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Plant Cell Laboratory Experiment-Preparation of Plant Tissue Culture Medium
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Objective: Preparation of plant tissue culture medium for development of callus and suspension culture.

Nutritional requirement for optimal growth of a tissue in vitro may vary with the species. Even tissue from different parts of a plant may have different restrictive requirements for satisfactory growth. A defined nutrient medium consists of inorganic salts, a carbon source, vitamins and growth regulators. A gelling agent is also used in static medium. Murashige- Skoog (Murashige- Skoog, 1962), Whites (White, 1963), Eriksson (Eriksson, 1965), B5 (Gamborg et al, 1968), are well-designed medium in plant tissue culture.

 

Materials:

1.  Double Distilled water

2.  Beakers

3.  Magnetic Stirrer

4.  Acid & Base for adjustment of pH

5.  Weighing balance

 

Protocol for preparation of MS Medium:

 

Stock Solutions: For the preparation of stock solutions, chemicals are dissolved in distilled water. Usually the stock solutions are prepared in higher concentrations according to following procedure.

 

1.  MS Major Salts (10X)                    g/l

     KNO3                                           33.0

     NH4NO3                                      38.0

     MgSO4. 7H2                                7.4

     CaCl2.2H2                                   8.8

     KH2PO4                                      3.4

 

2.  MS Minor Salts (400x)

     Kl                                                 0.16

     H3BO3                                        1.24

     MnSO4.4H2O                             4.46

     ZnSO4.7H2O                              1.72

     Na2SO4.2H2O                            0.05    

     CuSO4. 5H2O                             0.005

     CoCl2.6H2O                                0.005

 

3.  Iron Solution (200x)

     FeSO4.7H2O                               5.56

     Na2EDTA.2H2O                           7.46

 

4.  Vitamins Solution (400x)

     Inositol                                          20

     Nicotine acid                                 0.1

     Pyridoxine. HCl                             0.1

     Thiamine.HCl                                0.02

     Glycine                                          0.4

 

5.  Growth Hormone stock Solution

     Indole Butyric Acid (IBA)               0.2 mg/ml

     Butyric Acid (BA)                          0.2 mg/ml

     Surcose                                        30 g

     Agar                                              8 g

 

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