Tissue Culture Methods

The MS (Murashige and Skoog) medium is prepared by making stock solutions of various chemicals (both macro nutrients and micro nutrients) and later mixing them in required proportions in the distilled water to get desired concentrations.
Preparation of stock solutions of macro and micro nutrients of MS medium:
i) Stock solution A: 16.5 g of ammonium nitrate (NH4N03), 19 g of Potassium nitrate (KN03), 3.7 g of Magnesium sulphate (MgS04 7H20) and 1.7 g of Potassium dihydrogen phosphate (KH2P04) are weighed and dissolved in about 300 ml of distilled water. Then the final volume was made up to 500 ml with distilled water.
ii) Stock solution B: 41.5 mg of Potassium iodide (KI), 310 mg of Boric acid (H3B03), 111.5 mg of Manganese sulphate (MnS04 4H20), 430 mg of Zinc sulphate (Zn S04 7H20) and 12.5 g of Sodium molybdate (Na2 Mo04 2H20) were weighed and dissolved in 50 ml distilled water. 1.25 ml of stock solution of Copper sulphate (CuS04 5H20) and Cobalt chloride (CoCl2 6H20) is added. The final volume is made to 100 ml by adding distilled water.
Stock solutions of copper sulphate and cobalt chloride: 100 mg of Copper sulphate (Cu S04 5H20) and 100 mg of Cobalt chloride (CoCl2 6H20) is accurately weighed and dissolved in 50 ml distilled water. Then the final volume is made to 100 ml by adding distilled water.
iii) Stock solution C: 373 mg of Ethylene diamine tetra acetic acid disodium salt (Na2 EDTA) is weighed and dissolved in about 50 ml distilled water by constant heating and stirring. Then 278 mg of Ferrous sulphate (FeS04 7H20) is weighed and added slowly to the same solution with constant heating and stirring until completely dissolved. The final volume is then made to 100 ml with distilled water.

iv) Stock solution D: 200 mg Glycine, 50 mg of Nicotinic acid, 50 mg of Pyridoxine HCI and 10 mg of Thiamine- HCI are weighed and dissolved in 50 ml distilled water and the final volume is made to 100 ml by adding distilled water.
Preparation of stock solutions of hormones:
The Stock solutions (l mg/ml) of various hormones are prepared as follows-
i) Stock solutions of NAA and 2,4D : 100 mg powder of NAA or 2,4D is weighed into a 100 ml volumetric flask and dissolved in 2 to 5 ml of 1.0 N Sodium hydroxide (NaOH). After complete dissolution, the final volume is made to 100 ml by adding distilled water.
ii) Stock solutions of kinetin and benzyl adenine: 100 mg powder of BA or kinetin is weighed into a 100 ml volumetric flask and dissolved in 2 to 3 ml of 1.0 N Hydrochloric final volume is made to 100 ml by adding distilled water.
iii) Stock solutions of IAA and IBA: 100 mg powder of lAA or IBA weighed into a 100ml volumetric flask and dissolved in few drops of 90 per cent Ethyl alcohol. After complete dissolution, the final volume is made to 100 ml by adding distilled water.
All the stock solutions are stored in amber colored bottles and placed in refrigerator. The required quantities are pipetted out at the time of media preparation. The volume of stock
Steps followed during media preparation:
The medium can be prepared in glass beaker of 1000 ml and 2000 ml capacity depending on the requirement. The following steps are followed during the preparation of (one litre) medium:
1.30 g of Sucrose is weighed and dissolved in approximately 300 ml distilled water in a glass beaker.
2. 50 ml of stock solution 'A', 2 ml of stock solution 'B', 10 ml of stock solution 'C' and 1 ml of stock solution 'D' are added in sequence.
3. 440 mg of CaCl2 2H20 and 100 mg of Myo - inositol are added directly.
4. Required concentrations of hormone (s) are added to the medium and the volume is made approximately 650 ml.
5. 8.0 g of agar-agar is boiled in approximately 300 ml distilled water and added to the prepared solution.
6. The pH of the medium is adjusted to 5.8 with 0.1 N Sodium hydroxide (NaOH) or O.1N Hydrochloric acid (HCI) as needed and the volume is made up to 1.0 litre.
7. The medium is dispensed in culture vessels at the rate of 15-20 ml per tube or 25 to 30 ml per petri plate and the tubes are closed tightly with cotton plugs.
8. The culture vessels along with medium are autoclaved for 20 minutes after the pressure reached to 1.06 kg/cm2 at 121°C for sterilization.
9. The pressure is brought to normal slowly and the culture vessels are taken out of the autoclave and the tubes are kept for cooling in slanting position to get more surface area for explant inoculation. The culture vessels with medium are stored in sterile control room until use.
Surface sterilization of explants:
Mature seeds of the genotypes under study are surface sterilized with 70% ethanol for one minute followed by 0.1 % mercuric chloride for 10-15 minutes. Later the excess mercuric chloride is removed by rinsing the treated explants with sterile distilled water for 4-5 times. After rinsing, the seeds are blotted on sterile paper towels.
Germination of aseptic seedlings:

Filter paper boats are prepared and inserted into culture tubes and tightly closed with cotton plugs. These tubes were sterilized by autoclaving at 121°C and 15 lbs pressure for 20 minutes. In each culture tube, 15 ml of sterile water was poured under the laminar air flow and sterilized seeds were inoculated in culture tubes at the rate of 2-4 seeds/tube depending on the size of the seed and kept for germination under aseptic conditions.
Inoculation:
From the aseptically raised seedlings, explants Viz., root, hypocotyls, cotyledonary leaf, shoot tip etc., are excised with sterile scalpel blade under laminar air flow and used for inoculating in the corresponding callus induction medium.Cultures were scored for callus induction frequency at end of 4th week.
The morphological appearances of the callus is described in terms of its colourd and structure at 40 days after inoculation.