PLANT TISSUE CULTURE MEDIUM -continued

Preparation of the medium

The preparation of plant tissue culture medium requires clean glassware, water of high quality, pure chemicals and the careful measurement of  medium components

Stock solutions of nutrients

Stock solutions are concentrated solutions of single or groups of chemicals, portions of which will be subsequently combined to make the final medium. It is better to prepare these stock solutions ahead of time in relatively large batches.

Stock solutions are stored in the refrigerator Inorganic stock solutions are more stable than organic If sediments or contaminants appear the solutions should be discarded

As an example let us make the stock solutions of Murashige and Skoog medium The formula of this medium is given below 

                   

Components	mg/l
Ammonium nitrate	1650
Potassium nitrate	1900
Calcium chloride	333
Magnesium sulphate	181
Potassium dihydrogen orthophosphate	170
Iron Sodium EDTA	36.7
Boric acid	6.2
Manganese sulphate	16.9
Zinc sulphate	8.6
Potassium iodide	0.83
Sodium molybdate	0.25
Copper sulphate	0.025
Cobalt chloride	0.025
Inositol	100
Thiamine HCl	0.4
Pyridoxine HCl	0.5
Nicotinic acid	0.5

The minerals can be grouped into three categories

1  .Macronutrient stock

2.  Micronutrient stock

3.  Iron EDTA stock

1 Macronutrient Stock   Macronutrient salts are made into a 10 times concentrated stock solution. The amounts to be weighed are as follows 

 

Ammonium nitrate	16.5 gms
Potassium nitrate	19 gms
Calcium chloride	3.33 gms
Magnesium sulphate	1.81 gms
Potassium dihydrogen orthophosphate	1.70 gms

 

 These salts are dissolved in 1 litre of distilled water. 100 ml of this stock solution can be used to make 1 litre of MS medium

 

2.Micronutrient Stock   Micronutrient salts are made into a 100 times concentrated stock solution the amounts of various salts to be weighed are as follows

 

Boric acid	620 mg
Manganese sulphate	1690 mg
Zinc sulphate	860 mg
Potassium iodide	83 mg
Sodium molybdate	25 mg
Copper sulphate	25 mg
Cobalt chloride	25 mg

 

Of these copper sulphate and cobalt chloride are both dissolved in 10 ml of distilled water and 1 ml of this is added to 1 litre of distilled water to which the rest of the weighed chemicals are also added and dissolved. 10 ml of this micronutrient stock can be used while preparing 1 litre of MS medium.

 

3.Iron EDTA stock.    Since ferrous sulphate added alone will lead to precipitation of iron, a chelating agent such as EDTA (Ethylene diamine tetra acetic acid) is used. Their amounts to be used are as follows.

 

Ferrous sulphate	2.78 gms
Sodium EDTA	3.73 gms

 

The two salts are separately dissolved in 500 ml each of distilled water and then mixed together. 10 ml of this stock solution is used per litre of MS medium.

Stock solutions for vitamins and hormones are prepared as follows

4. Vitamin stock  The amounts of vitamins to be weighed for making stock solution are

Inositol	100 gms
Thiamine HCl	40 mg
Pyridoxine	50 mg
Nicotinic acid	50 mg

These are dissolved in 100 ml of distilled water. 1 ml of this stock will give the appropriate amounts per litre of MS medium.

2.Hormone Stock.

a) Auxin Stock  A convenient stock solution of 2,4-D for example contains 1 mg per ml. To prepare 100 ml of this stock weigh 100 mg of 2,4-D into a container and add few drops of a 1 N Sodium hydroxide (40 gms in I litre of water) till 2,4-D dissolves. Make up to 100 ml with distilled water.

b) Cytokinin Stock  Similarly a convenient stock solution of a cytokinin such as kinetin can be made by weighing 100 mg of kinetin into a container and adding about 5 ml of 0.1 N hydrochloric acid (Remember always add acid to water and not vice versa.) Add 95 ml of water to make up to 100 ml. 1 ml of this will contain 1 mg of kinetin.

Stock solutions of vitamins and hormones should be stored in the freezer compartment of the refrigerator.

Procedure for medium preparation

To prepare 1 litre of MS medium the following volumes of stock solutions are added

1.Add 100 ml of macronutrient stock

2.Add 10 ml of micronutrient stock

3.Add 10 ml of Iron EDTA stock

4.Add 1 ml of vitamin stock

5.Add appropriate amounts of hormones

6.Add 30 gms of sucrose

7.Make up the volume to 1 litre

8.Stir and dissolve sucrose

9.Adjust pH to 5.8

10.Weigh and add 8 gms of agar

11.Heat on a burner till the medium boils and the agar dispenses

12.Dispense medium into bottles or test tubes ( 50 ml per bottle or 15 ml per tet tube)

13.Cap the containers( plastic caps for bottles and non adsorbent cotton for test tubes)

14.Place the containers in an autoclave and sterilize at 121 C for 20 minutes

15.Take out the bottles and allow the medium to cool and solidify