratio when using. \x0d\ The following is the mother liquor preparation program of a ms culture medium is announced as follows: \x0d\ Massive elements: ammonium nitrate 66.0 grams; \x0d\ potassium nitrate 76.0 grams; \x0d\ anhydrous calcium chloride 13.3 grams; \x0d\ magnesium sulfate heptahydrate 14.8 grams; \x0d\ potassium dihydrogen phosphate 6.8 grams. \x0d\ The above are dissolved separately and combined and fixed to 1 liter, 25 ml for each liter of standard ms medium prepared. \x0d\ Iron salt: ferrous sulfate heptahydrate 5.56 grams; \x0d\ disodium ethylenediaminetetraacetic acid (edtana) 7.46 grams. \x0d\ The above two are dissolved separately by heating, mixed, and fixed to 1 liter, ph adjusted to below 5.5, and 5 ml taken for each liter of ms prepared. \x0d\ Trace elements and organic complexes are too minute to use, for simplicity we can substitute vegetable extracts or potato extracts. Usually we are accustomed to boiling 100 grams of spinach in 100 ml of water for 10 minutes, filtering and retaining the filtrate, and adding 20 to 50 ml of extract for every 1 liter of ms medium prepared. Similarly, you can use 100 grams of potatoes with skin plus 200 ml of water and boil for 15 minutes, filter and leave the filtrate, adding 50~100 ml per liter of ms. \x0d\ After adding the above components, you also need to add 30 grams of sugar and 7 grams of agar per liter of ms medium. \x0d\ It should be noted here that both sugar and agar are variable amounts and should be adjusted as needed. Also carrageenan for jelly making can be used instead of agar for better transparency. \x0d\ After each of the above ingredients has been added, set the volume to about 800 ml and microwave until all the agar has dissolved. At this point, add the desired plant hormone (usually formulated as a 0.1% solution, so that for every 1 ml taken, it is 1 ppm when converted to the medium). 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.\x0d\3. Preparation of culture medium using ms original powder:\x0d\ Some domestic chemical companies have now developed simple ms medium original 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 raw powder: ms culture medium raw powder, which includes macroelements, trace elements, iron salts, organic complexes, sugar and agar, is usually weighed according to the method of use, heated in the microwave oven to dissolve, added to the hormone, and fixed to 1100 ml (1.1 liters), and adjusted the ph value to 5.8~6.0. \x0d\ 4. Dispensing:\x0d\ Dispensing of prepared culture medium in culture containers Be careful to dispense the medium while it is still hot, as agar solidifies below 40 degrees Celsius. Each culture bottle contains about 1/6~1/5 of the volume of the bottle. Be careful not to hang the medium on the outer wall of the bottle, as this may cause contamination. After dispensing, seal the bottles with polypropylene film or an instant noodle bag, and secure them with an adhesive ring (try to choose the inner tube of a bicycle, which is more heat-resistant). \x0d\ 5. Sterilization: \x0d\ To sterilize the media in a home pressure cooker, it is recommended that you buy some "sterilizing effect test paper" from a medical equipment store, which can indicate the effect of sterilization, and will change color when the sterilization has been achieved. Place the test strips and media in the pot at the same time, heat until there is a lot of steam coming out, then add pressure to cut down and keep the heat low for 30 to 40 minutes. \x0d\ After sterilization is completed and natural cooling is achieved, remove the medium and place it in a cool, dust-free place for later use. \x0d\ 6. Preparation of inoculation box: \x0d\ The newly made inoculation box must be clean and as free from dead space as possible. 2 hours before each use, put all the items needed for the operation into the inoculation box, and then use a small beaker to add 3~5 grams of potassium permanganate into the inoculation box, in the box to the beaker to pour 2~3 ml of formaldehyde solution, about a few seconds after the formaldehyde vapors will be seen to appear, so that all the items in the inoculation box for the first time to be disinfected; and 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, it will stimulate the formation of oxygen ozone, ozone as the third line of defense for sterilization, so that after three times of sterilization, inoculation box can basically reach the standard of sterility. \x0d\ Within 15 minutes before operation, pour 5 ml of concentrated ammonia into the beaker just used to evaporate formaldehyde, because formaldehyde is toxic to the human body, and ammonia and formaldehyde can be formed into a solid substance called "hexamethylenetetetramine", which neutralizes the irritating vapors of formaldehyde. At this time, the UV lamp should not be turned off, but continue to irradiate until 5 minutes before the operation is turned off, 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 photo reactivation protein, which can restore the thymine tetramers under the environment of visible light and revive microorganisms, which is called "photo reactivation", so after turning off the ultraviolet light, it is necessary to maintain darkness for a few minutes to avoid photo reactivation. Therefore, after turning off the UV light, it is necessary to maintain darkness for a few minutes in order to avoid the emergence of photoreactivation. \x0d\\\\x0d\ How to carry out group culture at home \x0d\ \x0d\ Preparation \x0d\\ Home group culture because of the limitations of the conditions, it is not possible to configure a large number of preparation of the medium,,, generally 1 liter at a time to configure appropriate. After boiling and measuring with PH test paper, it is 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 it is necessary to increase the sterilization time appropriately. \x0d\\\\x0d\inoculation\x0d\inoculation\x0d\inoculation box will be sterilized with culture medium, sterile water, disinfectant water and the equipment used into the inoculation box, because the inoculation box in the volume of the relatively small, 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 cigarette 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. The inoculation work can only be started after the vapors in the box have dissipated, which takes about 5-10 hours. \x0d\ \x0d\ Inoculation, uncover the sealing in the inoculation box ventilation holes and operating holes on the seal, remove the fumigation of the magnetic cup, the inoculation of the material used to feed the inoculation box, turn on the ultraviolet lamp, fifteen minutes after the closure of ultraviolet lamps, turn on the lighting, you can begin to work. 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 box temperature is high, humidity is greater, the hand is 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. \x0d\ In fact, inoculation with an inoculation box is much more comfortable for people to feel when working compared to an ultra-clean bench. The contamination rate is also very low, and little contamination occurs after skillful operation. The state of healing tissue differentiation, seedling differentiation, and growth after inoculation is no different from that of inoculation on the ultra-clean bench. \x0d\\\x0d\\culture\x0d\culture in the amateur conditions of the family culture culture of the conditions can not be exclusively controlled, so to make full use of the natural conditions, after inoculation of culture bottles can be placed in a place with strong diffused light, such as bookcases against the window, writing desk, but to avoid direct daylight, the general room temperature of 22-28 degrees Celsius can be normal growth, when. 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 raise 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, it is undoubtedly a pleasure to place them in a bookcase or on a workbench and feel that you have cultivated a vibrant little creature. \x0d\ \x0d\ Cultivation\x0d\ When the seedlings grow to a certain point, you can configure some rooting with nutrient base to transfer the seedlings to cultivate roots, and when a certain amount of roots grow, you can plant them out of the bottle. Seedlings out of the bottle planting must pay attention to the following aspects, one is to properly reduce the temperature; two is to increase humidity; three 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 metatarsalite, 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. \x0d\ \x0d\\ family to carry out group cultivation in fact is not a very difficult thing, as long as more brains, think of some ways to do it is also very easy to do. Everything is difficult to begin with, and there are always more solutions than difficulties. 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 incredible that what they couldn't do could be done in a very closed mountainous area of ours. My point in saying this is not to brag about myself, but to tell friends who want to engage in this industry, do not look down on your own energy, do not be superstitious about authority, as long as you have confidence, and strictly in accordance with the laws of science, there is nothing that can not be done, there is nothing that can not be done. \x0d\ \x0d\\ The selection and effect evaluation of home culture medium for tissue culture \x0d\ \x0d\ The selection of suitable medium is the basis for the success of plant tissue culture. \x0d\ \x0d\\ Selection of suitable culture medium is mainly considered from the following two aspects: one is the basic medium; the other is the concentration and relative proportion of various hormones. \x0d\ \x0d\ 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. \x0d\ \x0d\ first try these mediums for preliminary experiments, can be less detour, greatly; reduce the consumption of time, labor and material resources. When through a series of initial trial, can then according to the actual situation of some of the components of the small adjustments. \x0d\ \x0d\\ 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