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MS media preparation:

Stock solution I (500ml10times): Component KNO3 NH4NO3 MgSO4.7H2 O KH2PO4 CaCl2.2H2 O Procedure: A beaker was taken and washed it with distilled water. 200ml distilled water was taken in a beaker. Then 9.5 gm KNO3, 8.25gm NH4NO3, 1.85 gm MgSO4.7H2 O, 8.5 gm KH2PO4, 2.2 gm CaCl2.2H2O were added by turns and properly dissolved by magnetic stirrer. The solution was then taken in a measuring cylinder. Sufficient distilled water was added to make final volume 500ml. Stock solution II (200ml1000 times) Component H3BO3 MnSO4,4H2O ZnSO4 .7H2O Na2MoO4. 2H2O CuSO4.5 H2O COCl2.6H2O KI 1L 6.2mg 22.3 mg 8.6 mg 0.25 mg 0.025 mg 0.025 mg 0.83 mg 200ml1000 times 1.24gm 3.12 gm 1.72 gm 0.05 gm 0.005 gm 0.005 gm 0.166 gm 1L 1900mg 1650mg 370mg 170mg 440mg 500ml10times 9.5gm 8.25gm 1.85 gm 8.5 gm 2.2 gm

Sub stock solution(100ml100000 times) Component CuSO4.5 H2O COCl2.6H2O 1L 0.025 mg 0.025 mg 100ml100000 times 0.25 gm 0.25 gm

A beaker was taken and washed it with distilled water. 50 ml distilled water was taken in a beaker. Then 0.25gm CuSO4.5 H2O, 0.25 gm COCl2.6H2O were added by turns and dissolved by magnetic stirrer. The solution was taken in a measuring cylinder and adjusts the water at 100ml.

For preparation of stock solution II a clean beaker was taken and washed thoroughly with distilled water. 50 ml distilled water was taken in a beaker. Then 1.24gm H3BO3, 3.12 gm MnSO4.4H2O, 1.72 gm ZnSO4.7H2O, 0.05 gm Na2MoO4. 2H2O, 0.166 gm KI, were added respectively and dissolved with magnetic stirer. Then 2 ml of sub stock solution were added. The solution was then taken in a measuring cylinder and adjusted the volume at 100ml. Stock solution III (100ml1000times) Component Na2 EDTA Fe2SO4.7H2O 1L 37.3mg 27.8mg 100ml1000times 0 .373gm 0 .278gm

A clean beaker was taken and washed it with distilled water. 50ml distilled water was taken in the beaker and then 0.373gm Na2 EDTA, 0.278gm Fe2SO4.7H2O, was added. The constitutes were properly dissolved by magnetic stirrer. Then the solution was taken in a measuring cylinder. Sufficient water was then added to adjust the volume at 100ml. Stock solution IV(100ml1000times) Component Meso inositol Nicotinic acid Pyrodoxin Thymin Glycine 1L 100 mg 0.5 mg 0.5 mg 0.4 mg 2.0 mg 100ml1000times 10 gm 0.05 gm 0.05 gm 0.01 gm 0.2 gm

A clean beaker was taken and washed thoroughly with distilled water. 50 ml distilled water was taken in a beaker and then 10 gm Meso inositol, 0.05 gm Nicotinic acid, 0.05 gm Pyrodoxin, 0.01 gm Thymin, 0.2 gm Glycine was added. The constitute were properly dissolved by magnetic stirrer. Then the solution was taken in a measuring cylinder. Sufficient water was then added to adjust the volume at 100ml. Finally the stock solution was lebelled and stored at 4oC.

Media preparation (MS I) Procedure: 1. 200ml distilled water was taken in a clean conical flask which was washed by distilled water.

2. Then 50ml stock solution I, 5 ml stock solution II. 5 ml stock solution III. 5 ml stock solution VI was added periodically. 3. 15 gm sucrose was added. 4. 5 ml NAA stock solution was added. 5. The volume was adjusted at 500 ml by adding sufficient amount of distilled water. 6. The pH of the solution was then adjusted at 5.8-6.0 by adding 2 drops of 1N NaOH. The media was then despenced 7. 4 gm agar powder was then added in the solution. 8. The agar was melted by heating the solution at 110o C. 9. The media was then dispensed into test tube(~20 ml). 10. The media containing test tube was then sterilized by autoclaving at 1210 C for 20 min. The preparation of rice seeds for callus induction Mature seeds were dehusked, surface sterilized with 70% ethylalcohol for 30 sec, and then for 1 min with 5% sodium hypochlorite with 2 drops Tween-20 (commercial bleach is mainly hypochlorite). Surface sterilized seeds were rinsed several times with sterile distilled water before inoculation on callus induction medium.