Precautions for MS media, formulation of MS media

1.The preparation method of mother liquor and mother liquor is as follows: after several components in each mother liquor are weighed, they are thoroughly dissolved with a small amount of distilled water, and then they are miscible and finally the volume is fixed to 1 l.

2.The preparation method of the mother liquor is as follows: put the weighed Feso4·7H2O and Na2-EDTA·2H2O into 450 ml of distilled water respectively, stir them continuously while heating to dissolve them, then mix the two solutions, adjust the pH to, and finally set the volume to 1 L, and keep them in a brown glass bottle.

Plant growth regulators such as 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (naa benzylpurine (6BA) were also added to MS medium, and the mother liquor (0 1 mg ml) was prepared respectively. The preparation method is as follows: weigh 10 mg of each of these three substances, pre-dissolve 2,4-D and NaA with a small amount (1 ml) of absolute ethanol, dissolve 6BA with a small amount (1 ml) of substances with a concentration of mol L of NaOH solution, the dissolution process needs to be heated in a water bath, and finally the volume is set to 100 ml respectively, that is, the mother liquor with a mass concentration of 0 1 mg ml is obtained.

4.The success of tissue culture depends primarily on the choice of medium. Different media have different characteristics and are suitable for different plant species and inoculation materials.

When working on a tissue culture project, it is important to understand and analyze the various media so that you can choose the right one to use. The culture medium used in tissue culture generally includes basal culture medium and hormones, but the types and quantities of plant hormones vary with different culture stages and different materials, so plant hormones are not included in the formula of each medium. In addition to MS medium, there are also B5 medium and N6 medium.

When preparing the culture medium, attention should be paid to:

When using the pre-prepared mother liquor, the bottle containing the mother liquor should be gently shaken before measuring various mother liquors, and if it is found that there are precipitation, suspended solids or microbial contamination in the bottle, the mother liquor should be eliminated immediately and re-prepared; In order to prevent the mother liquor from being contaminated by microorganisms, the organic mother liquor is stored in the refrigerator4;

Before measuring the medium mother liquor with a graduated cylinder or pipette, the graduated cylinder or pipette must be washed twice with the measured mother liquor;

When measuring the mother liquor, it is best to write down the various mother liquors on the paper in the order in which they will be measured, measure 1 kind, and cross out 1 type to avoid mistakes. Dissolve the agar.

Weigh the agar 9 separately with a coarse balance

g. 30g of sucrose, put in 1

ml of enamel measuring cup, then add 750 distilled water

ml, heat with an electric stove and stir with a glass rod while heating until the liquid is translucent. Then the prepared mixed culture medium was added to the boiled agar, and finally distilled water was added to set the volume to 1

ml, stir well.

It should be noted that in the process of heating the agar and preparing the medium, the operator must not leave, otherwise the boiling agar will overflow and need to be re-weighed and prepared. In addition, if an enamel measuring cup is not available, a large beaker can be used instead. However, it should be noted that the outer surface of the bottom of the large beaker should not be wet, otherwise the beaker will easily explode when heated, causing the solution to overflow and cause scalding.

Adjust the pH with a dropper to absorb the amount of substance at a concentration of 1

Mol L of NaOH solution was dropped into the melted medium drop by drop, stirred while dropping, and the pH of the medium was measured with pH test paper at any time until the pH of the medium was about 6. Aliquoting of culture media.

The melted medium should be aliquoted while hot. To aliquot, pour the medium into a beaker and then into an Erlenmeyer flask (50

ml or 100

ml). Be careful not to allow the medium to get on the mouth and walls of the bottle. The amount of medium in an Erlenmeyer flask is about 1 5 1 4 of the volume of the Erlenmeyer flask. Every 1

ml medium, can be divided into 25 to 30 bottles. After the medium is dispensed, the bottle should be sealed in time. Use 2 pieces of sulfuric acid paper (each piece is about 9 in size

cm 9cm) sandwich 1 layer of thin kraft paper to seal the bottle mouth, and tie it with a rope. Finally, a label is applied to the outer wall of the Erlenmeyer flask.

1. A large number of elements (mother liquor).

nh₄no₃、kno₃、cacl₂·2h₂o、mgso₄·7h₂o 、kh₂po₄。

2. Trace elements (mother liquor).

ki、h₃bo₃、mnso₄·4h₂o、znso₄·7h₂o、na₂moo₄·2h₂o、cuso₄·5h₂o、cocl₂·6h₂o。

3. Iron salt (mother liquor).

feso₄·7h₂o₅、na₂-edta·2h₂o。

4. Organic ingredients (mother liquor).

Inositol, B niacin, pyridoxine hydrochloride (vitamin B6), thiamine hydrochloride (vitamin B1), glycine.

The dosage of the above various nutrients, except for the mother liquor, which is 20 times the concentrate, the rest are 200 times the concentrate.

1. Instruments and equipment required for the experiment.

2. Experimental arrangement and requirements: Several groups (6-7 people) in the class will prepare 500 mlms solid medium for each group, and 3. Explain the experimental methods and steps while demonstrating.

1. Calculate and fill in the media composition list.

Calculation formula: Mother liquor Preparation Mother liquor multiple.

Hormone Preparation Hormone Concentration Required in Culture Medium Hormone Concentration of Mother Liquor.

2.Take a 500ml beaker for each group, add 300ml of distilled water, weigh 4g of agar with a balance and put it into distilled water, heat it on an electric stove to dissolve. Weigh 15 g of sucrose into the dissolved agar.

3.According to the formula, take out various mother liquors, put them into the dissolved agar and sucrose solution, stir and mix with a glass rod, and add distilled water to set the volume to 500ml

4.The pH value is adjusted to 1 mol L of NaOH or 1 mol of LHCL, which can be determined with pH precision test strips or acidity meters.

5.Aliquot 500 ml of medium into 15 flasks of your choice using a media dispensing utensil.

6.Wrap the mouth of the bottle with parafilm, tampons or other closures and mark it.

7.Put the divided medium and various utensils to be sterilized, distilled water, etc., into the sterilization bucket of the autoclave, and add water to the outer pot to the water level. Cover the pot, put on the screws, and turn on the power to heat up.

When the temperature in the pot reaches or about, open the exhaust valve to drain the cold air in the pot, then close the exhaust valve, continue to heat until the temperature reaches or or, maintain 20 30min to cut off the power supply, let the air pressure in the pot drop naturally, when the pressure drops to zero, open the exhaust valve, discharge the remaining steam, and then open the lid to take out the medium and sterilized items.

8.After the culture medium and sterilized items are naturally cooled at room temperature, store in a cool, dry and clean place for later use.

4. Summarize the principle and general operation steps of autoclave sterilization.

1. A large number of elemental components:

Potassium nitrate kNO3: 1900 mg L; Ammonium nitrate NH4NO3: 1650 mg L; Potassium phosphate monobasic KH2PO4: 170 mg L;

Magnesium sulfate MGSO4·7H2O: 370mg L; Calcium chloride CaCl2·2H2O: 440 mg L.

2. Trace element components:

Potassium iodide ki: ; Boric acid H3BO3:; Manganese sulfate MnSO4·4H2O:;

Zinc sulfate ZnSO4·7H2O:; Sodium molybdate Na2MoO4·2H2O:;

Copper sulfate CuSO4·5H2O:; Cobalt chloride CoCl2·6H2O:.

3. Iron salt: disodium ethylenediaminetetraacetic acid Na2-EDTA:; Ferrous sulfate FeSo4·7H2O:.

4. Organic Ingredients:

Inositol: 100 mg l; Glycine: 2 mg l; Thiamine hydrochloride VB1:; Pyridoxine hydrochloride VB6:;

Niacin VB5 or VPP:.

5. Sucrose: 30g l

6. Agar: 7 g l

Normal! The multiplied drug may be a lot of elements! Because some plant explants are more likely to survive in a low concentration medium! That kind of medium is called 1 2ms medium!

MS medium is the most commonly used medium today.

It has a high concentration of inorganic salts, which is very beneficial to ensure the mineral nutrients required for tissue growth and accelerate the growth of callus.

Due to the high concentration of ions in the formula, even if some ingredients are slightly different in the process of preparation, storage, disinfection, etc., the balance between ions will not be affected.

MS solid medium can be used to induce callus, or for embryo, stem segment, shoot tip and anther culture, and its liquid medium can be used with significant success in cell suspension culture.

The amount and proportion of inorganic nutrients in this medium are appropriate to meet the nutritional and physiological needs of plant cells. Therefore, in general, there is no need to add organic additives such as amino acids, casein hydrolysate, yeast extract and coconut water. MS medium is characterized by its high content of nitrate, potassium and ammonium compared to the basic components of other media.

Carbon sources are generally used to synthesize cellular matter and metabolites. Nitrogen sources are used to synthesize proteins, nucleic acids, and nitrogen-containing metabolites. Water is a common use.

Inorganic salts said above.