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

                   

                                       

                                                                                           

                                                                                                                                                

COMPONENTS OF A PLANT TISSUE CULTURE MEDIUM

The components of a plant tissue culture medium can be classified into

1.Macronutrients

2.Micronutrients

3.Vitamins

4.Sucrose

5.Growth regulators

6.Undefined components

7.Agar

1.Macronutrients These are nutrients required in large quantities. They include nitrogen, phosphorus, potassium, sulphur, calcium, magnesium, iron, carbon, hydrogen and oxygen

2.Micronutrients These are nutrients required in minute quantities.They are also called trace elements.They include Boron, Molybdenum, Manganese, Cobalt, Zinc, Copper and Iodine

3. Vitamins Mostly B complex vitamins such as thiamine, nicotinic acid, pyridoxine, pantothenic acid and inositol are used

4.Sucrose It is used as the principal energy source. It is used at a concentration of 3%.

5.Growth regulators Two classes of plant growth regulators are used viz. auxins and cytokinins

Auxins The natural auxin Indole acetic acid (IAA) is not used since it is photolabile. The primary role of auxins is in producing cell division and cell enlargement resulting in callus ( a mass of unorganized tissue) from explants. For this purpose  2,4 dichlorophenoxyacetic acid (2,4-D) is used in the medium. 2,4-D is also used in making a suspension culture ( liquid medium containing thousands of plant cells). The second role of auxins is in the promotion of rooting in Stage III of micropropagation.For this purpose either Indole butyric acid (IBA) or Naphthalene acetic acid (NAA) is used

Cytokinins They have two roles that are important in tissue culture. They stimulate cell division resulting in callus growth. They also promote proliferation of shoot buds.The natural cytokinins zeatin and Isopentenyl adenine are very expensive. Hence they are not usually used. The synthetic cytokinins usually used are Kinetin (6-furfuryl aminopurine) and Benyl aminopurine (BAP)

6.Undefined components Their composition varies from source to source They include

a) Coconut milk from tender coconuts used at a concentration of 10%

b) Casein hydrolysate ( a mixture of amino acids produced by the hydrolysis of casein, the milk protein)

c) Yeast extract ( a source of vitamins)

d) Activated charcoal which is used  to promote rooting since it adsorbs ethylene

7.Agar It is used to solidify the medium.It is a polysaccharide derived from red algae It is used at a  concentration of 0.8%

MEDIA FORMULATIONS

Through trial and error scientists have discovered various chemical combinations (formulas) for specific purposes. The best known standard formula is that developed by Murashige and Skoog in 1962. It is primarly designed for micropropagation of herbaceous plants. A formula developed by Brent McGown and Grey Lloyd is called the Woody Plant medium (WPM) meant for woody plants. 

Other commonly used media include Gamborgs (B-5) medium (primarly used for suspension cultures), Nitsch and Nitsch medium (used for anther culture), White's medium ( for root culture), Lin and Staba medium (for production of secondary metabolites) and Vacin and Went medium (for orchid seed culture.)

Tissue culture techniques continued

Inoculation of the explant

This is done in the inoculation room in the sterile atmosphere of a laminar flow hood. The sterile air gently streaming through the hood allows you to freely open sterile culture tubes/ bottles and do the inoculation. 

 

 If the hood has ultraviolet germicidal lamp it should be turned on at least 15 minutes before working in the hood. Turn it off while working in the hood to avoid exposure of eyes and skin to the harmful uv rays. Except for the filter the hood should be wiped down gently with a disinfectant such as 70% alcohol or isopropanol. The air flow should be turned on 15 minutes before using the hood. Wash your hands thoroughly with soap and water and wipe them with 70% ethanol or isopropanol. wear a lab coat if possible. 

Tools used for inoculation such as forceps and scalpels are first wrapped in aluminium  foil and sterilized in an autoclave  Later they are sterilized by flaming them periodically over a Bunsen burner or a spirit lamp. Alternately they can be sterilized by keeping them in a glass bead sterilizer in between use

                                                                  Glass bead sterilizer

Points to remember while using the laminar flow hood

1.Avoid any obstruction of the laminar air flow because it will change the air flow pattern and so cause contamination  Do not have items such as beakers or racks of test tubes between the HEPA (high efficiency particulate) filter and the exposed cultures

2.Work at arms length as far back in the hood as it is practical

3.Do not make wide sweeping arm movements over the work area since contaminants may drop from the arms or sleeves

The more careful you are the lesser the chance of contamination