Preparation of Medium Mother Liquor

Before preparing the culture medium according to the culture medium formula, for the convenience of use and accurate dosage, the the macroelements, trace elements, iron salts, organic matter, and hormones are often prepared into mother liquors several times more than the amount required by the culture medium formula, and then the mother liquor is absorbed according to the pre-calculated amount to prepare the culture medium.

Procedures

1. Preparation of Macroelement Mother Liquor

The mother liquor of macroelements in inorganic salts is prepared into a 10-fold solution, that is, the amount of various compounds is increased by 10 times according to the medium formula, taking the MS formula as an example (Table 1). Using an electronic balance with a sensitivity of 1 mg, weigh various compounds except CaCl₂ in a 200 mL beaker, dissolve them in deionized water, and pour them into a 1000 mL volumetric flask after dissolution. Rinse the 200 mL beaker with a small amount of deionized water 3 times, and pour all the rinsing solution into the volumetric flask each time. Then add separately dissolved CaCl₂ (because CaCl₂ reacts with KH₂PO₄ to form water-insoluble precipitates such as calcium phosphate), and then dilute to 1000 mL with deionized water. Pour the prepared mixture into a 1000 mL narrow-mouth bottle, label it and store it in a refrigerator at 4°C. When preparing the culture medium according to the medium formula, take 100 mL of this solution for every 1000 mL of culture medium.

Table 1. MS Medium Macroelement Mother Liquor Preparation

2. Preparation of Trace Element Mother Liquor

The mother liquor of trace elements in inorganic salts is prepared into 100 times liquid, that is, the dosage of various compounds is expanded 100 times according to the medium formula, taking MS formula as an example (Table 2). Use an electronic balance to weigh various compounds in a 100 mL beaker, dissolve them in deionized water, and pour them into a 500 mL volumetric flask after dissolution. Rinse the 100 mL beaker with a small amount of deionized water 3 times, and pour all the rinsing solution into the volumetric flask each time. Then dilute it to 500 mL with deionized water, pour it into a 500 mL narrow mouth bottle, label it and store it in a refrigerator at 4°C. When preparing the culture medium according to the medium formula, take 10 mL of this solution for every 1000 mL of culture medium.

Table 2. MS Medium Trace Element Mother Liquor Preparation

3. Preparation of Iron Salt Mother Liquor

FeSO₄ and EDTA-Na₂ in inorganic salts are combined to form a 200-fold mother liquor, taking the MS formula as an example (Table 3). Weigh 27.9 mg FeSO₄ and 3.73 g EDTA with an electronic balance with a sensitivity of 1 mg, and dissolve them in deionized water respectively. Pour into a 500 mL volumetric flask, dilute to 500 mL, then pour into a 500 mL brown narrow mouth bottle, label it and store it in a refrigerator at 4°C. When preparing medium, take 5 mL of this solution for every 1000 mL of medium.

Table 3. MS Medium Iron Salt Mother Liquor Preparation

4. Preparation of Organic Mother Liquor

Vitamins and organic substances with small dosage are used to make 100 times mother liquor, taking MS formula as an example (Table 4). Use an electronic balance with a sensitivity of 0.1 mg to weigh various organic substances in a 100 mL beaker, dissolve them in deionized water, and then pour them into a 500 mL volumetric flask. Rinse the 100 mL beaker with a small amount of deionized water for 3 times, pour all the rinsing solution into the volumetric flask each time, and finally set the volume to 500 mL, pour it into a 500 mL narrow mouth bottle, label it and store it in a refrigerator at 4°C. When preparing medium, take 10 mL of this solution for every 1000 mL of medium.

Table 4. MS Medium Organic Mother Liquor Preparation

5. Preparation of Hormone Master Liquor

The concentration of the hormone mother solution is flexibly determined according to the requirement of the medium formula. The commonly used concentration of mother liquor is 0.2-2 mg/mL. Such substances must be prepared separately and weighed with an electronic balance with a sensitivity of 0.1 mg or 0.01 mg. The prepared hormone mother solution can be stored in a refrigerator at 4°C for several months. If precipitation or mildew is found in the mother liquor, it cannot be used any longer.

Note: Different solvents are used to dissolve various hormones. Auxin substances, such as 2,4-D, naphthaleneacetic acid, indoleacetic acid, etc., should be dissolved in 1-2 mL of absolute ethanol first, and then dilute to volume with deionized water. Cytokinin was first dissolved with a small amount of 1 mol/L HCl or 1 mol/L NaOH, and then made up to volume with deionized water. Folic acid was first dissolved with a small amount of dilute ammonia water, and then made to volume with deionized water.