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Tissue Culture Medium: Types and 5 Steps of Selection

Posted by Anjali Singh on 7th Jul 2020

Tissue Culture Medium: Types and 5 Steps of Selection

Do you know if you are using the right medium for your culture?

The right selection of media is a crucial step for the in-vitro culture of plant cells. Many of us are not aware of what type of culture medium exist, what are their applications, and which medium will be most suitable for the culture.

It is essential to understand what medium you are using and how to select suitable media for your cultures. You should understand the requirement of your plant and then accordingly prepare the food it requires for its growth.

In the previous article “ Major components of the culture media”, all the major components and their concentration range required for suitable plant growth were discussed.

This article will introduce you to the different types of tissue culture media and will present you with a “broad spectrum of experiment” that will help you in media selection suitable for your culture.

Types of Media

There are a number of culture mediums known to date such as MS medium, B5 medium, LS medium, White’s medium, etc. This section will give you a brief of five widely used culture medium in labs worldwide.

  1. Murashige and Skoog (MS) medium

    This medium was invented by two scientists named Toshio Murashige and Folke K. Skoog in 1962 while the two scientists were working on the discovery of plant growth regulators. It is the most commonly used medium in the tissue culture lab.

    Sometimes you may observe some numbers behind the MS, which indicate the concentration of sucrose in the medium. For example, MS0 indicates sucrose absence and MS10 indicated the presence of 10g/l sucrose in the medium. The formulation is a blend of nutrients like inorganic salts, vitamins, and amino acids.

    Purpose: This medium is used to induce organogenesis, callus culture, micropropagation, and cell suspension.

  2. Linsmaier and Skoog (LS) medium

    This medium was developed by Linsmaier and Skoog in 1965. It was first used to optimize the organic supplements of the tobacco culture. The medium has a similar component as Murashige and Skoog with a twist of Linsmaier and Skoog vitamins.

    He found that the increased concentration of thiamine hypochlorite (0.4mg/l rather than 0.1mg/l) compensated for the absence of vitamins except for inositol. Inositol is an enzymatic cofactor in glycolysis and TCA cycle and it is also involved in primary and secondary metabolism of the plants.

    Purpose: It is used for the purpose of organogenesis, callus culture, cell suspension, and micropropagation.

  3. Gamborg (B5) medium

    This medium was developed by O. L. Gamborg in 1968. He used the media for the callus and cell suspension culture of Glycine max belonging to the family of Fabaceae. This medium is a blend of nutrients like inorganic salts, vitamins, and carbohydrates.

    The medium has a higher concentration of nitrate and potassium and a lower concentration of ammonia. Potassium nitrate is useful in inducing the soybean root callus formation and ammonium sulfate plays an essential role in cell growth.

    Purpose: It is used for the purpose of protoplast culture.

  4. Nitsch and Nitsch (NN) medium

    The medium was developed by J. P. Nitsch in 1969 for the establishment of the in vitro anther culture of Nicotiana, from family Solanaceae. It contains a high concentration of thiamine, biotin, and folic acid that supports anther callus.

    Purpose: To establish the in vitro anther culture.

  5. White’s Medium

    The medium was developed by P. R. White in 1963 for the establishment of the root culture of tomato. This was the earliest plant tissue culture media developed for root culture. It has a lower salt concentration and a higher concentration of MgSO4. The concentration of nitrate is 19% lesser than the MS media.

    Purpose: White’s medium can be used for the purpose of shoot culture and callus culture. It is suitable for culture Musa and Daucus species.

Media Selection

It is a very crucial step in tissue culture but there isn't one step to formulate a suitable media for the establishment of the culture. However, you can start working with three media having different salt concentrations, such as high salt concentration, medium salt concentration, and low salt concentration, to get the desired response from the culture.

A combination of different ratio of auxin and cytokinin can be used for shoot proliferation or adventitious shoot formation. Furthermore, a range of sucrose concentration (2-6%) can be tested to establish the culture. So, there are various opportunities to make the desired medium suitable for your culture by simply manipulating the concentration of nutrient salt and plant growth regulators.

A broad spectrum experiment is described by De Fosard et al to create the suitable media for your culture, especially if the system is untested.

  1. Divide the medium into four broad categories: (a) minerals, (b) auxins, (c) cytokinins, and (d) organic nutrients (sucrose, amino acids, inositol, etc.)
  2. For each group of the component, choose three concentrations: high, medium, and low.
  3. Try various combinations of the four groups of the components into three different concentrations. This will lead to an experiment with 81 treatments.
  4. Denote the best of 81 treatments with four-letter code. For example, the treatment with medium salts, low auxin, medium cytokinin, and high organic nutrients would be represented as MLMH.
  5. After reaching this stage different concentrations of auxin and cytokinin can be tested to determine the suitable concentration of growth regulators.

References

  1. Tissue culture media. (1996). Studies in Plant Science, 39–62. DOI:10.1016/s0928-3420(96)80005-x.
  2. Nandkishore Jha. Plant Tissue Culture Media: Types, Constituents, Preparation, and Selection. https://www.biologydiscussion.com/plants/plant-tissue-culture/plant-tissue-culture-media-types-constituents-preparation-and-selection/10656
  3. Abobkar I.M. Saad and Ahmed M. Elshahed (October 17th 2012). Plant Tissue Culture Media, Recent Advances in Plant in vitro Culture, Annarita Leva and Laura M. R. Rinaldi, IntechOpen, DOI: 10.5772/50569