Standard Operating Procedures for MS Medium Preparation

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 ℃.

2 MS medium mother liquor concentration table:

(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

(5) MS-E: 100 times the concentration of working fluid

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-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-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-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

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

(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.

(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.

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