Detailed Explanation of Culture Medium Preparation Methods and Operating Steps

Stock solutions are generally prepared in advance at 10 to 100 times the required concentration, stored in the refrigerator during normal times, and only need to be measured out proportionally when preparing the culture medium. Taking MS medium as an example, according to the characteristics of the various chemicals, the stock solutions can be prepared separately as 6 types (see table) ┌───┬───────────────┬───────────────┐ │ │Preparation │Amount to take │ ├───┼───────────────┼───────────────┤ │Stock solution 1 │NH4NO3 82.5 g │When preparing 1 liter of medium with the 50x solution, take 20 mL │ │ │KNO3 950 g │ │ │ │MgSO4. 7H2O 18.5 g │ │ │ │Add water to make up to 1000 mL │ │ ├───┼───────────────┼───────────────┤ │Stock solution 2 │CaCl2 22.0 g │When preparing 1 liter of medium with the 100x solution, take 10 mL │ │ │Add water to make up to 500 mL │ │ ├───┼───────────────┼───────────────┤ │Stock solution 3 │KH2PO4 8.5 g │When preparing 1 liter of medium with the 100x solution, take 10 mL │ │ │Add water to make up to 500 mL │ │ ├───┼───────────────┼───────────────┤ │Stock solution 4 │Na2-EDTA 3.73 g │When preparing 1 liter of medium with the 100x solution, take 10 mL │ │ │FeSO4· 7H2O 2.78 g │ │ │ │Add water to make up to 1000 mL │ │ ├───┼───────────────┼───────────────┤ │Stock solution 5 │H3BO3 620 mg │ │ │ │MnSO4· 4H2O 2230 mg │When preparing 1 liter of medium with the 100x solution, take 10 mL │ │ │ZnSO4· 7H2O 860 mg │ │ │ │KI 83 mg │ │ │ │Na2MoO4·2H2O 12.5 mg │ │ │ │CuSO4·5H2O 1.25 mg │ │ │ │CoCl2·6H2O 1.25 mg │ │ │ │Add water to make up to 1000 mL │ │ ├───┼───────────────┼───────────────┤ │Stock solution 6 │Inositol 5 g │When preparing one liter of medium with the 100x solution, take 10 mL │ │ │Glycine 100 mg │ │ │ │Nicotinic acid 25 mg │ │ │ │Pyridoxine hydrochloride 25 mg │ │ │ │Thiamine hydrochloride 5 mg │ │ │ │Add water to make up to 500 mL │ │ └───┴───────────────┴───────────────┘ The prepared stock solution bottles should each be labeled, indicating the stock solution number, the preparation multiple, the date, and the amount to take when preparing 1 liter of stock solution. The prepared stock solutions can be stored in the refrigerator for later use (they can be kept for several months at low temperature). If mold and precipitate are found, they can no longer be used. When preparing the culture medium, various preparations must be made first: first arrange the stock solutions in order, then prepare the various required glassware, weigh out the required agar and sucrose, and prepare the required growth regulators. During preparation, first place a certain amount of water in a graduated cylinder or other type of container, to avoid splashing when adding the chemical solutions or precipitation caused by too high a concentration during mixing. Then, following the order of the stock solutions, measure out the specified amounts, add a certain amount of growth regulators as needed, and finally add the agar and sucrose; after adding, pour into a pot and heat to melt. The prepared culture medium should be adjusted for pH using 0.1 N HCl (or NaOH), generally maintained within the range of 5.1-6.0. The prepared culture medium should be dispensed while hot, poured into test tubes, Erlenmeyer flasks, and other culture vessels. After the culture medium is prepared, it should be sterilized under high pressure in a timely manner, and after cooling and solidifying, it can be used for inoculation. 3. Use of Growth Regulators Growth regulators are key substances in the culture medium and play a decisive role in plant tissue culture. (1) Auxins include NAA (naphthaleneacetic acid), IAA (indoleacetic acid), IBA (indolebutyric acid), etc. It is very sensitive in promoting root growth, and secondly it promotes the growth of shoots and stems. In rooting culture in tissue culture, it is the main promoting substance. (2) Cytokinins include BA (6-benzylaminopurine), KT (kinetin), ZT (zeatin), etc. It promotes cell division and promotes the growth of lateral buds; combined with a small amount of auxin, it is often used to induce massive proliferation and growth of buds. (3) Natural extracts. Some natural substances have a growth-promoting effect on plant tissues and cells. (Wang Huaiyu)