Plant Tissue Culture Protocols

Media Preparation

 
Preparation from Packaged Powder
Preparation from Basal Salt Solutions
Banana Powder Preparation and Use
Coconut Water Preparation and Use

Preparation from Packaged Powder

 
Powdered media are extremely hygroscopic and must be protected from atmospheric moisture. If possible the entire contents of each package should be used immediately after opening. Preparing the medium in a concentrated form is not recommended as some salt complexes may precipitate. Supplements that are added to the medium may affect shelf life and storage conditions. The basic steps for preparing the culture medium are listed below:


  1. Measure out approximately 90% of the final required volume of tissue culture grade water (Product No.W 3500), e.g. 900 ml for a final volume of 1000 ml. Select a container twice the size of the final volume.
  2. While stirring the water add the powdered medium and stir until completely dissolved. Heating may be required to bring powders into solution.
  3. Rinse the original container with a small volume of tissue culture grade water to remove traces of the powder. Add to the solution in Step 2.
  4. Add desired heat stable supplements (e.g. sucrose, gelling agent, vitamins, auxins, cytokinins, etc.)
  5. Add additional tissue culture grade water to bring the medium to the final volume.
  6. While stirring, adjust medium to desired pH using NaOH, HCl or KOH.
  7. If a gelling agent is used, heat until the solution is clear.
  8. Dispense the medium into the culture vessels before (or after) autoclaving according to your application. Add heat labile constituents after autoclaving.
  9. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C, for the time period described under Sterilization of Media Protocol.
  10. Allow medium to cool prior to use.

POWDERED MEDIA AND BASAL SALT MIXTURES ARE FOR LABORATORY USE ONLY. NOT FOR DRUG, HOUSEHOLD OR OTHER USES.

MATERIALS NOT PROVIDED

  • Deionized tissue culture grade water (Product No. W 3500)
  • 1 N Hydrochloric Acid (HCl) (Product No. H 9892)
  • 1 N Sodium Hydroxide (NaOH) (Product No. S 2770)

 

STORAGE
Store dry medium in a desiccator at 0-5 °C. Deterioration of powdered medium may be recognized by: 1) color change; 2) granulation, clumping, or particulate matter throughout the powder; 3) insolubility; 4) pH change; or 5) inability to promote growth when properly used.

PRECIPITATION IN MEDIA
Precipitates are known to occur, with time, in plant tissue culture media. The precipitates have been analyzed (Sigma, unpublished data; Dalton, et al. 1983). They are composed of small, pale yellow-white particles. Analysis of precipitates indicated a predominance of iron, phosphate, and zinc. The probable cause of the precipitates is the inevitable oxidation of ferrous ions to ferric ions and the presence of unchelated ferric ions. When the solubility of ferric phosphate is exceeded precipitation occurs. There are no reports of detrimental effects on growth and development in plant tissue culture due to the precipitates.

 


 

Preparation from Basal Salt Solutions

Liquid 10X solutions are offered for your convenience. To avoid precipitation over long term storage, Sigma has formulated two solutions which when mixed at the proper dilution make a solution with the appropriate salt concentration. The basic steps for preparing 1 liter of culture medium are listed below. CAUTION: Do not autoclave product in bottle. The bottle is NOT autoclavable.


  1. Measure approximately 700 ml of tissue culture grade water (Product No. W 3500).
  2. While stirring the water, add 100 ml of Macronutrient Solution.
  3. Continue stirring the mixture while adding 100 ml of Micronutrient Solution.
  4. Add desired heat stable supplements (e.g. sucrose, gelling agent, vitamins, auxins, cytokinins, etc.).
  5. Add additional tissue culture grade water to bring the medium to the final volume.
  6. While stirring, adjust medium to desired pH using NaOH, HCl or KOH.
  7. If a gelling agent is used, heat until the solution is clear.
  8. Dispense the medium into the culture vessels before (or after) autoclaving according to your application. Add heat labile constituents after autoclaving.
  9. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C, for the time period described under Sterilization of Media Protocol.
  10. Allow medium to cool prior to use.

BASAL SALT SOLUTIONS ARE FOR LABORATORY USE ONLY. NOT FOR DRUG, HOUSEHOLD OR OTHER USES.

MATERIALS NOT PROVIDED

  • Deionized tissue culture grade water (Product No. W 3500)
  • 1 N Hydrochloric Acid (Product No. H 9892)
  • 1 N Sodium Hydroxide (Product No. S 2770)
  • Medium additives as required

STORAGE
Store basal salt solutions at 0-5 °C. Deterioration of basal salt solutions may be recognized by: 1) color change; 2) pH change; 3) precipitation of components; or 4) inability to promote growth when properly used.

 


 

Banana Powder (Product No. B 4032)

Sigma offers a banana powder for use in orchid and other plant cell culture. Product number B4032 is a powder from a spray-dried mixture of banana and dextrin. Use this product at approximately 40 g/L. To reduce clumping, add powder slowly to the culture medium with constant stirring. The presence of banana solids is common in medium containing both of these products.

 


 

Coconut Water (Product No. C 5915)

Coconut water has been shown to stimulate shoot proliferation in many species of plants. Coconut water is prepared from selected coconuts and processed to remove most of the protein. The product is then filter sterilized and frozen prior to shipment. Remaining protein levels in the water may vary from one lot to the next and may result in precipitate when the product is frozen. This precipitation should not effect the growth of the plant tissue. The precipitate can be removed by filtering or by allowing it to settle to the bottom of the bottle and then decanting. Coconut water can be divided into smaller aliquots, corresponding to your standard medium batch size, and refrozen until needed. Coconut water should be used at a concentration of 5-20% (v/v).

 

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