Preparing Murashige and Skoog (MS) media with gellan gum for plant tissue culture involves several steps. Gellan gum is commonly used as a gelling agent in plant tissue culture media to provide a solid substrate for the growth of plant tissues. Here's a general procedure for preparing MS media with gellan gum:
Ingredients:
- -Murashige and Skoog (MS) basal salts
- -Sucrose or other carbohydrate source
- -Gellan gum
- -pH adjusters (potassium hydroxide or hydrochloric acid)
- -Plant growth regulators (if required)
- -Distilled water(if hard water,you should reduce the ca2+)
Procedure:
Weighing and Mixing:
- Weigh the appropriate amount of Murashige and Skoog basal salts according to the desired concentration. Add the salts to a large container.
Sucrose Addition:
- Weigh the desired amount of sucrose or other carbohydrate source and add it to the container with the MS salts. Mix the components thoroughly.
Gellan Gum Dispersion:
- Weigh the required amount of gellan gum separately. Gellan gum is usually added at a concentration of around 2 to 3 grams per liter of medium. To disperse gellan gum, it is recommended to sprinkle it onto the surface of the liquid medium while stirring continuously to prevent clumping.
Autoclaving:
- Once the gellan gum is dispersed, add distilled water to the mixture while stirring to reach the final volume. Adjust the pH of the medium using potassium hydroxide or hydrochloric acid. The pH should typically be around 5.7.
- Autoclave the medium to sterilize it. The autoclaving process helps to melt the gellan gum and dissolve it in the medium.
Cooling and Pouring:
- After autoclaving, allow the medium to cool to around 50-55°C. At this temperature, the gellan gum will start to solidify and gel. While the medium is still in a liquid state, pour it into sterile culture vessels (e.g., culture flasks or Petri dishes) to solidify.
Solidification and Sterilization:
- Allow the medium to solidify at room temperature. Once solid, the gellan gum provides a gel-like consistency for the plant tissues to grow on.
- Optionally, if you need to add heat-sensitive components like plant growth regulators, you can wait until the medium cools to a temperature where these components won't be degraded and then aseptically add them to the medium.
Storage:
- Seal the culture vessels and store them in a sterile environment until needed for plant tissue culture experiments.
It's important to note that the specific concentrations of MS salts, gellan gum, and other components may vary based on the plant species and the specific requirements of your tissue culture experiment. Always follow aseptic techniques and work in a clean and sterile environment when preparing plant tissue culture media. Additionally, refer to literature specific to your plant species for any adjustments to the standard MS medium formulation.
