Annexure-III

Preparation Of Plant Tissue Culture Stock Solutions And Media

 

Plant tissue culture media is prepared mainly using five different stock solutions. A general protocol for the preparation of nutrient stock solutions based on MS medium is given below:

  1. Preparation of Plant tissue Culture Stock Solutions

    • Preparation of macro stock Murashige and Skoog (MS-I)

    Macro stock Murashige and Skoog (MS-I) in 1000 ml

    S. No. Name of the chemical

    Molecular formula

    Quantity (g)
    (10X) (40X)
    1. Potassium nitrate KNO3 19.0 76.0
    2. Ammonium nitrate NH4NO3 16.5 66.0
    3. Potassium di-hydrogen phosphate KH2PO4 1.7 6.8
    4. Magnesium sulphate hepta- hydrate MgSO4.7H20 3.7 14.8

    • Take 500 ml double distilled water in a 2.0 liter beaker, weigh, add and keep on dissolving each salt sequentially in a descending order as listed above (dissolve by magnetic stirring), and finally make up the volume to 1000 ml by distilled water. Store at 4°C. Always check for precipitation in macro stock before using it for medium preparation.

     

    • Preparation of calcium stock Murashige and Skoog (MS-II)

      Calcium stock Murashige and Skoog (MS-II) in 1000 ml

      S. No. Name of the chemical

      Molecular formula

      Quantity (g)
      (10X) (40X)
      1. Calcium chloride dihydrate CaCl2.2H2O 4.4 17.6

      Take 500 ml double distilled water in a 2.0 liter beaker, weigh and add CaCl2.2H2O, dissolve by magnetic stirring, and finally make up the  volume to 1000 ml by distilled water. Store at 4°C.

       

    • Preparation of micro stock Murashige and Skoog (MS-III)

      Micro stock Murashige and Skoog (MS-III) in 1000 ml

      S. No. Name of the chemical

      Molecular formula

      Quantity (mg)
      (10X) (40X)
      1. Boric acid H3BO3 62.0 248.0
      2. Manganese sulphate mono-hydrate MnSO4.H20 169.0 676.0
      3. Zinc sulphate hepta-hydrate ZnSO4.7H20 86.0 344.0
      4. Potassium iodide KI 8.3 33.2
      5. *Sodium molybdate di-hydrate Na2MoO4.2H2O 2.5 10.0
      6. **Copper sulphate pentahydrate CuSO4.5H2O 0.25  1.0
      7. **Cobalt chloride hexa-hydrate CoCl2.6H20 0.25 1.0

      * Should be dissolved separately and then added to the stock solution

      ** Since it is difficult to weigh small quantities, prepare separate stocks of these salts @1.0 mg ml-l and then add required quantity. Take 500 ml of double distilled water in a 2.0 liter beaker, weigh, add and keep on dissolving each salt sequentially in descending order as

      ** Since it is difficult to weigh small quantities, prepare separate stocks of these salts @1.0 mg ml-l and then add required quantity. Take 500 ml of double distilled water in a 2.0 liter beaker, weigh, add and keep on dissolving each salt sequentially in descending order as listed above (dissolve by magnetic stirring), and finally make up the volume to 1000 ml by distilled water. Store at 40C.

      Note: Na2MoO4.2H2O should be dissolved separately in approximately 20ml of distilled water and added to the stock and MnSO4.H20 takes time todissolve.

     

    • Preparation of iron stock Murashige and Skoog (MS-IV)

      Iron-EDTA stock (MS-IV) in 1000 ml (10 X)
      S. No. Name of the chemical

      Molecular formula

      Quantity (mg)
      (10X) (40X)
      1. Sodium EDTA di-hydarte Na2EDTA.2H20 373.0 1492.0
      2.

       Ferrous sulphate heptahydrate

      		 FeSO4.7H20 278.0 1112.0

            Take 1000 ml double distilled water in a 2.0 liter amber colored bottle, and warm the water near boiling. Now weigh and add Na2EDTA.2H20 while stirring under a magnetic    stirrer; after Na2EDTA.2H20 has been  dissolved, add gradually FeSO4.7H20 while still under mild magnetic  stirring. This will yield a clear yellow solution. Immediately after adding FeSO4.7H20  close the bottle, keep on stirring at least for an hour and store at 40C.

     

    • Preparation of vitamin stock Murashige and Skoog (MS-V)
    S. No. Name of the chemical

    Quantity (mg)
    (10X) (40X)
    1. myo-Inositol 1000.0 4000.0
    2. Glycine 20.0  80.0
    3. *ThiamineHCI 1.0 4.0
    4. *Nicotinicacid 5.0 20.0
    5.

    *PyridoxineH CI

     

    		 5.0 20.0

    * Since it is difficult to weigh small quantities, prepare separate stocks of these salts @1.0 mg ml-l and then add required quantity.

    Take 500 ml double distilled water in a 2.0 liter beaker, keep on adding and dissolving the vitamins sequentially in descending order as listed above, and finally make up the volume to 1000 ml by adding distilled water. Store at 0-40C. Vitamin stock is very prone to microbial contamination in storage. Therefore, always check the stock before using.

     

  2. Media preparation

    Routine preparation of culture media using stock solutions is simple and involves following steps. The method has exemplified for preparing 1.0 liter of MS basal medium.

    Murashige and Skoog (MS) media for 1000ml
    S. No. Name of the chemical

    Quantity (ml)
    (10X) (40X)
    1. MS-I 100.0 25.0
    2. MS-II 100.0 25.0
    3. MS-III 100.0 25.0
    4. MS-IV 100.0 25.0
    5. MS-V 100.0 25.0

     
  3. Sterilization Techniques
    • 3.1 Steam sterilization

      • Plant tissue culture media are generally sterilized by autoclaving at 1210C (15 psi). The time required for sterilization depends on the volume of the medium in the vessel.

      • Always dispense medium in small aliquots whenever possible because many media components are broken down on prolonged exposure to heat and pressure.

      • Media exposed to temperatures in excess of 1210C may not properly gel or may result in poor culture growth.

      • Thermolabile components are prepared and filter-sterilized through a 0.2 μ filter in a sterile container.

      • The filtered chemicals is aseptically added to the culture medium, which has been autoclaved and allowed to cool to approximately 35-450C.

      • The medium is then dispensed under aseptic conditions in the laminar flow.

    • 3.2 Filter sterilization:

      For sterilization of small volume (50 ml), use syringe filters. Generally syringe filters are available in 4, 13 and 25 mm diameters. Readymade PTFE or cellulose ester membranes (0.2μ) are convenient to use since they are packed sterile, and manufactured with female luerlokTM inlet and male outlet as a standard; the filter housing can easily be connected to a syringe.

     

    • 3.3 Surface sterilization of explants:

      To prevent bacterial and fungal growth, tissue explants are surface sterilized before they are used to establish axenic/in vitro cultures. The most common disinfectants are listed below with the concentration and exposure. There is no universal working procedure for disinfecting plant tissue explants; the procedure varies from tissue to tissue and species to species. It is recommended that the experimenter standardize his/ her own protocol based on the following guidelines:

      • Wash the tissue explants with mild detergent (Tween-20) before treatment with the disinfectant solution.

      • Rinse the explants thoroughly under running tap water for 10-30min.

      • Submerge the explants into the disinfectant solution, seal the bottle and gently agitate.

      • Under sterile conditions, decant the disinfectant solution and rinse the explants several times with sterile distilled water.

        Commonly used disinfectants for plant tissue culture
        S. No.

         Name of the Disinfectants

        Concentration(%)

        Exposure time(min)
        1. Calcium hypochlorite 9-10 5-30
        2. *Sodium hypochlorite 0.5-5.0 5-30
        3. Hydrogen peroxide 3-12 5-15
        4. Ethyl alcohol 70-95 1-5.0
        5. Silver nitrate 1.0 5-30
        6. Mercuric chloride 0.1-1.0 1-5
        7.

        Benzalkonium chloride

        		  0.01-0.1  5-20

        *Commercial bleach contains about 5% sodium hypochlorite and thus may be used at a concentration of 10-20% which is equivalent to 0.5-1.0% sodium hypochlorite.

      • Take into account that the time required to sterilize liquids in autoclave depends on many factors. The most important is the volume of fluid that is being sterilized. In general, the time is the following:

        Volume (ml)

        Time (minutes)
        < 75 15
        75- 100 20
        250-500 25
        1000 30
        1500  35
        2000 40

       

      • Once the sterilization with autoclave is done, the items should be stored in optimal conditions. The culture media once they are cold are packed in plastic bags and refrigerated. Dry materials (petri dishes,cardboards and tools) are maintained in the oven dryer, erlenmeyers and sterile double-distilled water are placed on the respective shelf. When there is doubt about the sterility of any element to use, this should be considered contaminated and the process should be redone.