1 Preparation of the mother liquid of the MS medium mother liquid Due to the different tissue culture plants, different mediums need to be prepared. In order to reduce the amount of workpieces, the medicine can be formulated as a concentrated liquid. The concentration of some trace elements and organic ingredients is extremely low. Therefore, the mother liquor is first prepared into a mother liquor. The recommended concentration is as follows: Drug Name Final concentration Prepare the total mass of 50ml (g) niacin 0.01g / ml 0.5 VB6 0.1g / ml 5 Glycine 0.2g / ml 10 NaMoO4.2H2O 0.05g / ml 2.5 CuSO4.5H2O 0.05g / ml 2.5 KI 0.1g / ml 5 H3BO3 0.062g / ml 3.1 MnSO4. H2O 0.169g / ml 8.45 ZnSO4. 7H2O 0.172g / ml 8.6 CoCl.6H2O 0.05g / ml 2.5 VB1 0.1g / ml 5 The process of preparing mother liquor: (1) According to the concentration in the table, weigh the mass of the medicine in a volume of 50ml. (2) Add to the corresponding solution and make up to 50ml. (3) Place in a sealed 50ml plastic tube and store at -20 ℃. (1) MS-A (macro): 50 times the concentration of working fluid NH4NO3 41.25g / 500ml KNO3 47.5g / 500ml MgSO4 · 7H2O 9.25g / 500ml KH2PO4 9.25g / 500ml (2) MS-B (micro): 100 times the concentration of working fluid H3BO3 0.124g / 200ml MnSO4 · 4H2O 0.338g / 200ml ZnSO4 · 7H2O 0.172g / 200ml CuSO4 · 5H2O 0.0005g / 200ml CoCl2 · 6H2O 0.0005g / 200ml KI 0.0166g / 200ml Na2MoO4 · 2H2O 0.005g / 200ml (3) MS-C: 100 times the concentration of working fluid CaCl2 · 2H2O 22.0g / 500ml (4) MS-D: 100 times the concentration of working fluid niacin 0.01g / 200ml VB6 0.01g / 200ml VB1 0.002g / 200ml Glycine 0.04g / 200ml Na2-EDTA 3.725g / 500m FeSO4 · 7H2O 2.875g / 500m 3 Preparation of mother liquor MS-A: Weigh 41.25g of NH4NO3, 47.5g of KNO3, 9.25g of MgSO4 · 7H2O, 9.25g of KH2PO4, dissolve in distilled water, and bring the volumetric flask to 500ml. Store in a refrigerator at 4 ° C. MS-B: The mother liquor from which the mother liquor is drawn is as follows, and the volume of distilled water is adjusted to 200 ml. Store in a refrigerator at 4 ° C. Mother liquor volume Final concentration of mother liquor H3BO3 0.124g / 200ml MnSO4 · 4H2O 0.338g / 200ml ZnSO4 · 7H2O 0.172g / 200ml CuSO4 · 5H2O 0.0005g / 200ml CoCl2 · 6H2O 0.0005g / 200ml KI 0.0166g / 200ml Na2MoO4 · 2H2O 0.005g / 200ml MS-D: The mother liquor from the mother liquor is as follows, and the volume of distilled water is adjusted to 200ml. Store in the refrigerator at 4 ℃. Mother liquor volume Final concentration of mother liquor niacin 0.01g / 200ml VB6 0.01g / 200ml VB1 0.002g / 200ml Glycine 0.04g / 200ml MS medium basic working fluid quality table Composition Quantity (mg / l) Composition Quantity (mg / l) NH4NO3 1650 Na2-EDTA 37.3 KNO3 1900 FeSO4 · 7H2O 27.8 CaCl2 · 2H2O 440 CuSO4 · 5H2O 0.025 MgSO4 · 7H2O 370 sucrose 30 KH2PO4 170 Inositol 100.0 KI 0.83 niacin 0.5 H3BO3 6.2 Pyridoxine hydrochloride 0.5 MnSO4 · 4H2O 22.3 Glycine 2.0 ZnSO4 · 7H2O 8.6 Ammonium hydrochloride 0.4 Na2MoO4 · 2H2O 0.25 CoCl2 · 6H2O 0.025 pH 5.8 (2) Add 30g of solid sucrose (adjust the concentration according to the specific experiment) and dissolve. (3) Add the required hormones. Make up to 1000ml. (4) 0.1-1mol / l HCl or NaOH adjusts the pH value. (5) If a solid medium is prepared, add a certain concentration of agar. The agar concentration is 10% to 15%. (6) The liquid culture medium can choose two methods: high temperature and high pressure sterilization and filter sterilization. The solid medium is sterilized by high temperature and high pressure. Pour into flasks and other culture vessels, seal and sterilize. (7) Precautions for working fluid preparation: Because the culture medium is rich in nutrients, extremely beneficial bacteria and fungi multiply, causing pollution and affecting the success of tissue culture, there are generally two methods of high temperature and high pressure disinfection and filter disinfection. High temperature and high pressure sterilization: Generally use a sterilization pot to sterilize. Note that the sterilization pot cannot be overfilled. Keep the pressure in the pot at 1.1Kg / cm2. The temperature is about 120 ℃, about 15-20 minutes. Filter sterilization: Some medium components that are easily damaged by high temperature, such as IAA, IBA, ZT, etc., should not be sterilized by high temperature and high pressure method. Filter sterilization can be used. Filter sterilization can be added to the medium that has been sterilized by high temperature and high pressure. It is carried out in a clean room or on an ultra-clean work piece table to avoid contamination of the culture medium. 1 Tier Iron With Powder Coating Dish Rack 1 Tier Iron With Powder Coating Dish Rack,1 Tier Powder Coated Dish Rack,Metal Dish Rack With Drain Tray,Standing Metal Dish Rack Jiangmen Jianghai Jianshang Houseware Co.,LTD. , https://www.jm-jianshang.com
2 MS medium mother liquor concentration table:
(5) MS-E: 100 times the concentration of working fluid
MS-C: Weigh CaCl2 · 2H2O 22.0g distilled water to dissolve, the volumetric flask is made up to 500ml. Store in a refrigerator at 4 ° C.
MS-E: Weigh 3.725g of Na2-EDTA and 2.875g of distilled water of FeSO4 · 7H2O to dissolve, the volumetric flask is made up to 500ml. Store in a refrigerator at 4 ° C protected from light.
4 Preparation of basic working solution of MS medium
(1) Prepare 1 liter of working solution, put about 700ml of distilled water in the beaker, and draw 20ml of MS-A mother liquor, 10ml of MS-B mother liquor, 10ml of MS-C mother liquor, 10ml of MS-D mother liquor, and MS-E mother liquor in order. 10ml.
Experimental procedure