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Preparing Murashige-Skoog Media: Step by Step Procedure
Preparing Murashige-Skoog Media: Step by Step Procedure

Preparing Murashige-Skoog Media: Step by Step Procedure

Anjali Singh

4th Aug 2020

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Can you imagine in-vitro culturing without using media?

“No way!” Right?

But, why?

The answer to the question is simple, because plants need nutrients for their growth and survival, like all other organisms. In a natural environment, they fulfill their needs by getting it through the atmosphere, soil, and by associating with other organisms.

However, when plants are cultured inside the lab, the support and nutrients for their growth are provided to the plants through media.

In the article “Tissue Culture Medium: Types and 5 Steps of Selection” you can learn about the various types of media used to culture in-vitro plants. You will also learn about the purposes served by different mediums, and how to select the right media for your plant in just 5 steps!

You must know, of all the media, the Murashige-Skoog (MS) medium is one of the most extensively used in plant tissue culture labs, worldwide. So, what is its recipe? What concentration of nutrients is required for the media?

This article will answer all of the above questions, with a short description of components of the media.

You will also find a handy chart that you can keep with you while preparing the media in your lab.

Major Components of MS media

The media involves the following four major components:

  1. Inorganic nutrient: It includes mineral salts that are important for the growth and development of the plants. It is categorized into two groups: Macronutrients (Calcium, magnesium, nitrogen) and micronutrients (copper, iron, and zinc).
  2. Organic nutrient: It mainly includes vitamins and amino acids, required for the growth and differentiation of the cultures.
  3. Growth hormones: It includes auxins, cytokinins, and gibberellins. It is essential for the growth and development of tissues and organs.
  4. Gelling agents: It includes agar and gelatin. It provides support to the cultures for their establishment.

You can refer to the article “Major Components of Tissue Culture Media” to read more about the components of the media.


Media Preparation

Preparation of stock

1. Micronutrient Stock (100X)

  • Take 400 ml double-distilled water in a 1L beaker, then weigh the salts given in the table below and dissolve it in the water.
  • Transfer the solution to the 1L volumetric flasks, and make up the volume to 1L. Pipette out 10 ml of the solution to make 1L MS media.

SaltsConcentration - 100X (mg/l)
MnSO4.4H2O2230
ZnSO4.4H2O860
H3BO3620
KI83
Na2MoO4.2H2O25
CuSO4.5H2O2.5
COCl2.6H2O2.5

2. Iron Stock (20x)

  • Take 80 ml double-distilled water in a 100 ml beaker, weigh the components given in the table and dissolve it completely (in the same order as given in the table).
  • Transfer the solution to a volumetric flask of 100 ml and makeup to the final volume.
  • Pipette 5 ml of the stock solution for 1L of MS media.

ComponentsConcentration-20X (mg/100 ml)
Na2EDTA672
FeSO4.7H2O556


3. Vitamin Stock (100x)

  • Take a 100 ml beaker and add 50 ml double-distilled water in it. Weigh the vitamins given in the table below and dissolve it completely in the water.
  • Transfer the solution to a volumetric flask of 100 ml and makeup to the final volume.
  • Pipette out 1 ml of the vitamin stock solution for 100ml of MS media.
  • Add vitamins after the media is autoclaved to protect it from heat degradation. Vitamins can be sterilized by ultrafiltration technique.

ComponentsConcentration-100X (mg/100ml)
Glycine20
Nicotinic acid5
pyridoxine.HCl5
Thiamine.HCl1

NOTE: Discard the vitamin solution after 30 days.


3. Cytokinin Stock (100X)

  • Weigh “10mg kinetin” and dissolve it into a few drops of 1N HCl.
  • Add a few ml of double-distilled water to the above solution and transfer it to the 100 ml volumetric flask and makeup to the volume.
  • Store the stock in the refrigerator.
  • Use 1 ml of the stock for 1L of the MS media.


Steps of the Preparation

Take 400 ml double-distilled water in a 1L beaker. Weigh the macronutrients given in the table below, and dissolve them completely into the water.


MacronutrientsConcentration-100X (mg/L)
(NH4)NO31650
KNO31900
CaCl2.2H2O440
MgSO4.7H2O370
KH2PO4170
  1. From the prepared stock solutions, pipette out 5ml iron, 10 ml micronutrient, and 1ml kinetin to the 1L beaker of the media.
  2. Weigh 100 mg Myo-inositol and dissolve it in the previous mixture.
  3. Weigh 10 mg IAA and dissolve it into a few drops of 1N NaOH. Then, transfer the solution to the previous mixture.

NOTE: The stock of IAA is not prepared because of its oxidative degradation.

  1. Add 800 ml of double-distilled water in the beaker and adjust the pH of the media to 5.7.
  2. Transfer the prepared solution to a 1L volumetric flask and make up the final volume to 1L.
  3. Keep the solution in the refrigerator.

Final steps

  1. Sterilize all the equipment andglass culture jars used for the tissue culture process.
  2. Weigh 0.8g of supreme grade agar and 3.0g reagent-grade sucrose and transfer them to 250 ml Erlenmeyer flask.
  3. Add 100 ml of the stored MS media, in the flask and seal the cap with aluminum foil.
  4. Sterilize the flask with the media.
  5. After sterilizing the media for 15-20 minutes, add 1 ml vitamin solution.
  6. Swirl the flask for the dissolution of the vitamin, agar, and sucrose into the media, before pouring it into the culture bottles.
  7. Pour the media into culture jars and store them in the refrigerator for 1 hour, before the culturing process.

Now, your culture bottles are ready for the tissue-culture processes.


Here is the handy chart of the MS media recipe for your experiments:


ComponentsConcentration for all-100X (mg/L)

except

For iron (20X): (mg/100ml)

For vitamin: mg/100ml

1. Macronutrients

(NH4)NO31650
KNO31900
CaCl2.2H2O440
MgSO4.7H2O370
KH2PO4170

2. Micronutrients

MnSO4.4H2O2230
ZnSO4.4H2O860
H3BO3620
KI83
Na2MoO4.2H2O25
CuSO4.5H2O2.5
COCl2.6H2O2.5

3. Iron (20X) (mg/100ml)

Na2EDTA672
FeSO4.7H2O556

4. Vitamins (100X)(mg/100ml)

Glycine20
Nicotinic acid5
pyridoxine.HCl5
Thiamine.HCl1

5. Cytokinin (mg/100ml)

Kinetin100
Myo-inositol10
IAA10
Sucrose10
Agar30,000
Anjali

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