Preparation of Cytokinin Stock Solution
Materials and Equipments
1.. One litre capacity flask. 2. Test tube or jar. 3. Sucrose. 4. Water. 5. Agar. 6. PGR (cytokinin).
Stock solution N1, N2, N3, N4, and N5. 8. Myo inositol. 9. NaOH. 10. HCL. 11. Cotton cap or plug. 12. Autoclave.
Procedure
Take 50 mg of cytokinin in a clean dry test tube.
Dissolve in 1 – 2 ml of 1N NaOH.
Slightly warm it.
Add more distilled water to make the volume 100 ml.
The final concentration of this stock will be 0.5 mg/ml.
Steps involved in medium preparation (MS 1 litre medium)
Step 01 of 16….
Take 1 liter capacity flask.
Step 02 of 16….
Add 50 ml of stock solution number 1.
Step 03 of 16….
Add 20 ml each of stock solutions number 2 and 3.
Step 04 of 16….
Add 20 ml each of stock solutions number 4 and 5.
Step 05 of 16….
Add 100 mg of Myo inositol.
Step 06 of 16….
Add 30 g of sucrose.
Step 07 of 16….
Now add disttiled water to make volume to 900 ml.
Step 08 of 16….
Dissolve sucrose and myo inositol by continuous stirring or shaking the flask.
Step 09 of 16….
Add require concentration of growth regulator.
Step 10 of 16….
Adjust the pH of medium to 5.8 with 1 N NaOH/HCL.
Question 11 of 16
Add 7 g of agar (solidifying agent).
Step 12 of 16
Make final volume to 1 litre with distilled water.
Step 13 of 16….
Boil the medium and stir while boiling to dissolve the agar.
Step 14 of 16….
Pour the boiled medium (15 – 20 ml) in the tube of jar and plug with the cotton cap, respectively.
Step 15 of 16….
Autoclave the media at 121 °C, at 15 lbs/square inch pressure for 20 – 25 minutes.
Step 16 of 16….
Store the media after autoclaving at room temperature for 2 – 3 days, before inoculation.
Precaution:
Avoiding precipitation
Cause of precipitation: It is due to mixing of mono and divalent salts.
Solution: Prepare stock solutions (no. 1 – 5) as recommended.
Storing stock solution
Store stock solution in dark cloud bottles.
Labelling bottles
Cause: Wrong labelling of bottles.
Solution: Properly label the bottles before storing in refrigerator.
Media preparation
Cause: Using precipitated stock solution.
Solution: Never use precipitated stock solution in media preparation.
pH level of of Media
Adjust the pH level of the media carefully otherwise media may not solidify after autoclaving.
Using media
Cause: Early is use of media.
Solution
For inoculation, always use the media at least 3 days after autoclaving.