There are two ways to prepare the culture medium. One is to buy all the chemicals in the culture medium and prepare them by yourself as needed. The second is to buy commercial mixed powder of basic components of culture medium, such as MS and B5.

self-preparation can save costs, but it wastes time and manpower, and sometimes it brings trouble to the experiment because of the quality problems of drugs. As far as the current domestic situation is concerned, most of them are still prepared by themselves. For convenience, the main process of preparing culture medium is introduced by taking MS culture medium as an example.

1. prepare several kinds of mother liquor

1. prepare the mother liquor of Ms. A large number of elements

Generally, a large number of elements are prepared into mother liquors of 1 times respectively, and then diluted by 1 times respectively when used.

weigh

nh4no3 165g kh2po4 17g

kno3 19g CaCl2 2h2o 44g

mgso4 7h2o 37g

and prepare 1L mother liquor respectively. Pour into 1L reagent bottle and store in the refrigerator.

2. prepare MS trace element mother liquor

trace elements are generally prepared into 1 times mother liquor.

weigh

ki.83g na2moo4 2h2o .25g

h3bo3 .62g cuso4 5h2o .25g

mnso4 h2o1.69gcocl2 6h2o.25g

znso4 7h2o.86g <

CuSO 4 5h2o and CoCl2·6H2O are weighed very little, so if the accuracy of the balance does not reach 1/1, they can be prepared into adjustment solution first.

weigh

CuSO4 5h2o .5g CoCl 2 6H2O .5g

respectively to make 1ml adjustment solution, and then take 5ml, and there is .25g left.

3. preparing MS organic mother liquor

generally, it is prepared into 1 times MS organic mother liquor.

weigh

inositol 1g thiamine hydrochloride (VB1) .1g

nicotinic acid .5g glycine .2g

pyridoxine hydrochloride .5g

in turn to make 1L mother liquor, pour it into 1L reagent bottle and store it in the refrigerator.

4. preparation of MS iron salt mother liquor

generally, 1 times of MS iron salt mother liquor is prepared.

weigh

disodium p>EDTA 3.73g feso4 7h2o 2.78g

in turn to make 1L mother liquor, pour it into 1L reagent bottle and store it in the refrigerator.

therefore, there are five kinds of macro-element mother liquor in MS mother liquor, plus eight kinds of mother liquor, namely, MS trace element mother liquor, MS organic mother liquor and MS ferric salt mother liquor.

preparation of hormone mother liquor

all kinds of auxins and cytokinins should be prepared separately, and cannot be mixed together. Generally, auxins should be dissolved with a small amount of 95% alcohol or 1 equivalent of NaOH, and cytokinins should be dissolved with 1 equivalent of hydrochloric acid first, and then distilled water should be added to constant volume. Generally, 1mg is taken to make 1ml mother liquor.

2. Preparing culture medium

Take the preparation of 1L MS culture medium as an example, and do the following operations in sequence:

1. First, put some distilled water in the beaker.

2. Take 1ml of the above eight mother liquors and pour them in.

3. Generally, 3g sucrose is weighed and poured in, and stirred to dissolve.

4. add distilled water and dissolve it to 1L with a measuring cylinder.

5. Add various hormones according to the designed scheme, because the dosage of hormones is very small and hormones are very important for the growth of tissue culture plants. Therefore, if possible, it is best to use a micro-adjustable pipette to absorb and reduce errors.

6. adjust the PH to 5.7 ~ 5.8 with precision test paper or acid meter. (Use an acid meter if possible, which is more accurate) 1 equivalent of HCL and 1 equivalent of NaOH can be used to adjust the PH value of the solution.

preparation of 1 equivalent HCL: measure 8.3ml with a measuring cylinder to prepare 1ml solution.

1 equivalent NaOH preparation: weigh 4g NaOH to prepare 1ml solution.

7. Weigh about 5g of agar powder (good quality agar powder), pour it into the solution prepared above, and heat it on the electric stove until agar powder melts.

8. After cooling slightly, put them into culture containers. The culture container without cover should be sealed with sealing film or kraft paper and tied tightly with rubber band or rope.

9. Sterilize in a sterilization pot for about 2 minutes.

1. after sterilization, take out the culture medium from the sterilization pot and lay it flat on the experimental platform to cool and solidify it. Sterilization is one of the important tasks in tissue culture. Beginners should be clear about the categories of bacteria and sterility. The category of bacteria is: all objects exposed to the air, objects in contact with natural water, at least its surface is bacteria. From this point of view, the untreated places such as aseptic room, the surface of ultra-clean table, simple boiled culture medium, the knives we use, the scissors before treatment, the whole appearance of our body and the inner surface connected with the outside world, such as the whole digestive tract and respiratory tract, that is, the gas we exhale, the culture container, no matter how clean it is, are all bacteria.

The bacteria mentioned here include bacteria, fungi, actinomycetes, algae and other microorganisms. The characteristics of bacteria are: extremely small and invisible to the naked eye. Everywhere, all the time, all the time. Under natural conditions, it has strong endurance, simple living conditions and strong fertility, and can breed in large quantities when conditions are suitable.

Sterility refers to: the objects that have been burned at high temperature or cooked for a certain period of time, the objects that have been treated by other physical or chemical sterilization methods (of course, these methods must have been proved to be effective), the upper atmosphere, the interior of rocks, the tissues of healthy animals and plants that are not in contact with the outside, and the surfaces and interiors of strong acids and alkalis, chemical element disinfectants, etc. are sterile. It can be seen from the above that the sterile world on the earth's surface is much smaller than the bacterial world.

sterilization refers to killing all microorganisms or organisms on the surface and pores of an object by physical or chemical methods, that is, killing all living substances. A related concept is disinfection, which refers to killing, eliminating or fully inhibiting some microorganisms so that they will no longer be harmful. Obviously, after disinfection, many bacterial spores and fungal chlamydospores will not be completely killed, that is, because there are still living microorganisms in the disinfected environment and articles, there are no living microorganisms in the strictly sterilized operating space (inoculation, ultra-clean table, etc.) and the utensils used, as well as the clothes and hands of the operators. The operation carried out under such conditions is called aseptic operation.

The requirements for aseptic conditions in plant tissue culture are very strict, even exceeding the requirements for microbial culture. This is because the culture medium is rich in nutrients, which will cause mixed bacteria pollution if it is not careful. In order to achieve the goal of complete sterilization, different practical and effective methods must be adopted according to different objects, so as to ensure that the culture is not affected by miscellaneous bacteria and the test-tube seedlings can grow normally.

The commonly used sterilization methods can be divided into physical and chemical categories, namely, physical methods such as dry heat (baking and burning), damp heat (normal pressure or high pressure cooking), radiation treatment (ultraviolet, ultrasonic and microwave), filtration, cleaning and washing with a large amount of sterile water. The chemical method is to use mercuric chloride, formaldehyde, hydrogen peroxide, potassium permanganate, lysol, bleaching powder, sodium hypochlorite, antibiotics and alcohol chemicals. These methods and agents should be properly selected according to different materials and different purposes in the work.

1. Sterilize the culture medium with moist heat

Sterilize the culture medium within 24 hours after preparation. The principle of high-pressure sterilization is: in a closed steamer, the steam can't overflow, and the pressure keeps rising, so that the boiling point of water keeps rising, and the temperature in the steamer also increases. Under the pressure of .1MPa, the temperature in the pot reaches 121℃. At this steam temperature, all kinds of bacteria and their highly heat-resistant spores can be killed quickly.

pay attention to completely eliminate the air in the pot, so that the pot is full of steam, and sterilization can be complete. There are several different ways of high-pressure sterilization and deflation, but the purpose is to exhaust the air, so that the temperature in the pot can be raised evenly and the sterilization can be ensured completely. Common methods are: close the bleeder valve, after electrifying, when the pressure rises to .5MPa, open the bleeder valve to release air, and then close the bleeder valve after the pressure gauge pointer returns to zero.

when the pressure gauge rises to .1MPa after the valve is closed and the power is turned on again, start timing and maintain the pressure at .1～.15MPa for 2min.

the holding time varies according to the container size, as shown in the table. The figures listed in this table are very safe for complete sterilization. If the container is large in size, but the number of containers is small, the time can also be reduced. Because there is an open process during inoculation, it is very easy to cause pollution, which is mainly caused by bacteria in the air and the staff themselves, so the inoculation room should be strictly disinfected. In the inoculation room, equipment, walls, floors, etc. should be regularly scrubbed with 1% ~ 3% potassium permanganate solution. In addition to sterilization with ultraviolet rays and formaldehyde before use, it can also be sprayed with 7% alcohol or 3% lysol during use to make dust particles in the air settle down. The aseptic operation can be carried out according to the following steps:

1. Fumigate the inoculation room with formaldehyde 4 hours before inoculation, and turn on the ultraviolet lamp in it for sterilization;

2. Turn on the fan in clean bench and the ultraviolet lamp on the stage 2 minutes before inoculation;

3. Inoculators should wash their hands first, change into special lab clothes and slippers in the buffer room;

4. After getting on the workbench, wipe your hands, especially your nails, with alcohol cotton balls. Then wipe the countertop;

5. Wipe the inoculation tool with an alcohol cotton ball first, then fire the tweezers and scissors from beginning to end, and then repeatedly fire the tip to dry the Petri dish;

6. During inoculation, the vaccinator should not leave the workbench with his hands, talk, walk and cough.

7. After inoculation, clean the workbench, and sterilize it with ultraviolet lamp for 3 minutes. If inoculation is continuous, sterilize it with great intensity every 5 days.

Inoculation is the process of cutting or cutting sterilized organs such as roots, stems and leaves into small pieces and putting them into culture medium. Now the procedures before and after vaccination are introduced coherently.

aseptic inoculation steps:

1. Put the preliminarily washed and cut materials into a beaker, bring them to an ultra-clean table, sterilize them with disinfectant, then rinse them with sterile water, and finally drain off the water, then take them out and put them on sterilized gauze or filter paper.

2. after the material is sucked dry, take tweezers in one hand, scissors or scalpel in the other, and cut the material properly. For example, the leaves are cut into small pieces of .5cm square; The stem is cut into small pieces containing a node. The micro-stem tip should be stripped to the size of the stem tip with only 1 ~ 2 young leaves. In the process of inoculation, the inoculation equipment should be burned frequently to prevent cross-contamination.

3. Implant or place the cut explants on the culture medium with the instruments that have been burnt and disinfected. The specific operation process (taking the test tube as an example) is as follows: firstly, unwrap the wrapping paper, hold the test tube almost horizontally, make the mouth of the test tube close to the flame of alcohol lamp, and rotate the nozzle above the flame to make the nozzle burn inside and outside for several seconds. If you cover the mouth with a cotton plug, you can first burn it outside the nozzle, remove the cotton plug and then burn it inside the nozzle. Then take a cut explant with tweezers and send it into a test tube, and gently insert it into the culture medium. If the leaves are directly attached to the culture medium, it is advisable to put 1 ~ 3 pieces. As for the material placement method, there is no uniform requirement except that the stem tip and stem segment should be placed upright (the tip is upward). After inoculation, burn the nozzle on the flame for several seconds. And use a cotton plug. After plugging, wrap it in wrapping paper, and the inside of wrapping paper should be overdone. Culture refers to the process of putting culture materials in a culture room (no light, suitable temperature, sterile) to make them grow, divide and differentiate into callus, and then further differentiate into regenerated plants under light conditions.

1. culture method

1. solid culture method

that is, the method of cultivating plant materials with agar solidified medium. Is the most commonly used method now. Although the equipment of this method is simple and easy to operate, the nutrient distribution is uneven, the growth rate is uneven, and browning poisoning often occurs.

2. liquid culture method

that is, the method of cultivating plant materials in liquid culture medium without curing agent. Due to the low oxygen content in the liquid, it is usually necessary to ensure the supply of oxygen by stirring or vibrating the culture solution. The reciprocating table or rotary table is used for culture, and the speed is generally 5 ~ 1 r/min. This method of regular immersion can not only make the culture medium uniform, but also ensure the supply of oxygen.

2. culture steps

1. primary culture

primary culture aims to obtain sterile materials and clonal lines. That is, after inoculating some explants, the first few generations of culture. In primary culture, induction or differentiation medium is often used, that is, the medium contains more cytokinins and a small amount of auxin. The clonal propagation lines established by primary culture include stem tips, buds, embryoids and protocorms. According to the development direction of primary culture, it can be divided into:

1) the development of terminal buds and axillary buds < P > Using exogenous cytokinins can promote the dormant lateral buds with terminal buds or without axillary buds to start growing, thus forming a miniature shrub-like structure with many branches and buds. In a few months, one branch of this clustered seedling can be transferred to subculture, and the propagation culture of bud seedlings can be repeated, and most tender stems can be obtained quickly. Then a part of the tender stems are transferred to the rooting medium, and a complete small plant which can be planted in the soil can be obtained. Some woody plants and a few herbs can also reproduce in this way, such as rose, camellia, chrysanthemum, carnation and so on. This propagation method, also known as micropropagation, is regenerated without callus, so it is the best propagation method to keep the original variety of clonal offspring.

When sampling plants suitable for this kind of regeneration, only terminal buds, lateral buds or stem segments with buds can be used. Others, such as taking branches after seeds germinate, can also be used.

Shoot tip culture can be regarded as a special way in this respect. It uses the apical meristem of extremely tender terminal bud as explant for inoculation. In practice, some tissues, including shoot apex meristem, are used for culture, which ensures convenient operation and easy survival.

Generally, the culture obtained by shoot-fixing by culture has a long stem node with an upright stem tip, and the method of cutting stem segments is mainly used for propagation, such as carnation, Petunia and chrysanthemum. However, under special circumstances, adventitious buds will also be born and form a bud cluster.

2) Development of adventitious buds

In culture, adventitious buds are produced from explants, and usually the cells of callus are formed by dedifferentiation. Then, after redifferentiation, the organ primordium is formed from these meristems, which shows unidirectional polarity on the longitudinal axis of the organ (which is different from embryoids). In most cases, it forms buds and then roots.

Another way is to produce adventitious buds directly from organs. Some plants have the ability to grow adventitious buds from various organs, such as Petunia, Fluoco, Rubus, etc. When cultured in vitro, the culture medium provides nutrients, especially continuous supply of plant hormones, which greatly stimulates the ability of plants to form adventitious buds. The surface of many kinds of explants is almost completely covered with adventitious buds. Species that cannot reproduce asexually in many conventional methods.